Calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes.

Abstract

Calnexin (CNX) and calreticulin (CRT) are molecular chaperones that bind preferentially to monoglucosylated trimming intermediates of glycoproteins in the endoplasmic reticulum. To determine their role in the maturation of newly synthesized glycoproteins, we analyzed the folding and trimerization of in vitro translated influenza hemagglutinin (HA) in canine pancreas microsomes under conditions in which HA's interactions with CNX and CRT could be manipulated. While CNX bound to all folding intermediates (IT1, IT2 and NT), CRT was found to associate preferentially with the earliest oxidative form (IT1). If HA's binding to CNX and CRT was inhibited using a glucosidase inhibitor, castanospermine (CST), the rate of disulfide formation and oligomerization was doubled but the overall efficiency of maturation of HA decreased due to aggregation and degradation. If, on the other hand, HA was arrested in CNX‐CRT complexes, folding and trimerization were inhibited. This suggested that the action of CNX and CRT, like that of other chaperones, depended on an ‘on‐and‐off’ cycle. Taken together, these results indicated that CNX and CRT promote correct folding by inhibiting aggregation, preventing premature oxidation and oligomerization, and by suppressing degradation of incompletely folded glycopolypeptides.

Pathophysiological Roles of Calreticulin in Autoimmune Disease

“…One of the major functions of calreticulin within the ER is to act, along with calnexin, as a quality controller of glycoprotein formation [67]. Calreticulin appears to preferentially bind to monoglucosylated trimming intermediates of glycoproteins, suggesting that it associates with newly synthesized viral proteins in infected cells.…”

“…Calreticulin appears to preferentially bind to monoglucosylated trimming intermediates of glycoproteins, suggesting that it associates with newly synthesized viral proteins in infected cells. For example, calreticulin binds transiently to the HIV envelope glycoprotein, gp160 [68] and aids in the correct folding of influenza haemagglutinin [67]. The role of calreticulin in viral glycoprotein maturation and expression in host cells might be significant in autoimmune disease.…”

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells

“…Inhibition of CRT association with MUC2 by TUN therefore suggests that Nglycans on the mucin are necessary for the chaperone-mucin interaction. This is not surprising because CRT binding to a number of proteins is dependent on the presence of monoglucosylated N-oligosaccharides generated in the ER after trimming of the core glycan by glucosidases I and II [22,24]. Accordingly, we attempted to identify the stage of glycosylation processing of MUC2 that was required for CRT interaction.…”

“…These observations imply that, in the presence of CAS, immature MUC2 was not being processed correctly and remained bound to CRT in the ER. In the above experiments, the LS180 cells were treated with 1 mM CAS, a concentration that is commonly used in chaperone studies [22,24,34]. However, since 1 mM CAS strongly impaired MUC2 processing and secretion, the experiments were repeated after lowering the CAS concentration from 1 to 0.56 mM, the minimum level required to inhibit glucosidase I almost completely, or after decreasing the preincubation time with CAS from 45 to 10 min.…”

“…CLN and CRT function as chaperones in a similar manner, although CLN is a type 1 membrane protein, whereas CRT is soluble, is present in the ER lumen, and contains a Cterminal KDEL (Lys-Asp-Glu-Leu) ER retrieval signal [21]. However, CRT shares extensive sequence identity with the luminal domain of CLN [21], and both proteins are unique as chaperones, since they preferentially behave as lectins, interacting specifically with partially trimmed monoglucosylated N-linked oligosaccharides on newly synthesized protein molecules [22][23][24]. They recognize glycan chains of the form Glc " Man * GlcNAc # and bind with similar affinities [25].…”