Quantitative Measurements of Recombinant HIV Surface Glycoprotein 120 Binding to Several Glycosphingolipids Expressed in Planar Supported Lipid Bilayers

Abstract

The interaction of recombinant HIV-1 surface glycoprotein gp120 (rgp120) with natural isolates of lactosylceramide (LacCer), glucosylceramide (GlcCer), and galactosylceramide (GalCer) has been quantitatively measured under equilibrium conditions using total internal reflection fluorescence (TIRF) spectroscopy. The binding affinity (K(a)) of rgp120 to these glycosphingolipids (GSLs), reconstituted at 5 mol % in supported planar lipid bilayers composed of 95 mol % POPC, is ca. 10(6) M(-1) for dissolved rgp120 concentrations greater than 25 nM. In contrast, at concentrations of rgp120 between 0.2 and 15 nM, rgp120 does not bind significantly to LacCer and GlcCer, but has a high affinity for GalCer with a measured K(a) value of 1.6 x 10(9) M(-1). However, protein surface coverage measurements show that this strong binding process accounts for very little of the total protein adsorbed over the entire concentration range studied. At a protein concentration of ca. 20 nM, the surface coverage is only 3% of that achieved at apparent saturation (i.e., when the protein concentration is ca. 220 nM). Thus the "high affinity" binding sites comprise only a small fraction of the total number of binding sites. Several other variables were investigated. Rgp120 binding behavior at membranes doped with alpha-hydroxygalactosylceramide (alpha-GalCer) was very similar to that observed with GalCer, showing that the presence/absence of an alpha-hydroxy moiety does not significantly affect galactosylceramide recognition. Phase segregation of GalCer, which occurs when the mole fraction of this GSL in a POPC bilayer exceeds ca. 0.1, was also investigated and showed no effect on binding affinity at low rgp120 concentrations. To investigate the influence of fatty acid chain length, GSLs with monodisperse C(18) and C(24) chain lengths, both with and without an alpha-hydroxy moiety, were synthesized, and their binding affinity to rgp120 was examined. Relative to the natural isolates (which contain a mixture of chain lengths), minimal differences were observed; thus among the compounds tested, fatty acid chain length does not affect GSL recognition. The results of this work should aid efforts to design anti-HIV-1 agents based on membrane-tethered, carbohydrate-based receptors for rgp120.

Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide

“…In our assay, the Galcer binding on-rate was on the order of 10 3 M Ϫ1 s Ϫ1 and the off-rate was on the order of 10 Ϫ4 to 10 Ϫ5 s Ϫ1 with K d values in nanomolar range (17 to 75 nM). These rate constantderived K d values are consistent with those from earlier studies that used different techniques to determine the rgp120-Galcer interaction quantitatively (8,20,21).…”

“…The ability of HIV-1 gp120 to interact with Galcer supports the view that Galcer might play the role of an alternate receptor for HIV-1 (7)(8)(9)(10)(20)(21)(22)(23)(24). With Galcer present in the apical membrane of the epithelial cells lining the mucosal surfaces, including colonic (10) and vaginal and mammary epithelium (16)(17)(18), the HIV-1 Env-Galcer interaction could play an important role in establishing initial adhesion of HIV-1 virions/infected cells.…”

“…Antibodies against Galcer have been reported to inhibit epithelial cell-HIV-1 virion binding (7,8,19), and previous biochemical studies showed that the HIV-1 envelope (Env) gp120 interacts with Galcer, supporting the view that Galcer might play the role of an alternate receptor for HIV-1 (7)(8)(9)(10)(20)(21)(22)(23)(24). Enhanced expression of Galcer on an endo-cervical epithelial cell line, following infection with Chlamydia trachomatis, increased binding of HIV-1 and levels of virus in cocultures (25), and endosomal uptake of HIV-1 by Galcer-expressing mammary epithelial cells has been reported to facilitate infection of CD4 T cells (18).…”

“…Previous studies used Galcer in multilamellar liposomes (22) and planar mono-or bilayer forms (20,21,24) to detect an Env-Galcer interaction and did not provide time-resolved binding data. In our assay, the Galcer binding on-rate was on the order of 10 3 M Ϫ1 s Ϫ1 and the off-rate was on the order of 10 Ϫ4 to 10 Ϫ5 s Ϫ1 with K d values in nanomolar range (17 to 75 nM).…”

Packing Density of Glycolipid Biosurfactant Monolayers Give a Significant Effect on Their Binding Affinity Toward Immunoglobulin G

“…57, (8) 415-422 (2008) desirable approach without troublesome synthesis is the use of self-assembling systems 7,8) . However, only a few carbohydrate molecules are known to self-assemble into highly ordered structures which give effective molecular recognition properties 9,10) : this has limited the widespread application of carbohydrate ligands.…”