Human alpha(1,3/1,4)-fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment.
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.
Legault
D J
DJ
Department of Medicine, Howard Hughes Medical Institute, Ann Arbor, Michigan 48109-0650, USA.
Kelly
R J
RJ
Natsuka
Y
Y
Lowe
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002460:Cell Line; D017931:DNA Primers; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D017931:DNA Primers; D007527:Isoenzymes; D009844:Oligosaccharides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase