Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Abstract

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra-and intermolecular disulfide bonds, a ratelimiting step of protein folding in the endoplasmic reticulum (ER).Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexinassociated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.Polypeptide asparagines emerging in the ER 4 lumen as part of an Asn-X-Ser/Thr motif are covalently modified with preassembled glycans containing 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues (1). N-Glycosylation prepares nascent chains for association with the two lectin-like molecular chaperones calnexin and calreticulin and the glycoprotein-dedicated oxidoreductase ERp57 (2-5).Deletion of individual members of the calnexin chaperone system, namely calreticulin (6), UGT1 (7), and ERp57 (8), is embryonic lethal in mice. Deletion of calnexin does not result in embryonic lethality but causes a severe progressive pathology leading to premature death (9). The suboptimal glycoprotein folding efficiency upon chaperone deletion might be detrimental for organism viability, but all of these deletions are well tolerated at the cellular level. In fact, only a restricted number of endogenous, recombinant, or virus-encoded glycoproteins (e.g. the major histocompatibility complex class I peptide loading complex (8, 10), the influenza virus HA (11), and the ADAM1/ADAM2 fertilin complex (12)) have so far been shown to strongly depend on the calnexin chaperone system for maturation. Consistently, and as previously shown for calnexin, calreticulin, and UGT1 deletions, cells lacking ERp57 show normal viability and proliferation rates and unperturbed maturation and transport of several surface glycoproteins containing disulfide bonds (8). Conditional deletion of ERp57 in B lymphocytes/ plasma cells revealed no defect in the production of immunoglobulin chains, the most abundant N-glycosylated/disulfide-bonded products of these cells (8). The thorough analysis by Garbi et al. showed that ERp57 deletion specifically affected assembly and stability of the major histocompatibility class I p...

N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control

“…In the case of influenza virus hemagglutinin, which is one of the best-studied models of chaperone-assisted glycoprotein folding in the mammalian ER, GII/UGT1-driven cycles of release from and reassociation with CNX are required for efficient folding (M Molinari et al, unpublished). However, this protein appears to be one of the very few proteins that obligatorily depend on assistance from members of the CNX chaperone system for efficient maturation [8][9][10]. For the bulk of glycoproteins expressed in mammalian cells, deletion of UGT1 does not affect maturation ( [11 •• ]; T Soldà and M Molinari, unpublished).…”

Protein Folding in the Endoplasmic Reticulum

“…Calnexin, a type I membrane protein, and calreticulin, its soluble paralogue, both possess a singular globular carbohydrate-binding domain (Schrag et al 2001). Calnexin and calreticulin promote the efficient folding of glycoproteins by: (1) stabilizing folding events or slowing the folding process in a domain specific manner (Hebert et al 1996(Hebert et al , 1997Daniels et al 2003); (2) preventing aggregation and turnover (Hebert et al 1996;Vassilakos et al 1996); (3) retaining nonnative substrates in the ER to support additional attempts for proper folding (Rajagopalan et al 1994); (4) facilitating the formation of disulfide bond formation through their association with the oxidoreductase ERp57 (Oliver et al 1997;Zapun et al 1998;Solda et al 2006); and (5) perhaps facilitating Pro isomerization through association with the PPIase CypB (Kozlov et al 2010). More information on how oxidoreductases catalyze the formation of disulfide bonds can be found below (PDIs or oxidoreductases) and in Bulleid (2012).…”

N-glycan processing in ER quality control

“…Although most polypeptides mature normally in cells lacking calnexin or calreticulin, there are at least two relevant exceptions: influenza virus hemagglutinin (HA) exhibits substantial folding defects when expressed in cells lacking calnexin Pieren et al, 2005); and MHC class I molecules show loading with suboptimal peptides and premature release at the surface of calreticulin-deficient cells (Gao et al, 2002). Similarly, in ERp57-deleted cells, influenza HA (Soldà et al, 2006) and class I MHC molecules ) are the only model glycoproteins, among those analyzed so far, whose folding/export is significantly affected. Analyses of ERp57-deleted cells have led to the identification of at least one PDI family member, ERp72, which can act as a surrogate chaperone that catalyses intra-and inter-molecular disulfide bond formation when ERp57 is absent (Soldà et al, 2006).…”

“…However, the association of ERp57 with calnexin and calreticulin makes it the primary contender for the PDI that assists glycopolypeptide maturation Oliver et al, 1997;Zapun et al, 1998). Importantly, ERp57 is the first oxidoreductase for which knockout mammalian cell lines have been reported Soldà et al, 2006) (see below).…”

“…In recent years, murine fibroblasts lacking calreticulin (Mesaeli et al, 1999), calnexin (Denzel et al, 2002), UGT1 or ERp57 Soldà et al, 2006) have been reported. All of these knockouts result in embryonic lethality in mice, except for the calnexin knockout, which causes a severe and progressive pathology characterized by impaired growth and motor disorders that eventually lead to premature death (Denzel et al, 2002).…”