A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.
We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the second dimension. Under these conditions CB48 separated into two major (a and b) and one minor isoform (c); a was the most basic of the three isoforms. Two-dimensional chymotryptic peptide maps derived from each individual isoform were virtually identical. The charge differences between the isoforms were due in part to differential H3(32)PO4 incorporation by the protein. CB48 bound to intact collagen type IV and the collagenous region of collagen type IV, but not to the globular NC1 domain. Cell-surface labelling and indirect immunofluorescence experiments localized the bulk of CB48 intracellularly in the endoplasmic reticulum Golgi region, with a minor population of molecules on the cell surface. A specific rabbit polyclonal anti-CB48 serum did not inhibit the attachment or spreading of BAEC to collagen type IV in an 'in vitro' adhesion assay, suggesting that the cell-surface population of CB48 is not involved in BAEC adhesion. We conclude that CB48 is a collagen-binding phosphoprotein that interacts with the collagenous domain of collagen type IV and may be involved in intracellular transport of collagen molecules.
Yannariello-Brown
J
J
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
Madri
J A
JA
D000818:Animals; D001011:Aorta; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D003094:Collagen / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D038981:Receptors, Collagen
D007106:Immune Sera; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D038981:Receptors, Collagen; D003094:Collagen; D002918:Chymotrypsin