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, 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.
Silver
Gary M
GM
Heska Corporation, Fort Collins, Colorado 80525, USA.
Gaines
Patrick J
PJ
Hunter
Shirley W
SW
Maddux
Joely D
JD
Thomas
Rex E
RE
Wisnewski
Nancy
N
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D002371:Cat Diseases / Q000469:parasitology; D002415:Cats; D002460:Cell Line; D003001:Cloning, Molecular; D004064:Digestive System / Q000201:enzymology; D004478:Ectoparasitic Infestations / Q000469:parasitology / Q000662:veterinary; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005423:Siphonaptera / Q000201:enzymology* / Q000235:genetics
D001798:Blood Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases