Identification of human complement Factor H as a ligand for L-selectin.
The selectin family of adhesion molecules (E-, P- and L-selectins) is involved in leukocyte recruitment to sites of inflammation and tissue damage. Recently it has been shown that L-selectin is involved not only in leukocyte tethering and rolling, but also plays an important role in leukocyte activation. For example, glycosylation-dependent cell-adhesion molecule 1 (GlyCAM-1), a known ligand for L-selectin, has been shown to enhance beta2-integrin function. GlyCAM-1 is a secreted protein and is present in mouse serum at a concentration of approx. 1.5 microg/ml. There is no obvious GlyCAM-1 homologue in man and, to date, L-selectin ligand(s) from human serum have not been characterized. Therefore we have used L-selectin affinity chromatography, followed by ion-exchange chromatography, to isolate specific ligand(s) for L-selectin. Using this procedure, we have isolated three major glycoproteins of apparent molecular masses 170 kDa, 70kDa and 50 kDa. The 170 kDa protein band was digested with trypsin and peptides were analysed by delayed extraction matrix-assisted laser desorption ionization MS and protein database searching. The 170 kDa protein was identified as the human complement protein Factor H. Human Factor H, isolated by a different method, was shown to bind specifically to L-selectin in the presence of CaCl2, and binding was inhibited by anti-L-selectin antibodies, fucoidan and lipopolysaccharide. Only a part of the purified Factor H preparation bound to immobilized L-selectin. The interaction of Factor H with leukocyte L-selectin was shown to induce the secretion of tumour necrosis factor-alpha (TNF-alpha). Pretreatment of Factor H with sialidase reduced both the binding of L-selectin to Factor H and the Factor H-induced L-selectin-mediated TNF-alpha secretion by leukocytes. Taken together, these results demonstrate that a post-translationally modified form of human plasma Factor H is a potential physiological ligand for L-selectin.
Malhotra
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Cellular Biochemistry Unit, Glaxo-Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. RM18326@glaxowellcome.co.uk
Ward
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Sim
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Bird
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D001798:Blood Proteins / Q000302:isolation & purification; D002846:Chromatography, Affinity; D017242:Complement Factor H / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019041:L-Selectin / Q000378:metabolism*; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D001798:Blood Proteins; C493495:CFH protein, human; D006023:Glycoproteins; D008024:Ligands; D011134:Polysaccharides; D014409:Tumor Necrosis Factor-alpha; D019041:L-Selectin; D017242:Complement Factor H; C007789:fucoidan; D019158:N-Acetylneuraminic Acid