title: Changes in calcium and collagen IV binding caused by mutations in the EF hand and other domains of extracellular matrix protein BM-40 (SPARC, osteonectin).
abstract: Recombinant expression in a human kidney cell-line was used to prepare mutant human BM-40 with deletions including the N-terminal acidic domain, a central alpha-helical domain and the C-terminal EF hand domain. Two putative EF hand motifs were altered by point mutations. Only elimination of the whole EF hand domain or its single disulfide bond decreased production and secretion indicating that the C-terminal region of BM-40 is essential for correct folding. Deletions in the alpha-helical domain changed a single disulfide bond in this domain and caused oligomerization. Several mutations resulted in significant conformational changes as shown by CD spectroscopy and epitope analysis. Fluorescence titration demonstrated a single high-affinity (Kd = 0.08 microM) calcium-binding site with a low dissociation rate constant. A Glu to Lys mutation in the -Z position of the C-terminal EF hand motif and lack of a stabilizing disulfide bridge caused a 30 to 100-fold reduction in calcium affinity, while an Asp to Lys mutation in the X position had only a small effect. Deletions in the alpha-helical domain caused an even more dramatic reduction in calcium binding and abolished calcium-dependent binding of BM-40 to collagen IV. Both binding properties are critically dependent on a conformational interaction between the EF hand and the alpha-helical domain, which contains the collagen-binding site.
authors.affiliation: Department of Dermatology, University, Köln, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Pottgiesser
authors.forename: P
authors.initials: P
authors.lastname: Maurer
authors.forename: U
authors.initials: U
authors.lastname: Mayer
authors.forename: R
authors.initials: R
authors.lastname: Nischt
authors.forename: K
authors.initials: K
authors.lastname: Mann
authors.forename: R
authors.initials: R
authors.lastname: Timpl
authors.forename: T
authors.initials: T
authors.lastname: Krieg
authors.forename: J
authors.initials: J
authors.lastname: Engel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D015676:Osteonectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D015676:Osteonectin; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D003094:Collagen; D008274:Magnesium; D002118:Calcium