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Protein Conjugates of Polysaccharide from Cryptococcus Neoformans¹

Department of Experimental Chemistry, Division of Research, National Jewish Hospital at Denver, Denver, Colorado

Abstract

Soluble polysaccharide from culture filtrates of Cryptococcus neoformans can be made potently antigenic in mice if it is coupled to a heterologous protein by a reaction sequence that permits retention of the ester functions (specifically identified as acetate) in the native polysaccharide.

The key step in the reaction sequence which was developed rests on the utility of dimethylsulfoxide (DMSO) as an aprotic homogeneous reaction solvent for the polysaccharide. This allows controlled nitrocarbanilation of the carbohydrate without cleavage of the acetyl functions. The aminocarbanilate derived from the nitro compound was diazotized and coupled to bovine -globulin (BGG) to yield gelled protein-azocarbanilate-polysaccharide conjugates. Homogenized conjugates dispersed in Freund's adjuvant were potent antigens in mice and stimulated production of humoral anti-BGG and antipolysaccharide. De-acetylated polysaccharide coupled to BGG and polysaccharide-mouse globulin conjugate were essentially inert.

The polysaccharide and its derivatives were subject to ready insolubilization by what is judged to be an intermolecular crosslinking. It is suggested that the physical integrity of the cryptococcal capsule may be a consequence of a similar behavior in vivo. In addition, DMSO is an effective decapsulating agent for this yeast.

Footnotes

This investigation was supported by Public Health Service research Grants AI-03455, AI-06538 and in part by Grant AI-05840, from the National Institutes of Health.

² Present address: Department of International Medicine, University of Maryland, School of Medicine, Baltimore, Maryland.

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