Abstract
CCN1 is a secreted protein and belongs to the CCN family of matricellular proteins. CCN1 binds to various cell surface receptors; thus, CCN1 has important functions in cell proliferation, migration and angiogenesis through a variety of signaling pathways. We have reported that CCN1 is O-fucosylated and that this O-fucosylation regulates the secretion of CCN1 into the extracellular region. In this study, we detected collagen-like glycosylation and hydroxylation at Lys203 of recombinant CCN1 by mass spectrometry. We then examined the role of collagen-like glycosylation in the functions of CCN1. As a result, we found that a deficiency in collagen-like glycosylation decreased the secretion of CCN1 using wild-type CCN1- and collagen-like glycosylation-defective mutant CCN1-overexpressing cell lines. Further, knockout of lysyl hydroxylase3, a multifunctional protein with hydroxylase and glucosyltransferase activities, impaired the secretion and glycosylation level of recombinant CCN1. Previous studies reported that collagen glycosylation of Lys residues mediated by lysyl hydroxylase3 is glucosyl-galactosyl-hydroxylation, presuming that this collagen-like glycosylation detected at Lys203 of recombinant CCN1 in this study might be glucosyl-galactosyl-hydroxylation. Taken together, our results demonstrate the novel function of the collagen-like glycosylation of CCN1 and suggest that lysyl hydroxylase3-mediated glycosylation is important for CCN1 secretion.