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

Abstract

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 ␤-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel ␤-strands. In addition to this domain structure, HB-GAM contains the Nand 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 ␤-sheet domains of the protein. Search of sequence data bases shows that the ␤-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the ␤-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 ␤-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.Heparin-binding growth-associated molecule (HB-GAM) 1 (p18) was originally isolated from rat brain as an 18-kDa neurite outgrowth-promoting protein, the expression of which in brain tissue peaks during the perinatal stage of rapid axon growth and synapse formation (1). HB-GAM is highly homologous with the midkine (MK) sequence (2-4), and these proteins thus form a two-member family of small extracellular proteins that are conserved in vertebrates.In developing tissues HB-GAM associates with extracellular matrix of axonal tracts and of synapses (5, 6). It is also clearly expressed in developing basement membranes outside of brain (7) and in the cartilage matrix (8). N-syndecan (syndecan-3) acts as a receptor of HB-GAM in brain neurons in vitro (9) and localizes in many anatomical areas to the same developing fiber tracts as HB-GAM (10, 11). The heparan sulfate structure of brain N-syndecan is exceptionally heparin-like, especially in its high content of 2-0-sulfo-iduronic acid residues, which is of importance in the HB-GAM binding carbohydrate epitope, the minimal size of which appears to be 10 monosaccharide residues (12). The neurite outgrowth-promoting effect, based on HB-GAM/N-syndecan interaction, was very recently shown to be mediated by the cortactin/src-kinase signaling pathway to the cytoskeleton of neurites (13). These findings have led to the concept that N-syndecan mediates HB-GAM-induced neurite growth (for r...

C-Mannosylation and O-Fucosylation of Thrombospondin Type 1 Repeats

“…However, both are all-␤ proteins with a central ␤-sheet and three disulfide bridges. TSR modules are likely to have a ␤-sheet structure, as well, based on their homology with the all-␤ proteins HB-GAM (heparin-binding growth-associated molecule) and midkine (43). If this proposition is correct, the O-fucosylation site in TSRs is located in a loop between the first two ␤-sheets.…”

WISP-1 Binds to Decorin and Biglycan

“…The interaction of heparin binding growth-associated molecule with heparin is mediated primarily by ␤-sheet domains homologous to the thrombospondin type I repeat. Because it contains similar domains, those conclusions were also extended to midkine (41). For the midkine carboxyl-terminal domain, it has been suggested that the conserved tryptophan in the first ␤-strand and downstream basic amino acids in the second ␤-strand are involved in the formation of a heparin-binding surface (40).…”

“…This motif is thought to be involved in binding to sulfated glycoconjugates (8) and could constitute a dermatan sulfate or a second heparin binding domain for the CCN family. Recently, the three-dimensional structure of the heparin-binding growthassociate molecule and midkine were resolved (40,41). The interaction of heparin binding growth-associated molecule with heparin is mediated primarily by ␤-sheet domains homologous to the thrombospondin type I repeat.…”

“…The secondary structure of WISP-1 is not known, but the thrombospondin domain is well conserved among the CCN family. It was suggested that the thrombospondin type I repeat of different proteins share structural features that could be responsible for their heparin binding (41).…”

Dominant Negative Effectors of Heparin Affin Regulatory Peptide (HARP) Angiogenic and Transforming Activities

“…NMR experiments have clearly demonstrated that the HARP molecule is organized into two ␤-sheet domains linked by a flexible linker, and this structure is maintained through five intrachain disulfide bonds (19). Each ␤-sheet domain contains a heparin-binding site, which plays a role in the modulation of HARP mitogenic activity (20).…”