IDENTIFICATION, CHARACTERIZATION AND GENOMIC CLONING OF A O-LINKED N-ACETYLGLUCOSAMINE-CONTAINING CYTOPLASMIC LEISHMANIA GLYCOPROTEIN

Antibodies against Leishmania major wheat germ agglutinin-binding glyc oproteins were used to select from a genomic lambda gtl1 expression li brary a clone coding for a L. major glycoprotein. The partial DNA sequ ence indicated the presence of a mosaic of repetitive sequences. South ern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to ch romosome band 18. Northern blot analysis of L. major mRNA detected a m ajor transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies af finity-purified on the fusion protein identified a complex of two wate r-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polype ptides in other Leishmania species, in Crithidia lucilliae and very we akly in Leptomonas. The apparent molecular weight of these polypeptide s, while conserved within each species, varied between species. A pept ide map of the two polypeptides from L. major generated an identical p attern suggesting a close relatedness at the protein level. This prote in complex was not hydrolysed by N-glycanase and was not affected by t unicamycin, but treatment with anhydrous hydrogen fluoride suggested t hat it is O-glycosylated. The glycan moiety appears to be N-acetylgluc osamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was ca pable of adding [H-3]galactose to it. This was susceptible to beta eli mination and beta-galactosidase treatment. Taken together, the data in dicates that gp96/92 belongs to the newly described class of cytoplasm ic and nuclear glycoproteins containing O-linked N-acetylglucosamine.