Abstract
Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.
Highlights
In the human genome, ~2% of the genes encode proteases, among which trypsin-like serine proteases are a major group (Overall and Blobel, 2007)
The results indicate that lacking N-glycosylation site at Asn1022 (N1022) glycosylation reduces corin cell surface expression with or without the activation cleavage at R801
N-glycosylation at N1022 was found to be critical for corin cell surface expression, but the underlying mechanism was unknown (Wang et al, 2015)
Summary
In the human genome, ~2% of the genes encode proteases, among which trypsin-like serine proteases are a major group (Overall and Blobel, 2007). N-glycosylation has been shown to regulate the extracellular expression, secretion and activation of trypsin-like serine proteases, the underlying mechanisms are not elucidated (Bolt et al, 2007; Gladysheva et al, 2008; Jiang et al, 2014; Lai et al, 2015; Liao et al, 2007; Miyake et al, 2010; Wu and Suttie, 1999). It is unclear if N-glycans at the conserved sites have a general role in the biosynthesis of the trypsin-like serine proteases. We found that N-glycosylation in the protease domain of corin, EK and prothrombin has a common role in regulating the extracellular expression of these proteases, which involves calnexin-assisted protein folding and ER exiting
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