Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule.
To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G.I., Bliss, G.A., Newman, P.J., and McEver, R.P. (1990) J. Biol. Chem. 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets. Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747-760). In contrast, platelet P-selectin reacted with both antibodies. S-Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species. From platelets, two virtually identical species were separated. Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site. From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified. In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence. Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.
Ishiwata
N
N
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Takio
K
K
Katayama
M
M
Watanabe
K
K
Titani
K
K
Ikeda
Y
Y
Handa
M
M
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D019007:P-Selectin; D010455:Peptides / Q000737:chemistry; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D012995:Solubility
D001798:Blood Proteins; D019007:P-Selectin; D010455:Peptides; D010980:Platelet Membrane Glycoproteins