Mapping the ER Interactome: The P Domains of Calnexin and Calreticulin as Plurivalent Adapters for Foldases and Chaperones.

The lectin chaperones calreticulin (CRT) and calnexin (CNX) contribute to the folding of glycoproteins in the ER by recruiting foldases such as the protein disulfide isomerase ERp57 and the peptidyl prolyl cis-trans isomerase CypB. Recently, CRT was shown to interact with the chaperone ERp29. Here, we show that ERp29 directly binds to the P domain of CNX. Crystal structures of the D domain of ERp29 in complex with the P domains from CRT and calmegin, a tissue-specific CNX homolog, reveal a commonality in the mechanism of binding whereby the tip of the P domain functions as a plurivalent adapter to bind a variety of folding factors. We show that mutation of a single residue, D348 in CNX, abrogates binding to ERp29 as well as ERp57 and CypB. The structural diversity of the accessory factors suggests that these chaperones became specialized for glycoprotein folding through convergent evolution of their P-domain binding sites.

Glycoprotein quality control-related proteins effectively inhibit fibrillation of amyloid beta 1-42.

Formation of amyloid beta (Aß) aggregates is a risk factor for Alzheimer's disease. Accumulation of Aß aggregates on the cell surface causes oxidative stress, and ultimately results in cell death. Consequently, inhibition of aggregate formation is predicted to be beneficial. Recently, translocation of glycoprotein quality control-related (GPQC) proteins such as chaperones and protein disulfide-isomerase (PDI) family members was reported under oxidative stress conditions. Therefore, it is possible that GPQC proteins contact Aß peptides on the cell membrane during stress conditions. Here, we examined the effect of ER resident proteins on Aß aggregation. Our results show that minimal expression of GPQC proteins enables Aß to effectively avoid aggregation. Moreover, further analyses show that Aß structure remains in the monomer state in the presence of ER proteins. Thus, our findings show that GPQC proteins have strong affinity for Aß monomers, and suggests that the interaction between them repeatedly associates and dissociates in a short period of time.

Glycan specificity of a testis-specific lectin chaperone calmegin and effects of hydrophobic interactions.

BACKGROUND: Testis-specific chaperone calmegin is required for the generation of normal spermatozoa. Calmegin is known to be a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin. Although functional similarity between calnexin and calmegin has been predicted, detailed information concerned with substrate recognition by calmegin, such as glycan specificity, chaperone function and binding affinity, are obscure. METHODS: In this study, biochemical properties of calmegin and calnexin were compared using synthetic glycans and glycosylated or non-glycosylated proteins as substrates. RESULTS: Whereas their amino acid sequences are quite similar to each other, a certain difference in secondary structures was indicated by circular dichroism (CD) spectrum. While both of them inhibited protein heat-aggregation to a similar extent, calnexin exhibited a higher ability to facilitate protein folding. Similarly to calnexin, calmegin preferentially recognizes monoglucosylated glycans such as Glc1Man9GlcNAc2 (G1M9). While the surface hydrophobicity of calmegin was higher than that of calnexin, calnexin showed stronger binding to substrate. We reasoned that lectin activity, in addition to hydrophobic interaction, contributes to this strong affinity between calnexin and substrate. CONCLUSIONS: Although their similarity in carbohydrate binding specificities is high, there seems to be some differences in the mode of substrate recognition between calmegin and calnexin. GENERAL SIGNIFICANCE: Properties of calmegin as a lectin-chaperone were revealed in comparison with calnexin.