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. 1998 Nov 24;95(24):14435-40.
doi: 10.1073/pnas.95.24.14435.

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

A Puri  1 , P Hug, K Jernigan, J Barchi, H Y Kim, J Hamilton, J Wiels, G J Murray, R O Brady, R Blumenthal
Affiliations

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

A Puri et al. Proc Natl Acad Sci U S A. .

Abstract

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4(+) or GSL-depleted human CD4(+) cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4(+) cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4(+) nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(alpha1-->4)Gal(beta1-->4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or alpha-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.

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Figures

Figure 1
Figure 1
Recovery of fusion after addition of various GSL fractions isolated from the crude human erythrocyte mixture. (A) TLC analysis of erythrocyte GSLs. GSLs were isolated as described in Materials and Methods. Total lipid (25–50 μg) was spotted on a 5 × 20-cm silica-gel TLC plate. The plate was developed in CHCl3/MeOH/H2O (65:25:4, vol/vol). At the end of the run, the plate was air dried, sprayed with Bial’s reagent, heated to develop the spots, scanned with a Hewlett–Packard 4P scanner, and photographed. GSLs isolated from human erythrocytes are in lane 1, and GSLs isolated from bovine erythrocytes are in lane 2; GSL standards are in lane 3. (B) TLC of various GSL fractions purified by silica-gel column. The crude GSL mixture from human erythrocytes (shown in A, lane 1) was fractionated by silica-gel column chromatography. GSLs in the fractions were analyzed by chromatography on silica-gel TLC as in A. Lanes 1–7 are fraction numbers. (C) Recovery of fusion of CD4+ nonhuman cells by GSL fractions. GP4F cells were plated on microwells and infected with vCB3 to express human CD4 on the cell surface. Liposomes containing different GSL fractions were incorporated into the membranes of the target cells and the cells were labeled with CMTMR. DiO-labeled TF228 cells were cocultured with GSL-supplemented target cells for 4–6 h at 37°C. Images were collected, and fusion was calculated as described in Materials and Methods. Data presented here are normalized to the recovery of fusion by the crude human GSL fraction.
Figure 2
Figure 2
(A) Chemical structure of globo-series GSLs. Cer, ceramide; I, glucosylceramide; II, lactosyl ceramide {Gb2; [Gal(β1→4)Glc-Cer]}; III, Gb3 [Gal(α1→4)Gal(β1→4)Glc-Cer]; and IV, globoside [Gb4;GalNAc(β1→3)Gal(α1→4)Gal(β1→4)Glc-Cer]. (B) TLC of Fr. 3. GSLs were chromatographed on silica-gel TLC plates (as shown in Fig. 1A) and sprayed with orcinol. Lane 1, Fr. 3; lane 2, Gb3 (top spot); lane 3, the same neutral GSL standards as shown in Fig. 1 A and B. (C) HPTLC immunostaining. GSLs (5 μg) were chromatographed on silica-gel HPTLC plates. The plates were air dried and sprayed with orcinol (Left), incubated with 38.13 antibody (Center), or incubated with asialo-GM2 antibody (Right). Lane 1, Fr. 3; lane 2, asialo-GM2; lane 3, Gb3. (D) Mass spectrum of 24:0 Gb3 detected from Fr. 3. (E) Mass ion chromatogram of various molecular species of Gb3 present in Fr. 3. (F) 1H NMR proton spectrum of Fr. 3 in 98:2 DMSO-d6/D2O at 298 K. Relevant peaks are labeled with arrows. Numbers in parentheses refer to the position of the particular sugar in the trisaccharide with glucose attached to the ceramide at position 1. The peak marked with an asterisk is thought to arise from minor variations in the length of the ceramide fatty-acid chains.
Figure 2
Figure 2
(A) Chemical structure of globo-series GSLs. Cer, ceramide; I, glucosylceramide; II, lactosyl ceramide {Gb2; [Gal(β1→4)Glc-Cer]}; III, Gb3 [Gal(α1→4)Gal(β1→4)Glc-Cer]; and IV, globoside [Gb4;GalNAc(β1→3)Gal(α1→4)Gal(β1→4)Glc-Cer]. (B) TLC of Fr. 3. GSLs were chromatographed on silica-gel TLC plates (as shown in Fig. 1A) and sprayed with orcinol. Lane 1, Fr. 3; lane 2, Gb3 (top spot); lane 3, the same neutral GSL standards as shown in Fig. 1 A and B. (C) HPTLC immunostaining. GSLs (5 μg) were chromatographed on silica-gel HPTLC plates. The plates were air dried and sprayed with orcinol (Left), incubated with 38.13 antibody (Center), or incubated with asialo-GM2 antibody (Right). Lane 1, Fr. 3; lane 2, asialo-GM2; lane 3, Gb3. (D) Mass spectrum of 24:0 Gb3 detected from Fr. 3. (E) Mass ion chromatogram of various molecular species of Gb3 present in Fr. 3. (F) 1H NMR proton spectrum of Fr. 3 in 98:2 DMSO-d6/D2O at 298 K. Relevant peaks are labeled with arrows. Numbers in parentheses refer to the position of the particular sugar in the trisaccharide with glucose attached to the ceramide at position 1. The peak marked with an asterisk is thought to arise from minor variations in the length of the ceramide fatty-acid chains.
Figure 3
Figure 3
Enzymatic digestion of Fr. 3. (Left) Fr. 3 was digested with αGalA in parallel with standard GSLs, and the results were analyzed by HPTLC. Lane S, mixed GSL standards; lane 1, Gb2 with no enzyme; lane 2, Gb2 + αGalA; lane 3, Gb3 with no enzyme; lane 4, Gb3 + αGalA; lane 5, Fr. 3 with no enzyme; and lane 6, Fr. 3 + αGalA. (Right) Fusion activity after addition of αGalA-digested Fr. 3. Digested lipid was extracted, incorporated into liposomes, and transferred to HeLa-CD4/GSL−. Fusion activity was monitored as described for Fig. 1C. Controls represent untreated Hela-CD4 cells. Gb2 was added as an additional control.
Figure 4
Figure 4
Recovery of fusion activity by addition of various GSLs. The lipids were incorporated into liposomes and transferred to HeLa-CD4/GSL−, and fusion activity was monitored as described in Fig. 1C. Controls represent untreated Hela-CD4 cells. Data are from one of three similar experiments. GD3 is NeuNAcα2→8NeuNAcα2→3Galβ1→4Glcβ1→1Cer.
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