Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.21 (beta-glucosidase)
 3,280 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (HAG1) indicated sequence homology with genes for fungal beta-glucosidases. To understand the biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether H. capsulatum contained a beta-glucosidase enzyme activity and whether this activity was encoded by the HAG1 gene. Substrate gels with H. capsulatum culture supernatants revealed beta-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted beta-glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the H. capsulatum classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of beta-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of HAG1. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.
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PMID:Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid. 1037 7

The Histoplasma capsulatum H antigen is a major secreted glycoprotein of this pathogenic fungus that is a target of humoral and cell-mediated host responses. Its predicted protein sequence displays homology to beta-glucosidases of other organisms, but a recombinant antigen expressed in a prokaryotic system showed no enzymatic activity. We expressed a recombinant form of the protein carrying a carboxyl-terminus oligohistidine tag in the native fungal background to facilitate proper glycosylation and folding of a product that could then be purified from culture supernatants using nickel affinity chromatography. The recombinant protein was expressed and secreted by a transformant carrying the modified gene under the control of its native promoter. The purified protein from the native expression system showed beta-glucosidase enzymatic activity in substrate gels and quantitative microplate assays. This activity was blocked by glucosidase-specific inhibitors. These results are the first direct demonstration of the function of this protein, and show the utility of expression in a native system to achieve post-translational modification necessary for structural and functional integrity.
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PMID:Determination of beta-glucosidase enzymatic function of the histoplasma capsulatum H antigen using a native expression system. 1077 59