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Identification, characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea, Ctenocephalides felis
Author(s) -
Silver Gary M.,
Gaines Patrick J.,
Hunter Shirley W.,
Maddux Joely D.,
Thomas Rex E.,
Wisnewski Nancy
Publication year - 2002
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.10059
Subject(s) - biology , biochemistry , microbiology and biotechnology , protease , benzamidine , affinity chromatography , proteases , peptide sequence , amino acid , serine protease , complementary dna , enzyme , gene
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.