Structural Basis for Acceptor Substrate Recognition of a Human Glucuronyltransferase, GlcAT-P, an Enzyme Critical in the Biosynthesis of the Carbohydrate Epitope HNK-1

Shinako Kakuda,Tomoo Shiba,Masji Ishiguro,Hideki Tagawa,Shogo Oka,Yasuhiro Kajihara,Toshisuke Kawasaki,Soichi Wakatsuki,Ryuichi Kato

doi:10.1074/jbc.m400622200

Abstract

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

Highlights

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules
The HNK-1 carbohydrate epitope is synthesized in a stepwise manner through the addition of ␤-1,3-linked glucuronic acid (GlcA)1 by glucuronyltransferase(s) to precursor N-acetyllactosamine followed by the addition of sulfate group by sulfotransferase(s)
We succeeded in obtaining crystals of GlcAT-P in a variety of conditions: apo, with a donor substrate and manganese, and another with additional acceptor substrate

Summary

The abbreviations used are

GlcA, glucuronic acid; UDP-GlcA, uridine diphosphoglucuronic acid; r.m.s.d., root mean square deviation; ASOR, asialo-orosomucoid; MES, 4-morpholineethanesulfonic acid. A Complex 1 contains Mn2ϩ, UDP, and N-acetyllactosamine. B Complex 2 contains Mn2ϩ, and UDP. C Complex 3 contains Mn2ϩ, UDP, and asparagine-linked bi-antennary nonasaccharide. GlcAT-I transfers GlcA to Gal␤1–3Gal␤1– 4Xyl␤1-O-Ser in a biosynthesis pathway of proteoglycan [19, 26]. The substrate binding and the reaction mechanisms of GlcAT-I have been discussed at an atomic level based on its crystal structure in complex with donor and acceptor substrate [31,32,33]. We solved crystal structures of GlcAT-P in four different forms: [1] apoform, [2] with UDP-GlcA and Mn2ϩ, [3] with UDP-GlcA, Mn2ϩ, and an acceptor substrate analogue, N-acetyllactosamine, and [4] with UDP-GlcA, Mn2ϩ, and a natural acceptor substrate, an asparagine-linked biantennary nonasaccharide. Based on the structural and biochemical results, we describe the molecular mechanism of substrate recognition of HNK-1 associated glucuronyltransferase, GlcAT-P

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

CONCLUSIONS