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J. Biol. Chem., Vol. 275, Issue 26, 19469-19474, June 30, 2000

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Structural Characterization of the Fibroblast Growth Factor-binding Protein Purified from Bovine Prepartum Mammary Gland Secretion^*

Rene Lametsch, Jan T. Rasmussen, Laust B. Johnsen, Stig Purup^§, Kristen Sejrsen^§, Torben E. Petersen, and Christian W. Heegaard^¶

From the  Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, DK-8000 Aarhus C and the ^§ Danish Institute of Animal Science, Research Centre Foulum, DK-8830 Tjele, Denmark

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 Lys²⁴ as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg³¹-Gly³² and a secondary site between Arg⁵¹-Ser⁵². 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 Asn¹⁵⁵ and Ser¹⁷², respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys⁷¹-Cys⁸⁸, Cys⁹⁷-Cys¹³⁰, Cys¹⁰⁶-Cys¹⁴², Cys¹⁹⁸-Cys²³⁴, and Cys²¹⁴-Cys²²². 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF271896.

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