Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites

Abstract

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galβ(1,4)-GlcNAcβ(1,3)-]n glycan extensions. They are found on both N- and O­-glycoproteins and glycolipids, and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+, and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galβ(1,4)-GlcNAcβ(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ co-evolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

How to extend your (polylactosamine) antennae

“…Kadirvelraj et al. ( 7 ) also determined kinetics constants for both substrates for the wild-type and for ten variants in active site key residues, supporting the proposed mechanism. Hao et al.…”

“…( 6 ) and Kadirvelraj et al. ( 7 ) report the structure of the GT domain of B3GNT2, coupled with an in-depth analysis of the enzyme’s mechanism. Usefully, the groups used different approaches at several key junctures, so the papers provide nicely complementary insights.…”

“…Kadirvelraj et al. ( 7 ) cocrystallized B3GNT2 as UDP and as UDP plus trisaccharide acceptor complexes; they then modeled UDP-GlcNAc binding. Hao et al.…”

“…Using detailed comparisons to other GT-A structures, Kadirvelraj et al. ( 7 ) argue that GT-As generally evolve recognition to acceptors by extending and restructuring these hypervariable loops, allowing an enormous variety of acceptors to be recognized.…”

Peters plus syndrome mutations affect the function and stability of human β1,3-glucosyltransferase

“…B3GLCT belongs to the glycosyltransferase (GT)-31 family based on the Carbohydrate Active enZYmes (CAZy) classification ( 7 ). Currently, there are over 30 vertebrate subfamilies in the GT31 family ( 8 ), but structures of only two have been solved—mouse Manic Fringe (MFNG) ( 9 ) and human β1,3-N-acetylglucosaminyltransfease 2 (B3GNT2) ( 10 , 11 ). A recent phylogenetic study demonstrated that B3GLCT is a unique member of the GT31 family because it harbors two putative GT domains, which are more similar to each other than the rest of the members in the GT31 family ( 8 ).…”