Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues

Abstract

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

Limited Cleavage of Extracellular Matrix Protein BM-40 by Matrix Metalloproteinases Increases Its Affinity for Collagens

“…Several extracellular ligands have been identified for BM-40 including some collagen types and cytokines (1). This indicated various binding sites on BM-40 in agreement with a previous proposal of a mosaic structure for the protein (2). The most recent interpretation of the domain structure based on recombinant deletion mutants and a crystal structure of a fragment encompassing the C-terminal 150 residues (3,4) demonstrated an acidic and flexible N-terminal domain I (ϳ50 residues) followed by a follistatin-like (FS) 1 module (ϳ75 residues) and a novel extracellular calcium-binding (EC) module (ϳ150 residues).…”

“…The insert was then restricted with NheI and XhoI and cloned into the episomal expression vector pCEP-Sh (25) that contained the puromycin instead of the phleomycin resistance gene. 2 The correctness of the insert was verified by DNA cycle sequencing. Transfection of human EBNA-293 cells (Invitrogen) and purification of the recombinant mutant followed established protocols (3,25).…”

Structure and biological activity of basement membrane proteins

“…Studies with BM-40 demonstrated 25 -30% a-helical conformation, which was distinctly reduced after removal of calcium [147]. The data also indicated the cooperative binding of up to seven calcium ions which is close to the nine binding sites predicted from the sequence.…”

“…Reproduced with permission from [40] before considered to be a bone-specific protein with binding activities for calcium, hydroxyapatite and collagen I [I 511. Sequence data [40,41,147] indicates that the protein consists of four domains I -IV (Fig. 7) and that the two terminal domains (I, IV) represent potential calcium-binding sites.…”

“…The central region (domain 11) is rich in disulfide bonds and shows similarity to ovomucoid and other protease inhibitors. This domain contains phosphoserine [147].…”