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

Abstract

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. E, 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 '~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-~tm structures on the upper cell surface and as longer (up to 20 ~tm) 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 wholemount 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.

Phosphorylation of NG2 Proteoglycan by Protein Kinase C-α Regulates Polarized Membrane Distribution and Cell Motility

“…In well spread, quiescent cells NG2 is localized to short membrane protrusions from the cell surface, reminiscent of NG2-rich microspikes found on the surface of human melanoma cells (29). Ezrin and PKC-␣ are co-localized within very similar processes in fibrosarcoma cells (26), consistent with our observation that NG2, ezrin, and ␣ 3 ␤ 1 integrin all appear to be present in membrane protrusions on the surface of U251/NG2 cells.…”

“…Ezrin and PKC-␣ are co-localized within very similar processes in fibrosarcoma cells (26), consistent with our observation that NG2, ezrin, and ␣ 3 ␤ 1 integrin all appear to be present in membrane protrusions on the surface of U251/NG2 cells. It has been proposed that membrane protrusions of this sort serve as specialized scaffolds for presentation of molecules that mediate cell-cell and cell-matrix interactions (29). These protrusions appear to be distinct from retraction fibers, another type of microdomain that contains NG2 under certain conditions (25).…”

Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis

“…Fang et al, 1999). The localization of MCSP and CD8/MCSP on keratinocytes is also in good agreement with an earlier report that NG2 is found on cell surface microspikes and microvilli (Garrigues et al, 1986). The cytoplasmic domain of NG2 binds a multiple-PDZ-domain-containing protein called MUPP1; MUPP1 might be involved in linking NG2 to actin filaments or to cytoplasmic signalling cascades (Barritt et al, 2000).…”

Tumor-Reactive CD4+ T Cell Responses to the Melanoma-Associated Chondroitin Sulphate Proteoglycan in Melanoma Patients and Healthy Individuals in the Absence of Autoimmunity

“…Association of NG2 with CD44 and ␣ 4 ␤ 1 integrin, cell-surface components involved in melanoma progression, mediates an anti-adhesive effect on melanoma cells and, therefore, increases the metastatic potential of melanoma cells (8). The ability of MCSP to enhance the migratory potential of melanoma cells is further supported by the finding that expression of MCSP is restricted to cell surface microspike domains on migrating melanoma cells in vitro (9).…”