Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

Abstract

We have investigated the significance of protein glycosylation for metabolism of acetylcholine receptors (AcChoR) in primary cultures of embryonic chicken muscle cells. Tunicamycin, a specific inhibitor of the glycosylation of asparagine residues on glycoproteins, decreased AcChoR accumulation and accelerated its degradation. In contrast, there was no evidence that tunicamycin treatment affected AcChoR biosynthesis, intracellular transport, or incorporation into surface membranes. Leupeptin, an inhibitor of intracellular proteases, markedly increased accumulation of AcChoR on the external surface of muscle cells treated with tunicamycin. Our findings indicate that impairment of protein glycosylation prevents accumulation of AcChoR by increasing its susceptibility to degradation by cellular proteases.is strongly impaired by TM treatment. However, the proteolytic degradation of underglycosylated AcChoR is substantially increased under these conditions, resulting in marked diminution of AcChoR accumulation. The protease inhibitor leupeptin substantially reverses the decrease in AcChoR in TM-treated cultures, suggesting that glycosylation stabilizes AcChoR against proteolytic degradation. The possibility that the carbohydrate on AcChoR may function in regulating its stability is supported by our findings that: (i) impaired protein glycosylation results in accelerated degradation of AcChoR, and (ii) concanavalin A (Con A), a ligand of AcChoR (2,3), increases AcChoR stability in control cultures but not in TM-treated cells.The acetylcholine receptor (AcChoR) of skeletal muscle cells is an integral membrane glycoprotein that is uniquely well characterized both pharmacologically and biochemically (for recent reviews see refs. 1-3). Thus AcChoR may serve as a model for detailed study of the function of the carbohydrate components of glycoproteins in the regulation of well-defined plasma membrane properties. AcChoR is of particular interest because its synthesis, distribution on the muscle cell surface, and degradation are tightly regulated during differentiation and upon innervation (3). The striking effects induced under neuronal influence include the disappearance of AcChoR from extrasynaptic regions of the muscle cell surface and a marked decrease in the degradation rate of synaptic AcChoR (3). The regulation of AcChoR metabolism has recently been intensively studied in cell cultures of differentiating skeletal muscle (3, 4) using 25I-labeled a-bungarotoxin ('25I-a-Bgt), a specific ligand that binds to AcChoR with high specificity and low reversibility (5).The function of carbohydrate components of glycoproteins can be conveniently studied in cultured cells by the use of tunicamycin (TM), an antibiotic that specifically inhibits protein glycosylation (6-17). Treatment of cells with TM results in the synthesis of glycoproteins deficient in asparagine-linked oligosaccharides (6,18,19). TM has been utilized to study the contribution of the oligosaccharide components to the posttranslational processing (16, 17), i...

Expression and function of a nonglycosylated major histocompatibility class I antigen.

“…In accordance with these results, most, if not all, glycoproteins have been shown to exhibit reduced surface expression upon tunicamycin treatment. However, in contrast to our conclusion, in some cases reduction was explained by enhanced degradation; e.g., nonglycosylated acetylcholine receptor (35) and fibronectin (36) reportedly degrade more rapidly, although data are conflicting for the latter protein (37). Hastened degradation has been indicated also for the T25 glycoprotein (8).…”

Role of glycosylation in cell surface expression and stability of HERG potassium channels

“…Decreased stability of nonglycosylated proteins has been reported for other ion channel proteins. Inhibition of N-linked glycosylation has been shown to reduce stability of acetylcholine receptor and Shaker K ϩ channel proteins (2,12,23). Although it is generally believed that N-linked glycosylation protects proteins from proteolytic degradation, the specific proteolytic system involved in the rapid degradation of nonglycosylated proteins is not fully understood.…”

“…Although it is generally believed that N-linked glycosylation protects proteins from proteolytic degradation, the specific proteolytic system involved in the rapid degradation of nonglycosylated proteins is not fully understood. The accelerated degradation of nonglycosylated acetylcholine receptor has been shown to involve an intracellular lysosomal proteolytic system (23). Recently, it was reported that the rapid degradation of nonglycosylated Shaker K ϩ channel protein involves cytoplasmic proteasomes (12).…”

Effect of diabetes on the sugar nucleotides in several tissues of the rat

“…In considering what effect this enhanced glycosylating ability of diabetic kidney might have on basement membrane collagen formation, the question of the importance of the addition of carbohydrate to the peptide chain is relevant. Several recent reports have indicated that, for many newly synthesized proteins, the addition of carbohydrate provides protection against intracellular proteolysis and that molecules not glycosylated turn over more rapidly [47][48][49]. Degradation prior to secretion has been shown to be an important regulator of fibroblast procollagen production [50]; if basement membrane collagen synthesis is similarly controlled, a higher degree of carbohydration might lead to a greater net production of this protein in diabetes.…”