Abnormal glycosylation of human cellular fibronectin in the presence of swainsonine

• Published in: The Journal of biological chemistry 1983-10, Vol.258 (19), p.11883-11889
• Main Authors: Arumugham, R G, Tanzer, M L
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Cell physiology; Fibroblasts - drug effects; Fibroblasts - metabolism; Fibronectins - genetics; Fundamental and applied biological sciences. Psychology; Glycopeptides - isolation & purification; Glycosides - biosynthesis; Humans; Molecular and cellular biology; Oligosaccharides - genetics; Oligosaccharides - isolation & purification; Protein Processing, Post-Translational - drug effects; Secretion. Exocytosis; Skin - metabolism; Swainsonine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)44315-8

The relationship between post-translational modifications of macromolecules and their intracellular routing is of fundamental importance. The availability of the indolizidine alkaloid, swainsonine, which interferes with glycoprotein processing, provides a new probe for studying relationships between glycosylation of proteins and their cellular routing. Using fibronectin as a model glycoprotein, we have explored the effect of swainsonine upon oligosaccharide structure, glycoprotein synthesis, and secretion. Confluent human fibroblasts were labeled with radioactive mannose or glucosamine in the presence or absence of swainsonine. Fibronectin was secreted into the medium of swainsonine-treated cultures and found to contain endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides, instead of the normal complex endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides. Pronase-released glycopeptides and hydrazine-released oligosaccharides of isolated fibronectin from the culture media were analyzed using endo-beta-N-acetylglucosaminidase H and specific exoglycosidase digestions in conjunction with calibrated gel filtration chromatography. The structure of the swainsonine-modified endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide was found to be a hybrid type, Gal beta leads to GlcNAc beta leads to Man alpha leads to [Man alpha leads to (Man alpha leads to) Man alpha leads to]Man beta leads to GlcNAc beta leads to (+/- Fuc alpha leads to)-GlcNAc. Other experiments showed that the synthesis and secretion of fibronectin were not affected by its change in glycosylation. The results also infer that swainsonine inhibits Golgi mannosidase II in intact cells, and that removal of two mannose residues by that mannosidase is not a prerequisite for addition of galactose to the existing peripheral nonreducing GlcNAc or for the addition of fucose to the innermost reducing GlcNAc. The composite results indicate that significant changes in oligosaccharide structure had little effect upon the routing and cellular release of a typical N-asparagine-linked glycoprotein.