title: Modular arrangement and secretion of a multidomain serine protease. Evidence for involvement of proline-rich region and N-glycans in the secretion pathway.
abstract: The Limulus Factor C (FC), a multidomain glycoprotein that binds bacterial endotoxin with high affinity, belongs to the serine protease family of the complement and blood coagulation cascade. Here, we provide compelling evidence for the importance of modular arrangement and relevance of the proline-rich region (PRR) and N-glycosylation to the secretion and function of FC. We propose that PRR could be a universal conformational domain that regulates protein folding and targeting. FCs lacking PRR preceding the serine protease domain, were localized intracellularly. Misfolded conformers of the intracellular FCs were more susceptible to trypsin digestion. Glycosylation inhibition studies indicate that the presence but not the exact structure of the N-glycans affects the secretion of FC, although the complexity of glycosylation may influence its endotoxin-induced proteolytic cleavage with resultant enzymatic activity. Disruption of specific N-glycan sites at positions 740, 767, and 912, downstream of the PRR, at or near the serine protease domain, blocks its secretion. Co-expressed molecular chaperones like canine calnexin associates with glycosylated FCs to increase its solubility and secretion level but did not alter their expression profiles. Our results clearly demonstrate that the folding and secretion of a multidomain serine protease like FC are determined by its modular domain arrangement and site-specific N-glycans. The secreted FCs containing the N-terminal portion of FC are able to detect lipopolysaccharide with high sensitivity. We also identified the lectin-like and sushi 4 domains to contribute to the binding of lipopolysaccharide.
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Nguan Soon
authors.initials: NS
authors.lastname: Tan
authors.forename: Bow
authors.initials: B
authors.lastname: Ho
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037281:Calnexin / Q000737:chemistry; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004285:Dogs; D006031:Glycosylation; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D008050:Lipid A / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
chemical_list: D008050:Lipid A; D008070:Lipopolysaccharides; D011134:Polysaccharides; D037281:Calnexin; D011392:Proline; D012697:Serine Endopeptidases