Structural and Mechanistic Characterization of Leukocyte-Type Core 2 β1,6-N-Acetylglucosaminyltransferase: A Metal-Ion-Independent GT-A Glycosyltransferase

Pak, John E.; Satkunarajah, Malathy; Seetharaman, J.; Rini, James M.

doi:10.1016/j.jmb.2011.10.039

Cited by 17 publications

(30 citation statements)

References 63 publications

(63 reference statements)

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“…In the mammalian GT6, the metal binding D X D motif is at the junction of the two subdomains but, in the bacterial enzyme, it is replaced by the Asn 95 -Ala-Asn 97 sequence. The D X D motif is a shared feature of all GT families with GT-A folds (19, 20) with the exception of the metal-independent GT14 family (21). …”

Section: Resultsmentioning

confidence: 99%

“…The GT14 and GT6 families differ in catalyzing inverting and retaining reactions, respectively, but GT14 members also differ in having no conserved motif corresponding to D X D (or N X N). The crystallographic structures of one GT14, leukocyte type core 2 β1,6- N -acetylglucosaminyl-transferase (C2GnT-L), in the apo-form and in complexes with either acceptor substrate or UDP, have been determined (21, 30). The enzyme is a disulfide-bonded dimer and, in the complex with UDP, the two molecules of each dimer are in “open” and “closed” conformations.…”

Section: Resultsmentioning

confidence: 99%

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Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

Pham¹,

Stinson²,

Thiyagarajan³

et al. 2014

Journal of Biological Chemistry

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Background: Bacterial homologues of human blood group synthases (glycosyltransferase family GT6) differ in being metal-independent.Results: The structure has been determined of a GT6 from Bacteroides ovatus in a complex with the substrate UDP-GalNAc.Conclusion: Interactions with the polypeptide replace substrate-metal interactions in metal-dependent mammalian homologues.Significance: Metal independence in GT6 is attainable because the metal acts in substrate binding but not directly in catalysis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

Pham¹,

Stinson²,

Thiyagarajan³

et al. 2014

Journal of Biological Chemistry

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“…Although both C2GnT1 and 2 have a SPDE sequence resembling a DxD motif which was shown for C2GnT1 to contain the catalytic base Glu320 [45,46], a metal ion cofactor was not required for catalysis. Interestingly, the presence of 12.5 mM Mn 2+ in the assay led to an 83–85% reduction of C2GnT2 activity using core 1 or core 3 substrates.…”

Section: Resultsmentioning

confidence: 99%

“…The crystal structures of human T2 and T10 [49,50] as well as mouse T1 [51] reveal a lectin binding domain, which suggests that the enzymes can bind a glycopeptide as the acceptor substrate. Of the four enzymes that extend the Tn antigen, only the crystal structure of mouse C2GnT1 is known, with and without the acceptor or UDP [45,46]. It appears that the peptide moiety of glycopeptides is not bound to the enzyme but extrudes into the solution.…”

Section: Discussionmentioning

confidence: 99%

Acceptor specificities and selective inhibition of recombinant human Gal- and GlcNAc-transferases that synthesize core structures 1, 2, 3 and 4 of O-glycans

Gao

Aryal

et al. 2013

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior. Methods We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors. Results Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme–substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-D-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs. Conclusions This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4. General significance These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.

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“…4A, other two amino acids identified by magenta asterisks) playing a role in acceptor substrate. In particular, the K251 and R254 residues were found to make key hydrogen bonds and a van der Waals interaction with the bound disaccharide acceptor (37). Thus, alternative splicing removes key amino acids involved in the acceptor substrate binding site and could therefore affect the activity of pBo17.…”

Section: Resultsmentioning

confidence: 99%

Bovine Herpesvirus 4 Modulates Its β-1,6- N -Acetylglucosaminyltransferase Activity through Alternative Splicing

Lété

Markine-Goriaynoff

Machiels

et al. 2016

J Virol

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Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein ␤-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus. IMPORTANCEViruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme which is a key enzyme for the synthesis of complex O-glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection.

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Structural and Mechanistic Characterization of Leukocyte-Type Core 2 β1,6-N-Acetylglucosaminyltransferase: A Metal-Ion-Independent GT-A Glycosyltransferase

Cited by 17 publications

References 63 publications

Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

Acceptor specificities and selective inhibition of recombinant human Gal- and GlcNAc-transferases that synthesize core structures 1, 2, 3 and 4 of O-glycans

Bovine Herpesvirus 4 Modulates Its β-1,6- N -Acetylglucosaminyltransferase Activity through Alternative Splicing

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