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, the p70 chitinase from Brugia malayi was cloned and expressed in two forms: a full-length product of approximately 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 K(m) 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.
Venegas
A
A
Department of Biology, Tufts University, Medford MA, USA.
Goldstein
J C
JC
Beauregard
K
K
Oles
A
A
Abdulhayoglu
N
N
Fuhrman
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth / Q000235:genetics; D001483:Base Sequence; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D017179:Brugia pahangi / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D002688:Chitinases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D007120:Immunochemistry; D016056:Immunodominant Epitopes / Q000235:genetics; D007700:Kinetics; D008842:Microfilariae / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D016056:Immunodominant Epitopes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D002688:Chitinases