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J Biol Chem, Vol. 274, Issue 29, 20725-20732, July 16, 1999

A Convenient Oxidation of Natural Glycosphingolipids to Their "Ceramide Acids" for Neoglycoconjugation
BOVINE SERUM ALBUMIN-GLYCOSYLCERAMIDE ACID CONJUGATES AS INVESTIGATIVE PROBES FOR HIV gp120 COAT PROTEIN-GLYCOSPHINGOLIPID INTERACTIONS

Murugesapillai Mylvaganam and Clifford A. Lingwood

From the Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada and the  Departments of Biochemistry and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

A new method to cleave the double bond of sphingolipids has been developed. Using limited concentrations of KMnO₄ and an excess of NaIO₄, in a neutral aqueous tert-butanol solvent system gave nearly quantitative yields of the oxidized product. A variety of natural glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb₃C, Gb₄C, Gg₄C, Gb₅C, and GM₁C, gave the corresponding acids: 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. "glycosyl ceramide acids" (GSL·^CCOOH) in excellent yields (80-90%). Deacyl GSLs (dGSLs) were oxidized to acids containing the oligosaccharides devoid of hydrocarbon chains, i.e. "ceramide oligosaccharides" (dGSL·NRR₁^CCOOH, where R = R₁ = H; R = H, R₁ = CH₃CO; or R = R₁ = Me). The efficacy of this method was demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC, Gb₃C, and Gb₄C into neoglycoproteins via coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of GalC-BSA conjugates, (GalC·CONH)_nBSA and (GalS·NHCO)_nBSA gave a value of 9 ± 1 and 16 ± 2 for n. Neoglycoconjugates derived from GlcC, GalC (type I and II and the behenic analog), SGC, LC, and Gb₃C were recognized by the recombinant human immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate showed significantly reduced binding, and the Gb₄C conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires a terminal galactose and/or glucose residue. Terminal N-acetylgalactosamine containing GSLs are not bound. The ceramide acid conjugates provide a more effective scaffold for presentation of glycone for rgp120 binding than those derived from dGSLs. The retention of receptor specificity of the glycoconjugates was validated by retention of the expected binding specificity of VT1 and VT2_e for Gb₃C and Gb₄C conjugates, respectively. These studies open a new vista in the generation of glycoconjugates from GSLs and further emphasize the role of aglycone in glycolipid recognition.

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