Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein.
Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.
Handman
E
E
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Barnett
L D
LD
Osborn
A H
AH
Goding
J W
JW
Murray
P J
PJ
D000117:Acetylglucosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D016054:DNA, Protozoan / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D017125:Genes, Protozoan; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics*; D007891:Leishmania / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D018320:Leishmania major / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D013045:Species Specificity; D014158:Transcription, Genetic
D016054:DNA, Protozoan; D004274:DNA, Recombinant; D006023:Glycoproteins; D015800:Protozoan Proteins; D000117:Acetylglucosamine