Relationship between Calnexin and BiP in Suppressing Aggregation and Promoting Refolding of Protein and Glycoprotein Substrates

Victoria S Stronge,Yoshiro Saito,Yoshito Ihara,David B Williams

doi:10.1074/jbc.m107091200

Victoria S Stronge, Yoshiro Saito + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m107091200

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Abstract

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.

Highlights

Calnexin (CNX)1 is a type I membrane protein of the endoplasmic reticulum (ER) that interacts transiently with newly synthesized Asn-linked glycoproteins
How do these functions compare with those of other “classical” molecular chaperones such as those of the Hsp70 family? Does the existence of both lectin and polypeptide-binding sites confer an advantage to CNX over other molecular chaperones that bind solely through polypeptide-based interactions, e.g. either by increasing the avidity of CNX for glycoprotein substrates or by increasing the number of potential substrates that are able to bind to CNX? is it possible to identify other molecular chaperones of the ER that are capable of replacing rabbit reticulocyte lysate (RRL) and collaborating with CNX in the refolding of denatured proteins?
soluble form of canine CNX (S-CNX) and BiP Are Effective in Suppressing the Aggregation of a Non-glycosylated Protein—We recently demonstrated that the soluble ER luminal domain of CNX (S-CNX)

Summary

Introduction

Calnexin (CNX)1 is a type I membrane protein of the endoplasmic reticulum (ER) that interacts transiently with newly synthesized Asn-linked glycoproteins. Is capable of suppressing the aggregation of non-glycosylated proteins in vitro, suggesting the presence of a polypeptidebinding site that allows S-CNX to function as a bona fide molecular chaperone in addition to its functions as a lectin [15].

Results

Conclusion