Summary
HIV-1 infection in CD4-negative cells is now a well-established alternative route of infection for several cell types, including mucosal epithelial cells. Among different proposed mechanisms for HIV-1 entry into CD4-negative cells, the glycosphingolipid galactosylceramide (GalCer) has been characterized as a specific receptor for the surface envelope glycoprotein gp120 in cell lines of neural and epithelial origin. In this report we describe a protocol for identifying the GalCer receptor on the surface of CD4-negative cells. The development of this assay has been motivated by the unavailability of GalCer-specific antibodies, rendering the unambiguous immunological detection of this glycolipid on the cell surface impossible. For instance, we show that the commercially available anti-GalCer mAb (the R-mAb) can bind to the surface of both GalCer-positive and GalCer-negative human colon epithelial cell lines. This lack of specificity is due to a cross-reaction with several intestinal glycolipids that are not recognized by gp120. By contrast, gp120 only binds to the surface of GalCer-positive colon cells and the binding is specifically abolished by the R-mAb, as well as by compounds previously characterized as inhibitors of the GalCer-gp120 interaction. The specificity of gp120 binding was further demonstrated by incorporating sulfatide (the sulfated derivative of GalCer) in the membrane of GalCer-negative cells: in this case, gp120 binding was easily detectable by indirect immunofluorescence. Based on these data, we suggest the following protocol for assessing the expression of functional GalCer/sulfatide receptors on the surface of CD4-negative cells: (i) binding of gp120 revealed by indirect immunofluorescence; and (ii) inhibition of gp120 binding by the R-mAb. This would be preferable to using the R-mAb directly as a probe for GalCer/sulfatide expression.
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Delézay, O., Yahi, N. & Fantini, J. Detection of functional galactosylceramide (GalCer) receptors on CD4-negative HIV-1 target cells. Perspectives in Drug Discovery and Design 5, 192–202 (1996). https://doi.org/10.1007/BF02174014
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DOI: https://doi.org/10.1007/BF02174014