title: Structural and functional characterization of the extracellular calcium-binding protein BM-40/secreted protein, acidic, rich in cysteine/osteonectin from the nematode Caenorhabditis elegans.
abstract: Caenorhabditis elegans BM-40 (positions 19-264) and its extracellular calcium-binding domain (positions 139-264) were obtained in recombinant form from human kidney cells using an episomal expression vector. The purified proteins showed single bands of 33 kDa [BM-40-(19-264)-peptide] or 14 kDa [BM-40-(139-264)-peptide] on electrophoresis, contained internal disulfide bonds and a helices and were relatively resistant to matrix metalloproteinases. Hexosamine analysis indicated substitution by one N-linked and two O-linked oligosaccharides and recombinant BM-40 was indistinguishable in its immunological epitopes from nematode tissue-derived BM-40, suggesting that it was obtained in native form. Both recombinant C. elegans proteins showed a distinct binding activity for human collagens I and IV in solid-phase and surface-plasmon-resonance assays with an affinity (Kd = 1-2 microM), comparable to that of mammalian BM-40. However, calcium-binding studies revealed only a low-affinity site (Kd = 6.2 mM) and failed to show the characteristic conformational change upon addition of EDTA. These and a few other differences are apparently due to two extra disulfide bonds and two deletions/insertions in C. elegans BM-40 and can be partly interpreted from the X-ray structure of a large part of human BM-40. The immunological assays available and the predictions of the location of the collagen-binding epitope should facilitate a molecular and genetic approach to understand the function of BM-40 in the development of C. elegans.
authors.affiliation: Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Maurer
authors.forename: T
authors.initials: T
authors.lastname: Sasaki
authors.forename: K
authors.initials: K
authors.lastname: Mann
authors.forename: W
authors.initials: W
authors.lastname: Göhring
authors.forename: J E
authors.initials: JE
authors.lastname: Schwarzbauer
authors.forename: R
authors.initials: R
authors.lastname: Timpl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D015676:Osteonectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D015801:Helminth Proteins; D015676:Osteonectin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D008666:Metalloendopeptidases