The transient association of ERp57 with N‐glycosylated proteins is regulated by glucose trimming

Abstract

The thiol-dependent reductase ERp57 has been shown to interact specifically with in vitro synthesised glycoproteins imported into canine pancreatic microsomes. On this basis, it was proposed that ERp57 forms part of a glycoprotein-specific folding 'machinery', present in the lumen of the endoplasmic reticulum (ER). In this study, we have investigated the interaction of ERp57 with newly synthesised proteins using semi-permeabilised mammalian cells (SP cells), in which the ER remains essentially intact and, hence, resembles that of a living cell. We demonstrate that ERp57 interacts preferentially with the glycosylated versions of soluble and membrane proteins, and that this interaction occurs in combination with calnexin and calreticulin. For the first time, we have performed a detailed analysis of the kinetics of ERp57 binding to newly synthesised glycoproteins. We find that ERp57 associates transiently with glycoproteins Ϫ a characteristic of molecular chaperones. Using mutant SP cells deficient in glucosidase I, we confirm that the binding of ERp57 to glycoproteins depends upon glucose trimming. We also demonstrate, for the first time, that the release of ERp57 from glycoprotein substrates is dependent upon glucose trimming. These data are combined to present a unified model for the role of ERp57/ER lectin complexes during glycoprotein folding in vivo.Keywords : endoplasmic reticulum ; ERp57/GRP58 ; molecular chaperone ; N-linked glycosylation; protein disulphide isomerase.The endoplasmic reticulum (ER) membrane is a primary site of protein synthesis in mammalian cells. Newly synthesised proteins are inserted into, or translocated across, the ER membrane, from where they can reach their final destination in one of the organelles of the secretory pathway, the plasma membrane or the cell exterior.To facilitate protein maturation, the ER is equipped with a number of membrane and soluble proteins involved in the processing, modification, folding and oligomeric assembly of newly synthesised polypeptides. ER proteins involved in protein maturation include folding enzymes, such as peptidylprolyl cisϪtrans isomerase and members of the protein disulphide isomerase (PDI) family, as well as molecular chaperones of the Hsp70 and Hsp90 class heat-shock proteins, such as immunoglobulin heavy-chain-binding protein (BiP/GRP78) and GRP94 [1].In addition to these folding enzymes and chaperones, the ER contains two proteins with lectin-like properties, namely the type-I membrane protein calnexin, and its soluble homologue, calreticulin. Calnexin and calreticulin associate transiently with a wide variety of N-glycosylated proteins, and were proposed to influence folding, oligomerisation and ER retention. Together with several enzymes that modify N-linked oligosaccharides, calnexin and calreticulin constitute an ER quality-control machinery for glycoproteins [2].Calnexin and calreticulin display a specificity for monoglucosylated oligosaccharides [2Ϫ4] that are generated in the ER by the combined actions of two A-glucosidases and a...

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

“…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].…”

“…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].…”

“…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].…”

The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

“…In this model, CNX and CRT do not function as classical molecular chaperones that prevent aggregation by binding to hydrophobic polypeptide segments. Rather, they are thought to promote folding by recruiting other chaperones and folding enzymes, such as the thiol oxidoreductase ERp57 (7,8), to the glycoprotein substrate.The concept that CNX and CRT associate with glycoproteins solely through lectin-oligosaccharide interactions is based primarily on experiments wherein cultured cells were treated with tunicamycin to block Asn-linked oligosaccharide addition or with the glucosidase I and II inhibitors castanospermine or deoxynojirimycin to prevent the conversion of the Glc 3 Man 9 GlcNAc 2 precursor to the monoglucosylated Glc 1 Man 9 GlcNAc 2 species. Subsequent immunoprecipitation with anti-CNX or anti-CRT antibodies frequently revealed a dramatic reduction in the amounts of various glycoproteins co-isolating as complexes with these chaperones (2, 9 -14).…”

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.…”