pH-dependent function, purification, and intracellular location of a major collagen-binding glycoprotein.

Abstract

Abstract. 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. Doublestaining 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 shockregulated 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.ECENT studies have characterized a major collagenbinding protein of Mr 47,000 D that is present in a wide variety of cultured cell types (12,15,16,21). This membrane-associated glycoprotein binds to gelatin (denatured collagen types I and ~ and to native collagen types I, M, and IV (12,15,16,21). It represents the major protein besides fibronectin that binds to collagen-affinity columns under physiological salt conditions, and it can be the major concanavalin A-binding protein of membrane preparations (6,12,15,16,21).Previously, we reported (16) that this 47-kD collagen/ gelatin-binding protein corresponds to a previously described membrane protein (6,19), and that its synthesis and phosphorylation are regulated in opposing directions by an oncogene product. In addition, we found that this protein is a novel heat-shock protein (15). Studies of its specificity of binding indicated that it is relatively specific for the collagens, although it can bind to fetuin (16; Nagata, K., S. Saga, and K. M. Yamada, unpublished); whether such interactions with noncollagenous proteins provide a mechanism for reguDr. Saga's present address is The Second Department of Pathology, Nagoya University School of Medicine, Nagoya 466, Japan. Dr. Nagata's present address is Chest Disease Research Institute, Kyoto University, Kyoto 606, J...

Osteogenesis imperfecta

“…Further modifi cations occur during transport from the rER to the Golgi apparatus in special coat protein com plex vesicles that contain melanoma inhibitory activity protein 3 (also known as TANGO1) and through the Golgi stack by cisternal maturation. Serpin H1 also has binding sites along the helical portion of the molecule and assists shuttling of folded collagen into the cis Golgi [25][26][27] . These biosynthetic steps depend on a proper rER environment (for example, optimal calcium levels and redox potential), and the quality control mech anisms can lead to the activation of the unfolded protein response using the ER associated degrad ation pathway or the autophagy mediated lyso somal degradation sys tem to eliminate molecules that were not properly folded.…”

“…3). Serpin H1 stabilizes the folded pro collagens in the rER and assists their shuttling into the cis Golgi [25][26][27] . FKBP65 also has PPIase activity 24 .…”

Localization of HSP47 mRNA in murine bleomycin-induced pulmonary fibrosis

“…HSP47 has a specific role in the intracellular processing of procollagen production as a collagen-specific molecular chaperone [9][10][11][12].…”

Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum

“…Rabbit serum raised against rat type I collagen was obtained from Advance, Co. Ltd+ (Tokyo, Japan). Rabbit polyclonal and rat monoclonal (11DI0) IgG against chick HSIM7 was generated previously (Saga et al, 1987), and used after affinity purification by chick HSP47-cottpled Sepharose 4B column. Rabbit polyclonal IgG against protein disulfide isomerase was a kind gift from Dr.…”

“…HSP47 is a novel 47,000-D heat shock protein which also exists in the ER of collagen-secreting cells 1988a;Saga et al, 1987). This protein binds specifically to collagen (type I and IV) in vitro .…”