Expression of recombinant microfilarial chitinase and analysis of domain function

A family of chitinase isozymes was previously characterized from the microfilariae of Brugia malayi and Brugia pahangi. The expression of these enzymes correlates with the onset of microfilarial infectivity for the mosquito vector. To study the role of chitinase activity in filarial transmission, th...

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Bibliographic Details
Published in:	Molecular and biochemical parasitology Vol. 78; no. 1-2; pp. 149 - 159
Main Authors:	Venegas, Ana, Goldstein, Joshua C., Beauregard, Kathryn, Oles, Anita, Abdulhayoglu, Nur, Fuhrman, Juliet A.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-06-1996
Subjects:	Animals Antibodies, Monoclonal Antigens, Helminth - genetics Base Sequence Brugia Brugia malayi Brugia malayi - enzymology Brugia malayi - genetics Brugia malayi - immunology Brugia pahangi - enzymology Brugia pahangi - genetics Brugia pahangi - immunology chemical structure chitin Chitinase Chitinases - genetics Chitinases - immunology Chitinases - metabolism cloning Cloning, Molecular complementary DNA disease vectors DNA, Complementary - genetics DNA, Helminth - genetics enzyme activity Filariasis gene expression Immunochemistry Immunodominant Epitopes - genetics Kinetics Microfilaria microfilariae Microfilariae - enzymology Microfilariae - genetics Molecular Sequence Data NC6 domain Oligosaccharides pathogenicity pET vectors recombinant proteins Recombinant Proteins - genetics Recombinant Proteins - immunology Recombinant Proteins - metabolism structural genes Substrate Specificity Chitinase PBS-Tw Filariasis 4MUGlcN-Ac2 Brugia NC6 domain Microfilaria 4MUGlc-NAc3 pET vectors GlcNAc 4MU β-ME
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Summary:	A family of chitinase isozymes was previously characterized from the microfilariae of Brugia malayi and Brugia pahangi. The expression of these enzymes correlates with the onset of microfilarial infectivity for the mosquito vector. To study the role of chitinase activity in filarial transmission, the p70 chitinase from Brugia malayi was cloned and expressed in two forms: a full-length product of ∼ 62 kDa and a truncated product of 43 kDa containing only the N-terminal catalytic domain. Two epitopes defined by monoclonal antibodies were preserved only in the full-length recombinant enzyme. It was found that deletion of the cysteine-rich C-terminal domain increased the yield of the recombinant expression product, and did not affect the Km for di- or trisaccharide substrates. However, affinity for high molecular weight chitin was specific to the full-length molecule, and is apparently mediated by the cysteine-rich domain, suggesting a role for this part of the protein in targeting the secreted enzyme to its substrate.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0166-6851 1872-9428
DOI:	10.1016/S0166-6851(96)02620-5