Identification, characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea, Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood‐fed adult C. felis midguts was examined. SDS‐PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, includin...

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Published in:	Archives of insect biochemistry and physiology Vol. 51; no. 3; pp. 136 - 150
Main Authors:	Silver, Gary M., Gaines, Patrick J., Hunter, Shirley W., Maddux, Joely D., Thomas, Rex E., Wisnewski, Nancy
Format:	Journal Article
Language:	English
Published:	New York Wiley Subscription Services, Inc., A Wiley Company 01-11-2002
Subjects:	Amino Acid Sequence Animals Baculoviridae Binding Sites Blood Proteins - metabolism bloodmeal Cat Diseases - parasitology cat flea Cats Cell Line Cloning, Molecular Digestive System - enzymology Ectoparasitic Infestations - parasitology Ectoparasitic Infestations - veterinary Escherichia coli Gene Expression Genetic Vectors IgG immunoglobulin Immunoglobulin G - metabolism Molecular Sequence Data Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Serine Endopeptidases - genetics Serine Endopeptidases - isolation & purification Serine Endopeptidases - metabolism serine protease Siphonaptera - enzymology Siphonaptera - genetics
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Summary:	The degradation of cat immunoglobulin G (IgG) in blood‐fed adult C. felis midguts was examined. SDS‐PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31‐kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30‐ and 40‐kDa were observed when the purified protease was incubated with protein A purified cat IgG. N‐terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host’s immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N‐terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem. Physiol. 51:136–150, 2002. © 2002 Wiley‐Liss, Inc.
Bibliography:	ArticleID:ARCH10059 istex:3B4DCC984A70D26715BC487266331C18A2E87AF6 ark:/67375/WNG-V8N1S6NZ-D ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0739-4462 1520-6327
DOI:	10.1002/arch.10059