Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion

A novel heparin-binding protein was purified to homogeneity from bovine pre partum mammary gland secretion using heparin-Sepharose chromatography and r everse-phase high performance liquid chromatography successively. Structura l information obtained by N-terminal amino acid sequencing of a series of p roteolytically generated peptides permitted the cloning of the correspondin g cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-b ase pair 5'-untranslated region followed by a 702-base pair coding region a nd a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234-amino acid residues, including a signal sequenc e. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a p rimary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Se r(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF- BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP b ound FGF-2. The theoretical mass of the protein predicted from the cDNA seq uence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis, T he apparent molecular weight differences are most likely due to post-transc riptional modifications, shown to involve N- and O-glycosylation of Asn(155 ) and Ser(172), respectively. All 10 cysteine residues in the protein parti cipated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(8 8), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cy s(222). As the 10 cysteines of the three known FGF-BPs are positionally con served, the disulfide bond pattern of bovine FGF-BP may be regarded as repr esentative for the FGF-BP family.