Decorin Core Protein Secretion Is Regulated by N-Linked Oligosaccharide and Glycosaminoglycan Additions

Abstract

Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms: a proteoglycan substituted with a single chondroitin sulfate chain and N-linked oligosaccharides and a core protein glycoform substituted with N-linked glycans but without a glycosaminoglycan chain. In this report, we have addressed two distinct questions. What is the ratelimiting step in glycosaminoglycan synthesis? Is glycosylation with either N-linked oligosaccharides or glycosaminoglycan required for secretion of decorin? N-terminal sequencing of the core protein glycoform, the addition of benzyl-␤-D-xyloside, and a UDP-xylose: core protein ␤-D-xylosyltransferase activity assay show that xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis. Decorin can be efficiently secreted with N-linked oligosaccharides alone or with a single chondroitin sulfate chain alone; however, there is severely impaired secretion of core protein devoid of any glycosylation. A decorin core protein mutant devoid of N-linked oligosaccharide attachment sites will not be secreted by Chinese hamster ovary cells deficient in xylosyltransferase or by parental Chinese hamster ovary wild type cells if the xylosyltransferase recognition sequence is disrupted. This finding suggests that quality control mechanisms sensitive to an absence of N-linked oligosaccharides can be abrogated by interaction of the core protein with the glycosaminoglycan synthetic machinery. We propose a model of regulation of decorin secretion that has several components, including appropriate substitution with N-linked oligosaccharides and factors involved in glycosaminoglycan synthesis.

Regulation of intracellular decorin via proteasome degradation in rat mesangial cells

“…The N-linked oligosaccharide moieties of these proteins play a crucial role for protein folding and secretion [Helenius, 1994]. It is known that a DCN core protein mutant devoid of N-linked oligosaccharide attachment sites is not be secreted by CHO cells [Seo et al, 2005], and such mutant glycoproteins with improper folding are either retained in the endoplasmic reticulum lumen until they become properly folded or retro-translocated into the cytosol and degraded by the 26S proteasome [Ellgaard et al, 1999;Rö misch, 2005;Moremen and Molinari, 2006]. In this respect, the mechanism of ERassociated degradation through UPS remains to be elucidated.…”

“…Since N-linked oligosaccharide regulates DCN stability [Seo et al, 2005], we investigated whether ubiquitination of DCN is influenced by its glycosylation state. After being treated with tunicamycin, an N-glycosylation inhibitor, DCN ubiquitination increased in immunoprecipitation, while the DCN protein is decreased in MC on Western blot (Fig.…”

Expression of the Metastasis Suppressor KAI1 in Decidual Cells at the Human Maternal-Fetal Interface

“…Decorin is produced as a core protein migrating as a doublet around 46 kd apparent molecular mass. The mature proteoglycan decorin is formed by attachment of a glycosaminoglycan chain 40 and migrates between 70 and 100 kd in our cell system. Silencing with siKAI1 effectively abrogated KAI1 protein expression in all cultures.…”

Regulation of Xylosyltransferase I Gene Expression by Interleukin 1β in Human Primary Chondrocyte Cells

“…The XT-I enzyme has received much attention because it plays a central role in GAG synthesis. Indeed, this enzyme catalyzes the first and rate-limiting step in the biosynthesis pathway (16,17) and is therefore considered as a regulatory factor of GAG biosynthesis process. Accordingly, it has been shown that loss of XT-I expression was associated with strong decrease in GAG synthesis during arthritis development in rats, whereas high expression of XT-I was associated with high rate of GAG synthesis during cartilage repair in a rat model of cartilage regen-eration, suggesting that XT-I regulates GAG synthesis during cartilage destruction and repair (18).…”