Introduction
The recent study published in Nature explores the structural mechanisms underlying the regulated N-glycosylation process at the secretory translocon, a crucial aspect of protein synthesis and processing in eukaryotic cells. N-glycosylation, a common post-translational modification, significantly influences protein stability, folding, and function. Approximately 20% of human proteins undergo this modification, and defects in the N-glycosylation pathway can lead to congenital disorders of glycosylation (CDG). The research identifies key components and interactions within the oligosaccharyltransferase (OST) complexes, specifically OST-A and OST-B, that modulate glycosylation at the endoplasmic reticulum (ER).
Regulatory Mechanisms of N-Glycosylation
The study highlights the roles of two OST complexes encoded by metazoan genomes: OST-A and OST-B. OST-A is positioned at the ER membrane, where it associates with ribosomes and the secretory translocon complex (SEC61 and TRAP). This localization enables OST-A to preferentially modify nascent polypeptide chains as they enter the ER lumen. In contrast, OST-B can modify sites that are not engaged by OST-A but does not associate with the ribosome-translocon complex.
Importantly, the presence of an acceptor sequon (Asn-X-Thr/Ser) does not guarantee modification, as various factors such as sequence context, folding kinetics, and cellular metabolic state influence glycosylation outcomes. These variations can lead to differential glycosylation patterns across different cells and tissues, with significant implications for protein function and stability.
Key Proteins and Their Interactions
The research identifies GRP94, a prominent ER chaperone, as a critical player in this regulatory network. While GRP94 is typically glycosylated at one site, under conditions of ER stress, additional glycosylation at facultative sites can occur, potentially leading to protein degradation via the ER-associated degradation (ERAD) pathway. The study also reveals that the proteins CCDC134 and FKBP11 play essential roles in regulating GRP94's glycosylation status during its synthesis.
Using genome-wide CRISPR-Cas9 screening, the researchers discovered that CCDC134 is crucial for suppressing hyperglycosylation of GRP94's facultative sites. Disruption of this pathway leads to GRP94 hyperglycosylation and degradation, subsequently affecting the maturation and trafficking of its client proteins, such as IGF1R and LRP6, which are integral to various signaling pathways.
Structural Insights from Cryo-EM
The study employs cryo-electron microscopy (cryo-EM) to elucidate the structure of the translocon-bound GRP94. The findings reveal how the nascent GRP94 interacts with OST-A and CCDC134, forming a stable complex that occludes the OST-A active site, thereby preventing inappropriate glycosylation. The pre-N segment of GRP94 acts as a pseudo-substrate inhibitor, blocking access to the acceptor site during translation.
Furthermore, the structural analysis demonstrates the dynamic nature of the translocon, emphasizing how different substrate proteins can influence its composition and function. The researchers propose that the interactions between nascent GRP94, OST-A, and CCDC134 create a lumenal vestibule that shields the downstream domains of GRP94 from glycosylation by OST-B, thereby facilitating proper protein folding.
Conclusion
This comprehensive study provides significant insights into the mechanisms regulating N-glycosylation at the secretory translocon, highlighting the critical roles of specific proteins and their interactions. The findings underscore the importance of precise regulation in protein synthesis and modification, which is essential for maintaining cellular function and preventing diseases linked to glycosylation defects. The research opens avenues for further exploration of how translocon dynamics and glycosylation processes are coordinated, potentially leading to novel therapeutic strategies for managing congenital disorders of glycosylation and related conditions.