Enhanced Catalysis of Ribonuclease B Folding by the Interaction of Calnexin or Calreticulin with ERp57

Abstract

The endoplasmic reticulum is the site of folding, disulfide bond formation, and N-glycosylation of secretory proteins. Correctly folded proteins are exported from the endoplasmic reticulum, whereas incorrectly folded proteins are retained by a quality control system. The type I membrane-protein calnexin and its soluble homologue calreticulin are constituents of this system that recognize monoglucosylated N-linked glycans that are present on unfolded glycoproteins. Although several components of the quality control apparatus are well characterized, it is not known whether and how they interact with enzymes that catalyze protein folding. The endoplasmic reticulum protein ERp57 is homologous to protein-disulfide isomerase and can be cross-linked to the same monoglucosylated glycoproteins that bind to calnexin and calreticulin. The present study demonstrates that the disulfide isomerase activity of ERp57 on the refolding of monoglucosylated ribonuclease B is much greater when this glycoprotein is associated with calnexin or calreticulin. This result is in contrast to protein-disulfide isomerase, whose activity on monoglucosylated ribonuclease B is decreased in the presence of these lectins. No direct binding of monoglucosylated ribonuclease B or monoglucosylated glycans to ERp57 could be detected, but we show that ERp57 interacts directly with calnexin.

The protein disulphide-isomerase family: unravelling a string of folds

“…Ferrari and H.-D. So$ ling, unpublished work), and although ERp72, ERp57, PDIp and ERp28 have been reported to interact with peptides and\or malfolded proteins [10,[102][103][104][105][106][107][108], only for PDIp has the interaction been indicated to be direct [108].…”

“…This is in contrast with PDI, which interacts with proteins independently of their glycosylation status [65,104,107]. Furthermore, it is interesting that this calnexin-or calreticulin-mediated interaction greatly increases the disulphide-isomerase activity of ERp57 on a monoglucosylated protein, whereas the same type of interaction has been indicated to reduce PDI activity and peptide binding [100,106,149]. ERp57 exhibits a lower redox activity than PDI, as seen in a glutathione-insulin transhydrogenase assay [150,151], and cannot substitute for PDI in P4H [116].…”

“…Redox-active-site [60,144] pendent manner [104,105], but probably only indirectly via interactions with calnexin or calsequestrin [106,107], as ERp57 itself lacks lectin-like properties [106]. This is in contrast with PDI, which interacts with proteins independently of their glycosylation status [65,104,107].…”

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).…”

Transglutaminase Activity of Human ER-60

“…[9][10][11][12][13] In addition, ER-60 homologues of nematodes ( Diroˆlaria immitis and Caenorhabditis elegans), and bovine and giardial PDIs have been shown to have transglutaminase (TGase; EC 2.3.2.13) activity. [14][15][16] The TGases comprise a family of enzymes that catalyze the formation of isopeptide bonds between the g-carboxamide groups of peptidebound glutamine residues and the e-amino groups of lysine residues.…”