EDEM 1 redox sensitive binding and catalytic activity 1 EDEM 1 ’ s mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity

Endoplasmic reticulum (ER) degradationenhancing α-mannosidase-like 1 protein (EDEM1) is a protein quality control factor that was initially proposed to recognize N-linked glycans on misfolded proteins through its mannosidase-like domain (MLD). However, recent studies have demonstrated that EDEM1 binds to some misfolded proteins in a glycan-independent manner, suggesting a more complex binding landscape for EDEM1. In this study, we have identified a thiol-dependent substrate interaction between EDEM1 and the α-1 antitrypsin ERassociated protein degradation (ERAD) clients, Z and NHK, specifically though the single Cys residue on Z/NHK (Cys256), required for binding under stringent detergent conditions. In addition to the thiol-dependent interaction, the presence of weaker protein-protein interactions was confirmed, suggestive of bipartite client binding properties. About four reactive thiols on EDEM1 were identified and were not directly responsible for the observed redox-sensitive binding by EDEM1. Moreover, a protein construct comprising the EDEM1 MLD had thiol-dependent binding properties along with its active glycantrimming activities. Lastly, we identified an additional intrinsically disordered region (IDR) located at the C-terminus of EDEM1 was identified in addition to its previously identified N-terminal IDR. We also determined that both IDRs are required for binding to the ERAD component ERdj5, as an interaction with ERdj5 was not observed with the MLD alone. Together, our findings indicate that EDEM1 employs different binding modalities to interact with ERAD clients and ER quality control (ERQC) machinery partners and that some of these properties are shared with its homologues EDEM2 and EDEM3. _______________________________________________________ In the ER lumen, nascent polypeptides gain access to an array of molecular chaperones and foldases such as BiP, lectin molecular chaperones such as calnexin (CNX) and calreticulin (CRT), and oxidoreductases like PDI and ERp57, all of which comprise part of the ERQC machinery (1– 4). The ERQC attempts to fold and rescue aberrantly folded proteins by protecting exposed hydrophobic regions (BiP), binding to N-linked glycans (CNX and CRT), and catalyzing the http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.004183 The latest version is at JBC Papers in Press. Published on July 18, 2018 as Manuscript RA118.004183 by gest on A uust 2, 2018 hp://w w w .jb.org/ D ow nladed from EDEM1 redox sensitive binding and catalytic activity 2 formation of the correct disulfide bonds (PDI, ERp57 etc.). Terminally misfolded proteins are extracted from futile folding cycles, prevented from reengaging the ERQC machinery and ultimately targeted for degradation via the ER-Associated Degradation (ERAD) pathway (5, 6). Numerous ERAD components have previously been characterized that recognize exposed hydrophobic regions, mispaired disulfide bonds, or specific Nlinked glycan structures. For instance, mannosidases contribute to the extensive glycan trimming that occurs on ERAD-bound clients, and oxidoreductases are proposed to facilitate retrotranslocation by unfolding and reducing ERAD clients to make them translocation competent (7–9). The ER Degradation Enhancing MannosidaseLike protein-1 (EDEM1) has been proposed to select and sequester terminally misfolded proteins away from productive folding cycles and target them for degradation. Evidence for these roles include preferential interaction with misfolded ERAD clients, direct interaction with ERAD machinery components such as Sel1L and ERdj5, and acceleration of the degradation of misfolded clients when overexpressed (10–14). Although EDEM1 and its role in ERAD has been extensively studied and characterized since its discovery in 2001 (11, 13), much of the protein’s properties remain enigmatic. For instance, early EDEM1 studies suggested that EDEM1 lacked mannosidase activity although it shares a mannosidase-like domain similar to that of the active ER mannosidase, Man1b1 (ER ManI) (11). However, several groups have demonstrated contradictory cellular findings showing EDEM1 has weak mannosidase activity (15–17). Additionally, attempts to ascertain on which glycan branch EDEM1 acts have been conducted by determining the glycan profile of cells in which EDEM1 was overexpressed or knocked out, but in vitro analyses using recombinant EDEM1 or its MLD are lacking, likely due to difficulty in expressing recombinant EDEM1 and its MLD (17, 18). EDEM1 has been implicated to play a role in ERAD and function in the ER lumen; however, how the protein is retained in the ER lumen remains undetermined. EDEM1 exhibits dual topology as a result of inefficient cleavage of its signal peptide, which yields a membrane-bound protein that is able to act on membrane-associated ERAD clients (19). However, EDEM1 also exists as a soluble luminal protein that remains in the ER while lacking a known ER retention/retrieval sequence. EDEM1 interacts with numerous ERlocalized binding partners and is proposed to exist in a multiprotein complex; which may mediate its ER retention, although this has yet to be shown. The clientor machinery-binding properties of EDEM1 appear multifaceted and specific to each binding-partner. For instance, the Lederkremer group showed that N-terminal truncation of EDEM1 possessing a portion of the MLD is sufficient for binding to H2a (20). Petrescu and colleagues identified an N-terminal IDR that is required for interaction with a unnatural soluble form of tyrosinase, further supporting the nonessential role of the MLD in the interaction between EDEM1 and H2a or soluble tyrosinase (21). Likewise, the role of glycans in EDEM1 client binding is perplexing and appears substratespecific, as in some cases (NHK, H2a, and SHH) interactions with EDEM1 were independent of substrate glycosylation; however, in other cases glycans appeared required (BACE457) (10, 12, 20, 22). Interactions between EDEM1 and ER machinery binding partners have been reported and characterized, most notably being that the EDEM1 MLD was involved in the interaction with Sel1L, as mutating the putative catalytic triad or using mannosidase inhibitors abolished this interaction (10, 23). Combined, these results illustrate a much more complex binding landscape of EDEM1 that involves distinct protein-dependent modes of interactions. In this study, we have identified bimodal interactions between EDEM1 and the α1antitrypsin (A1AT) disease-associated and ERAD variants Z and NHK. The interactions between EDEM1 and Z/NHK involve covalent and weaker protein-protein interactions that were identified under different detergent conditions. We also provided evidence that the MLD of EDEM1 possesses glycosidase activity. Altogether, these findings broaden the scope of the EDEM substrate-binding properties and provide further evidence supporting cellular mannosidase activity of the EDEM1 MLD. RESULTS by gest on A uust 2, 2018 hp://w w w .jb.org/ D ow nladed from EDEM1 redox sensitive binding and catalytic activity 3 The binding of EDEM1 to misfolded A1AT is bipartite and involves oxidation-dependent and weak protein-protein interactions EDEM1 preferentially interacts with misfolded A1AT variants NHK and Z in a glycanindependent manner (10). Given the oxidative nature of the ER lumen, we determined whether this preferential interaction is oxidationdependent. A pulse-chase experiment was performed to compare the protein interactions in the absence or presence of the reducing agent dithiothreitol (DTT) (Fig. 1A). EDEM1-FLAG was co-expressed in HEK293T cells with wild type (WT) A1AT, Z or NHK. The proteins were radiolabeled for 30 min, followed by a 15-min chase, to allow EDEM1 to reach its mature folded state. A 30-min chase with or without 5 mM DTT was performed (+/lanes) following the initial 15 min chase (Fig. 1A). Cells were lysed with 0.5% Triton X-100 in MNT buffer (TX) or with 2% 3[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS or CH) in HBS. Cell lysates were divided into equal fractions and subjected to anti-A1AT (Fig. 1A, lanes 1-12) and anti-FLAG (Fig. 1A, lanes 13-24) immunoprecipitations. Immunoprecipitations obtained from “TX” lysates were washed under stringent buffer conditions containing 0.1% SDS while those obtained from “CH” lysates were washed under milder conditions using 0.5% CHAPS. The DTT treatment did not affect the maturation or secretion of WT A1AT as the protein appears to have reached its mature state as indicated by the presence of complex sugars acquired in the Golgi as demonstrated by the higher molecular weight species (Fig. 1A, lanes 14). Additionally, the DTT treatment did not appear to affect the inherent binding properties of EDEM1 as it did not bind to WT A1AT regardless of the of the addition of DTT using either TX/SDS or CHAPS washes (Fig. 1A, lanes 1-4 and 13-16). Under mild detergent conditions (CHAPS), the interaction between EDEM1 and NHK (Fig. 1A, lanes 5, 6, 17, and 18) or Z (Fig. 1A, lanes 9, 10, 21, and 22) was maintained regardless of the whether DTT was added. However, under stringent detergent conditions (TX/SDS), the interaction between EDEM1 and NHK (Fig. 1A, lanes 7, 8, 19, and 20) or Z (Fig. 1A, lanes 11, 12, 23, and 24) was disrupted upon addition of DTT indicating the presence of an oxidation requirement, as well as the involvement of weaker protein-protein interactions. WT A1AT and the misfolded variants NHK and Z possess a single cysteine (Cys or C) residue at position 256 and three N-linked glycosylation sites (Fig. 1B). We have previously determined that the interaction between EDEM1 and NHK is glycan-independent under stringent detergent conditions (10). It was next determined if the interaction between A1AT and EDEM1 involved A1AT Cys256. Cys256Ser mutants of Z and NHK were generated and tested for their binding to EDEM1 (Fig. 1C). EDEM1-FLAG was co-expressed in HEK293T cells with WT A1AT, Z, Z C256S, NHK, or NHK C256S.