LMAN1

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using a selective hook (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

Protein-carbohydrate interactions play a crucial role in mediating several biomolecular recognition events. We attempt to unravel its intricacies by understanding how carbohydrate-binding proteins interpret the glycan code. We aim to decipher lectin-mediated recognition in the endoplasmic reticulum (ER), which plays a crucial role in ER-mediated quality control (ER-QC). The ER-QC functions in three phases-protein folding, transport, and degradation. Altered protein QC leads to ER-related storage disorders. Cargo transport proteins-Ergic53 and Vip36-necessary for maintaining cellular homeostasis-are our primary focus. They recognize monoglucosylated/high mannose N-glycans on the folded glycoproteins. This article reports on the first dynamic investigation of the ER cargo lectins in complex with the high mannose glycans using an advanced sampling technique-replica exchange molecular dynamics to decipher the inherent conformational heterogeneity and the binding mechanism. The study involves simulations for the proteins complexed with three high mannose glycans-Man8B, Man9, and mono-glucosylated glycan. The recognition process is captured using MD simulations to achieve mechanistic insights and characterize the dynamics of glycans in their native and bound states via dihedral angle analysis. Results indicate that the flipped conformation of the glycans was crucial in differentiating their interaction with the proteins. Similar conformers of the glycans are preferred for Ergic53 and Vip36 in their glycan recognition events. Ergic53 preferred Man8B while it was Man9 for Vip36, in coherence with the previous experimental reports. These simulations provide a computational microscopic purview of the mechanism at both spatial and temporal scales. The results correlate with the published experimental data on the specificities of these lectins.