Abstract
We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation.
Highlights
Can CN bind its substrates through an N-linked glycosylation-independent mechanism? Several studies found that CN interactions with proteins occur after enzymatic removal of glycans [5,6,7] and that glycosylation or glucosidase inhibitors did not completely inhibit associations with CN (8 –13)
We measured 125I-Bgt binding to the total cellular pool of acetylcholine receptors (AChRs), which is a measure of the maturation of AChR subunits during their assembly in the endoplasmic reticulum (ER) (Fig. 1C). 48 h after the temperature shift CAS treatment caused a 74% decrease in cellular 125I-Bgt binding, similar to the CAS-induced decrease observed for cell surface 125I-Bgt binding
TUN completely abolished total cellular 125I-Bgt binding. These data indicate that CAS and TUN treatment inhibit new AChR assembly because intracellular 125I-Bgt binding site formation occurs during AChR assembly in the ER
Summary
Can CN bind its substrates through an N-linked glycosylation-independent mechanism? Several studies found that CN interactions with proteins occur after enzymatic removal of glycans [5,6,7] and that glycosylation or glucosidase inhibitors did not completely inhibit associations with CN (8 –13). Recent results using various CN deletion or point mutations found regions of CN that can prevent aggregation of non-glycosylated proteins [17, 18] These results support a role for a lectin-independent association between CN and its substrates. Mutations of vesicular stomatitis virus (VSV) G protein that remove N-linked glycosylation sites associate with CN but, it was concluded that the glycan-independent associations were a result of nonspecific aggregation of mutant VSV G and CN [11]. Another possible explanation for glycosylation independent CN associations is that the observed polypeptide associations are with chaperones that are part of a larger scaffold of chaperones that includes CN. Our data indicate that CN can associate with AChR subunits without interacting with N-linked glycans, and that CN has no role in subunit assembly events that require N-linked glycosylation
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