EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.

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.

Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.