Human α(1,3/1,4)-Fucosyltransferases Discriminate between Different Oligosaccharide Acceptor Substrates through a Discrete Peptide Fragment

Daniel J. Legault,Yuko Natsuka,Robert J. Kelly,John B. Lowe

doi:10.1074/jbc.270.36.20987

Abstract

Five different human alpha(1,3)-fucosyltransferase (alpha(1,3)-Fuc-T) genes have been cloned. Their corresponding enzymes catalyze the formation of various alpha(1,3)- and alpha(1,4)-fucosylated cell surface oligosaccharides, including several that mediate leukocyte-endothelial cell adhesion during inflammation. Inhibitors of such enzymes are predicted to operate as anti-inflammatory agents; in principle, the isolation or design of such agents may be facilitated by identifying peptide segment(s) within these enzymes that interact with their oligosaccharide acceptor substrates. Little is known, however, about the structural features of alpha(1,3)-Fuc-Ts that dictate acceptor substrate specificity. To begin to address this problem, we have created and functionally characterized a series of 21 recombinant alpha(1,3)-Fuc-T chimeras derived from three human alpha(1,3)-Fuc-Ts (Fuc-TIII, Fuc-TV, and Fuc-TVI) that maintain shared and distinct polypeptide domains and that exhibit common as well as idiosyncratic acceptor substrate specificities. The in vivo acceptor substrate specificities of these alpha(1,3)-Fuc-T chimeras, and of their wild type progenitors, were determined by characterizing the cell surface glycosylation phenotype determined by these enzymes, after expressing them in a mammalian cell line informative for the synthesis of four distinct alpha(1,3)- and alpha(1,4)-fucosylated cell surface oligosaccharides (Lewis x, sialyl Lewis x, Lewis a, and sialyl Lewis a). Our results indicate that as few as 11 nonidentical amino acids, found within a "hypervariable" peptide segment positioned at the NH2 terminus of the enzymes' sequence-constant COOH-terminal domains, determines whether or not these alpha(1,3)-Fuc-T can utilize type I acceptor substrates to form Lewis a and sialyl Lewis a moieties.

Highlights

Cell surface ␣(1,3)- and ␣(1,4)-fucosylated oligosaccharides have received a substantial amount of attention because some are thought to be essential to the initiation of immune cell adhesion to vascular endothelium during the inflammatory process
Interactions between sialyl Lewis x-bearing glycoconjugates on leukocytes and P- and E-selectin expressed by activated vascular endothelium [5]
Analysis of COOH-terminal Subdomain Exchanges; Type I Substrate Utilization Is Determined by Sequences in the NH2terminal Hypervariable Segment—The amino acid sequences of human Fuc-TIII and Fuc-TVI differ from each other at only

Summary

Introduction

Cell surface ␣(1,3)- and ␣(1,4)-fucosylated oligosaccharides have received a substantial amount of attention because some are thought to be essential to the initiation of immune cell adhesion to vascular endothelium during the inflammatory process (reviewed in Refs. 1–5 and 6 –15). Animal intervention studies indicate that such fucosylated oligosaccharide molecules can act as anti-inflammatory agents by inhibiting leukocyte-endothelial cell interactions [15,16,17,18] Absence of such molecules on the leukocytes of individuals with the rare human leukocyte adhesion II syndrome is associated with profound defects in leukocyte-endothelial cell adhesion and with nonpyogenic infections [19, 20]. These observations suggest that compounds capable of inhibiting leukocyte sialyl Lewis x expression might represent candidates for anti-inflammatory pharmacologic agents. These molecules may exhibit distinct biologic functions, including those involving selectin-dependent cell adhesion

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