Golgi Metal Ion Homeostasis in Human Health and Diseases

The Golgi apparatus is a membrane organelle located in the center of the protein processing and trafficking pathway. It consists of sub-compartments with distinct biochemical compositions and functions. Main functions of the Golgi, including membrane trafficking, protein glycosylation, and sorting, require a well-maintained stable microenvironment in the sub-compartments of the Golgi, along with metal ion homeostasis. Metal ions, such as Ca2+, Mn2+, Zn2+, and Cu2+, are important cofactors of many Golgi resident glycosylation enzymes. The homeostasis of metal ions in the secretory pathway, which is required for proper function and stress response of the Golgi, is tightly regulated and maintained by transporters. Mutations in the transporters cause human diseases. Here we provide a review specifically focusing on the transporters that maintain Golgi metal ion homeostasis under physiological conditions and their alterations in diseases.

Unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating Ethylene and Abscisic acid pathway

Submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates Galactosyltransferase (GALT9) and their expression is inversely affected at 24 hours of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild type and MIM775 Arabidopsis shoot. A similar recovery phenotype of galt9 mutant indicates the role of miR775-GALT9 module in post-submergence recovery. We predicted Golgi-localized GALT9 to be potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29, and ORE1), ethylene signalling (EIN2 and EIN3) and ABA biosynthesis (NCED3) pathway genes in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role of miR775-GALT9 module in post-submergence recovery through a crosstalk with ethylene and ABA pathway.

Mammalian brain glycoproteins exhibit diminished glycan complexity compared to other tissues

Glycosylation is essential to brain development and function, but prior studies have often been limited to a single analytical technique and excluded region- and sex-specific analyses. Here, using several methodologies, we analyze Asn-linked and Ser/Thr/Tyr-linked protein glycosylation between brain regions and sexes in mice. Brain N-glycans are less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose and fucosylated/bisected structures. Most brain O-glycans are unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern is observed between regions, and sex differences are minimal compared to those in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of glycosylation genes in humans show a global downregulation in the brain compared to other tissues. We hypothesize that this restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.

SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders

Introduction The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination. Objectives To assess (1) the effects of different Spikes on human primary MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation. Methods Human primary MΦ were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts. Results LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation. Conclusions Glycosylated Spike per se is not pro-inflammatory for human MΦ, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination. Graphical abstract

Characterization of the O-Glycoproteome of Porphyromonas gingivalis

Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability.

Synthesis of Dolichols in Candida albicans Is Co-Regulated with Elongation of Fatty Acids

Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.

Glycosylation-on-a-Chip: A Flow-Based Microfluidic System for Cell-Free Glycoprotein Biosynthesis

In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.

Elevated N-Glycosylation Contributes to the Cisplatin Resistance of Non-Small Cell Lung Cancer Cells Revealed by Membrane Proteomic and Glycoproteomic Analysis

Chemoresistance is the major restriction on the clinical use of cisplatin. Aberrant changes in protein glycosylation are closely associated with drug resistance. Comprehensive study on the role of protein glycosylation in the development of cisplatin resistance would contribute to precise elucidation of the complicated mechanism of resistance. However, comprehensive characterization of glycosylated proteins remains a big challenge. In this work, we integrated proteomic and N-glycoproteomic workflow to comprehensively characterize the cisplatin resistance-related membrane proteins. Using this method, we found that proteins implicated in cell adhesion, migration, response to drug, and signal transduction were significantly altered in both protein abundance and glycosylation level during the development of cisplatin resistance in the non-small cell lung cancer cell line. Accordingly, the ability of cell migration and invasion was markedly increased in cisplatin-resistant cells, hence intensifying their malignancy. In contrast, the intracellular cisplatin accumulation was significantly reduced in the resistant cells concomitant with the down-regulation of drug uptake channel protein, LRRC8A, and over-expression of drug efflux pump proteins, MRP1 and MRP4. Moreover, the global glycosylation was elevated in the cisplatin-resistant cells. Consequently, inhibition of N-glycosylation reduced cell resistance to cisplatin, whereas promoting the high-mannose or sialylated type of glycosylation enhanced the resistance, suggesting that critical glycosylation type contributes to cisplatin resistance. These results demonstrate the high efficiency of the integrated proteomic and N-glycoproteomic workflow in discovering drug resistance-related targets, and provide new insights into the mechanism of cisplatin resistance.