The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation.
Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.
Isordia-Salas
Irma
I
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad St, Philadelphia, PA 19140, USA.
Pixley
Robin A
RA
Parekh
Hemant
H
Kunapuli
Satya P
SP
Li
Fengling
F
Stadnicki
Anthony
A
Lin
Yingzhang
Y
Sartor
R Balfour
RB
Colman
Robert W
RW
D000818:Animals; D001920:Bradykinin / Q000378:metabolism / Q000493:pharmacokinetics; D016466:CHO Cells; D006224:Cricetinae; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D016264:Dextran Sulfate / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D007249:Inflammation; D007616:Kaolin / Q000378:metabolism; D007704:Kininogens / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010457:Peptidoglycan / Q000494:pharmacology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051381:Rats; D011917:Rats, Inbred Lew; D013045:Species Specificity; D013997:Time Factors
D007704:Kininogens; D010457:Peptidoglycan; D011134:Polysaccharides; D007616:Kaolin; D016264:Dextran Sulfate; D004262:DNA Restriction Enzymes; D001920:Bradykinin