Malectin participates in a backup glycoprotein quality control pathway in the mammalian ER.

Malectin is a conserved, endoplasmic reticulum (ER)-resident lectin that recognizes high mannose oligosaccharides displaying terminal glucose residues. Here we show that Malectin is an ER stress-induced protein that selectively associates with glycopolypeptides without affecting their entry and their retention in the Calnexin chaperone system. Analysis of the obligate Calnexin client influenza virus hemagglutinin (HA) revealed that Calnexin and Malectin associated with different timing to different HA conformers and that Malectin associated with misfolded HA. Analysis of the facultative Calnexin clients NHK and α1-antitrypsin (α1AT) revealed that induction of Malectin expression to simulate conditions of ER stress resulted in persistent association between the ER lectin and the model cargo glycoproteins, interfered with processing of cargo-linked oligosaccharides and reduced cargo secretion. We propose that Malectin intervention is activated upon ER stress to inhibit secretion of defective gene products that might be generated under conditions of aberrant functioning of the ER quality control machinery.

Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates.

Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L(S) substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L(M) substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide.

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.

EDEM contributes to maintenance of protein folding efficiency and secretory capacity.

A stringent quality control process selects misfolded polypeptides generated in the endoplasmic reticulum (ER) for ER-associated degradation (ERAD). Here we assessed the maintenance of efficient glycoprotein folding in cells with defective ERAD caused by lack of adaptation of the intralumenal level of ER degradation-enhancing alpha-mannosidase-like protein (EDEM) to an increase in the ER cargo load. When these cells were converted into factories for production of high levels of human beta-secretase, maturation of this N-glycosylated aspartic protease progressed as in wild-type cells initially to gradually become less efficient. Up-regulation of EDEM to strengthen the ERAD machinery (but not up-regulation of calnexin to reinforce the folding machinery) was instrumental in maintaining folding efficiency and secretory capacity. Our data underscore the important role that the degradation machinery plays in maintaining a functional folding environment in the ER.

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.

Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle.

The mechanisms that determine how folding attempts are interrupted to target folding-incompetent proteins for endoplasmic reticulum-associated degradation (ERAD) are poorly defined. Here the alpha-mannosidase I-like protein EDEM was shown to extract misfolded glycoproteins, but not glycoproteins undergoing productive folding, from the calnexin cycle. EDEM overexpression resulted in faster release of folding-incompetent proteins from the calnexin cycle and earlier onset of degradation, whereas EDEM down-regulation prolonged folding attempts and delayed ERAD. Up-regulation of EDEM during ER stress may promote cell recovery by clearing the calnexin cycle and by accelerating ERAD of terminally misfolded polypeptides.