Expression of the alpha(1,3)fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of L-selectin ligands.

Lymphocyte homing to lymph nodes and Peyer's patches is mediated, in part, by adhesive interactions between L-selectin expressed by lymphocytes and L-selectin ligands displayed at the surface of the cuboidal endothelial cells lining the post-capillary venules within lymphoid aggregates. Candidate terminal oligosaccharide structures thought to be essential for effective L-selectin ligand activity include a sulfated derivative of the sialyl Lewis x tetrasaccharide. Cell type-specific synthesis of this oligosaccharide is presumed to require one or more alpha(1,3)fucosyltransferases, operating upon common 3'-sialylated and/or sulfated N-acetyllactosamine-type precursors. The identity of the alpha(1,3)fucosyltransferase(s) expressed in cells that bear L-selectin ligands has not been defined. We report here the molecular cloning and characterization of a murine alpha(1,3)fucosyltransferase locus whose expression pattern correlates with expression of high affinity ligands for L-selectin. In situ hybridization and immunohistochemical analyses demonstrate that this cDNA and its cognate alpha(1,3)fucosyltransferase are expressed in endothelial cells lining the high endothelial venules of peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. These expression patterns correlate precisely with the expression pattern of L-selectin ligands identified with a chimeric L-selectin/IgM immunohistochemical probe and by the high endothelial venule-reactive monoclonal antibody MECA-79. Transcripts corresponding to this cDNA are also detected in isolated bone marrow cells, a source rich in the surface-localized ligands for E- and P-selectins. Sequence and functional analyses indicate that this murine enzyme corresponds to the human Fuc-TVII locus. These observations suggest that Fuc-TVII participates in the generation of alpha(1,3)fucosylated ligands for L-selectin and provide further evidence for a role for this enzyme in E- and P-selectin ligand expression in leukocytes.

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.

Molecular cloning, nucleotide sequence, and expression of the gene encoding a trypsin-like protease from Streptomyces erythraeus.

Streptomyces erythraeus produces an extracellular mammalian-type serine protease bearing trypsin-like substrate specificity. The gene encoding the protease was cloned and sequenced as an initial step for investigating its structure-function relationship by site-specific mutagenesis. The cloned gene is composed of an 816-bp open reading frame encoding 272 amino acid residues, suggesting that it is synthesized as a precursor protein containing a 42-residue prepropeptide. In the N-terminal prepropeptide portion, the tract of 30 residues from the initiator methionine has a typical signal sequence for Streptomyces and the remaining 12 residues are thought to comprise a propeptide. The cloned gene was replaced downstream of a strong promoter in a high expression plasmid, pSEV2, and expressed in Streptomyces lividans TK24. The gene product was secreted extracellularly and identified as an inactive precursor which consists of the mature enzyme and the 12-residue N-terminally extended peptide chain. The precursor protein was converted to a fully active mature form by limited proteolysis with alpha-chymotrypsin at the Phe-(-1)-Ile-1 bond. Protein sequence analysis revealed that, except for the C-terminal three residues, recombinant SET is identical with the native enzyme.