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The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Jul 1;105(1):517–527. doi: 10.1083/jcb.105.1.517

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

PMCID: PMC2114926  PMID: 3038929

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. 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.

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Selected References

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  1. Akiyama S. K., Hasegawa E., Hasegawa T., Yamada K. M. The interaction of fibronectin fragments with fibroblastic cells. J Biol Chem. 1985 Oct 25;260(24):13256–13260. [PubMed] [Google Scholar]
  2. Akiyama S. K., Yamada K. M. Comparisons of evolutionarily distinct fibronectins: evidence for the origin of plasma and fibroblast cellular fibronectins from a single gene. J Cell Biochem. 1985;27(2):97–107. doi: 10.1002/jcb.240270204. [DOI] [PubMed] [Google Scholar]
  3. Chen W. T., Singer S. J. Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7318–7322. doi: 10.1073/pnas.77.12.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen W. T., Singer S. J. Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol. 1982 Oct;95(1):205–222. doi: 10.1083/jcb.95.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen W. T., Wang J., Hasegawa T., Yamada S. S., Yamada K. M. Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides. J Cell Biol. 1986 Nov;103(5):1649–1661. doi: 10.1083/jcb.103.5.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dubbelman T. M., Yamada K. M. A survey of differences between membrane polypeptides of transformed and nontransformed chick embryo fibroblasts. Biochim Biophys Acta. 1982 Dec 8;693(1):177–187. doi: 10.1016/0005-2736(82)90485-0. [DOI] [PubMed] [Google Scholar]
  7. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galfrè G., Milstein C., Wright B. Rat x rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG. Nature. 1979 Jan 11;277(5692):131–133. doi: 10.1038/277131a0. [DOI] [PubMed] [Google Scholar]
  9. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  10. Hedman K. Intracellular localization of fibronectin using immunoperoxidase cytochemistry in light and electron microscopy. J Histochem Cytochem. 1980 Nov;28(11):1233–1241. doi: 10.1177/28.11.7000891. [DOI] [PubMed] [Google Scholar]
  11. Hughes R. C., Taylor A., Sage H., Hogan B. L. Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2+-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum. Eur J Biochem. 1987 Feb 16;163(1):57–65. doi: 10.1111/j.1432-1033.1987.tb10736.x. [DOI] [PubMed] [Google Scholar]
  12. Kurkinen M., Taylor A., Garrels J. I., Hogan B. L. Cell surface-associated proteins which bind native type IV collagen or gelatin. J Biol Chem. 1984 May 10;259(9):5915–5922. [PubMed] [Google Scholar]
  13. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  14. Nagata K., Saga S., Yamada K. M. A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein. J Cell Biol. 1986 Jul;103(1):223–229. doi: 10.1083/jcb.103.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nagata K., Yamada K. M. Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts. J Biol Chem. 1986 Jun 5;261(16):7531–7536. [PubMed] [Google Scholar]
  16. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  17. Platt J. L., Michael A. F. Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine. J Histochem Cytochem. 1983 Jun;31(6):840–842. doi: 10.1177/31.6.6341464. [DOI] [PubMed] [Google Scholar]
  18. Robbins P. W., Wickus G. G., Branton P. E., Gaffney B. J., Hirschberg C. B., Fuchs P., Blumberg P. The chick fibroblast cell surface after transformation by Rous sarcoma virus. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 2):1173–1180. doi: 10.1101/sqb.1974.039.01.135. [DOI] [PubMed] [Google Scholar]
  19. Saraste J., Hedman K. Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface. EMBO J. 1983;2(11):2001–2006. doi: 10.1002/j.1460-2075.1983.tb01691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Taylor A., Hogan B. L., Watt F. M. Biosynthesis of EGF receptor, transferrin receptor and colligin by cultured human keratinocytes and the effect of retinoic acid. Exp Cell Res. 1985 Jul;159(1):47–54. doi: 10.1016/s0014-4827(85)80036-7. [DOI] [PubMed] [Google Scholar]
  21. Terasaki M., Song J., Wong J. R., Weiss M. J., Chen L. B. Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell. 1984 Aug;38(1):101–108. doi: 10.1016/0092-8674(84)90530-0. [DOI] [PubMed] [Google Scholar]
  22. Virtanen I., Ekblom P., Laurila P. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. J Cell Biol. 1980 May;85(2):429–434. doi: 10.1083/jcb.85.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yamada S. S., Yamada K. M., Willingham M. C. Intracellular localization of fibronectin by immunoelectron microscopy. J Histochem Cytochem. 1980 Sep;28(9):953–960. doi: 10.1177/28.9.6997370. [DOI] [PubMed] [Google Scholar]

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