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P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin-like glycoprotein expressed on the surface of myeloid cells and serves as the high affinity counterreceptor for P-selectin. The PSGL-1-P-selectin interaction is calcium dependent and requires presentation of sialyl-Lewisx (sLex)-type structures on the O-linked glycans of PSGL-1. We report here the identification of a non-carbohydrate component of the binding determinant that is critical for high affinity binding to P-selectin. Located within the first 19 amino acids, this anionic polypeptide segment contains at least one sulfated tyrosine residue. We propose that this sulfotyrosine-containing segment of PSGL-1, in conjunction with sLex presented on O-linked glycans, constitutes the high affinity P-selectin-binding site.

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We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP ؉ as a cofactor and, like its E. coli counterpart, is inhibited by GDPfucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.Fucose is found as a component of glycoconjugates such as glycoproteins and glycolipids in a wide range of species from humans to bacteria. For example, fucose is a component of the capsular polysaccharides and antigenic determinants of bacteria, while in mammals fucose is present in many glycoconjugates, the most widely known being the human blood group antigens. Fucose-containing glycoconjugates have been implicated as playing key roles in embryonic development in the mouse (1) and more recently in the regulation of the immune response, specifically as a crucial component of the selectin ligand sialyl Lewis X (reviewed in Refs. 1 and 2). In all cases, fucose is transferred from GDP-fucose to glycoconjugate acceptors by specific transferases. Thus, defects in GDP-fucose biosynthesis will affect all fucosylation within the cell. Recently, individuals deficient in the biosynthesis of GDP-fucose have been identified (3, 4) and suffer from the immune disorder leukocyte adhesion deficiency type II (LADII).1 These patients fail to synthesize fucosylated blood groups, and their leukocytes do not express the fucose containing carbohydrate sialyl Lewis X. The patient's leukocytes do not extravasate normally, which leads to recurrent infections.In his pioneering work in the early 1960s, Ginsberg (5, 6) elucidated the enzymatic pathway converting GDP-mannose to GDP-fucose. Later, Yurchenco and Atkinson (7) showed that this was the primary biosynthetic route to GDP-fucose. As shown in Fig. 1, GDP-mannose is converted to GDP-fucose by GDP-mannose 4,6-dehydratase via the oxidation of mannose at C-4 followed by the reduction of C-6 to a methyl group, yielding GDP-4-keto-6-deoxymannose. The reaction has been reported to proceed with transfer of a hydride fro...

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