X-ray Crystal Structure of Leukocyte Type Core 2 β1,6-N-Acetylglucosaminyltransferase

Abstract

Leukocyte type core 2 ␤1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Gal␤1-3[GlcNAc␤1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Gal␤1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Gal␤1-3GalNAc at 2.0 and 2.7 Å resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Gal␤1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside disphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.3 is a cis-medial Golgi resident glycosyltransferase that catalyzes the conversion of the core 1 O-glycan to that of the core 2 structure (1, 2). Core 2 O-glycans have been shown to be key ligands in selectin-mediated lymphocyte homing and leukocyte rolling; lymphocyte L-selectins bind to endothelial cell O-glycans containing 6-sulfo sialyl Lewis x on core 2 and extended core 1 structures, whereas neutrophils expressing sialyl Lewis x on core 2 O-glycans bind to E-and P-selectins on endothelial cells (3-5). C2GnT-L is also of considerable interest in the study of tumor metastasis given that its expression is highly correlated with tumor progression in a number of cancers. It is overexpressed in colorectal, lung, and prostate cancer, and recent work has shown that transfection of C2GnT-L into a prostate cancer cell line leads to increased tumor size in an experimental tumor model (6 -8).C2GnT-L is an inverting N-acetylglucosaminyltransferase belonging to family GT-14 (9). It transfers N-acetylglucosamine (GlcNAc), in ␤1,6 linkage, from UDP-GlcNAc to Gal␤1-3Gal-NAc-O-Ser/Thr (core 1) to generate Gal␤1-3[GlcNAc␤1-6]GalNAc-O-Ser/Thr (core 2). C2GnT-L (leukocyte-type or C2GnT-I) along with C2GnT-M (mucin-type or C2GnT-II) (10, 11) and C2GnT-T (thymus-type or C2GnT-III) (12) are the three ␤1,6 N-acetylglucosaminyltransferases involved in the biosynthesis of core 2 branched O-glycans. The three enzymes differ in tissue distribution, and the mammalian homologues show 40 -50% sequence similarity with each other. Divalent metal ions, which have been shown to be essential for catalytic activity in many glycosyltransf...

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

“…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.…”

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

“…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.…”

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…[195][196][197][198][199][200] Interestingly, there is a recent example of a GT-A enzyme that is metal-ion-independent and lacks the DXD motif. [201] In Scheme 14. Representative bacterial natural-product glycoforms.…”