Abstract
N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a β1-6-linked N-acetylglucosamine branch. β1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GnT-V a promising target for drug development. However, the molecular basis of GnT-V’s catalytic mechanism and substrate specificity are not fully understood. Here, we report crystal structures of human GnT-V luminal domain with a substrate analog. GnT-V luminal domain is composed of a GT-B fold and two accessary domains. Interestingly, two aromatic rings sandwich the α1-6 branch of the acceptor N-glycan and restrain the global conformation, partly explaining the fine branch specificity of GnT-V. In addition, interaction of the substrate N-glycoprotein with GnT-V likely contributes to protein-selective and site-specific glycan modification. In summary, the acceptor-GnT-V complex structure suggests a catalytic mechanism, explains the previously observed inhibition of GnT-V by branching enzyme GnT-III, and provides a basis for the rational design of drugs targeting N-glycan branching.
Highlights
N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a β1-6-linked N-acetylglucosamine branch. β1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GlcNAc transferases (GnTs)-V a promising target for drug development
Genetic ablation of the MGAT5 gene in mice resulted in reduced growth and metastasis of a mammary tumor[17], and overexpression of GnT-V in threedimensional (3D)-cultured mammary normal epithelial cells caused neoplastic change with abnormal cancerous cell morphology[18]
We tested a series of constructs truncating the N-terminal region and found T121-L741, named GnT-V luminal domain, is sufficient for catalytic reaction without impairing protein production in mammalian cells (Figs. 1c, d)
Summary
N-acetylglucosaminyltransferase-V (GnT-V) alters the structure of specific N-glycans by modifying α1-6-linked mannose with a β1-6-linked N-acetylglucosamine branch. β1-6 branch formation on cell surface receptors accelerates cancer metastasis, making GnT-V a promising target for drug development. The acceptor-GnT-V complex structure suggests a catalytic mechanism, explains the previously observed inhibition of GnT-V by branching enzyme GnT-III, and provides a basis for the rational design of drugs targeting N-glycan branching. Formation of another GlcNAc branch (bisecting GlcNAc) by GnT-III completely interferes with GnT-V action[11] (Fig. 1a) This regulated modification by GnT-V underscores the enzymatic basis of the complex and specific function of glycans, but as yet the underlying catalytic mechanisms have not been clarified. In contrast to the other mammalian GlcNAc transferases (GnTs) such as GnT-I, II, III IVa, and IVb, GnT-V lacks the conventional Asp-x-Asp (DXD) motif, which is a critical sugarbinding motif commonly found in many vertebrate glycosyltransferases, and its catalytic reaction is metal independent, with weak donor binding and tight acceptor binding[10] (Supplementary Figure 1).
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