pH-dependent function, purification, and intracellular location of a major collagen-binding glycoprotein.
A major collagen-binding heat shock protein of molecular mass 47,000 D was found to bind to collagen by a pH-dependent interaction; binding was abolished at pH 6.3. Native 47-kD protein could therefore be purified from chick embryo homogenates in milligram quantities by gelatin-affinity chromatography and gentle acidic elution. Rat monoclonal and rabbit polyclonal antibodies were generated against the purified 47-kD protein. Immunofluorescence microscopy of cultured chick embryo fibroblasts with these antibodies revealed bright, granular perinuclear staining as well as a weaker reticular network structure towards the cell periphery, suggesting that this protein was located in the endoplasmic reticulum. No immunofluorescence staining was detected on the cell surface. Double-staining experiments with these antibodies and fluorescently labeled wheat-germ agglutinin suggested that the 47-kD protein was absent from the Golgi apparatus. Localization of the 47-kD protein in the endoplasmic reticulum but not in the Golgi complex was confirmed by immunoelectron microscopy. In vivo localization studies using immunohistochemistry of cryostat sections of chick liver revealed that the 47-kD protein was present in fibrocytes, Kupffer cells, and smooth muscle cells. It was absent from hepatocytes and the epithelia of bile ducts or sinusoidal endothelium. This major transformation- and heat shock-regulated glycoprotein is thus localized intracellularly, is expressed in only certain cells, and functions in a pH-regulated manner. These findings suggest that this glycoprotein is not likely to be a general cell-surface collagen receptor, but may instead play roles in intracellular protein processing or translocation.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism*; D005347:Fibroblasts / Q000032:analysis / Q000648:ultrastructure; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000276:immunology / Q000302:isolation & purification*; D038981:Receptors, Collagen
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D038981:Receptors, Collagen; D003094:Collagen