Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako 351-0198, Japan.
Glycobiology. 2019 Sep 20;29(10):696-704. doi: 10.1093/glycob/cwz052.
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.
CCN1 是一种分泌蛋白，属于细胞基质蛋白的 CCN 家族。CCN1 与各种细胞表面受体结合；因此，CCN1 通过多种信号通路在细胞增殖、迁移和血管生成中具有重要功能。我们已经报道 CCN1 被 O-岩藻糖基化，这种 O-岩藻糖基化调节 CCN1 向细胞外区域的分泌。在这项研究中，我们通过质谱法检测到重组 CCN1 中赖氨酸 203 处的胶原样糖基化和羟化。然后，我们研究了胶原样糖基化在 CCN1 功能中的作用。结果发现，使用野生型 CCN1 和胶原样糖基化缺陷突变体 CCN1 过表达细胞系，胶原样糖基化缺陷会降低 CCN1 的分泌。此外，多功能蛋白赖氨酸羟化酶 3 的敲除会损害重组 CCN1 的分泌和糖基化水平，赖氨酸羟化酶 3 具有羟化酶和葡萄糖基转移酶活性。先前的研究报告称，赖氨酸羟化酶 3 介导的赖氨酸残基胶原糖基化是葡萄糖基-半乳糖基羟化，假定本研究中在重组 CCN1 的赖氨酸 203 处检测到的这种胶原样糖基化可能是葡萄糖基-半乳糖基羟化。总之，我们的结果表明 CCN1 胶原样糖基化的新功能，并表明赖氨酸羟化酶 3 介导的糖基化对 CCN1 分泌很重要。
