EDEM2 and OS-9 Are Required for ER-Associated Degradation of Non-Glycosylated Sonic Hedgehog

Abstract

Misfolded proteins of the endoplasmic reticulum (ER) are eliminated by the ER-associated degradation (ERAD) in eukaryotes. In S. cerevisiae, ER-resident lectins mediate substrate recognition through bipartite signals consisting of an unfolded local structure and the adjacent glycan. Trimming of the glycan is essential for the directional delivery of the substrates. Whether a similar recognition and delivery mechanism exists in mammalian cells is unknown. In this study, we systematically study the function and substrate specificity of known mammalian ER lectins, including EDEM1/2/3, OS-9 and XTP-3B using the recently identified ERAD substrate sonic hedgehog (SHH), a soluble protein carrying a single N-glycan, as well as its nonglycosylated mutant N278A. Efficient ERAD of N278A requires the core processing complex of HRD1, SEL1L and p97, similar to the glycosylated SHH. While EDEM2 was required for ERAD of both glycosylated and non-glycosylated SHHs, EDEM3 was only necessary for glycosylated SHH and EDEM1 was dispensable for both. Degradation of SHH and N278A also required OS-9, but not the related lectin XTP3-B. Robust interaction of both EDEM2 and OS-9 with a non-glycosylated SHH variant indicates that the misfolded polypeptide backbone, rather than a glycan signature, functions as the predominant signal for recognition for ERAD. Notably, SHH-N278A is the first nonglycosylated substrate to require EDEM2 for recognition and targeting for ERAD. EDEM2 also interacts with calnexin and SEL1L, suggesting a potential avenue by which misfolded glycoproteins may be shunted towards SEL1L and ERAD rather than being released into the secretory pathway. Thus, ER lectins participate in the recognition and delivery of misfolded ER substrates differently in mammals, with an underlying mechanism distinct from that of S. cerevisiae.

EDEM1's mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity

“…with Z or NHK when the Cys was mutated to a Ser (Fig. 1C, lanes [21][22][23][24], suggesting that the previously observed covalentlike interaction between the misfolded A1AT ERAD variants and EDEM1 involved the single Cys 256 from A1AT.…”

“…Petrescu and co-workers (21) identified an N-terminal IDR that is required for interaction with an unnatural soluble form of tyrosinase, further supporting the nonessential role of the MLD in the interaction between EDEM1 and H2a or soluble tyrosinase. Likewise, the role of glycans in EDEM1 client binding is perplexing and appears substrate-specific 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 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).…”

Glycosylation-directed quality control of protein folding

“…For some substrates, EDEM1 enhances degradation of the glycosylated species but has no effect on the degradation of their non-glycosylated variants 125,126 . Other studies reported a requirement of EDEM1 for degradation of some non-glycosylated protein s as well 127,128 .…”

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

“…Concordantly, the knock-down of EDEM2 acting upstream of EDEM3 in mannose chain trimming [42] has no effect on the expression level of del52 and ins5 mutants. EDEM3, like other members of the EDEM family, has previously been shown to interact with nonglycosylated substrates [43]. However, its role in the degradation of proteins has been limited to glycosylated substrates thus far.…”