Polypeptide GalNAc-transferases Research Articles

O-glycosylation of SARS-CoV-2 spike protein by host O-glycosyltransferase strengthens its trimeric structure.

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.

Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

ISOGlyP: O-Glycosylation Site Prediction Using Peptide Sequences and GALNTs.

Mucin-type O-glycosylation is one of the most common posttranslational modifications of proteins. The abnormal expression of various polypeptide GalNAc-transferases (GALNTs) which initiate and define sites of O-glycosylation is linked to many cancers and other diseases. Many current O-glycosylation prediction programs utilize O-glycoproteomics data obtained without using the transferase isoform(s) responsible for the glycosylation. With 20 different GALNTs in humans, having the ability to predict and interpret O-glycosylation sites in terms of specific GALNT isoforms is invaluable.To fill this gap, ISOGlyP (isoform-specific O-glycosylation prediction) has been developed. Using position-specific enhancement values generated based on GalNAc-T isoform-specific amino acid preferences, ISOGlyP predicts the propensity that a site would be glycosylated by a specific transferase. ISOGlyP gave an overall prediction accuracy of 70% against in vivo data, which is comparable to that of the NetOGlyc4.0 predictor. Additionally, ISOGlyP can identify the known effects of long- and short-range prior glycosylation and can generate potential peptide sequences selectively glycosylated by specific isoforms. ISOGlyP is freely available for use at https://ISOGlyP.utep.edu . The code is also available on GitHub ( https://github.com/jonmohl/ISOGlyP ).

RUNX3-Regulated GALNT6 Promotes the Migration and Invasion of Hepatocellular Carcinoma Cells by Mediating O-Glycosylation of MUC1.

Background Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Dysregulation of messenger RNAs (mRNA) has been recognized to be associated with HCC carcinogenesis and development. Polypeptide GalNAc Transferase 6 (GALNT6), an O-type glycosyltransferase, has been confirmed as tumor promoter in different cancers. However, the function of GALNT6 in HCC remains to be studied. Methods RT-qPCR and western blot experiments were, respectively, performed for evaluating RNA expressions and protein levels. Supported by bioinformatics analysis, mechanism assays were conducted for validating the potential relation between different genes. Functional assays were implemented to analyze HCC cell migration and invasion after different transfections. Results GALNT6 was aberrantly upregulated in HCC cells. Knockdown of GALNT6 could repress HCC cell migration and invasion. RUNX3 was verified to bind to GALNT6 promoter and activate GALNT6 transcription. GALNT6 depletion led to inhibited O-glycosylation and aggravated degradation of MUC1. MUC1 overexpression could rescue the impeded HCC cell migration and invasion induced by GALNT6 knockdown. Conclusion To sum up, GALNT6 transcriptionally activated by RUNX3 mediated the O-glycosylation of MUC1, thus exerting promoting influence on HCC cell migration and invasion.

Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling.

The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.

GALNT2 regulates ANGPTL3 cleavage in cells and in vivo of mice

Angiopoietin-like protein 3 (ANGPTL3) is an important inhibitor of lipoprotein lipase and endothelial lipase that plays critical roles in lipoprotein metabolism. It specifically expresses in the liver and undergoes proprotein convertase-mediated cleavage during secretion, which generates an N-terminal coiled-coil domain and C-terminal fibrinogen-like domain that has been considered as the activation step for its function. Previous studies have reported that the polypeptide GalNAc-transferase GALNT2 mediates the O-glycosylation of the ANGPTL3 near the cleavage site, which inhibits the proprotein convertase (PC)-mediated cleavage in vitro and in cultured cells. However, loss-of-function mutation for GALNT2 has no effect on ANGPTL3 cleavage in human. Thus whether GALNT2 regulates the cleavage of ANGPTL3 in vivo is unclear. In present study, we systematically characterized the cleavage of Angptl3 in cultured cells and in vivo of mice. We found that endogenous Angptl3 is cleaved in primary hepatocytes and in vivo of mice, and this cleavage can be blocked by Galnt2 overexpression or PC inhibition. Moreover, suppressing galnt2 expression increases the cleavage of Angptl3 in mice dramatically. Thus, our results support the conclusion that Galnt2 is a key endogenous regulator for Angptl3 cleavage both in vitro and in vivo.

ISOGlyP: de novo prediction of isoform-specific mucin-type O-glycosylation.

Mucin-type O-glycosylation is one of the most common posttranslational modifications of proteins. The abnormal expression of various polypeptide GalNAc-transferases (GalNAc-Ts) which initiate and define sites of O-glycosylation are linked to many cancers and other diseases. Current O-glycosyation prediction programs utilize O-glycoproteomics data obtained without regard to the transferase isoform (s) responsible for the glycosylation. With 20 different GalNAc-Ts in humans, having an ability to predict and interpret O-glycosylation sites in terms of specific GalNAc-T isoforms is invaluable. To fill this gap, ISOGlyP (Isoform-Specific O-Glycosylation Prediction) has been developed. Using position-specific enhancement values generated based on GalNAc-T isoform-specific amino acid preferences, ISOGlyP predicts the propensity that a site would be glycosylated by a specific transferase. ISOGlyP gave an overall prediction accuracy of 70% against in vivo data, which is comparable to that of the NetOGlyc4.0 predictor. Additionally, ISOGlyP can identify the known effects of long- and short-range prior glycosylation and can generate potential peptide sequences selectively glycosylated by specific isoforms. ISOGlyP is freely available for use at ISOGlyP.utep.edu. The code is also available on GitHub (https://github.com/jonmohl/ISOGlyP).

Predicting mucin-type O-Glycosylation using enhancement value products from derived protein features.

Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins. This glycosylation is initiated in the Golgi by the addition of the sugar N-acetylgalactosamine (GalNAc) onto protein Ser and Thr residues by a family of polypeptide GalNAc transferases. In humans there are 20 isoforms that are differentially expressed across tissues that serve multiple important biological roles. Using random peptide substrates, isoform specific amino acid preferences have been obtained in the form of enhancement values (EV). These EVs alone have previously been used to predict O-glycosylation sites via the web based ISOGlyP (Isoform Specific O-Glycosylation Prediction) tool. Here we explore additional protein features to determine whether these can complement the random peptide derived enhancement values and increase the predictive power of ISOGlyP. The inclusion of additional protein substrate features (such as secondary structure and surface accessibility) was found to increase sensitivity with minimal loss of specificity, when tested with three different published in vivo O-glycoproteomics data sets, thus increasing the overall accuracy of the ISOGlyP predictions.

Ser and Thr acceptor preferences of the GalNAc-Ts vary among isoenzymes to modulate mucin-type O-glycosylation.

A family of polypeptide GalNAc-transferases (GalNAc-Ts) initiates mucin-type O-glycosylation, transferring GalNAc onto hydroxyl groups of Ser and Thr residues of target substrates. The 20 GalNAc-T isoenzymes in humans are classified into nine subfamilies according to sequence similarity. GalNAc-Ts select their sites of glycosylation based on weak and overlapping peptide sequence motifs, as well prior substrate O-GalNAc glycosylation at sites both remote (long-range) and neighboring (short-range) the acceptor. Together, these preferences vary among GalNAc-Ts imparting each isoenzyme with its own unique specificity. Studies on the first identified GalNAc-Ts showed Thr acceptors were preferred over Ser acceptors; however studies comparing Thr vs. Ser glycosylation across the GalNAc-T family are lacking. Using a series of identical random peptide substrates, with single Thr or Ser acceptor sites, we determined the rate differences (Thr/Ser rate ratio) between Thr and Ser substrate glycosylation for 12 isoenzymes (representing 7 GalNAc-T subfamilies). These Thr/Ser rate ratios varied across subfamilies, ranging from ~2 to ~18 (for GalNAc-T4/GalNAc-T12 and GalNAc-T3/GalNAc-T6, respectively), while nearly identical Thr/Ser rate ratios were observed for isoenzymes within subfamilies. Furthermore, the Thr/Ser rate ratios did not appreciably vary over a series of fixed sequence substrates of different relative activities, suggesting the ratio is a constant for each isoenzyme against single acceptor substrates. Finally, based on GalNAc-T structures, the different Thr/Ser rate ratios likely reflect differences in the strengths of the Thr acceptor methyl group binding to the active site pocket. With this work, another activity that further differentiates substrate specificity among the GalNAc-Ts has been identified.

Molecular basis for fibroblast growth factor 23 O-glycosylation by GalNAc-T3.

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3's structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

UDP-N-acetyl-\u03b1-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase from the snail Biomphalaria glabrata \u2013 structural reflections

UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41) is the initiating enzyme for mucin-type O-glycosylation in animals. Members of this highly conserved glycosyltransferase family catalyse a single glycosidic linkage. They transfer an N-acetylgalactosamine (GalNAc) residue from an activated donor (UDP-GalNAc) to a serine or threonine of an acceptor polypeptide chain. A ppGalNAcT from the freshwater snail Biomphalaria glabrata is the only characterised member of this enzyme family from mollusc origin. In this work, we interpret previously published experimental characterization of this enzyme in the context of in silico models of the enzyme and its acceptor substrates. A homology model of the mollusc ppGalNAcT is created and various substrate peptides are modelled into the active site. We hypothesize about possible molecular interpretations of the available experimental data and offer potential explanations for observed substrate and cofactor specificity. Here, we review and synthesise the current knowledge of Bge-ppGalNAcT, supported by a molecular interpretation of the available data.

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs.

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

Exploring Regulation of Protein O-Glycosylation in Isogenic Human HEK293 Cells by Differential O-Glycoproteomics

Most proteins trafficking the secretory pathway of metazoan cells will acquire GalNAc-type O-glycosylation. GalNAc-type O-glycosylation is differentially regulated in cells by the expression of a repertoire of up to twenty genes encoding polypeptide GalNAc-transferase isoforms (GalNAc-Ts) that initiate O-glycosylation. These GalNAc-Ts orchestrate the positions and patterns of O-glycans on proteins in coordinated, but poorly understood ways - guided partly by the kinetic properties and substrate specificities of their catalytic domains, as well as by modulatory effects of their unique GalNAc-binding lectin domains. Here, we provide the hereto most comprehensive characterization of nonredundant contributions of individual GalNAc-T isoforms to the O-glycoproteome of the human HEK293 cell using quantitative differential O-glycoproteomics on a panel of isogenic HEK293 cells with knockout of GalNAc-T genes (GALNT1, T2, T3, T7, T10, or T11). We confirm that a major part of the O-glycoproteome is covered by redundancy, whereas distinct O-glycosite subsets are covered by nonredundant GalNAc-T isoform-specific functions. We demonstrate that the GalNAc-T7 and T10 isoforms function in follow-up of high-density O-glycosylated regions, and that GalNAc-T11 has highly restricted functions and essentially only serves the low-density lipoprotein-related receptors in linker regions (C6XXXTC1) between the ligand-binding repeats.

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

The GalNAc-type O-glycoproteome is orchestrated by a large family of polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with partially overlapping contributions to the O-glycoproteome besides distinct nonredundant functions. Increasing evidence indicates that individual GalNAc-Ts co-regulate and fine-tune specific protein functions in health and disease, and deficiencies in individual GALNT genes underlie congenital diseases with distinct phenotypes. Studies of GalNAc-T specificities have mainly been performed with in vitro enzyme assays using short peptide substrates, but recently quantitative differential O-glycoproteomics of isogenic cells with and without GALNT genes has enabled a more unbiased exploration of the nonredundant contributions of individual GalNAc-Ts. Both approaches suggest that fairly small subsets of O-glycosites are nonredundantly regulated by specific GalNAc-Ts, but how these isoenzymes orchestrate regulation among competing redundant substrates is unclear. To explore this, here we developed isogenic cell model systems with Tet-On inducible expression of two GalNAc-T genes, GALNT2 and GALNT11, in a knockout background in HEK293 cells. Using quantitative O-glycoproteomics with tandem-mass-tag (TMT) labeling, we found that isoform-specific glycosites are glycosylated in a dose-dependent manner and that induction of GalNAc-T2 or -T11 produces discrete glycosylation effects without affecting the major part of the O-glycoproteome. These results support previous findings indicating that individual GalNAc-T isoenzymes can serve in fine-tuned regulation of distinct protein functions.

Site-specific glycosylation of Ebola virus glycoprotein by human polypeptide GalNAc-transferase 1 induces cell adhesion defects

The surface glycoprotein (GP) of Ebola virus causes many of the virus's pathogenic effects, including a dramatic loss of endothelial cell adhesion associated with widespread hemorrhaging during infection. Although the GP-mediated deadhesion depends on its extracellular mucin-like domain, it is unknown whether any, or all, of this domain's densely clustered O-glycosylation sites are required. It is also unknown whether any of the 20 distinct polypeptide GalNAc-transferases (ppGalNAc-Ts) that initiate mucin-type O-glycosylation in human cells are functionally required. Here, using HEK293 cell lines lacking specific glycosylation enzymes, we demonstrate that GP requires extended O-glycans to exert its deadhesion effect. We also identified ppGalNAc-T1 as largely required for the GP-mediated adhesion defects. Despite its profound effect on GP function, the absence of ppGalNAc-T1 only modestly reduced the O-glycan mass of GP, indicating that even small changes in the bulky glycodomain can cause loss of GP function. Indeed, protein-mapping studies identified a small segment of the mucin-like domain critical for function and revealed that mutation of five glycan acceptor sites within this segment are sufficient to abrogate GP function. Together, these results argue against a mechanism of Ebola GP-induced cell detachment that depends solely on ectodomain bulkiness and identify a single host-derived glycosylation enzyme, ppGalNAc-T1, as a potential target for therapeutic intervention against Ebola virus disease.

The Multiplicity of Polypeptide GalNAc-Transferase: Assays, Inhibitors, and Structures.

Mucin-type O-glycosylation is the dominant form of glycosylation in eukaryotes and plays an important role in various physiological processes. The polypeptide GalNAc-transferase (GalNAc-T) catalyzes the first step in the attachment of mucin-type O-glycosylation. GalNAc-T was recently uncovered to be linked with cancer, atherogenic dyslipidemia, and X-linked hypophosphatemic rickets. Therefore, it has attracted increasing interest as a new target for exploring the underlying mechanism and developing new treatments for related diseases. Decades of studies on GalNAc-T have laid a stable foundation for understanding the catalytic mechanism, determining atom-resolution three-dimensional structures, and developing various types of biochemical assays as well as small-molecule inhibitor leads. Here, we systematically summarize this invaluable knowledge on GalNAc-T and cultivate new perspectives to foster breakthrough points for mucin-type O-glycosylation.

Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4.

Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a “naked” Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.

A study of the association of rs12040273 with susceptibility and severity of coronary artery disease in a Chinese Han population

BackgroundThe single nucleotide polymorphism (SNP) rs12040273, a variant of UDP-N–acetylgalactosamine, polypeptide GalNAc-transferase 2, has recently been reported to be significantly associated with development of carotid artery intima-media thickness (IMT) in a Chinese population based on a genome-wide association study. Because IMT is a potent marker of coronary artery disease (CAD), the aim of this study was to evaluate the relation of rs12040273 to susceptibility and severity of CAD in a Chinese Han population.MethodsWe performed a hospital-based case-control study. Three hundred and thirty-one individuals (199 CAD patients and 112 non-CAD controls) undergoing coronary angiography were consecutively enrolled in the study. The Gensini score results were used to assess the severity of CAD. The method of polymerase chain reaction-ligase detection reaction (PCR-LDR) was used to distinguish different genotypes at rs12040273.ResultsThe distribution of genotypes at rs12040273 was comparable between CAD patients and non-CAD controls (P > 0.05). The frequencies of the genotypes were also not significantly associated with the risk of CAD and its severity assessed by the Gensini score method, with the OR of 1.38 (95% CI = 0.80–2.40, P = 0.24) and 1.14 (95% CI = 0.69–1.86, P = 0.60) respectively. However, stratified analysis showed that the serum HDL-C levels of subjects with the CC genotype were significantly higher than those with CT/TT genotypes in non-CAD controls (P = 0.002).ConclusionOur results suggest that the rs12040273 variants might not be associated with the susceptibility of CAD or its severity in a Chinese Han population. Moreover, the CC genotype could be associated with elevated serum HDL-C levels.

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.