Excessive nitric oxide (NO) production in cytokine-activated β cells has been implicated in β cell disruption in type 1 diabetes. β cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 β cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor, S -nitroso- N -acetyl- d,l -penicillamine (SNAP). SNAP increased cytosolic Ca ²⁺ , and only agents depleting ER Ca ²⁺ induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca ²⁺ . Overexpression of calreticulin increased the Ca ²⁺ content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca ²⁺ , causes ER stress, and leads to apoptosis. Thus, ER Ca ²⁺ stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated β cell apoptosis.

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing α-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I α-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded α1-antitrypsin variant (null (Hong Kong)) and of TCRα. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded α1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent α1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of α1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has α1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a coimmunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong α₁-antitrypsin is enhanced by overexpression of the ER processing α1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded α₁-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415–422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded α₁-antitrypsin. We also show that misfolded α₁-antitrypsin NHK contains labeled Glc₁Man₉GlcNAc and Man_5–9GlcNAc released by endo-β-N-acetylglucosaminidase H in pulse-chase experiments with [2-³H]mannose. Overexpression of ER ManI greatly increases the formation of Man₈GlcNAc, induces the formation of Glc₁Man₈GlcNAc and increases trimming to Man_5–7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man₈GlcNAc₂ isomer B as previous studies have suggested.