title: Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion.
abstract: A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. 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 primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(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 bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence 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. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.
authors.affiliation: Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, DK-8000 Aarhus.
authors.forename: R
authors.initials: R
authors.lastname: Lametsch
authors.forename: J T
authors.initials: JT
authors.lastname: Rasmussen
authors.forename: L B
authors.initials: LB
authors.lastname: Johnsen
authors.forename: S
authors.initials: S
authors.lastname: Purup
authors.forename: K
authors.initials: K
authors.lastname: Sejrsen
authors.forename: T E
authors.initials: TE
authors.lastname: Petersen
authors.forename: C W
authors.initials: CW
authors.lastname: Heegaard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003126:Colostrum / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D015723:Gene Library; D006031:Glycosylation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008321:Mammary Glands, Animal / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011247:Pregnancy; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; C489453:Fgfbp1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016222:Fibroblast Growth Factor 2; C070699:FGFBP1 protein, human; D003545:Cysteine