Characterization of a yeast d-mannan with an α- d-glucosyl phosphate residue as an important immunochemical determinant

Abstract

Rabbit antiserum against whole heat-killed cells of the yeast Hansenula polymorpha gives with the isolated cell-wall d-mannan a precipitin reaction that shows a 50-percent inhibition by α- d-glucosyl phosphate. The antiserum does not cross-react with Streptococcus faecalis glycan phosphate, which contains a glucosyl phosphate antigenic determinant, indicating an α- d anomeric configuration for the determinant in the H. polymorpha mannan and β- d for that of the S. faecalis determinant. The homologous H. polymorpha immune reaction is also partly inhibited by large fragments, obtained from the mannan by partial acetolysis, a reaction that cleaves preferentially (1→6) linkages to yield oligosaccharides of mannose up to the pentasaccharide that contain mostly α- d-(1→2) linkages. Digestion of the intact mannan with a bacterial exo-α-mannanase releases 40 percent of the d-mannose, but the acetolysis pattern of the resulting enzyme-resistant fragment is not significantly altered from that of the undigested mannan, thus showing that the structure is different from that of the mannan of Saccharomyces cerevisiae, which has an α- d-(1→6)-linked backbone with α-(1→2)- and α-(1→3)-linked side chains. The H. polymorpha mannan appears to have a modified block-type structure in which the acetolysis-stable oligosaccharides are connected by (1→6) linkages, some of the (1→2)-linked units forming a part of the backbone. Methylation analysis showed that most of the (1→6)-linked d-mannose residues are otherwise unsubstituted, but the presence of a small amount of 3,4-di- O-methyl- d-mannose showed that the branch-points in the mannan involve positions 2 and 6 with an average chain-length of 8 mannose units. P.m.r. of the d-mannan with intact phosphate diester linkages showed the expected signal for the anomeric proton of an α- d-glucosyl phosphate group; the signal disappeared after cleavage of the d-glucosyl phosphate diester bond in acid.