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. 1986 Nov 1;103(5):1699–1710. doi: 10.1083/jcb.103.5.1699

The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface

PMCID: PMC2114375  PMID: 2430975

Abstract

A cell surface chondroitin sulfate proteoglycan associated with human melanomas and defined by mAb's F24.47 and 48.7 has been characterized biochemically and localized by indirect immunogold electron microscopy. These antibodies recognize distinct epitopes on the intact proteoglycan. In addition, mAb 48.7 also recognizes an epitope on a 250,000-D glycoprotein and is therefore similar to antibody 9.2.27 (described by Bumol, T.F., and R.A. Reisfeld, 1982, Proc. Natl. Acad. Sci. USA., 79:1245-1249). Furthermore, it was shown that the glycosaminoglycan chains released by alkaline borohydride treatment of the proteoglycan recognized by mAb 48.7 had a size of approximately 60,000 D. Since the intact proteoglycan was estimated to be 420,000 D, there are probably three chondroitin sulfate chains attached to the 250,000-D core glycoprotein. Furthermore, an oligosaccharide fraction containing 42% of the 3H activity (glucosamine as precursor) was isolated. Immunolocalization studies using whole-mount electron microscopy revealed that the chondroitin sulfate proteoglycan was present almost exclusively on microspikes, a microdomain of the melanoma cell surface. These processes were present as 1-2-micron structures on the upper cell surface and as longer (up to 20 micron) structures at the cell periphery. Peripheral microspikes were involved in the initial interactions between adjacent cells and formed complex footpads that made contact with the substratum. Immunogold-labeled cells were also thin sectioned and the specific localization of the chondroitin sulfate proteoglycan antigen was quantitated. The data confirmed the results of whole-mount microscopy and demonstrated a statistically significant association of the antigen with the microspike processes as compared with other areas of the cell surface. By using two different mAb's (48.7 and F24.47) that recognize epitopes on either the core glycoprotein or the intact proteoglycan, respectively, we have demonstrated that both molecules have the same restricted distribution at the cell surface. The specific localization of the antigen to microspikes at the cell surface suggests it may play a role in cell-cell contact and cell-substratum adhesion, which could be important in the metastatic process.

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Selected References

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  1. Albrecht-Buehler G. Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function? J Cell Biol. 1976 May;69(2):275–286. doi: 10.1083/jcb.69.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albrecht-Buehler G., Goldman R. D. Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells. Exp Cell Res. 1976 Feb;97(2):329–339. doi: 10.1016/0014-4827(76)90624-8. [DOI] [PubMed] [Google Scholar]
  3. Barnhart B. J., Cox S. H., Kraemer P. M. Detachment variants of Chinese hamster cells. Hyaluronic acid as a modulator of cell detachment. Exp Cell Res. 1979 Mar 15;119(2):327–332. doi: 10.1016/0014-4827(79)90360-4. [DOI] [PubMed] [Google Scholar]
  4. Beaumier P. L., Krohn K. A., Carrasquillo J. A., Eary J., Hellström I., Hellström K. E., Nelp W. B., Larson S. M. Melanoma localization in nude mice with monoclonal Fab against p97. J Nucl Med. 1985 Oct;26(10):1172–1179. [PubMed] [Google Scholar]
  5. Brennan M. J., Oldberg A., Hayman E. G., Ruoslahti E. Effect of a proteoglycan produced by rat tumor cells on their adhesion to fibronectin-collagen substrata. Cancer Res. 1983 Sep;43(9):4302–4307. [PubMed] [Google Scholar]
  6. Bumol T. F., Reisfeld R. A. Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1245–1249. doi: 10.1073/pnas.79.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bumol T. F., Walker L. E., Reisfeld R. A. Biosynthetic studies of proteoglycans in human melanoma cells with a monoclonal antibody to a core glycoprotein of chondroitin sulfate proteoglycans. J Biol Chem. 1984 Oct 25;259(20):12733–12741. [PubMed] [Google Scholar]
  8. Carlson D. M. Structures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins. J Biol Chem. 1968 Feb 10;243(3):616–626. [PubMed] [Google Scholar]
  9. Chang Y., Yanagishita M., Hascall V. C., Wight T. N. Proteoglycans synthesized by smooth muscle cells derived from monkey (Macaca nemestrina) aorta. J Biol Chem. 1983 May 10;258(9):5679–5688. [PubMed] [Google Scholar]
  10. Culp L. A., Murray B. A., Rollins B. J. Fibronectin and proteoglycans as determinants of cell-substratum adhesion. J Supramol Struct. 1979;11(3):401–427. doi: 10.1002/jss.400110314. [DOI] [PubMed] [Google Scholar]
  11. Dietrich C. P., Sampaio L. O., Toledo O. M., Cássaro C. M. Cell recognition and adhesiveness: a possible biological role for the sulfated mucopolysaccharides. Biochem Biophys Res Commun. 1977 Mar 21;75(2):329–336. doi: 10.1016/0006-291x(77)91046-4. [DOI] [PubMed] [Google Scholar]
  12. Dippold W. G., Lloyd K. O., Li L. T., Ikeda H., Oettgen H. F., Old L. J. Cell surface antigens of human malignant melanoma: definition of six antigenic systems with mouse monoclonal antibodies. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6114–6118. doi: 10.1073/pnas.77.10.6114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Forrester J. V., Wilkinson P. C. Inhibition of leukocyte locomotion by hyaluronic acid. J Cell Sci. 1981 Apr;48:315–331. doi: 10.1242/jcs.48.1.315. [DOI] [PubMed] [Google Scholar]
  14. Fransson L. A., Carlstedt I., Cöster L., Malmström A. Proteoheparan sulfate from human skin fibroblasts. Evidence for self-interaction via the heparan sulfate side chains. J Biol Chem. 1983 Dec 10;258(23):14342–14345. [PubMed] [Google Scholar]
  15. Furcht L. T., Mosher D. F., Wendelschafer-Crabb G. Effects of cell density and transformation on the formation of a fibronectin extracellular filamentous matrix on human fibroblasts. Cancer Res. 1978 Dec;38(12):4618–4623. [PubMed] [Google Scholar]
  16. Harper J. R., Reisfeld R. A. Inhibition of anchorage-independent growth of human melanoma cells by a monoclonal antibody to a chondroitin sulfate proteoglycan. J Natl Cancer Inst. 1983 Aug;71(2):259–263. [PubMed] [Google Scholar]
  17. Hassell J. R., Robey P. G., Barrach H. J., Wilczek J., Rennard S. I., Martin G. R. Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4494–4498. doi: 10.1073/pnas.77.8.4494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hatae Y., Atsuta T., Makita A. Glycosaminoglycans in human lung carcinoma. Gan. 1977 Feb;68(1):59–63. [PubMed] [Google Scholar]
  19. Hayman E. G., Oldberg A., Martin G. R., Ruoslahti E. Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells. J Cell Biol. 1982 Jul;94(1):28–35. doi: 10.1083/jcb.94.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hellström I., Garrigues H. J., Cabasco L., Mosely G. H., Brown J. P., Hellström K. E. Studies of a high molecular weight human melanoma-associated antigen. J Immunol. 1983 Mar;130(3):1467–1472. [PubMed] [Google Scholar]
  21. Hughes E. N., August J. T. Murine cell surface glycoproteins. Identification, purification, and characterization of a major glycosylated component of 110,000 daltons by use of a monoclonal antibody. J Biol Chem. 1982 Apr 10;257(7):3970–3977. [PubMed] [Google Scholar]
  22. Hök M., Kjellén L., Johansson S. Cell-surface glycosaminoglycans. Annu Rev Biochem. 1984;53:847–869. doi: 10.1146/annurev.bi.53.070184.004215. [DOI] [PubMed] [Google Scholar]
  23. Iozzo R. V., Bolender R. P., Wight T. N. Proteoglycan changes in the intercellular matrix of human colon carcinoma: an integrated biochemical and stereologic analysis. Lab Invest. 1982 Aug;47(2):124–138. [PubMed] [Google Scholar]
  24. Iozzo R. V. Proteoglycans and neoplastic--mesenchymal cell interactions. Hum Pathol. 1984 Jan;15(1):2–10. doi: 10.1016/s0046-8177(84)80326-3. [DOI] [PubMed] [Google Scholar]
  25. Kawakami H., Terayama H. Liver plasma membranes and proteoglycan prepared therefrom inhibit the growth of hepatoma cells in vitro. Biochim Biophys Acta. 1981 Aug 6;646(1):161–168. doi: 10.1016/0005-2736(81)90283-2. [DOI] [PubMed] [Google Scholar]
  26. Keller K. L., Keller J. M., Moy J. N. Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference. Biochemistry. 1980 May 27;19(11):2529–2536. doi: 10.1021/bi00552a035. [DOI] [PubMed] [Google Scholar]
  27. Knox P., Wells P. Cell adhesion and proteoglycans. I. The effect of exogenous proteoglycans on the attachment of chick embryo fibroblasts to tissue culture plastic and collagen. J Cell Sci. 1979 Dec;40:77–88. doi: 10.1242/jcs.40.1.77. [DOI] [PubMed] [Google Scholar]
  28. Koda J. E., Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells. Basal extracellular proteoglycan binds specifically to native type I collagen fibrils. J Biol Chem. 1984 Oct 10;259(19):11763–11770. [PubMed] [Google Scholar]
  29. Kojima J., Nakamura N., Kanatani M., Akiyama M. Glycosaminoglycans in 3'-methyl-4-dimethylaminoazobenzene-induced rat hepatic cancer. Cancer Res. 1982 Jul;42(7):2857–2860. [PubMed] [Google Scholar]
  30. Kojima J., Nakamura N., Kanatani M., Omori K. The glycosaminoglycans in human hepatic cancer. Cancer Res. 1975 Mar;35(3):542–547. [PubMed] [Google Scholar]
  31. Kraemer P. M., Tobey R. A. Cell-cycle dependent desquamation of heparan sulfate from the cell surface. J Cell Biol. 1972 Dec;55(3):713–717. doi: 10.1083/jcb.55.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Lark M. W., Culp L. A. Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. Affinity fractionation on columns of platelet factor 4, plasma fibronectin, and octyl-sepharose. J Biol Chem. 1984 Jun 10;259(11):6773–6782. [PubMed] [Google Scholar]
  34. Lark M. W., Laterra J., Culp L. A. Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix. Fed Proc. 1985 Feb;44(2):394–403. [PubMed] [Google Scholar]
  35. Mourão P. A., Michelacci Y. M., Toledo O. M. Glycosaminoglycans and proteoglycans of normal and tumoral cartilages of humans and rats. Cancer Res. 1979 Jul;39(7 Pt 1):2802–2806. [PubMed] [Google Scholar]
  36. Nakamura N., Kojima J. Changes in charge density of heparan sulfate isolated from cancerous human liver tissue. Cancer Res. 1981 Jan;41(1):278–283. [PubMed] [Google Scholar]
  37. Norling B., Glimelius B., Wasteson A. Heparan sulfate proteoglycan of cultured cells: demonstration of a lipid- and a matrix-associated form. Biochem Biophys Res Commun. 1981 Dec 31;103(4):1265–1272. doi: 10.1016/0006-291x(81)90259-x. [DOI] [PubMed] [Google Scholar]
  38. Noro A., Kimata K., Oike Y., Shinomura T., Maeda N., Yano S., Takahashi N., Suzuki S. Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide. J Biol Chem. 1983 Aug 10;258(15):9323–9331. [PubMed] [Google Scholar]
  39. Pacifici M., Boettiger D., Roby K., Holtzer H. Transformation of chondroblasts by Rous sarcoma virus and synthesis of the sulfated proteoglycan matrix. Cell. 1977 Aug;11(4):891–899. doi: 10.1016/0092-8674(77)90300-2. [DOI] [PubMed] [Google Scholar]
  40. Pal S., Strider W., Margolis R., Gallo G., Lee-Huang S. Isolation and characterization of proteoglycans from human chondrosarcomas. J Biol Chem. 1978 Feb 25;253(4):1279–1289. [PubMed] [Google Scholar]
  41. Pober J. S., Guild B. C., Strominger J. L., Veatch W. R. Purification of HLA-A2 antigen, fluorescent labeling of its intracellular region, and demonstration of an interaction between fluorescently labeled HLA-A2 antigen and lymphoblastoid cell cytoskeleton proteins in vitro. Biochemistry. 1981 Sep 15;20(19):5625–5633. doi: 10.1021/bi00522a042. [DOI] [PubMed] [Google Scholar]
  42. Roblin R., Albert S. O., Gelb N. A., Black P. H. Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells. Biochemistry. 1975 Jan 28;14(2):347–357. doi: 10.1021/bi00673a022. [DOI] [PubMed] [Google Scholar]
  43. Ross A. H., Cossu G., Herlyn M., Bell J. R., Steplewski Z., Koprowski H. Isolation and chemical characterization of a melanoma-associated proteoglycan antigen. Arch Biochem Biophys. 1983 Aug;225(1):370–383. doi: 10.1016/0003-9861(83)90042-5. [DOI] [PubMed] [Google Scholar]
  44. Ruoslahti E., Engvall E. Complexing of fibronectin glycosaminoglycans and collagen. Biochim Biophys Acta. 1980 Aug 13;631(2):350–358. doi: 10.1016/0304-4165(80)90308-6. [DOI] [PubMed] [Google Scholar]
  45. Saito H., Yamagata T., Suzuki S. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem. 1968 Apr 10;243(7):1536–1542. [PubMed] [Google Scholar]
  46. Sakashita S., Engvall E., Ruoslahti E. Basement membrane glycoprotein laminin binds to heparin. FEBS Lett. 1980 Jul 28;116(2):243–246. doi: 10.1016/0014-5793(80)80654-5. [DOI] [PubMed] [Google Scholar]
  47. Stamatoglou S. C., Keller J. M. Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen. J Cell Biol. 1983 Jun;96(6):1820–1823. doi: 10.1083/jcb.96.6.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stamatoglou S. C., Keller J. M. Interactions of cellular glycosaminoglycans with plasma fibronectin and collagen. Biochim Biophys Acta. 1982 Oct 28;719(1):90–97. doi: 10.1016/0304-4165(82)90311-7. [DOI] [PubMed] [Google Scholar]
  49. Takeuchi J., Sobue M., Sato E., Shamoto M., Miura K. Variation in glycosaminoglycan components of breast tumors. Cancer Res. 1976 Jul;36(7 Pt 1):2133–2139. [PubMed] [Google Scholar]
  50. Underhill C. B., Keller J. M. A transformation-dependent difference in the heparan sulfate associated with the cell surface. Biochem Biophys Res Commun. 1975 Mar 17;63(2):448–454. doi: 10.1016/0006-291x(75)90708-1. [DOI] [PubMed] [Google Scholar]
  51. Vaheri A., Kurkinen M., Lehto V. P., Linder E., Timpl R. Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4944–4948. doi: 10.1073/pnas.75.10.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wartiovaara J., Linder E., Ruoslahti E., Vaheri A. Distribution of fibroblast surface antigen: association with fibrillar structures of normal cells and loss upon viral transformation. J Exp Med. 1974 Dec 1;140(6):1522–1533. doi: 10.1084/jem.140.6.1522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wasteson A. A method for the determination of the molecular weight and molecular-weight distribution of chondroitin sulphate. J Chromatogr. 1971 Jul 8;59(1):87–97. doi: 10.1016/s0021-9673(01)80009-1. [DOI] [PubMed] [Google Scholar]
  54. Wilson B. S., Imai K., Natali P. G., Ferrone S. Distribution and molecular characterization of a cell-surface and a cytoplasmic antigen detectable in human melanoma cells with monoclonal antibodies. Int J Cancer. 1981 Sep 15;28(3):293–300. doi: 10.1002/ijc.2910280307. [DOI] [PubMed] [Google Scholar]
  55. Wilson B. S., Ruberto G., Ferrone S. Immunochemical characterization of a human high molecular weight--melanoma associated antigen identified with monoclonal antibodies. Cancer Immunol Immunother. 1983;14(3):196–201. doi: 10.1007/BF00205360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Winterbourne D. J., Mora P. T. Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation. J Biol Chem. 1981 May 10;256(9):4310–4320. [PubMed] [Google Scholar]
  57. Woodbury R. G., Brown J. P., Yeh M. Y., Hellström I., Hellström K. E. Identification of a cell surface protein, p97, in human melanomas and certain other neoplasms. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2183–2187. doi: 10.1073/pnas.77.4.2183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Woodley D. T., Rao C. N., Hassell J. R., Liotta L. A., Martin G. R., Kleinman H. K. Interactions of basement membrane components. Biochim Biophys Acta. 1983 Dec 27;761(3):278–283. doi: 10.1016/0304-4165(83)90077-6. [DOI] [PubMed] [Google Scholar]
  59. Woods A., Hök M., Kjellén L., Smith C. G., Rees D. A. Relationship of heparan sulfate proteoglycans to the cytoskeleton and extracellular matrix of cultured fibroblasts. J Cell Biol. 1984 Nov;99(5):1743–1753. doi: 10.1083/jcb.99.5.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamada K. M., Kennedy D. W., Kimata K., Pratt R. M. Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J Biol Chem. 1980 Jul 10;255(13):6055–6063. [PubMed] [Google Scholar]
  61. Yeh M. Y., Hellström I., Brown J. P., Warner G. A., Hansen J. A., Hellström K. E. Cell surface antigens of human melanoma identified by monoclonal antibody. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2927–2931. doi: 10.1073/pnas.76.6.2927. [DOI] [PMC free article] [PubMed] [Google Scholar]

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