Thrombospondin Type I Repeat Research Articles

Heparin-binding Growth-associated Molecule Contains Two Heparin-binding β-Sheet Domains That Are Homologous to the Thrombospondin Type I Repeat

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.

M-Spondin, a Novel ECM Protein Highly Homologous to Vertebrate F-Spondin, Is Localized at the Muscle Attachment Sites in theDrosophilaEmbryo

The muscle attachment site (MAS) inDrosophilaprovides a unique and excellent model system to study the mechanism of cell–matrix adhesion in developing organisms. Here, we report on the isolation and characterization of a novel extracellular matrix (ECM) molecule localized at the MAS, encoded by theM-spondin(mspo) gene. M-spondin protein contains a thrombospondin type I repeat (TSR) previously found in a variety of ECM molecules. Furthermore, it shares two conserved domains with F-spondin, a vertebrate ECM molecule with TSRs. The presence of TSR(s) and the two homologous domains thus defines a novel gene family of ECM molecules. ThemspomRNA was expressed by a large subset of muscles in the embryonic body wall. Secreted M-spondin protein diffused and eventually became immobilized at the MAS in late embryos. When expressed in S2 cells, the protein was secreted and became concentrated in the matrix on the surface of the culture dish. Genetic analysis revealed that both deletion mutants and misexpression mutants suffered no obvious developmental defects. We propose that M-spondin, although its function is redundant, is a component of the ECM and mediates mechanical linkage between the muscles and apodemes.