A single chain 19-kDa fragment from bovine thrombospondin binds to type V collagen and heparin.
Location of the type V collagen-binding domain within bovine thrombospondin (TSP) was investigated by using fragments of reduced and alkylated TSP. A fragment of relative molecular mass (19 kDa) was isolated, which inhibited binding of 125I-TSP to type V collagen in a solid-phase binding assay. A direct binding assay using the 125I-labeled fragment confirmed that the fragment actually bound to collagen. The fragment retained specificity for the native structure of type V collagen like the intact TSP molecule. Its binding to the collagen, however, was not inhibited by Ca2+ in contrast to intact TSP. Amino acid sequence analysis of the 19-kDa fragment suggested that this fragment corresponded to Val333-Lys412, a part of the stalklike region in the human TSP primary structure. It was found that the fragment also bound well to heparin in a specific and saturable manner and, furthermore, binding to type V collagen was inhibited by soluble heparin. Removal of the N-linked sugar chain from this fragment resulted in a 14-kDa fragment. The deglycosylated fragment retained the ability to bind to type V collagen as well as heparin. These results suggest that a type V collagen-binding site is present in the 80-residue portion of bovine TSP (Val333-Lys412) and is likely to be identical or lie very close to a heparin-binding site, which exists in the type I repeat structure.
Takagi
J
J
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan.
Fujisawa
T
T
Usui
T
T
Aoyama
T
T
Saito
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets; D002417:Cattle; D002853:Chromatography, Liquid; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D019699:Thrombospondins
D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D006493:Heparin; D003094:Collagen