POSTER ABSTRACTS - In Alphabetical Order
Poster 1
Normal Cell Expansion in Arabidopsis Requires a Specific Pectic Heteropolymer Containing Homogalacturonan Synthesized by GAUT1
Melani A. Atmodjoa,b,e, Robert A. Amosa,b, Li Tana, Ian M. Blacka, D.C. Amandaa, Ioana Petrascua, Joshua Indecha, Stefan Eberharda, Sindhu Kandemkavila, Sivakumar Pattathila, Michael G. Hahna,d, and Debra Mohnena,b,c
a. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA
b. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA
c. Center for Bioenergy Innovation
d. Department of Plant Biology, University of Georgia, Athens, GA, USA.
Presenting Author Email: matmodjo@ccrc.uga.edu
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Plant growth and development rely on cell expansion and cell wall extensibility. Here we report the characterization of an Arabidopsis T-DNA insertion mutant of GAUT1, an a-1,4-galacturonosyltransferase (GalAT) [1] that synthesizes pectic homogalacturonan (HG). Arabidopsis GAUT1 functions with its homolog GAUT7 in an enzyme complex [2] in vivo. In vitro the GAUT1:GAUT7 complex elongates HG acceptors into high molecular weight polymers and also initiates HG synthesis in the absence of exogenous acceptors [3]. Besides GAUT1, five other members of the 15-member Arabidopsis GAUT gene family have been biochemically shown to synthesize HG [4], leading to the question of why plants have a large gene family to synthesize the seemingly simple homoglycan HG.
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Arabidopsis gaut1-/- mutants are recovered as seedlings severely stunted in shoot and root growth due to significantly smaller cell size. The mutation also negatively affects reproduction by impeding pollen tube elongation and hindering normal seed development. To study the effects of the mutation on cell wall structure, we generated gaut1-/- suspension cell cultures and showed that those cells are also smaller and that they have reduced microsomal HG:GalAT activity and cell wall GalA content compared to WT. Analysis of wall fractions sequentially extracted from suspension culture cell walls using increasingly harsh solvents revealed that gaut1-/- has altered wall extractability compared to WT and other gaut mutants. Intriguingly, the largest reduction in GalA content in gaut1-/- versus WT occurs in the wall fraction produced by the harshest extraction step. Endopolygalacturonase digestion of this fraction resulted in a distinct band during high polyacrylamide gel electrophoresis and this band was substantially downregulated in the gaut1-/- sample compared to the WT. NMR analysis of the band suggests that it is an rhamnogalacturonan I (RG-I)-like structure that was branched or associated with neutral sugars. Based on these data we hypothesize that GAUT1 synthesizes a unique HG that is connected to branched RG-I in a pectic heteropolymer and that this polymer is tightly integrated into the wall and required for wall integrity and cell expansion. These findings support a unique role for GAUT1 and its synthesized HG in the plant cell wall.
Funding provided by The BioEnergy Science Center and The Center for Bioenergy Innovation, both U.S. Department of Energy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science.
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1. Sterling, J.D. et al. (2006) PNAS103, 5236-41.
2. Atmodjo, M.A. et al. (2011) PNAS 108, 20225-30.
3. Amos, R.A. et al. (2018) J. Biol. Chem. 293,19047-63.
4. Engle, K.A. et al. (2022) Plant J. 109:1441-56.
Poster 2
Two imaging mass spectrometry guided workflows for sialic acid isomer-targeted glycoprotein enrichment and direct glycopeptide identification in FFPE tumor tissues using the timsTOF-flex platform
Hongxia (Hellena) Bai, Peggi Angel, Richard Drake; MUSC, Charleston, SC
Presenting Author Email: hongxia.bai@bruker.com
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Our group has previously generated extensive N-linked glycan tissue maps of FFPE tumor tissues using N-glycan MALDI imaging workflows. These glycan distribution maps are now being used to identify the glycoprotein carriers present in distinct histological regions. Two strategies are being applied: (i) the direct analysis of intact glycopeptides from whole FFPE lysates, and (ii) the target-ed enrichment and subsequent characterization of α2,3-sialylated glycoproteins. For direct glyco-peptide analysis, lysates from one square centimeter of a 5 micron human prostate tumor or non-tumor tissue region were prepared and digested with trypsin. Glycopeptides were identified using an ion mobility (TIMS) and Parallel Accumulation Serial Fragmentation (PASEF) method with stepped low and high collision energies for differential fragmentation of peptides and glycans, targeting glycan-specific ions. Using MSFragger-Glyco for analysis, the prostate tumor section yielded 207 glycopeptides mapped to 47 proteins, while the adjacent non-tumor section revealed 82 glycopeptides correlating to 24 proteins. A spectrum of glycan structures, ranging from high mannose to complex, sialylated, and fucosylated forms were linked to the carrier proteins. For targeted α2,3-sialylated glycoprotein analysis, the same prostate tumor tissue was used with a biorthogonal derivatization approach specifically targeting glycoproteins with α2,3-sialylation that incorporates an azide linker. The azide was conjugated to magnetic alkyne beads, facilitating their selective capture. Glycoproteins bound to the beads were digested with trypsin to deter-mine a list of proteins captured, followed by PNGase F removal of bound peptides to identify spe-cific N-linked consensus sites. For prostate FFPE tissues, post-enrichment protein counts were 1,875 for tumor and 1,287 for non-tumor samples. Peptide analysis post-PNGase F deglycosyla-tion confirmed potential sialylation sites in 77 tumor and 61 non-tumor proteins. Direct lysate analysis identifies the glycopeptide landscape, while targeted enrichment identifies clinically rele-vant α2,3-sialylated glycoproteins. Additional work targeting other tumors, immune cells, and different protease digestion workflows is currently ongoing.
Poster 3
Drug Discovery for Sanfilippo Syndrome
Amrita Basu1, Madison Intermann2, Barry C. Maloney2, Antoinette C. Russell3, Christopher A. Rice4, Ryan J. Weiss1,2
1. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602
2. Department of Biochemistry and Molecular Biology, University of Georiga, Athens, GA 30602
3. Center for Tropical & Emerging Global Diseases, University of Georgia, Athens, GA, USA
4. Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, IN, USA
Presenting Author Email: Amrita.Basu@uga.edu
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Mucopolysaccharidosis Type IIIA (MPS IIIA), also known as Sanfilippo Syndrome Type A, is an inher-ited lysosomal storage disorder wherein patients are unable to catabolize cellular heparan sulfate due to inherited loss-of-function mutations in the gene encoding for the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH). Mutations in this enzyme lead to intra-lysosomal storage and accumulation of HS, which results in severe neuropathology, including regression of intellec-tual and motor abilities, behavioral problems, hearing loss, and dementia. Children born with this disorder exhibit developmental abnormalities, organ failure, and neurodegeneration, which often result in death within the first two decades of life. Unfortunately, there are no FDA-approved treatments for patients with MPS IIIA, and current strategies are focused primarily on symptom management. The main objective of this project is to identify small molecule therapeutic agents to lower the accumulation of HS in MPS IIIA as a form of substrate reduction therapy. We hypothe-size that small molecule agents that lower expression of the key HS biosynthetic enzyme, EXT1, could potentially reduce cellular HS levels and lysosomal accumulation in cells, thus restoring cell homeostasis. To explore this, we established a high-throughput drug screening assay using CRISPR-engineered EXT1 reporter cells and a library of FDA-approved drugs (2,320 compounds) to search for agents that could decrease EXT1 expression. Primary selection of screening hits was based on a cutoff of > 75% inhibition of EXT1 levels with minimal cytotoxicity. Intriguingly, many of the top hits from the screen shared common therapeutic targets and have previously been shown to cross the blood brain barrier. Treatment of patient-derived fibroblasts with the most promising com-pounds resulted in a significant reduction in EXT1 expression, intracellular HS levels, and lysoso-mal storage, as measured by the lysosomal membrane protein, LAMP1. Future studies will clarify the mechanisms of action for the repurposed drug candidates and test their efficacy in animal models of the disease. Overall, these studies have identified small molecule agents to reduce lyso-somal storage of HS and provide exciting targets to pursue for downstream drug development for MPS IIIA and related disorders.
Poster 4
Optimizing Glycan-Protein Interactions Through Molecular Dynamics Simula-tions: Insights from the Lectenz® Process
George N. Bendzunas1, Sheng-Cheng Wu1, Lu Meng1, Sylvain, Lehoux1, Robert J. Woods2, and Loretta Yang3
1Lectenz Bio, Athens, GA, USA; 2Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA; 3Lectenz Bio, San Diego, CA, USA
Presenting Author Email: gbendzunas@lectenz.com
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Abstract: Understanding and manipulating the intricate interactions between proteins and glycans is of paramount importance for advancing therapeutic and diagnostic innovations. However, the complexity and diversity of these molecular interactions pose significant chal-lenges. This study introduces an advanced application of molecular dynamics (MD) simulations aimed at optimizing the binding specificity and affinity of proteins to glycans, leveraging the innovative Lectenz® process. The Lectenz® methodology provides a strategic platform for the enhancement of enzyme, lectin, or glycan-binding protein (GBP) interactions with target-ed glycans, facilitating precise molecular recognition. Our approach harnesses MD simulations to explore the dynamic nature of protein-glycan interactions, enabling the identification of key structural modifications that enhance binding properties. Through a series of computa-tional analyses, we delineate the energetic and structural bases for improved glycan recogni-tion, leading to the rational design of proteins with superior binding characteristics. Here we delineate the computational framework employed in our MD simulations, highlighting the se-lection process for protein and glycan candidates and the evaluation metrics for binding effi-cacy. We present compelling case studies where this methodology has been successfully ap-plied to engineer proteins with significantly improved glycan-binding capacities, corroborated by subsequent experimental validations. Our findings not only demonstrate the potential of MD simulations in refining glycan-binding interactions but also underscore the versatility of the Lectenz® process in advancing glycoscience research. Supported by NIH grant OD035390.
Poster 5
Ian Black, Christian Heiss, Jiri Vlach, Parastoo Azadi.
Presenting Author Email: ianblack@ccrc.uga.edu
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Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize sam-ples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography–mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results because they are poorly soluble in DMSO and tend to undergo β-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides be-come much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more effi-ciently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This proto-col produces partially methylated alditol acetate derivatives in high yield with minimal β-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.
Poster 6
A Method for Identifying the Absolute Configuration of Carbohydrates Using Ion Mobility-Mass Spectrometry
Mya Brown, University of Georgia, mya.brown@uga.edu
Ron Orlando, University of Georgia, orlando@ccrc.uga.edu
Department of Chemistry, UGA
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Carbohydrates have multiple stereochemical forms and can exist in either a D- or an L- stereochemical configuration. For example, aldohexoses have four chiral centers and, thus, 24 = 16 isomers. There are eight D-isomers and eight L-isomers, with the D-configuration being the one typically found in nature for the hexoses. Stereochemical isomers create challenges when analyzing unknown glycans by mass spectrometry alone. We have developed a method to identify the absolute configuration of monosaccharides.
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In this method, the analyte is derivatized with an optically pure compound containing an additional stereochemical center, allowing the labeled species to be resolved by ion mobility mass spectrometry. Aldohexose monosaccharides were derivatized with amino acids by reductive amination with the reducing agent sodium cyanoborohydride. Monosaccharides derivatized in this manner showed separation in the gas phase by ion mobility. Preliminary results displayed that some amino acids provided better separations than others. For instance, monosaccharides tagged with amino acids containing a phenyl group, such as Tyrosine, were superior for resolving D-/L-aldohexoses. These results led us to predict that the larger the size of the tag, the better the ion mobility separation. Future work will extend these studies to slightly larger biopolymers as the tag, such as di-, tri-, tetra-saccharides/peptides, where one subunit will be in the D-/L-form. This work will describe a method using reductive amination and ion mobility mass spectrometry to determine the absolute configuration of carbohydrates without acid hydrolysis.
Poster 7
Probing O-mannosylated sites via biorthogonal Selective ExoEnzymatic Labelling (SEEL)
Ashley Carter1,2, Linda Zhao1, David Steen1,3, Jeremy Praissman1,, David Live1,3, Geert-Jan Boons1,2,4, Lance Wells1,3
1. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States
2. Department of Chemistry, University of Georgia, Athens, Georgia, United States
3. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States
4. Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands
Presenting Author Email: ashley.carter@uga.edu
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Defects in O-mannosylation of α-dystroglycan (α-DG) lead to dystroglycanopathies, which are congenital muscular dystrophies involving neurodevelopmental abnormalities. Interactions between α-DG and its extracellular matrix (ECM) ligands require α-DG to be extended by O-mannose structures based on the M3 core structure by the glycosyltransferase POMGNT2. The core M3 can be extended with matriglycan, which is a repeating disaccharide that binds laminin globular domain containing proteins in the ECM. The only available detection methods for the M3 sites are antibodies that recognize the disaccharide repeats of matriglycan. Therefore, it remains a possibility that unextended M3 structures exist, but there is currently no tool available to identify core M3 glycans without the presence of the repeating disaccharide. A route for enhanced detection of theses glycans was developed by employing bioorthogonal tagging, allowing for enrichment of O-mannosylated sites independently of matriglycan. Leveraging bioorthogonal selective exoenzymatic labelling (SEEL), O-mannose sites were enriched for on the surface of living cells. The O-mannose sites were extended by POMGNT1 and POMGNT2, glycosyltransferases that mediate the branching point of the O-mannosylation pathway, and an azido-modified form of UDP-GlcNAc (UDP-GlcNAz). Subsequentially, the azido-sugar modified O-mannosylated sites were clicked with a bioorthogonal biotin tag for neutravidin enrichment. Along with known O-mannosylated proteins α-DG and KIAA1549, adipocyte plasma membrane associated protein (APMAP) and laminin subunit beta 1 (LAMB1) were also enriched and identified as potentially novel O-mannosylated proteins.
Poster 8
Determining the Substrate Specificity of M3 Glycan Biosynthetic Enzymes
Wells Lab {Dr. Lance Wells, Dr. Linda Zhao, Dr. Trevor Adams, Robert Bridger, David Steen Jr., Jeffery Fairley}; Moremen Lab {Dr. Digant Chapla, Chin Huang}; Haltiwanger Lab {Dr. Bob Haltiwanger}; Barb Lab {Dr. Adam Barb}
Institution: Complex Carbohydrate Research Center, University of Georgia
Department: Biochemistry and Molecular Biology
Presenting Author Email: Terrell Carter - Terrell.Carter@uga.edu
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Dystroglycanopathy is a common subtype of congenital muscular dystrophy (CMD) that always presents with progressive muscle weakness but can also involve neurological complications. Dystroglycanopathy is so named because it results from the failure of extracellular matrix components, such as laminin, to bind properly glycosylated alpha-dystroglycan (α-DG). This binding is not a protein-protein interaction but instead is a specific protein-glycan interaction. Dystroglycanopathy results from mutations in the genes encoding the glycosyltransferases that build this specific glycan structure referred to commonly as the functional M3 (fM3) glycan. Biosynthesis of the fM3 glycan requires the activity of 11 enzymes and appears to only be present on one protein, α-DG, at 3 sites. We hypothesize that localization and specificity of the M3 pathway specific enzymes ensure that only α-DG is modified with these glycans that bind ECM proteins. Utilizing synthetic peptides, recombinant enzymes, and sugar nucleotides, we are currently attempting to build the entire fM3 glycan in vitro and examining specificity of the enzymes at each step. Completion of this work will provide a better understanding of the fM3 glycan pathway enzymes that when deficient lead to congenital muscular dystrophy.
Poster 9
Understanding the mechanisms of the Pgf glycosylation machinery
Nil Cortiella, M.Sc.1; Camilo Perez1; Christine Szymanski1; Jacqueline Abranches2
1. University of Georgia (Biochemistry and Molecular Biology Department)
2. University of Florida
Presenting Author Email: Nil Cortiella (nil.cortiella@uga.edu)
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Dental diseases are one of the most widespread afflictions worldwide, affecting over half of the planet’s population. It is a cause of discomfort, pain and, if the infection advances, septice-mia and endocarditis threaten the patient’s life. Even though it is true that dental diseases do not normally pose a risk, they are a huge problem in developing countries, as these infections tend to progress untreated. Bacteria, specifically Streptococcus sp., are the main characters in this infection, starting with tooth colonization where biofilms arise creating the perfect envi-ronment for them to start thriving and breaking the tooth surface by acidogenesis of the tissue1.
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One of the critical steps of this colonization is the secretion of glycosylated adhesins: extra-cellular wall-associated proteins that attach the bacteria to the tooth, normally acting as colla-gen binding proteins and conferring mechanical resistance against mouth cleaning and saliva movements. These adhesins require post-translational glycosylations that activate them. In some streptococcal strains, the Pgf glycosylation machinery2,3, oversees these modifications. Pgf proteins are members of an essential set of proteins described to modify adhesin proteins like Cnm and WapA, a key for their functionality and host colonization.
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In this project the structure and function of the proteins that form the Pgf machinery will be studied, characterizing their kinetic parameters and structural enzymatic domains. Embedding them in nanodiscs and with Cryo-EM imaging we will reveal the substrate binding residues of these proteins alongside their high-resolution structures. We have already started analyzing the first protein of the pathway, revealing a UDP-Hexose transferase activity. Understanding each individual protein will reveal the sequence of events within the Pgf machinery, allowing us to find new drug targets to stop Streptococcus on their infectious process.
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1. Daboor, S. M., Syed, F., Masood, S., Al-Azab, M. S. & Nori, E. A REVIEW ON STREPTOCOCCUS MU-TANS WITH ITS DISEASES DENTAL CARIES, DENTAL PLAQUE AND ENDOCARDITIS. Teaching Assistant of Microbiology vol. 2 (2015).
2. Avilés-Reyes, A. et al. Characterization of the pgf operon involved in the posttranslational modi-fication of Streptococcus mutans surface proteins. Sci Rep 8, (2018).
3. de Mojana di Cologna, N. et al. Post-translational modification by the Pgf glycosylation machin-ery modulates Streptococcus mutans OMZ175 physiology and virulence. Mol Microbiol (2023) doi:10.1111/mmi.15190.
Poster 10
The Characterization of Two Glycosyltransferases Involved in Xyloglucan Biosynthesis in Lemnaceae (Duckweed)
Charles J. Corulli1,2, Alexander S. Graf1,2, Pradeep K. Prabhakar1,2, Digantkumar G. Chapla1,2, Tasleem Javaid1,2, Kelley W Moremen1,2, Maria J. Peña 1,2, and Breeanna R. Urbanowicz1,2
1Complex Carbohydrate Research Center, University of Georgia
2Department of Biochemistry and Molecular Biology, University of Georgia
Presenting Author Email: cjc24633@uga.edu
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Plant biomass is an important and heavily utilized source for industrial materials, biofuels, and food. Duckweeds are aquatic monocots in the Lemnaceae family, taxonomically divided into 36 recognized species across five genera: Landoltia, Lemna, Spirodela, Wolffiella, and Wolffia. Duckweed is unique among emerging energy crops, as they are aquatic monocots that grow quickly and have a small concentration of lignin with a high proportion of non-cellulosic matrix polysaccharides in their plant cell walls. Gaining insight into how plant biopolymers are synthesized is the first step in understanding how to better enhance plant biomass production for our various purposes. We are developing duckweed as a new model to investigate the complex pathways involved in plant cell wall biosynthesis using glycosyltransferases (GTs) from the functionally diverse carbohydrate-active enzyme (CAZyme) GT47 family. The plant GT47 family is a prime target for characterization due to the large diversity of donor and acceptor substrates utilized in the family. In plants, the GT47 family has been reported to aid in the synthesis of every plant cell wall polysaccharide excluding cellulose. Here, we biochemically investigate two duckweed GT47s, MURUS3 (MUR3) and xyloglucan L-side chain galactosyltransferase position 2 (XLT2). These two enzymes have been previously characterized in vivo, but have not undergone in depth biochemical characterization. MUR3 and XLT2 are two regiospecific xyloglucan (XyG) modifying GTs which catalyze the addition of β-d-Gal using UDP-α-d-Gal as a donor to form the common ‘L’ side-chains of xyloglucan, β-d-Gal-(1,2)-α-d-Xyl. The biochemical characterization of these two enzymes exists as a proof of concept for evaluating and optimizing a methodology for the large-scale characterization of duckweed GTs from the GT47 family.
Poster 11
Developing a One-Pot Multienzyme Xylan Synthesis Platform for Improved In Vitro Characterization of Plant Cell Wall Glycosyltransferases
Thomas Curry1, Luiza Goncalves1, Emily Mathus1, Breeanna Urbanowicz1
1 Biochemistry and Molecular Biology, University of Georgia. Athens, USA
Presenting Author Email: thomas.curry@uga.edu
Keywords: Xylan, OPME, Glycosyltransferase, Nucleotide Sugar
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Xylan is the second most abundant polysaccharide in plant cell walls, yet is underutilized due to its molecular composition and structural complexity [1]. Biochemical characterization of pathway enzymes is vital to understand synthesis of complex glycopolymers to inform future synthetic biology, agricultural and biomaterial applications. However, current methods are often associated with experimental limitations, including insolubility of biologically relevant oligosaccharide acceptor substrates and inconsistent commercial availability of both donor and acceptor substrates. Thus, these restrictions hinder our ability to study the contributions of enzymes that dictate substituent patterning, and ultimately the polymer-polymer interactions within the plant cell wall [2]. To address these limitations, we are developing a modular, one-pot multienzyme (OPME) system to study the glycosyl- and acetyltransferases that synthesize xylan polysaccharides in an accessible, cost-effective manner. This method allows us to produce both nucleotide sugar donor substrates and xylan backbone acceptor polysaccharides from readily available substrates without complex chemistry or time-consuming purification steps. Recombinant glycosyltransferases and nucleotide sugar synthesis enzymes from a variety of organisms have been studied, demonstrating their capacity for synergistic xylan synthesis activity in vitro. Current work is focused on optimizing the system using high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry analyses to identify bottlenecks, which are addressed by adjusting reaction conditions, utilizing enzymes from other organisms, or engineering enzymes for better activity under established conditions. Future development of this platform will focus on expanding the range of OPME substrate generation pathways, integration of a variety of xylan biosynthetic enzymes for improved biochemical characterization, and scalable synthesis of engineered polysaccharides. The development of an improved system to study xylan synthesis is a significant step toward building a comprehensive view of complex polysaccharide biosynthesis, influencing the future development of designer biomass based on manipulation of carbohydrate active enzymes compatible with Design Build Test Learn (DBTL) cycle frameworks.
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[1] Curry T, Pena M, and Urbanowicz B (2023) An update on xylan structure, biosynthesis, and potential commercial applications. The Cell Surface, 9
[2] Alvarez-Martinez I, Ruprecht C, Senf D, Wang H, Urbanowicz B, and Pfrengle F (2023) Chemo-enzymatic synthesis of long-chain oligosaccharides for studying xylan-modifying enzymes. Chemistry, 29(26): e202203941
Poster 12
Understanding the teichoic acid polymerization mechanisms of Streptococcus pneumoniae
Eric Céster
Presenting Author Email: eric.cesterdiaz@uga.edu
Department and institution: University of Georgia, Davidson Life Sciences, Biochemistry and Molecular Biology
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Conjugate vaccines are a type of vaccine that combines a weak antigen with a strong antigen as a carrier, so that the immune system elicits a stronger immunological response against the weak one. The identification of potential bacterial polysaccharides (weak antigens) for the de-velopment of novel conjugate vaccines supposes an important research priority. Vaccines against Streptococcus pneumoniae are unable to cover the more than 100 existing bacterial serotypes. We hypothesize that two uncharacterized S. pneumoniae proteins, TarP and TarQ, work in close association to polymerize and control the chain length of teichoic acids, potential carbohydrate candidates for the development of conjugate vaccines. We propose to express and purify both proteins recombinantly to obtain a high-resolution structure of the complex that they form with Cryo-EM using nanodisc preparations. We further aim to characterize the activity of the complex through polymerization assays using an in vitro chemo-enzymatically synthetized teichoic acid repeating unit precursor. To achieve this, the glycosyltransferases in-volved in S. pneumoniae’s teichoic acid synthesis pathway will be purified and synthesis reac-tions will be set-up, tracking the resulting product with mass spectrometry experiments, sugar-radiolabeling and nuclear-magnetic resonance. Our findings will allow us to describe the mo-lecular polymerization mechanism of the TarP-TarQ complex, as well as to set-up a pipeline for the production of novel conjugate vaccines via a bioconjugation approach.
Poster 13
The role of heparan sulfate proteoglycans in the pathogenesis of synucleinopathies
Saumya Digraskar1, Jaewon Huh1, Piyali Das1, Micah Simmons1, Alana Colafrancesco1, Rita Cowell1, Jeffrey Esko2, Laura Volpicelli-Daley1, Patricia Aguilar Calvo1
1Department of Neurology, The University of Alabama at Birmingham, Birmingham, AL
2Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA
Presenting Author Email: sdigrask@uab.edu
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Synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies, are among the most prevalent neurodegenerative disorders globally. These conditions manifest with motor impairment, cognitive decline, and psychiatric disturbances. The aggregation and neuronal accu-mulation of the α-synuclein protein (α-syn) are central pathological features in synucleinopathies. Mounting evidence suggests that synuclein pathology advances by the cell-to-cell spread of α-syn aggregates through the brain. However, the molecular mechanisms driving the α-syn propagation and cell targeting are unknown. Heparan sulfate proteoglycans (HSPGs) are glycoproteins that colocalize with α-syn deposits in the brains of PD patients. HSPGs are receptors for the internali-zation of α-syn aggregates in immortalized cells. We hypothesize that HSPGs modulate α-syn pa-thology by mediating the neuronal internalization and aggregation of α-syn. Since HSPGs interact with ligands through the negatively charged sulfate groups in their HS chains, we investigated how decreasing HS sulfation impacts α-syn pathology and disease progression in mice injected with α-syn aggregates. First, we cultured neurons and astrocytes from mice conditional knockout for the N-Deacetylase/N-Sulfotransferase-1 (Ndst1f/f), the enzyme responsible for the N-sulfation of HS chains, to determine the role of HSPGs in α-syn cellular incorporation. Ndst1f/f cells were in-fected with lentivirus expressing Cre recombinase and incubated with 488-tagged α-syn aggre-gates. α-syn incorporation was measured using a confocal microscope. We found that depleting Ndst1 expression almost completely prevented the neuronal incorporation of α-syn aggregates. In contrast, depleting Ndst1 expression reduced astrocytic incorporation by 50%. Together, our in vitro studies indicate that sulfated HSPGs are the main receptors for the neuronal internalization of α-syn PFFs, and thus could modulate the in vivo propagation of α-syn. To explore this question, we injected α-syn aggregates into the striatum of mice that express lower neuronal HS sulfation and their wild-type (WT) littermates. Our preliminary behavioral studies at 7 weeks post-injection showed no evidence of motor impairment in either genotype. However, WT littermates showed higher levels of anxiety than their counterparts expressing lower HS sulfation, which suggests that decreasing HS sulfation might have slowed down α-syn pathology. Future studies will deter-mine whether manipulating the HS:syn interaction impacts the α-syn aggregation and spread, neurotoxicity, and synucleinopathy progression.
Poster 14
Genome-wide Regulation of Exostosin-1/2 for Drug Target Discovery in Multiple Hereditary Exostoses (MHE)
Xiaolin Dong1,2, Kavya Suryadevara2, Lance Wells1,2, Ryan Weiss1,2
1 Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30605, USA
2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA30605, USA
Presenting Author Email: Xiaolin.Dong@uga.edu
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Multiple Hereditary Exostoses (MHE) is an autosomal dominant skeletal disorder characterized by cartilage-capped bony outgrowths, also known as exostoses, that form near the growth plate of long bones and other skeletal elements. MHE affects approximately 1 in 50,000 people worldwide, and there are currently no approved therapies. 90% of MHE patients have heterozygous loss-of-function mutations in exostosin-1 (EXT1) or exostosin-2 (EXT2), which are genes that encode key glycosyltransferases involved in heparan sulfate (HS) assembly. A mutation in EXT1 or EXT2 leads to a significant reduction in HS levels, affecting various cell signaling pathways, and ultimately resulting in ectopic chondrogenesis and exostoses formation. Although the pathogenesis of MHE is linked to reduced HS levels, targets for developing new therapies remain unclear. To date, very little is known about the upstream and downstream factors that regulate the expression and function of EXT1 and EXT2 in human cells. We hypothesized that increasing the expression of the normal EXT gene copy by targeting regulatory factors that control it could correct EXT1/EXT2 haploinsufficiency and restore functionally normal levels of HS in cells. To identify novel regulatory factors for EXT1 and/or EXT2, we established reporter cell lines by integrating a green fluorescent protein (GFP) at the C-terminus of endogenous EXT1 and EXT2 in human chondrocytes via CRISPR/Cas9 gene editing. We subsequently conducted genome-wide CRISPR screening assays to identify regulatory factors that may affect EXT1 and/or EXT2 expression. The top hits from the EXT1-screen included β-1,3-galactosyltransferase 1 (C1GALT1) and its molecular chaperone, Cosmc (C1GALT1C1), both of which are involved in Core 1 mucin-type O-glycosylation. Our data reveals that loss of COSMC or C1GALT1 alters EXT expression, HS composition, and FGF-mediated cell signaling pathways, highlighting the intricate crosstalk between HS biosynthesis and mucin-type O-glycosylation. Interestingly, the knockout of COSMC or C1GALT1 in chondrocytes also selectively altered HS proteoglycan expression at the cell surface. Elucidating specific regulatory pathways controlled by O-glycosylation that impact HS biosynthesis may provide critical insights for developing targeted therapies for MHE.
Poster 15
Identification of a novel bacterial galactose-3/6-S preferring sulfatase using functional metagenomic screening
Julia E. Dreifus1, Léa Chuzel1, Christopher H. Taron2, and Jeremy M. Foster1
1New England Biolabs, Ipswich, MA 01938, USA
2Gloucester Marine Genomics Institute, Gloucester, MA 01930, USA (Current address)
Presenting Author Email: jdreifus@neb.com
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Glycans are involved in many biological processes and play a key role in numerous diseases. The biological functions of glycans are dictated both by the arrangement of saccharide residues and the presence or absence of specific chemical saccharide modifications (termed post-glycosylation modifications, or PGMs). PGMs encompass a range of diverse chemical groups, including sulfate, phosphate, acetyl, methyl, and zwitterions, and the distinct chemistry of each alters how glycans interact with surrounding molecules1. Consequently, changes in modification patterns can have severe impacts on health. For instance, mutations in some sulfotransferases cause congenital disorders, including muscular-skeletal abnormalities, deafness, and heart defects1. PGMs are technically challenging to identify and characterize, leaving them under-explored despite their importance. To this end, we are using a high-throughput functional metagenomics screening approach to discover novel bacterial enzyme specificities to facilitate the study of post glycosylation modifications.
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In this study, we screened a human gut microbiome DNA fosmid library for an enzyme that can remove sulfate from the third carbon of galactose, a common modification found on N- and O-linked glycans in mammals and the influenza virus2,3,4. We identified two candidates that act on galactose-3-sulfate. Further study determined that they both remove sulfate from the sixth carbon of galactose, and one also has low affinity for sulfate at the third position of glucosamine. These sulfatases are classified by bioinformatics as part of the S1_46 and S1_65 sulfatase subfamilies. The S1_46 subfamily has one other biochemically defined family member which removes sulfate solely from the third carbon of N-acetylglucosamine5,6. Thus, these enzymes exhibit unique specificities not previously observed in these subfamilies. Furthermore, one of these enzymes represents a subfamily of sulfatases with no previously characterized members, opening the door for characterization of this group. The galactose-3/6-sulfatases described in this work contribute a novel specificity to a powerful enzymatic toolbox that will be used to facilitate the study of post-glycosylation modifications.
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Muthana, S. M., Campbell, C. T. & Gildersleeve, J. C. Modifications of Glycans: Biological Significance and Therapeutic Opportunities. ACS Chem. Biol. 7, 31–43 (2012).
Van Rooijen, J.J, Kamerling, J.P., Vliegenthart, F.G. Sulfated di-, tri-, and tetraantennary N-glycans in human Tamm-Harsfall glycoprotein. Eur. J. Biochem. 256, 471-487 (1998).
Bai, X., Brown, J.R., Carki, A., Esko, J. D. Enhanced 3-O-sulfation of galactose in Asn-linked glycans and Maackia amurenesis lectin binding in a new Chinese hamster ovary cell line. Glycobiology. 11(8), 621-632 (2001).
She, Y., Li, X., Cyr, T.D. Remarkable structural diversity of N-glycan sulfation of influenze vaccines. Anal. Chem. 91, 5083-5090 (2019).
Barbeyron, T., Brillet-Gueguen, L., Carre, W., Carriere, C., Caron, C., Czjzek, M., Howbeke, M., Michel, G. Plos One. DOI:10.1371/journal.pone.0164846 (2016).
Luis, A. S., Bale, A., Byrne, D.P., Wright, G.SA., London, J., Chunsheng, J., Karlsson, N.G., Hansson, G.C., Wyers, P.A., Czjzek, M., Barbeyron, T., Yates, E. A., Martens, E.C., Cartmell, A. Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota. Nat. Chem. Biol. 18(8), 841-849 (2022).
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Poster 16
Network for Advanced NMR and CCRC NMR Facility: Opportunities for Studies of Biomolecules in Solution
Alexander Eletsky, Mario Uchimiya, John Glushka, John Grimes, Laura Morris, Arthur S. Edison. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 Presenting Author Email: aeletski@uga.edu The Network for Advanced NMR (NAN) is an NSF-funded partnership between Universities of Connecticut, Georgia, and Wisconsin.
Our goal is to provide distributed access to state-of-the-art NMR resources to the scientific community in biomedicine, materials science, and chemistry. This includes remote access to NMR instrument facilities of the member universities. A web portal is provided for instrument search, user management, and data archiving and retrieval. Knowledgebases covering biological and materials science solid-state NMR, solution-state structural biology, and NMR-based metabolomics are currently being developed. These will include training and educational materials, sample NMR data sets, calibration and experiment setup procedures, and data processing tools to suit the needs of users with limited NMR experience. A part of this project’s funding covers the installation of two 1.1 GHz NMR instruments, a solidstate instrument currently operational at NMRFAM (UW-Madison), and a solution-state instrument to be installed at CCRC UGA in summer 2024. Additionally, the CCRC NMR facility features five instruments in the 600-900 MHz range with special capabilities, such as sample changers for automation with large sample sets, cryogenic probes optimized for 1H, 13C, 15N or 19F detection, and a unique 1.7 mm 800 MHz system for small samples in capillary tubes. These instruments are suitable for probing structure and dynamics of biomolecules in solution, ligand binding studies, analysis of small molecules and glycans, and metabolomics applications. Here we present highlights from selected collaborative projects to illustrate the various uses of CCRC NMR facility instrumentation in studies of biomolecules in solution, including glycoproteins.
Poster 17
A Metabolome-Wide Screening Method for Protein-Metabolite Interactions by NMR Spectroscopy Conrad Epps
1,3, Safal Shrestha2 , Natarajan Kannan1,2 and Art Edison1,2,3 1Department of Biochemistry and Molecular Biology; 2 Institute of Bioinformatics; 3 Complex Carbohydrate Research Center Institution: University of Georgia Presenting Author Email: conrad.epps@uga.edu
The metabolome comprises all metabolically active small-molecule chemicals within an organism. It has the most direct effect on an organism’s phenotype and immediate biological state and influences and modulates other multiomic levels, such as the genome, epigenome, transcriptome and proteome. However, the ‘omics’ disciplines are often sequestered from one another, with metabolites frequently studied simply as biomarkers of gene and protein activity. There is much work to be done, especially concerning the integration of the metabolome and proteome in the study of allosteric regulations of enzymes. Even in well-annotated organisms, not every metabolite has been tested against every enzyme, limiting our total knowledge of metabolic pathways and mechanisms. We seek to develop a functional metabolomics methodology for integrating the metabolome and proteome in a more holistic way. Here, we utilize saturation transfer difference NMR (STD NMR), a technique commonly used in pharmaceutical research to screen drug target proteins against ligand libraries and identify binding ligands. By screening a kinase of interest against the entire metabolome extract of Caenorhabditis elegans, we are able to see exclusive and novel interactions between the protein and various different ligands. This suggests that the binding metabolites are either substrates, products, or allosteric effectors, and justifies further investigation. This work will provide us with a less isolated and more dynamic depiction of the functional metabolome. The methodology will also serve as a practical and relatively simple way to screen an organism’s metabolome for multiomics interactions and effector molecules.
Poster 18
Fraction Libraries for NMR Metabolomic Analysis
Christopher Esselman, (University of Georgia, IOB, CCRC), Alexis Molina, (University of Georgia, Genetics, CCRC), Ricardo M. Borges (Federal University of Rio de Janeiro), Amanda Shaver (Johns Hopkins University), Pam Kirby (University of Georgia, CCRC), Arthur Edison (University of Georgia, IOB, Genetics, CCRC) Institute of Bioinformatics, University of Georgia Presenting Author Email: cse94833@uga.edu
Mass spectrometry (MS) and nuclear magnetic resonance (NMR) each provide unique information in metabolomics studies; however, NMR data analysis tools for metabolomics are less developed than available MS tools. Two significant reasons are a lack of resolution due to peak overlap and lower sensitivity in NMR. To address these issues, we are developing an experimental and computational workflow for fractionating and analyzing highly concentrated biological mixtures. Fractionation reveals overlapping peaks by spreading them over time, and with multiple injections into the same fractions, we can distinguish low-concentration compounds. From preliminary data using our workflow, we can annotate known metabolites via database matching, and we have collected “fingerprints” of unknown compounds. We plan to do more intensive NMR analysis on specific fractions containing unknown compounds for novel metabolite discovery. Activity-guided fractionation is commonly used in natural product chemistry but is impractical for high-throughput metabolomics. Our methodology incorporates the benefits of fractionation while maintaining high throughput. We aim to apply this method to a study investigating the interplay of the Target of Rapamycin (TOR) and O-GlcNAc signaling regarding circadian rhythms and lifespan in the model organisms Neurospora crassa and Caenorhabditis elegans.
Poster 19
Glydentify, a deep learning tool for classifying glycosyltransferase function
1. Aarya Venkat, Department of Biochemistry and Molecular Biology, University of Georgia 2. Ruili Fang, Department of Computer Science, University of Georgia 3. Zhongliang Zhou, Department of Computer Science, University of Georgia 4. Shaan Gill, Department of Biochemistry and Molecular Biology, University of Georgia Natarajan Kannan, Department of Biochemistry and Molecular Biology, University of Georgia Presenting Author Email: ruili.fang@uga.edu
Protein language models have emerged as a powerful tool for predicting protein function by capturing the underlying grammar and syntax of protein sequences. Here, we introduce Glydentify, an open-source and user-friendly application that uses protein language models for the classification of glycosyltransferases (GTs) and donor prediction. Utilizing the state-of-the-art ESM2 protein language model, Glydentify extracts high-dimensional sequence embeddings to accurately classify GTs into fold A families with 92\% accuracy. The tool also predicts GT-A donor binding preferences with an accuracy of 91\%. Notably, Glydentify identifies key residues that contribute to a prediction, thereby adding an explainable component to the application. With an intuitive interface powered by Gradio, Glydentify requires no programming experience from the user, democratizing access to cutting-edge deep learning technologies for GT research. The application is freely available on GitHub and can be accessed directly through any web browser (https://huggingface.co/spaces/ arikat/Glydentify).
Poster 20
Characterization of polysialylated proteins in human CD4+ T cells
Gutiérrez-Valenzuela LD1,2 , Villanueva-Cabello TM1,2, Salinas-Marín R1,2, Martínez-Duncker I1 . 1. Laboratorio de Glicobiología Humana y Diagnóstico Molecular, Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México CP. 62209. 2. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México CP. 62209. Fax: +52-777-3297020; Tel: +52-777-3297000; e-mail: duncker@uaem.mx Presenting Author Email: lydiguva07@gmail.com
Introduction. Human naive CD4+ T cells are the subset of T lymphocytes responsible of modulating the adaptive immune response1 . We have previously reported that the activation of these cells is accompanied by increasing the de novo sialylation2 , allowing us to identify the overexpression of polysialyltransferases ST8Sia ll and ST8Sia lV and the resulting synthesis of polysialic acid (PSA), not previously described in these cells. By using the well characterized antiPSA 12E3 monoclonal antibody (mAb), expression of PSA was detected in the cell surface of resting and activated cells and in at least eight protein bands revealed through Western blot3 . In addition, shRNA mediated-gene silencing of ST8Sia ll and ST8Sia lV in activated T-cells, showed an increased immune response, indicating that PSA has a potential role in regulating activation3 . Objective. In this work we aim to identify the novel polysialylated proteins expressed in human CD4+ T cells, as well as to determine the type of glycan (N- or O-glycans) to which PSA is attached. Methods. Potential polysialylated proteins were immunoprecipitated by incubating the anti-PSA 12E3 mAb overnight with whole lysates obtained from activated CD4+ T cells and subsequently incubating the protein-antibody complex with protein L (Sigma). The association of the protein/ antibody complex was eluted with phosphate buffer and Tris-Gly buffer. Samples were subject to mass spectrometry MALDI-MS. The association of PSA to N-glycans was evaluated by enzymatic digestion of the same lysates with PNGase F and subsequent Western blotting using the anti-PSA 12E3 mAb. Results. The digestion with PNGase F indicated that at least three of the eight polysialylated glycoproteins have PSA bound to N-glycans. Mass spectrometry revealed different proteins with involvement in T cell function.
References
1. Pitfalls in determining the cytokine profile of human T cells.
2. Activation of human naive Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.
3. Polysialic acid is expressed in human naïve CD4+ T cells and is involved in modulating activation.
Poster 21
FUT10 and FUT11 are novel protein O-fucosyltransferases that modify EMI domains
Huilin Hao1, Youxi Yuan1, Atsuko Ito1, Benjamin M. Eberand2, Michelle Cielesh2, Mark Larance2and Robert S. Haltiwanger1
1Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30605, United States
2 Charles Perkins Centre, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
Presenting Author Email: hh84581@uga.edu
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Fucosylated glycan structures are commonly found in mammalian cells and play vital roles in diverse biological events. Among the 13 identified fucosyltransferases (FUTs) in humans, only Protein O-fucosyltransferases (POFUTs) 1 and 2 directly transfer fucose to protein serine or threonine residues via O-linkage. Both POFUT1 and POFUT2 require the recognition of specific consensus sequences within certain cysteine-rich domains: Epidermal Growth Factor-like (EGF) repeats and Thrombospondin Type 1 Repeats (TSRs), respectively. O-fucose glycans modulate protein function through direct intermolecular interactions with binding partners, regulating processes such as Notch-ligand binding. They also form intramolecular interactions that stabilize EGF repeats and TSRs, thereby aiding in proper folding. In addition to EGF repeats and TSRs, a recent platelet proteome study identified a novel O-fucose site on the elastin microfibril interface (EMI) domain of Multimerin-1 (MMRN1), a major platelet protein that supports platelet adhesion and thrombus formation. We found that neither POFUT1, nor POFUT2, was responsible for this modification, suggesting that a novel POFUT may exist for modifying protein EMI domains. In Alphafold2 screens of binding structures involving the EMI domain, the closely related FUT10 and FUT11 enzymes demonstrated significant EMI binding that was not observed with other FUTs. Robust O-fucosylation activity was observed in our in vitroassays using purified, recombinant FUT10/11 and EMI substrates combined with mass spectrometric analysis. Knocking out of either FUT10,or FUT11,in HEK293T cells substantially decreased the stoichiometry of O-fucosylated peptides of EMI. These findings imply that FUT10 and FUT11 function as POFUTs for EMI domains. EMI O-fucosylation remained unaffected in knockout of the Golgi GDP-fucose transporter, SLC35C1, in HEK293T cells compared to WT cells, which combined with subcellular localisation data demonstrate FUT10/11 function in the ER. Much like POFUT1 and POFUT2, both FUT10 and FUT11 rely on properly folded EMI structures for efficient modification. Notably, mutating the O-fucose sites of the MMRN1 EMI domain led to a ~50% reduction in MMRN1 secretion. These findings suggest that akin to POFUT1 and POFUT2, FUT10 and FUT11 participate in a non-canonical ER quality control pathway for EMI domains. This work was supported by NIH grant R35GM148433.
Poster 22
XLID OGT TPR Variants Have Altered Interactomes
Naomi L. Hitefield1, Hannah M. Stephen1, Philip A. Schroeder1, Jeremy L. Praissman1, Lance Wells1
1From the Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30605, United States of America
Presenting Author Email: nlhitefield@uga.edu
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One in 500 males are affected by X-Linked Intellectual Disability (XLID), which is a group of neurodevelopmental disorders attributed to mutations in the X-chromosome and present with intellectual disability (ID) and developmental delay. Our lab, and others, have reported mutations in the X-chromosome OGT gene found causal for an intellectual disability syndrome. OGT encodes the essential human O-GlcNAc transferase (OGT) which transfers Beta-N-acetylglucosamine (O-GlcNAc) onto serine and threonine residues of over 8,000 human nucleocytoplasmic proteins. OGT plays vital roles in many cellular processes including transcriptional regulation and neural development and function. The tetratricopeptide repeat (TPR) domain of OGT provides enzyme targeting and substrate specificity. Our lab previously characterized XLID causal TPR variants of OGT, and the variants had no uniform, appreciable defect in catalytic activity or thermal stability compared to wild-type (WT). Therefore, we hypothesize the interactome of OGT is affected by these OGT TPR variants. Our initial work in HeLa cells investigated the WT TPR interactome utilizing a BioID proximity labeling approach with BirA*, a promiscuous biotin ligase. HeLa cells were transfected with BirA* fused to a WT OGT TPR domain, alongside an eGFP-BirA* negative control, and lysates were neutravidin enriched. Shotgun proteomics revealed 115 proteins in HeLa cells interacting with the TPR domain of OGT, including enrichments for proteins involved in regulating gene expression and ID-related disorders. We then optimized this BioID approach with TurboID, a modified BirA*, to reduce time required for biotin labeling and allow for detection of transient OGT interactions. We analyzed interactomes of 5 XLID causal TPR variants alongside WT TPR OGT-TurboID fusion proteins in SH-SY5Y cells, a neuroblastoma cell line, under both polarized and depolarized conditions. We identified 136 interactors, but more significantly, a small subset of those had reduced interaction with variant TPR domains compared to WT OGT. Proteins of interest identified have roles in gene expression and neuronal function, and we are investigating the impact of loss of OGT-protein interactions on these proteins. These studies are geared at elucidating a molecular understanding of the impact of XLID missense mutations on OGT.
Poster 23
Marfan syndrome variant fibrillin-1 exhibits aberrant O-glucosylation within POGLUT2 and POGLUT3 modification sites
Nicholas R. Kegley, Atsuko Ito, Daniel B. Williamson, and Robert S. Haltiwanger
Presenting Author Email: nrk63756@uga.edu
Marfan syndrome (MFS) is a heritable autosomal dominant disorder that affects ~1 in 7500 people. The disorder is characterized by connective tissue problems in the aorta, lungs, and eyes. MFS is caused by defective, variant forms of the extracellular matrix protein fibrillin-1 (FBN1). Recently, our lab observed that FBN1 is a major target of the novel Protein O-GLUcosylTransferases, POGLUT2 and POGLUT3. These enzymes add an O-glucose modification to Epidermal Growth Factor-like repeats (EGFs), small cysteine-rich domains found across FBN1 that contain the putative consensus sequence C-X-N-T-X-G-S-F/Y-X-C, where ‘X’ denotes any amino acid, and the bolded ‘S’ indicates the O-glucosylated serine. While the putative consensus residues are common to modified EGFs, it is unknown if they are required for modification to occur. Relatedly, the role of O-glucose in FBN1 function and MFS is also unknown. To test the putative consensus requirements, FBN1 constructs that replace a conserved residue with an alanine or a MFS variant residue were expressed in HEK293T cells and analyzed using a semi-quantitative mass spectral method. This method showed that none of the alanine mutants significantly affected O-glucosylation, suggesting the conserved residues (other than the serine) are not required for modification. Some MFS variant EGF domains exhibited reduced O-glucosylation, while other EGFs display aberrant O-glucose elongation. These changes could be pathogenic. While the variant proteins exhibited a mix of glycosylation changes, very few lost their O-glycan entirely. Thus, a new POGLUT2 and 3 sequence is proposed: C-X-X-X-X-X-S-X-X-C, with only the modifiable serine and cysteines remaining as necessary for efficient POGLUT2 and 3 modification of EGF repeats. These results have helped to elucidate the specificity of POGLUT2 and POGLUT3 and are the first steps in understanding the modification’s role in the context of MFS. This work was supported by NIH GM061126 and HL161094, and Nick Kegley was partially supported by T32 GM107004.
Poster 24
N-GlyFindTM – a high-specificity affinity reagent for detection and enrichment of N-glycosylated proteins
Sheng-Cheng Wu1, Christian Gerner-Smidt1, Lu Meng1, George N. Bendzunas1, Sylvain Lehoux1, Robert J. Woods2, and Loretta Yang3
1Lectenz Bio, Athens, GA, USA; 2Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA; 3Lectenz Bio, San Diego, CA, USA
Presenting Author Email: slehoux@lectenz.com
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N-glycans play crucial roles in nearly every aspect of biological processes, and their distinct properties make them appealing as disease biomarkers and therapeutics targets. However, due to their highly branched and variably linked nature, N-glycans often remain a challenge to detect, purify, and analyze structurally. Despite improvement in analytical techniques and instrumentation, there is still a great need for reagents with well-defined carbohydrate specificity and high affinity that can be used to interrogate and enrich biological samples. Lectenz Bio has been engineering glycan-processing enzymes and glycan-binding proteins into high-affinity glycan-binding reagents with adjustable specificities. Here, we report the development of N-GlyFindTM, an asparagine-linked glycan (N-glycan) specific reagent engineered from mouse Fbs1 (Fbx2). We used computational structural modeling to guide the selection of specific amino acid variants to be expressed in Yeast Surface Display libraries for screening and enrichment. Binding properties of N-glycan-specific protein candidates were further evaluated by a panel of assays including Glycan Microarray, Western Blot, Bio-Layer Interferometry, ELISA, and Affinity Chromatography. N-GlyFindTM is an N-glycan pan-specific reagent exhibiting high specificity towards N-glycosylated peptides or proteins with no binding to the corresponding non-glycosylated peptide or proteins. Supported by NIH grant OD035390.
Poster 25
Utilizing Causal X-Linked Intellectual Disability Variants to Gain Insight into the O-GlcNAc Transferase Enzyme
Johnathan Mayfield1, Laura Holden1, Trevor Adams1, Hannah Stephens1, Lance Wells1
1From the Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30605, United States of America
Presenting Author Email: Jjman95@uga.edu
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X-linked intellectual disability (XLID) occurs in 1 in 500 males in the US with many cases being of unknown genetic etiology. Our laboratory was the first to biochemically characterize variants of O-GlcNAc Transferase (OGT), the sole enzyme responsible for the O-GlcNAc modification onto thousands of nucleocytoplasmic proteins, that are causal for an intellectual disability syndrome termed OGT-Congenital Disorder of Glycosylation (OGT-CDG). Initial OGT-CDG causal variants were identified in the Tetratricopeptide repeat (TPR) domain of OGT. While no common significant differences in biochemical characterization were observed for these variants, RNA-seq demonstrated alterations in genes regulating neurogenesis in CRISP/Cas9-edited male human embryonic stem cells. Further, we have established that TPR domain variants have altered protein-protein interactions. Currently, we are characterizing novel catalytic domain variants of OGT, T570A, Y835C, and A952V, discovered in new OGT-CDG families. Our studies have shown that all three variants are active O-GlcNAc transferases, but they all fail to recapitulate the activity of wildtype OGT when expressed in multiple cell types. This finding is in alignment with the molecular modeling of the catalytic domain variants, which suggested the variants likely have an altered Km for the donor sugar nucleotide, UDP-GlcNAc. Furthermore, one variant, Y835C, shows significantly decreased expression in multiple systems suggesting the variant results in decreased stability of the enzyme. Thus, these variants likely result in hypoglycosylation of key substrates. We are currently working to fully characterize the variants in regards to enzymatic activity towards protein and peptide substrates as well as the stability defects of Y835C using mammalian expressed full-length recombinant enzymes. We also aim to investigate if glucosamine supplementation can alleviate the glycosylation defect seen in cells as this may provide a therapeutic option for patients. Surprisingly, both TPR and catalytic domain variants present similarly in OGT-CDG patients. Thus, we are currently pursuing the hypothesis that all variants, despite different altered biochemical characteristics, have a common downstream impact on glycosylation of key transcriptional regulators. Overall, this project aims to provide insight into how these OGT-CDG variants in different domains of OGT impact function of the enzyme and lead to the same neurodevelopmental phenotype observed in patients.
Poster 26
Enzymatic, anchoring, and biological functions of the GAUT family in synthesis of multi-domain pectins
DEBRA MOHNEN1, CLIFFORD OKOYE1, MELANI ATMODJO1, ROBERT AMOS1 and KRISTEN A. ENGLE21 Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, USA2 Complex Carbohydrate Research Center and Department of Plant Biology, University of Georgia, Athens, USAPresenting Author Email: dmohnen@ccrc.uga.edu
Pectins are the most complex plant cell wall glycans. Their homogalacturonan (HG) and rhamnogalacturonan (RG) backbones are modules for mixed glycan domain polymers. Our goal is to define the number and type of complex modular pectin polymers and their functions. Individual pectic glycans may exist as homoglycans (e.g. HG), domains in heteroglycans (e.g., HG-RG-I-HG; HG-RG-II-HG) and proteoglycans (e.g. APAP1 and RG-I-AGP) (reviewed in1,2,3). It is important to determine which pectic polymers function in, for example, cell expansion, plant growth and shape, cell:cell adhesion and pollen tube elongation4. HG, a homopolymer of α-1,4-linked GalA and the most abundant pectin in growing cells is synthesized by the GAUT family of proven and putative HG biosynthetic galacturonosyltransferases (GalATs). Arabidopsis has 15 GAUTs. Six are confirmed HG α-1,4-GalATs (GAUTs 1, 4, 10, 11, 13, 14 and the GAUT1:GAUT7 complex) and three anchor GAUT1 to the Golgi (GAUTs 5, 6, 7). We have reviewed GAUT catalytic activity, gaut mutant and transgenic phenotypes, locations of GAUT transcript expression, predicted GAUT structural domains, transcripts co-expressed with the GAUTs, and GAUT evolution. From this analysis we propose that GAUTs synthesize HG in a minimum of five different pectic polymers with unique functions in plants.
References
Mohnen et al., 2024 In The Plant Cell Wall – Research Milestones and Conceptual Insights. Ed. Anja Geitmann, CRC Press/Taylor & Francis Group, LLC. Chapter 5, pages 94-126.2.
Delmer et al., 2024 The Plant Cell Advance access publication https://doi.org/10.1093/plcell/koad325.3.
Tan et al., 2023 Carbohydrate Polymers 301: 120340.4. Vissenberg and Höfte, 2024
In The Plant Cell Wall – Research Milestones and Conceptual Insights. Ed. Anja Geitmann, CRC Press/Taylor & Francis Group, LLC. Chapter 6, pages 127-146.
Poster 27
O-GlcNAc as a regulator of TOR and Circadian Rhythms in Neurospora crassa
University of Georgia, Genetics Department
Unpublished work. Chris Essleman, Dr. Jonathan Arnold, Dr. Arthur Edison
Presenting Author Email: Alexis Molina - alexis.molina@uga.edu
N. crassa is a model organism for thestudy of circadian rhythms because of its banding phenotype, which is responsive to light and dark cycles and regulation of clock genes. The TOR (target of rapamycin) pathway is highly conserved in eukaryotes from N. crassa to humans. The pathway regulates transcription in response to nutrient sensing and overlaps with biological clock regulation. This suggests we may be able to use N. crassa as a model to study how the TOR pathway regulates the biological clock. Previous in vitro research has shown that proteins in the mTOR pathway are O-GlcNAcylated (a post-translational modification) to modulate the TOR pathway. In N. crassa, there is evidence of an O-GlcNAc transferase analog, which adds O-GlcNAc; however, an O-GlcNAcase analog has not been studied yet. The first aim of this project is to use western blotting to prove that all components of O-GlcNAc are present and functional in N. crassa. Next, we will use a dual specificity aptomer (Hart and Zhu, 2023) to O-GlcNAcylate individual proteins in the TOR pathway and observe changes in rhythmicity and transcription of clock regulation genes. As a control, we will have a wild-type strain that does not contain our dual specificity aptamer. An inducible promoter will be placed in our mutant strain upstream of our dual specificity aptamer. The inducible promoter will be dependent on quinic acid media, which has not been shown to alter rhythmicity. The strains will be grown in race tubes so banding patterns can be observed to study changes in the cell’s circadian rhythms in vivo. When a banding strain of N. crassa is inoculated into a race tube, banding patterns will correspond to the clock to show disruptions, changes in rhythms, and longevity as individual TOR pathway proteins are O-GlcNAcylated. RNA seq analysis can complement this visual data to look for upor down-regulation in the transcription of gene clusters involved in regulating the clock, such as the White Collar complex and the frq-oscillator, which regulates clock control genes.
Poster 28
Genome Wide CRISPR Screening to Bioengineer a Safer, Recombinant Heparin
1University of Georgia, Biochemistry and Molecular Biology,
2University of Georgia, Complex Carbohydrate Research Center
Haruki Takeuchi1,2, Caitlien Nguyen1,2, and Ryan Weiss1,2
Presenting Author Email: Jack Moore - jcm62815@uga.edu
Heparin is one of the most widely used drugs in the world, with approximately 12 million people in the US alone being administered heparin annually. Heparin acts as a potent anticoagulant due to its ability to activate antithrombin III (AT), a serine protease naturally found in the blood responsible for regulating the coagulation response. This interaction is facilitated by heparin’s highly negative overall charge as well as a critical pentasaccharide sequence present along its repeating disaccharide backbone. While an effective anticoagulant, a major drawback to the use of heparin arises from the life-threating side effect of heparin induced thrombocytopenia (HIT), which occurs from an off-target interaction between heparin and the chemokine, platelet factor 4 (PF4). Interestingly, heparin is only produced in mast cells and is predominately sourced from pig mucosa in China. Although heparin is exclusively produced in mast cells, a structurally analogous molecule known as heparan sulfate (HS) is ubiquitously expressed on the surface of all metazoan cells. Heparin and HS share the same repeating disaccharide backbone; however, they differ significantly in the quantity and patterning of various sulfation modifications. Due to their differences in composition, HS does not possess the same anticoagulant capabilities of heparin. Both heparin and HS share the same biosynthetic machinery, yet the regulation of these enzymes and the assembly of heparin versus HS remains poorly understood. In this study, we developed genome-wide CRISPR screening assays to identify factors that regulate the assembly of HS to enhance its interaction with AT and reduce its interaction with PF4, as a method to bioengineer a safer, recombinant form of anticoagulant heparin. From the screens, we identified previously studied genes essential for HS/heparin formation as well as novel candidate genes whose function is unknown relative to HS/heparin assembly. Regulatory factors identified from the genetic screens will be used to bioengineer a cell line that can produce a safer form of the drug heparin and will help elucidate the regulatory mechanisms for this vital complex polysaccharide.
Poster 29
Integrating Computational Modeling and Mutagenesis to Study the Pectic Homogalacturonan de novo Biosynthetic Activity of GAUT13 and GAUT14 and its Biological Function in Arabidopsis
CLIFFORD OKOYE1(cliffordokoye@uga.edu), ROBERT AMOS1 and DEBRA MOHNEN1
1 Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, USA
The GAUT (GALACTURONOSYLTRANSFERASE) gene family encodes proteins involved in the synthesis/elongation of homogalacturonan (HG)1. GAUT13 and GAUT14 have two activities, they elongate pectic exogenous HG acceptors in vitro and also de novo synthesize HG using UDP-GalA as both an acceptor and donor substrate2. These dual activities pose challenges both functionally and enzymatically. The HG de novo synthesis activity competes with the acceptor-dependent activity for UDP-GalA making it difficult to measure enzyme kinetics. Also, the biological function of the high HG de novo synthesis activity of GAUTs 13 and 14 is unclear since most HG isolated from cell walls is a heteroglycan with rhamnogalacturonan I or II3. To begin to address these challenges, we modeled GAUT14 with bound Mn2+ and donor and acceptor substrates, identifying key residues predicted to be involved in acceptor-substrate interactions. Using an AlphaFold-modeled protein structure and Molecular Docking and Molecular Dynamic simulations, we identified and mutated amino acids predicted to be involved in the two different enzyme activities. Tests of these mutated proteins showed we successfully knocked down GAUT14's HG de novo synthesis activity while largely preserving its HG acceptor-dependent elongation activity. The results provide a foundation for dissecting the functional intricacies of these pectin biosynthetic enzymes.
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References
1. Sterling et al. 2006. Proc Natl Acad Sci U S A.103:5236–5241.
2. Engle et al. 2022.Plant Journal. 109:1441-1456.
3. Mohnen et al 2024. CRC Press/Taylor & Francis Group.5:94-126.
Poster 30
Regulation of heparan sulfate assembly by the non-canonical EZH2/TRIM28/SULF1 axis in melanoma
Neil G. Patel1,2, Amrita Basu1, Farhan Valummel, Alexandra Drakaki3, Marten Hoeksema3, Ryan J. Weiss1,2
1Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602
2Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602
3Amsterdam University Medical Center, Amsterdam, Netherlands
Presenting Author Email: ngp94529@uga.edu
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Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and in the extracellular matrix. Each HSPG consists of a core protein with one or more covalently linked linear heparan sulfate (HS) polysaccharides. These HS chains are composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated, enabling key interactions with various extracellular signaling molecules and other ligands. The fine structure of the HS chains mediates various cellular interactions which are crucial to maintaining proper cell growth and homeostasis. Dysregulation of HS expression and assembly has been implicated in the progression of various cancers, including melanoma. Recently, our group discovered that the regulation of SULF1, an extracellular HS 6-O sulfatase, can attenuate melanoma cell growth in vitro. In this study, we aimed to understand the epigenetic regulatory mechanisms of HS biosynthesis and how these regulatory mechanisms may be involved in the progression of melanoma growth. To investigate this, we conducted an in silico analysis of the cis-regulatory regions of all genes involved in HS assembly using publicly available bioinformatic tools and genomic datasets. Intriguingly, enrichment analysis revealed multiple members of the polycomb repressive complex 2 (PRC2), a set of chromatin remodeling proteins which are frequently mutated in cancer, as potential regulators of HS biosynthesis. To investigate the epigenetic regulation of HS biosynthesis, we targeted the primary catalytic subunit of PRC2, the histone methyltransferase enhancer of zeste 2 (EZH2), which is commonly overexpressed and/or mutated in human melanoma cells. We found that EZH2 regulates the expression of multiple enzymes involved in HS biosynthesis, which leads to downstream effects on HS-ligand interactions and cell homeostasis. Pharmacological inhibition of EZH2-mediated histone methylation and targeting other prominent members of the PRC2 revealed non-canonical mechanisms for controlling HS assembly. Co-immunoprecipitation proteomics studies revealed an interaction between EZH2 and TRIM28, a nuclear scaffolding protein typically upregulated in human melanoma. This interaction seems essential for the expression of SULF1. Overall, these studies provide insight into the molecular mechanisms by which HS biogenesis is differentially regulated in physiology and disease and may provide insight regarding potential therapeutic targets for melanoma.
Poster 31
Sparse isotope labeling for structural analysis of glycoproteins
James H Prestegard1,2,3, Monique J Rogals3, Chin Huang1,2, Alexander Eletsky2, Robert V Williams2,3 and Kelley W Moremen1,2
1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA
2Complex Carbohydrate Research Center, University of Georgia, Athens, GA
3Department of Chemistry, University of Georgia, Athens, GA
J. Prestegard, presenter, jpresteg@uga.edu
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Structural and functional analysis of glycoproteins presents special problems for NMR. Producing natively glycosylated glycoproteins with uniform isotopic labeling in mammalian cell cultures usually requires isotopically labeled versions of amino acids, rather than the inexpensive substrates required by E. coli cultures. Moreover, mammalian cells do not typically tolerate perdeuteration. However, uniform labeling is primarily required for de novo structure determination. Structural validation of computational models and functional characterization of proteins may require only sparse labels. We have been exploring a simple way of producing glycoproteins labeled in all alanine methyl groups and all residues of attached glycans. Production is based on supplementation of media with 13C1-glucose. The resulting resonances are well dispersed and of high sensitivity, but they require an alternate assignment strategy. Combining easily collected data, such as 13C-edited NOESY and chemical shifts from 13C-detected HSQCs, with paramagnetic data from tagged proteins and a computational model, provides a path to these assignments. This path has been integrated into a software package, ASSIGN_SLP. We will illustrate application of this package to a cell-surface adhesion and signaling molecule, CEACAM1. We also will illustrate the use of the paramagnetic data in assessing conformation preferences of the glycans attached to CEACAM1.
Poster 32
GlyGen: Computational and Informatics Resources for Glycoscience
René Ranzinger
Presenting Author Email: rene@ccrc.uga.edu
Complex Carbohydrate Research Center, University of Georgia, Athens, GA-30602, USA
Advancing our understanding of the roles that glycosylation plays in development and disease is frequently hindered by the diversity of the data that must be integrated to gain insight into these complex phenomena. GlyGen is an initiative with the goal of democratizing glycoscience research by developing and implementing a data repository that integrates diverse types of data, including glycan structures, glycan biosynthesis enzymes, glycoproteins, along with genomic and proteomic knowledge. To achieve this integration, GlyGen has established international collaborations with database providers from different domains (including but not limited to EBI, NCBI, PDB, and GlyTouCan) and glycoscience data producers. Information from these resources and groups are standardized and cross-linked to allow queries across multiple domains. To facilitate easy access to this information, an intuitive, web-based interface (https://glygen.org) has been developed to visually represent the data.
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For each glycan and glycoprotein in the dataset, GlyGen provides a details page that displays information from the integrated resources in a concise representation. Individual details pages are interlinked with each other allowing easy data exploration across multiple domains. For example, users can browse from the webpage of a glycosylated protein to the glycan structures that have been described to be attached to this protein, and, from there, to other proteins that carry the same glycan. All information accessed through GlyGen is linked back to original sources, allowing users to easily access and browse through information pages in these resources as well. The GlyGen portal itself provides multiple different search interfaces for users to find glycans and proteins based on their properties or annotations. Beyond the data on glycans and proteins, GlyGen also provides multiple tools for studying glycosylation pathways, investigating relationships between glycans based on incomplete structures or mapping of different ID namespace.
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Our goal is to provide scientists with an easy way to access the complex information underlying state-of-the-art knowledge that describes the biology of glycans and glycoproteins. To schedule an individual demo of GlyGen or add your data to GlyGen contact Rene Ranzinger (rene@ccrc.uga.edu).
Poster 33
Understanding the structure and function of pectin O-acetyltransferases using in silico approaches
Lubana Shahin1,2, Emily Mathus2,3, Liang Zhang1,2, Debra Mohnen1,2, and Breeanna Urbanowicz1,2
1 Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA
2 Complex Carbohydrate Research Center, University of Georgia, Athens, GA
3 Department of Biological Sciences, University of Georgia, Athens, GA
Presenting Author Email: lubana.shahin@uga.edu
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Pectins comprise up to 35% of the primary plant cell wall (PCW) and play a vital role in cell-cell adhesion/interaction and defense mechanisms. Pectin O-acetylation influences both its physiochemical properties and its role in environmental adaptation. Pectin O-acetyltransferases (POATs) have been identified as members of the trichome birefringence-like (TBL) gene family in Arabidopsis thaliana; however, no POATs have been studied to derive structure-function relationships. Moreover, the substrate selectivity of many POATs (rhamnogalacturonan-I (RG-I) vs. homogalacturonan (HG)) remains enigmatic. Here, we aim to study POATs’ structure and function with a focus on identifying substrate-specific amino acids (RG-I/HG) using computational approaches. In this study, computational approaches like AlphaFold2 are used to predict POATs structures (Jumper et al., 2021). Protein sequence, structural alignment, and molecular docking are used to predict and identify amino acids involved in substrate binding. The role of identified amino acids on POATs enzymatic activity will be studied by introducing point mutations into the protein and conducting biochemical reactions with various pectin acceptor substrates. Understanding the function and catalytic activity of POATs will be an important step toward understanding the roles of the enzymes involved in polysaccharide acetylation.
Poster 34
Non-POMT1/2 O-Mannosylated Proteins Avoid POMGNT1 Extension
David Steen1, Sally Riewe Boyd1, Lorenzo Povolo2, Adnan Halim2, Lance Wells1
1Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, 30605, United States of America; 2Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark
Presenting Author Email: David.Steen@uga.edu
Protein O-linked mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a GlcNAc transferase that catalyzes the addition of GlcNAc to an O-linked mannose in the beta-1,2 position forming the core M1 structure. The M1 structure can be branched to form core M2 structures, and both can be further extended by enzymes in mammals. Despite recombinant POMGNT1 being promiscuous on multiple synthetic O-mannose peptides derived from alpha-dystroglycan (alpha-DG), multiple non-POMT1/2 O-mannosylated glycoproteins appear to only contain core M0 structures (i.e. the O-mannose monosaccharide) thus apparently avoiding extension by POMGNT1 in cells. These M0 modified glycoproteins include the cadherins and the plexins. In order to investigate this further, we expressed and purified all glycoproteins in HEK293F POMGNT1 KO cells: both known M0 O-mannosylated glycoproteins E-cadherin, N-cadherin, and c-MET as well as alpha-DG (to generate M0 structures at sites that would normally be M1). We confirmed that the M0 modified glycoproteins were indeed not extended and that the O-hexose observed via mass spectrometry was in fact an O-mannose based on mannosidase sensitivity. We also demonstrated that recombinant POMGNT1 extended O-mannose peptides derived from trypsin digests of all four of the glycoproteins. However, only alpha-DG served as an efficient acceptor substrate for POMGNT1 at the protein level. Given that O-mannosylation and protein folding occur in the ER while POMGNT1 is localized to the cis-Golgi, our results suggest a steric or electrostatic hindrance of POMGNT1 preventing it from acting on cadherins and plexins which is not present on alpha-DG.
Poster 35
Bordetellae Colonization oligosaccharide (b-Cool), a novel glycan essential for nasal colonization of Bordetella bronchiseptica.
Yang Su1,2, Maiya Callender3, Colleen Sedney3, Jillian Master3, John Glushka2, Evgeny Vinogradov4, Andrew Preston5, Thomas Krunkosky7, Eric T. Harvill3 and Maor Bar-Peled1,2
1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 2Complex Carbohydrate Research Center, University of Georgia, Athens, GA 3Department of Infectious Disease, University of Georgia, Athens, GA 4National Research Council Canada, Human Health Therapeutics Centre, Ottawa 5Department of Biology and Biochemistry, University of Bath, Bath, U.K. 6Department of Biomedical Sciences, University of Georgia, Athens, GA
Presenting Author Email: Yang Su, yang.su@uga.edu
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The Bordetella species (bordetellae) are highly contagious upper respiratory pathogens in both animals and humans. Despite worldwide vaccination, bordetellae continue to transmit due to their ability to colonize, proliferate, and persist in their hosts. Bordetellae efficiently colonize the hosts starting from a small number of bacteria, but bacterial factors involved in this important early step of infection are not well understood. Here we report a glycan factor that significantly contributes to the early nasal colonization of Bordetella bronchiseptica. This glycan is conserved in bordetellae, including recent clinical isolates of Bordetella pertussis. Thus, we named the glycan as bordetellae Colonization oligosaccharide (b-Cool). We have identified a 9-gene cluster encoding enzymes involved in b-Cool formation. LC-MS and NMR analyses revealed an abundant oligosaccharide present in B. bronchiseptica wild type but absent from the Δb-Cool mutant. Using mouse models, B. bronchiseptica Δb-Cool mutant showed 80% reduction in nasal colonization compared to wild type, starting as early as 6h post inoculation. Interestingly, the mutant showed colonization defects only in the nasal cavity but not in the trachea. To better understand this organ-specific colonization defect, we utilized primary nasal and trachea epithelial cells in air-liquid interface as infection models. We observed a significant colonization defect of the Δb-Cool compared to the wild type in primary nasal epithelia but not in primary trachea epithelia. These findings in cellular infection models are consistent with our findings in mouse models and indicate involvement of nasal-specific factors in the early colonization. As a consequence of the nasal colonization defect, the Δb-Cool showed 70% decreased ability to transmit amongst mice compared to the wild type. The significant role of b-Cool in nasal colonization and transmission makes this glycan a candidate for glyco-conjugate vaccine to combat the ongoing transmission of pertussis.
Poster 36
Development of High Sensitivity MS Methods for Identification and Quantification of Glycosphingolipids (GSLs)
Mehrnoush Taherzadeh Ghahfarrokhi, Stephanie Archer-Hartmann, Christian Heiss, Parastoo Azadi
Complex Carbohydrate Research Center (CCRC), University of Georgia, Athens, GA
Presenting Author Email: mehrnoush.taherzadeh@uga.edu
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Glycosphingolipids (GSLs) are a group of glycoconjugates with a glycan head that is glycosidically linked to the C-1 hydroxyl group of a ceramide tail. While ceramides exhibit variations, the primary structural and functional categorizations are based upon the diverse glycans. GSLs play pivotal roles in various biological processes including cell adhesion, signaling, proliferation, and apoptosis depending on their structural features. Thus, precise identification and quantification of GSLs hold significant importance. Mass spectrometric characterization of glycosphingolipids poses analytical challenges stemming from the multiplication of the structural complexity of the glycan and the ceramide. Therefore, it could be executed with different objectives. In this study two versatile, sensitive, and easy to adapt methods have been developed for identification and quantification of GSLs. In the first experiment, isolated intact GSLs were permethylated and analyzed using nESI-MS/MS. In the second experiment, GSL glycans were first cleaved with endoglycoceramidase (EGCase II) prior to analysis. The released glycans were labeled with procainamide and analyzed with UPLC-FLR-MS/MS. Notably, chromatographic separation of enzymatically released glycans enabled resolution of isomeric glycoforms that were indistinguishable utilizing permethylation methodology. Furthermore, this approach demonstrated superior sensitivity, identifying two additional glycoform masses. The EGCase was able to release both neutral and sialylated glycans with varying sizes. Optimization of the reactions, cleanup, chromatographic separation, and mass spectrometric method improved the coverage and sensitivity of the analyses. Since permethylation and procainamide labeling changes the m/z value of GSLs and glycoforms, the limited existing databases and tools for lipids and metabolites are of small use for data interpretation. Compilation of the m/z database, as well as utilization of a MATLAB algorithm for database search significantly decreased the time and effort needed for data interpretation. MS/MS was used for structure assignment confirmation in both methods. Finally, we investigated utilization of an internal standard for quantification of GSLs in both methods.
Poster 37
Elucidating the interaction mechanisms between the proteins involved in chondroitin sulfate biosynthesis
Daniel Tehrani
Biochemistry and Molecular Biology Department
University of Georgia
Dr. Kelley Moremen (PI)
Presenting Author Email: Daniel.Tehrani@uga.edu
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Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) attached to a core proteoglycan (PG) that is found in the extracellular matrix of connective tissues like cartilage, bone, skin, and ligaments. There it serves several varied roles including membrane flexibility, strength, and cell signaling. CS, and a related PG heparan sulfate (HS), are repeat disaccharide polymers that are both initiated through the formation of a tetrasaccharide linkage region attached to a variety of PG core proteins, like bikunin, syndecan, and glypican. The committed step in CS polymer synthesis results from the addition of a single β1,4-N-acetylgalactosamine (GalNAc) to the linker tetrasaccharide by the enzymes CSGALNACT1 or CSGALNACT2, instead of being initiated down the HS pathway by the addition of an α1,4-N-acetylglucosamine (GlcNAc) with EXTL3. Following this committed step, the CS polymer is elongated by alternating addition of β1,4GalNAc and β1,3-glucuronic acid (GlcA) residues by four proteins, CHPF1, CHPF2, CHSY1, and CHSY3, to yield CS chains as long as 100 monosaccharide units. However, their direct role in CS elongation is unknown. Sequence alignments and structural insight using AlphaFold Multimer reveal that all CHPFs and CHSYs contain both a Glycosyltransferase Family 7 (GT7)-like GlcA transferase domain and a GT31-like GalNAc transferase domain separated by a cystatin-like domain. Here, we create and analyze various heterodimer complex models to predict which oligomeric complexes might exist in the CS pathway. We then express these complexes to show that a combination of one CHPF and one CHSY can form viable complexes with one another that are capable of elongating CS chains, while individual proteins are not stably expressed. Furthermore, we will generate domain-specific catalytically inactive mutants to elucidate the co-polymerase activity of these proposed bifunctional, multidomain proteins. We also propose that heterocomplex formation is essential to provide stability and mechanistic features required for CS elongation. Further kinetic analysis on these enzyme complexes will provide an understanding of the roles of these proteins in CS biosynthesis and an improved understanding of the protein-specific interactions involved in CS elongation.
Poster 38
Using High-Resolution Ion Mobility to separate and identify α-Gal containing glycans from their non-α-Gal isomers
Chemistry Department at University of Georgia
Supervisor/Co-author: Ron Orlando, University of Georgia, orlando@ccrc.uga.edu
Presenting Author Email: Hoang Kim Ngan Thai, nht95736@uga.edu
Severe allergic reactions can happen from administering biotherapeutics containing non-human α-Gal (galactose-α-1,3-galactose) glycans to humans. Detection of α-Gal glycans remains a challenging task due to the presence of isomeric species. For instance, A2G1(α-Gal)F has an identical mass to A2G2F, one of the most common N-glycans found in antibody theraputics, making detection of A2G1(α-Gal)F difficult. Cetuximab is known to have N-glycans containing α-Gal, including A2G1(α-Gal)F, and was chosen for the study. The released N-glycans from Cetuximab were first run on a HILIC-MS/MS system. The system struggled to separate and identify A2G1(α-Gal)F from the A2G2F isomer. An Ion Mobility Spectrometer (IMS) was added to the system to improve resolving power. Glycans released from Cetuximab were then run on the HILIC-IMS-MS system. The mobiligram of A2G2F (m/z 1003.9) from Cetuximab contained two peaks whose collision cross section (CCS) values were 440±2Ų and 445±2Ų which suggested the existence of a secondary species. Exoglycosidase digestions were performed to confirm the Gal linkages in the two species. It was found that A2G2F gave rise to the peak at 440Ų, and the 445Ų peak corresponds to A2G1(α-Gal)F. The study revealed the ability of IMS as a powerful analytical technique that allows the separation and detection of α-Gal from their non-α-Gal isomers.
Poster 39
Network for Advanced NMR and CCRC NMR Facility: Opportunities for Metabolomics
Mario Uchimiya1, *, Alexander Eletsky1, John Glushka1, John Grimes1, Laura Morris1, Arthur S. Edison1
1Complex Carbohydrate Research Center, University of Georgia, Athens GA 30602
The Network for Advanced NMR (NAN) is an NSF-funded partnership between the University of Georgia, the University of Connecticut, and the University of Wisconsin at Madison (UW-Madison). Our goal is to provide access to state-of-the-art NMR resources for the scientific community. This includes a web portal for instrument search, user management, and data archiving and retrieval, and knowledgebases for biological and materials sciences, especially for users with limited NMR experience. This project includes the installation of two 1.1-GHz NMR instruments, a solid-state instrument currently operational at UW-Madison, and a solution-state instrument to be installed at CCRC in the summer of 2024. The CCRC NMR facility also features several instruments in the 600-900 MHz range with unique capabilities, including sample changers for automation, high-sensitivity cryogenic probes with 1H, 13C, 15N and 19F detection, and a unique 1.7-mm 800-MHz system for small samples in capillary tubes. The knowledgebases for metabolomics provides protocols and optimized NMR experiment parameter sets for metabolomics. It also includes example data sets, data processing tools, training, and educational materials. Researchers who are interested in applying metabolomics to their specific research projects can make use of these resources at NAN and the CCRC. This work is supported by NAN, the Edison Lab at UGA, and the Georgia Research Alliance.
Poster 40
Functional analysis of structural changes in del177 isoform of the CMP-Sia transporter
Brenda I. Velázquez Dodge1, Martínez-Duncker Iván1and Salinas-Marín Roberta1*
1Laboratorio de Glicobiología y Diagnóstico Molecular, Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, México.
Presenting Author Email: brenda.velazquez@uaem.edu.mx; * rsm@uaem.mx
Sialylation is the addition of sialic acid moieties to the terminal position on a variety of glycoconjugates in the medial and Trans-Golgi Network (TGN) through 20 sialyltransferases (STs) involving CMP-sialic acid (CMP-Sia) and CMP-Sia transporter (CST). In humans, the CST (hCST) consists of 337 amino acids, and is encoded by SLC35A1 that expresses 5 different isoforms. One isoform named del177 retains the C-terminal region and is functional despite losing 59 amino acids encoded by exon 6 of SLC35A1. The del177 isoform is also expressed in Chinese hamster ovary cells, but unlike the human, the del177-haCST variant is not functional due to probably to differences in amino acid residues respect to human isoform.
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Mutations in SLC35A1 can disrupt sialylation, leading to Congenital Disorders of Glycosylation (CDGs). These disorders present a range of symptoms, including developmental and intellectual disabilities, hypotonia, ataxia, and epilepsy. Sialic acid is essential for cellular processes like cell signaling and adhesion, emphasizing the importance of understanding CST functionality and its amino acid involvement.
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Our research focuses on the del177-hCST isoform, aiming to characterize its topology and identify key amino acids involved in its functionality. We conducted point mutations in the del177-haCST variant and assessed its function through resistance assays to RCA lectin I and MTT proliferation assays. Additionally, we analyzed the glycophenotype using SNA lectin staining and flow cytometry.
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Preliminary findings indicate promising results. The p. C263S mutation and insertion of G267 in del177-haCST partially restored sialylation, with recoveries of 44% and 22%, respectively. These findings were consistent with increased resistance to RCA I lectin and MTT proliferation assays.
Understanding the dynamics of CST isoforms offers potential insights into developing targeted therapies for CDGs. Further research into CST functionality is crucial for addressing the unmet need in CDG treatment and advancing precision medicine approaches.
Poster 41
Unleashing [NiFe]-Hydrogenases: Probing Catalytic Mechanisms with Breakthrough Selenocysteine Insertion Technology
Rhiannon M. Evans,1 Natalie Krahn,2 Joshua Weiss,2 Kylie A. Vincent,1 Dieter Söll3,4 and Fraser A. Armstrong1
1Department of Chemistry, University of Oxford, South Parks Road, Oxford, UK, OX1 3QR; 2Department of Biochemistry and Molecular Biology, B122 Life Sciences Bldg. University of Georgia, Athens, GA 30602, 3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA; 4Department of Chemistry, Yale University, New Haven, CT 06520, USA
Presenting Author Email: joshua.weiss@uga.edu
Hydrogenases are a class of redox enzymes with applications in complex carbohydrate-mediated hydrogen production in cell-free systems, wastewater treatment, biofuel cells, antioxidant therapies, energy metabolism, and more. [NiFe]-hydrogenases are a subtype of hydrogenases with a valuable, rare property: electrochemical reversibility. This allows efficient, reversible conversion of molecular hydrogen to protons and electrons, making [NiFe]-hydrogenases an ideal candidate to uncover hydrogenase mechanisms. [NiFe]-hydrogenases importantly demonstrate long-range proton-coupled electron transfer (PCET) in their active sites with a Ni-coordinated cysteine (Cys) which are hallmarks of enzymatic electrochemical reversibility. Specifically, the Cys-S atoms’ function in [NiFe]-hydrogenases are usually probed by substituting similar amino acids, impacting the structure of the active site. Although selenocysteine (Sec) is an almost identical amino acid to Cys, limitations in site-specific Sec insertion into proteins prohibited its use as a probe. Here, we utilize recent advancements in Sec insertion technology to substitute key catalytic Cys residues with Sec in E. coli [NiFe]-Hydrogenase-2 (Hyd-2). Through protein film voltammetry, Sec substitution was shown to 1) greatly enhance proton reduction compared to H2 oxidation and 2) bidirectionally increase overpotential. This data provides compelling evidence that a Ni-coordinating Cys contributes to [NiFe]-hydrogenase electrocatalytic reversibility. This work experimentally validates a long-standing mechanistic hypothesis surrounding [NiFe]-hydrogenase functioning which will aid in future hydrogenase engineering and points to a broader evolutionary mechanism in microbial energy processing efficiency.
Poster 42
Developing enzymatic tools to analyze Rhamnogalacturonan I (RG-I) structure
Liang Zhang1, Jiri Vlach1, Stephanie Archer-Hartmann1, Ian Black1, Parastoo Azadi1, and Breeanna Urbanowicz1
1Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30601
Presenting Author Email: lzhang417@uga.edu
Rhamnogalacturonan I (RG-I) is a structurally complex pectic polysaccharide found in plant cell walls. The RG-I backbone is formed by a disaccharide repeat of (1,2)-α-L-rhamnose-(1,4)-α-D-galacturonic acid. The rhamnose residues in the backbone can be substituted at O-4 with monosaccharides and linear or branched arabinan, galactan, and arabinogalactan side chains. The galacturonic acid residues in the backbone can also be acetylated at O-2 or O-3. RG-I plays important roles in seed germination, root growth, stem and pollen tube elongation, fruit ripening, and cell anisotropic growth. However, the relationship between RG-I structure and its role in plant cell wall architecture is not well understood, due to a lack of analytic methods. We prepared RG-I from different plant tissues. We expressed an RG-I specific lyase (SRG-PL) and applied this enzyme to study RG-I structure. To achieve this, RG-I was first analyzed by NMR, and then hydrolyzed with SRG-PL. The resultant fragments were analyzed by high-performance mass spectrometry. Taken together, our data showed RG-I from different plant tissues was dissimilar in terms of the presence of side chains and O-acetylation, which indicated RG-I structure may influence its function in different tissues. During our work, we also found that the fragments from the enzymatic digestion of RG-I could be well separated on a size exclusion column, providing a way to prepare diverse RG-I oligosaccharides that can be used as substrates to study enzymes involved in RG-I synthesis.
Poster 42 [Stand-In]
The close cousins of O-GlcNAc-transferases: the O-fucose-transferases
Megna Tiwari-Crowe1,2,3, Elisabet Gas Pascual1,2,3, Msano Mandalasi1,2,3, Ana-Maria Garcia3, Manish Goyal4, Marla Popov 2, Ron Orlando2,3, John Samuelson4, and Christopher M. West1,2,3
1Center for Tropical and Emerging Global Diseases, 2Complex Carbohydrate Research Center, 3Department of Biochemistry and Molecular Biology, University of Georgia, Athens GA; 4Department of Molecular and Cell Biology, Boston University School of Medicine, Boston MA
Toxoplasma gondii, the causative agent of Toxoplasmosis, infects over 30% of the human population worldwide. This parasite is highly successful in adapting to highly varied environment as it transits among tissues and invades cells. Of interest here is the O-fucosyltransferase (OFT TgSPY, named after the plant OFT named SPINDLY, that modifies Ser/Thr residues of 33 nucleocytoplasmic proteins of tachyzoites with a single fucose residue. OFT-like genes occur in the parasites Cryptosporidium and Acanthamoeba and social amoeba Dictyostelium. This process is related to the O-GlcNAcylation of nucleocytoplasmic proteins in humans and plants, which has been implicated in mediating stress and nutritional responses. SPY is required for optimal growth of Toxoplasma and Dictyostelium in vitro, and the high degree of conservation of OFT with OGT suggests that OFT may also mediate responses to stress. To facilitate investigation of O-Fuc in T. gondii and other protists, we developed antibodies specific for fucose-O-Ser or fucose-O-Thr (anti-FOS/T). Unlike AAL that cross-reacts with terminal fucose on N- and O-linked glycans of ER, Golgi, secreted, and plasma membrane proteins, these anti-FOS/T antibodies only bind to nucleocytosolic proteins modified by the OFT. Anti-FOS/T labeling in Toxoplasma, Cryptosporidium, Acanthamoeba, and Dictyostelium is reminiscent of O-GlcNAc in humans. Interestingly, anti-FOS/T detects overlapping sets of proteins by western blot analysis, with a subset of what is recognized by AAL in Arabidopsis. Furthermore, the O-fucome of Dictyostelium includes proteins that overlap with the O-fucome of Toxoplasma and is highly response to starvation-induced development. The anti-FOS/T antibodies promise a revolution in the identification and localization of nucleocytosolic proteins modified with O-fucose in protists, plants, and bacteria, which cannot be studied with AAL because of the presence of terminal fucose on secreted proteins.
To facilitate further investigation of these differences in Toxoplasma and other protists containing the OFT,we present here the development of rabbit antibodies specific for fucose-O-Ser and fucose-O-Thr (anti-FOS/T). Unlike AAL that cross-reacts with terminal fucose on N- and O-linked glycans of ER, Golgi, secreted, and plasma membrane proteins, these anti-FOS/T antibodies only bind to nucleocytosolic proteins modified by the OFT. We show that anti-FOS/T antibody labels Toxoplasma nuclei without cross-reacting with host secreted proteins, and labeling is inhibited by fucose and lost in spyΔ tachyzoites. The anti-FOS/T antibodies promise a revolution in the identification and localization of nucleocytosolic proteins modified with O-fucose in protists, plants, and bacteria, which cannot be studied with AAL because of the presence of terminal fucose on secreted proteins.