Structural and functional characterization of the extracellular calcium-binding protein BM-40/secreted protein, acidic, rich in cysteine/osteonectin from the nematode Caenorhabditis elegans

Patrik Maurer, Takako Sasaki, Karlheinz Mann, Walter Göhring, Jean E. Schwarzbauer, Rupert Timpl

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

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 (K(d) = 1-2 μM), comparable to that of mammalian BM-40. However, calcium-binding studies revealed only a low-affinity site (K(d) = 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.

Original languageEnglish (US)
Pages (from-to)209-216
Number of pages8
JournalEuropean Journal of Biochemistry
Volume248
Issue number1
DOIs
StatePublished - Jan 1 1997

All Science Journal Classification (ASJC) codes

  • Biochemistry

Keywords

  • Calcium binding
  • Collagen binding
  • Evolution
  • Extracellular matrix

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