Biosynthetic and Structural Studies on Thy-1 in a Rat Neuronal Tumor Cell Line

Abstract

Because of the limited information available about the synthesis of N-linked glycoproteins in nerve cells, in regard to both processing steps and enzyme characterization, the biosynthetic processing of Thy-1 of the rat neuronal tumor cell line BN-1010-1 has been investigated using several inhibitors of biosynthesis and transport. (i) Tunicamycin completely inhibited mannose incorporation into Thy-1. Unglycosylated Thy-1 was not transported to the cell surface and was probably degraded rapidly following synthesis. (ii) Brefeldin A completely inhibited the transport of all [3H]mannose-labeled proteins releasable by phosphatidylinositol-specific phospholipase C, including Thy-1, to the surface of BN-1010-1 cells. Removal of the inhibitor led to rapid reversal of the inhibition. Pulse-chase experiments demonstrated that ≍50% of Thy-1 was degraded after 4 h in the presence of brefeldin A. (iii) Castanospermine treatment slowed the appearance of Thy-1 on the cell surface. The surface Thy-1 contained mainly normal Man5, Man6, and Man7 oligosaccharides, suggesting that Golgi endo-α-D-mannosidase effected the removal of glucose. (iv) Treatment with deoxymannojirimycin resulted in the synthesis of Thy-1 that contained Man8 and Man9 oligosaccharides compared to Man5, Man6, and Man7 in the control. Neither the rate of appearance nor the level of surface expression was affected by the drug. (v) Swainsonine did not affect either the rate of appearance or the level of surface expression of Thy-1. The HPLC elution profile of neutral oligosaccharides resulting from Endo-H digestion of Thy-1 synthesized in the presence of swainsonine was indistinguishable from controls. The lack of an effect of swainsonine is explained by the unexpected absence of a complex type oligosaccharide in Thy-1 of BN-1010-1 cells, as shown in experiments with a variety of lectins as well as digestions with Endo-H or glycopeptidase F, or digestions with both enzymes in sequence. The fact that, after [3H]fucose-labeling, Endo-H digestion produced Thy-1 still labeled with fucose indicates that hybrid oligosaccharide is present in Thy-1.