title: The complete primary structure of glycosylated porcine platelet factor 4.
abstract: We have purified platelet factor 4 from porcine platelets and shown that it is glycosylated. The purified protein migrated as a broad band at approximately 14,000 Da, characteristic of glycoproteins. Electrospray mass spectroscopy of the intact protein gave a predominant mass of 11,111 Da, with a minor component of 10,804 Da. Sialidase digestion reduces both forms to a single mass of 10,497 Da. Upon Edman degradation, the amino terminus was found to be blocked by the presence of a pyroglutamate residue. We have determined the complete primary structure of platelet factor 4 by peptide mapping and Edman degradation, thereby completing information on the amino-terminal and carboxy-terminal regions which is missing in the previously published partial sequence. Sequencing of the intact and deglycosylated protein show that the glycosylation site is at Thr8. The amino acid composition accounts for a mass of 9623 Da, and the carbohydrate moeity was found to contribute 1490 Da. The biological activity of the porcine protein has been compared to recombinant human platelet factor 4 in an endothelial cell proliferation assay; both inhibit at a concentration giving half the maximal inhibition of 0.1 microM. Removal of the 19 amino-terminal residues carrying the carbohydrate moiety results in no change in the biological activity.
authors.affiliation: Glaxo Institute for Molecular Biology, Geneva, Switzerland.
authors.forename: A E
authors.initials: AE
authors.lastname: Proudfoot
authors.forename: E
authors.initials: E
authors.lastname: Magnenat
authors.forename: T M
authors.initials: TM
authors.lastname: Haley
authors.forename: T E
authors.initials: TE
authors.lastname: Maione
authors.forename: T N
authors.initials: TN
authors.lastname: Wells
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010978:Platelet Factor 4 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D013552:Swine
chemical_list: D010978:Platelet Factor 4