Polypeptide-GalNAc-Transferase-13 Shows Prognostic Impact in Breast Cancer.
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ABSTRACT:
SUBMITTER: Fernandez E
PROVIDER: S-EPMC8616257 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:O-Linked ?-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.
| S-EPMC6813768 | biostudies-literature
Project description:N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.
| S-EPMC4245906 | biostudies-literature
Project description:It is estimated that >50% of proteins are glycosylated with sugar tags that can modulate protein activity through what has been called the sugar code. Here we present the first QM/MM calculations of human GalNAc-T2, a retaining glycosyltransferase, which initiates the biosynthesis of mucin-type O-glycans. Importantly, we have characterized a hydrogen bond between the ?-phosphate of UDP and the backbone amide group from the Thr7 of the sugar acceptor (EA2 peptide) that promotes catalysis and that we propose could be a general catalytic strategy used in peptide O-glycosylation by retaining glycosyltransferases. Additional important substrate-substrate interactions have been identified, for example, between the ?-phosphate of UDP with the attacking hydroxyl group from the acceptor substrate and with the substituent at the C2' position of the transferred sugar. Our results support a front-side attack mechanism for this enzyme, with a barrier height of ~20 kcal mol(-1) at the QM(M05-2X/TZVP//BP86/SVP)/CHARMM22 level, in reasonable agreement with the experimental kinetic data. Experimental and in silico mutations show that transferase activity is very sensitive to changes in residues Glu334, Asn335 and Arg362. Additionally, our calculations for different donor substrates suggest that human GalNAc-T2 would be inactive if 2'-deoxy-Gal or 2'-oxymethyl-Gal were used, while UDP-Gal is confirmed as a valid sugar donor. Finally, the analysis herein presented highlights that both the substrate-substrate and the enzyme-substrate interactions are mainly concentrated on stabilizing the negative charge developing at the UDP leaving group as the transition state is approached, identifying this as a key aspect of retaining glycosyltransferases catalysis.
| S-EPMC4744471 | biostudies-literature
Project description:BackgroundThe family of polypeptide N-acetylgalactosaminyl transferases is responsible for the altered O-linked glycosylation occurring during the development of various cancers and their progression via altering O-glycan biosynthesis. Our studies were designed to investigate the expression and prognostic values of GalNAc-T5 and improve the risk stratification in patients with gastric cancer.MethodsTissue samples from a training set and a validation set of patients with gastric adenocarcinoma from China were used for analyses. GalNAc-T5 expression was retrospectively analysed by immunohistochemistry. Results were assessed for association with clinicopathological parameters and overall survival by using Kaplan-Meier analysis. Prognostic values of GalNAc-T5 expression and clinical outcomes were evaluated by Cox regression analysis. A molecular prognostic stratification scheme incorporating GalNAc-T5 expression was determined by using receiver operating characteristic analysis.ResultsGalNAc-T5 expression was markedly reduced in gastric cancer tissues compared with non-malignant gastric mucosa. Low intratumoral GalNAc-T5 density, which was associated with tumour cell differentiation, T classification, N classification, and TNM stage in the two independent sets, was an independent prognosticator for poor prognosis of gastric cancer patients. Applying the prognostic value of intratumoral GalNAc-T5 density to the conventional clinicopathologic TNM stage system showed a better prognostic value in patients with gastric cancer.ConclusionsIntratumoral GalNAc-T5 expression was recognised as an independent prognostic marker for the overall survival of gastric cancer patients. Detection of GalNAc-T5 expression in gastric cancer tissues might add some prognostic information for patients with this disease and lead to a more accurate classification under the TNM stage system.
| S-EPMC3992513 | biostudies-literature
Project description:O-linked glycans of secreted and membrane-bound proteins have an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues and suppression of GalNAc-T3 significantly attenuates the growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine the nucleotide-binding protein, α-transducing activity polypeptide-1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1 and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers.
| S-EPMC3373266 | biostudies-literature
Project description:UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltrans- ferases (ppGalNAc Ts) comprise a large family of glycosyltransferases that initiate mucin-type protein O-glycosylation, transferring alpha-GalNAc to Thr and Ser residues of polypeptide acceptors. Families of ppGalNAc Ts are found across diverse eukaryotes with orthologs identifiable from mammals to single-cell organisms. The peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly understood. Previously, we reported a series of oriented random peptide substrate libraries for quantitatively determining the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 (Gerken TA, Raman J, Fritz TA, Jamison O. 2006. Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 & T2 (ppGalNAc T1 & T2) derived from oriented random peptide substrates. J Biol Chem. 281:32403-32416). With these substrates, previously unknown features of the transferases were revealed. Utilizing these and a new lengthened set of random peptides, studies have now been performed on PGANT5 and PGANT2, the Drosophila orthologs of T1 and T2. The results from these studies suggest that the major peptide substrate determinants for these transferases are contained within 2 to 3 residues flanking the site of glycosylation. It is further found that the mammalian and fly T1 orthologs display very similar peptide substrate preferences, while the T2 orthologs are nearly indistinguishable, suggesting similar peptide preferences amongst orthologous pairs have been maintained across evolution. This conclusion is further supported by sequence homology comparisons of each of the transferase orthologs, showing that the peptide substrate and UDP binding site residues are more highly conserved between species relative to their remaining catalytic and lectin domain residues.
| S-EPMC2574660 | biostudies-literature
Project description:Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large number of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.
| S-EPMC3409716 | biostudies-literature
Project description:Initial glycosylation of mucin-type O-linked protein is catalysed by one of the UDP-GalNAc: polypeptide N-acetyl-galactosaminyl transferase-3 (GalNAc-T3). O-glycosylation is important in the binding of cell adhesion molecules, cell differentiation, invasion, and metastasis in tumours. This study was designed to detect GalNAc-T3 expression in lung adenocarcinoma by using immunohistochemical staining, and to evaluate the relationship between the GalNAc-T3 expression level and prognosis and recurrence in completely resected lung adenocarcinoma patients. A low expression of GalNAc-T3 was detected in the cytoplasm of tumour cells in 79 of 148 patients (53.4%) with lung adenocarcinoma. The low expression of GalNAc-T3 was associated with poorly differentiated tumour (P<0.0001), poor pathologic stage (P<0.0001), lymph node metastasis (P<0.0001), and tumour recurrence (P=0.016). The lung carcinoma patients with low GalNAc-T3 expression had a poorer prognosis than those with high GalNAc-T3 expression, using both univariate and multivariate analyses (overall survival: P<0.0001 and P=0.011, respectively). In addition, multivariate analysis of the clinicopathological characteristics of stage I lung adenocarcinoma indicated that the low expression of GalNAc-T3 was a significant independent factor for predicting poor prognosis and early recurrence (P=0.006, rr=2.87 and P=0.019, rr=3.05, respectively). The low expression of GalNAc-T3 may be a useful marker for predicting poor prognosis and early recurrence in completely resected lung carcinoma patients, particularly patients with stage I diseases.
| S-EPMC2409559 | biostudies-other
Project description: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.
| S-EPMC6314128 | biostudies-literature
Project description:Mammalian mucin-type O-glycosylation is initiated by a large family of ∼20 UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer α-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the -1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.
| S-EPMC3077648 | biostudies-literature