Initiation of the decorin glycosaminoglycan chain in the endoplasmic reticulum-golgi intermediate compartment

Abstract

We have transiently expressed decorin with a C-terminal KDEL endoplasmic reticulum retention signal peptide in COS-7 cells to study initiation of galactosaminoglycan synthesis in the endoplasmic reticulum-Golgi intermediate compartment. All decorin-KDEL molecules were substituted with N-linked oligosaccharides sensitive to endoglycosidase H, indicating that the core protein was located proximal to the medial-Golgi. O-Linked glycosylation was only initiated in a minor fraction of the molecules. The O-linked saccharides were characterized by gel filtration after stepwise degradations using chondroitin ABC/AC-I lyases, β1-3-glycuronidase, β-galactosidase, and alkaline phosphatase. The major O-linked saccharide was the linkage region pentasaccharide GalNAcβ1-4GlcUAβ1-3Galβ1-3Galβ14Xyl-2-phosphate, demonstrating initiation of chondroitin synthesis in the endoplasmic reticulum-Golgi intermediate compartment. In the presence of brefeldin A, partial elongation of a chondroitin chain took place, indicating retrieval of polymerases but not of sulfotransferases.