Role of glycosylation in corin zymogen activation.
The cardiac serine protease corin is the pro-atrial natriuretic peptide convertase. Corin is made as a zymogen, which is activated by proteolytic cleavage. Previous studies showed that recombinant human corin expressed in HEK 293 cells was biologically active, but activated corin fragments were not detectable, making it difficult to study corin activation. In this study, we showed that recombinant rat corin was activated in HEK 293 cells, murine HL-1 cardiomyocytes, and rat neonatal cardiomyocytes. In these cells, activated corin represented a small fraction of the total corin molecules. The activation of recombinant rat corin was inhibited by small molecule trypsin inhibitors but not inhibitors for matrix metalloproteinases or cysteine proteases, suggesting that a trypsin-like protease activated corin in these cells. Glycosidase digestion showed that rat and human corin proteins contained substantial N-glycans but little O-glycans. Treatment of HEK 293 cells expressing rat corin with tunicamycin prevented corin activation and inhibited its pro-atrial natriuretic peptide processing activity. Similar effects of tunicamycin on endogenous corin activity were found in HL-1 cells. Mutations altering the two N-glycosylation sites in the protease domain of rat corin prevented its activation in HEK 293 and HL-1 cells. Our results indicate that N-linked oligosaccharides play an important role in corin activation.
Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Xudong
X
Liao
Wei
W
Wang
Shenghan
S
Chen
Qingyu
Q
Wu
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004792:Enzyme Precursors / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism*
D004792:Enzyme Precursors; D011134:Polysaccharides; D011994:Recombinant Proteins; C119812:CORIN protein, human; D012697:Serine Endopeptidases