A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.

Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices.

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo.

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

Post-translational Modifications of α-Synuclein, their Therapeutic Potential and Crosstalk in Health and Neurodegenerative Diseases.

α-Synuclein (α-Syn) aggregation in Lewy bodies and Lewy neurites has emerged as a key pathogenetic feature in Parkinson's disease (PD), Dementia with Lewy Bodies and Multiple System Atrophy. Various factors, including post-translational modifications (PTMs), can influence the propensity of α-Syn to misfold and aggregate. PTMs are biochemical modifications of a protein that occur during or after translation and are typically mediated by enzymes. PTMs modulate several characteristics of proteins including their structure, activity, localization, and stability. α-Syn undergoes various post-translational modifications, including phosphorylation, ubiquitination, SUMOylation, acetylation, glycation, O-GlcNAcylation, nitration, oxidation, polyamination, arginylation, and truncation. Different PTMs of a protein can physically interact with one another or work together to influence a particular physiological or pathological feature in a process known as PTMs crosstalk. The development of detection techniques for the co-occurrence of PTMs in recent years has uncovered previously unappreciated mechanisms of their crosstalk. This has led to the emergence of evidence supporting an association between α-Syn PTMs crosstalk and synucleinopathies. In this review, we provide a comprehensive evaluation of α-Syn PTMs, their impact on misfolding and pathogenicity, the pharmacological means of targeting them, and their potential as biomarkers of disease. We also highlight the importance of the crosstalk between these PTMs in α-Syn function and aggregation. Insight into these PTMS and the complexities of their crosstalk can improve our understanding of the pathogenesis of synucleinopathies and identify novel targets of therapeutic potential. Significance Statement α-Synuclein as a key pathogenic protein in Parkinson's disease and other synucleinopathies, making it a leading therapeutic target for disease modification. Multiple post-translational modifications occur at various sites in α-Synuclein and alter its biophysical and pathological properties, some interacting with one another to add to the complexity of the pathogenicity of this protein. This review details these modifications, their implications in disease and potential therapeutic opportunities.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

The FAM114A proteins are adaptors for the recycling of Golgi enzymes.

Golgi resident enzymes remain in place whilst their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. We used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

N-Glycosylation Profiles of Immunoglobulin G and Future Cardiovascular Events.

BACKGROUND: Posttranslational glycosylation of IgG can modulate its inflammatory capacity through structural variations. We examined the association of baseline IgG N-glycans and an IgG glycan score with incident cardiovascular disease (CVD). METHODS: IgG N-glycans were measured in 2 nested CVD case-control studies: JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin; NCT00239681; primary prevention; discovery; Npairs=162); and TNT trial (Treating to New Targets; NCT00327691; secondary prevention; validation; Npairs=397). Using conditional logistic regression, we investigated the association of future CVD with baseline IgG N-glycans and a glycan score adjusting for clinical risk factors (statin treatment, age, sex, race, lipids, hypertension, and smoking) in JUPITER. Significant associations were validated in TNT, using a similar model further adjusted for diabetes. Using least absolute shrinkage and selection operator regression, an IgG glycan score was derived in JUPITER as a linear combination of selected IgG N-glycans. RESULTS: Six IgG N-glycans were associated with CVD in both studies: an agalactosylated glycan (IgG-GP4) was positively associated, while 3 digalactosylated glycans (IgG glycan peaks 12, 13, 14) and 2 monosialylated glycans (IgG glycan peaks 18, 20) were negatively associated with CVD after multiple testing correction (overall false discovery rate <0.05). Four selected IgG N-glycans comprised the IgG glycan score, which was associated with CVD in JUPITER (adjusted hazard ratio per glycan score SD, 2.08 [95% CI, 1.52-2.84]) and validated in TNT (adjusted hazard ratio per SD, 1.20 [95% CI, 1.03-1.39]). The area under the curve changed from 0.693 for the model without the score to 0.728 with the score in JUPITER (PLRT=1.1×10-6) and from 0.635 to 0.637 in TNT (PLRT=0.017). CONCLUSIONS: An IgG N-glycan profile was associated with incident CVD in 2 populations (primary and secondary prevention), involving an agalactosylated glycan associated with increased risk of CVD, while several digalactosylated and sialylated IgG glycans associated with decreased risk. An IgG glycan score was positively associated with future CVD.

Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

Systematic Investigation of the Trafficking of Glycoproteins on the Cell Surface.

Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.

Multiplexed Antibody Glycosylation Profiling Using Dual Enzyme Digestion and Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method.

Antibody glycosylation plays a crucial role in the humoral immune response by regulating effector functions and influencing the binding affinity to immune cell receptors. Previous studies have focused mainly on the immunoglobulin G (IgG) isotype owing to the analytical challenges associated with other isotypes. Thus, the development of a sensitive and accurate analytical platform is necessary to characterize antibody glycosylation across multiple isotypes. In this study, we have developed an analytical workflow using antibody-light-chain affinity beads to purify IgG, IgA, and IgM from 16 µL of human plasma. Dual enzymes, trypsin and Glu-C, were used during on-bead digestion to obtain enzymatic glycopeptides and protein-specific surrogate peptides. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used in order to determine the sensitivity and specificity. Our platform targets 95 glycopeptides across the IgG, IgA, and IgM isotypes, as well as eight surrogate peptides representing total IgG, four IgG classes, two IgA classes, and IgM. Four stable isotope-labeled internal standards were added after antibody purification to calibrate the preparation and instrumental bias during analysis. Calibration curves constructed using serially diluted plasma samples showed good curve fitting (R2 > 0.959). The intrabatch and interbatch precision for all the targets had relative standard deviation of less than 29.6%. This method was applied to 19 human plasma samples, and the glycosylation percentages were calculated, which were comparable to those reported in the literature. The developed method is sensitive and accurate for Ig glycosylation profiling. It can be used in clinical investigations, particularly for detailed humoral immune profiling.

ST3GAL1 promotes malignant phenotypes in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) has poor prognosis and elucidation of the molecular mechanisms underlying iCCA malignancy is of great significance. Glycosylation, an important post-translational modification, is closely associated with tumor progression. Altered glycosylation, including aberrant sialylation resulting from abnormal expression of sialyltransferases (STs) and neuraminidases (NEUs), is a significant feature of cancer cells. However, there is limited information on the roles of STs and NEUs in iCCA malignancy. Here, utilizing our proteogenomic resources from a cohort of 262 iCCA patients, we identified ST3GAL1 as a prognostically relevant molecule in iCCA. Moreover, overexpression of ST3GAL1 promoted proliferation, migration and invasion and inhibited apoptosis of iCCA cells in vitro. Through proteomic analyses, we identified the downstream pathway potentially regulated by ST3GAL1, which was the NF-κB signaling pathway and further demonstrated that this pathway was positively correlated with malignancy in iCCA cells. Notably, glycoproteomics showed that O-glycosylation was changed in iCCA cells with high ST3GAL1 expression. Importantly, the altered O-glycopeptides underscored the potential utility of O-glycosylation profiling as a discriminatory marker for iCCA cells with ST3GAL1 overexpression. Additionally, miR-320b was identified as a post-transcriptional regulator of ST3GAL1, capable of suppressing ST3GAL1 expression and then reducing the proliferation, migration and invasion abilities of iCCA cell lines. Taken together, these results suggest ST3GAL1 could serve as a promising therapeutic target for iCCA.

Genotype-dependent N-glycosylation and newly exposed O-glycosylation affect plasmin-induced cleavage of histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is an abundant plasma protein harboring at least three N-glycosylation sites. HRG integrates many biological processes, such as coagulation, antiangiogenic activity, and pathogen clearance. Importantly, HRG is known to exhibit five genetic variants with minor allele frequencies of more than 10%. Among them, Pro204Ser can induce a fourth N-glycosylation site (Asn202). Considerable efforts have been made to reveal the biological function of HRG, whereas data on HRG glycosylation are scarcer. To close this knowledge gap, we used C18-based LC-MS/MS to study the glycosylation characteristics of six HRG samples from different sources. We used endogenous HRG purified from human plasma and compared its glycosylation to that of the recombinant HRG produced in Chinese hamster ovary cells or human embryonic kidney 293 cells, targeting distinct genotypic isoforms. In endogenous plasma HRG, every N-glycosylation site was occupied predominantly with a sialylated diantennary complex-type glycan. In contrast, in the recombinant HRGs, all glycans showed different antennarities, sialylation, and core fucosylation, as well as the presence of oligomannose glycans, LacdiNAcs, and antennary fucosylation. Furthermore, we observed two previously unreported O-glycosylation sites in HRG on residues Thr273 and Thr274. These sites together showed more than 90% glycan occupancy in all HRG samples studied. To investigate the potential relevance of HRG glycosylation, we assessed the plasmin-induced cleavage of HRG under various conditions. These analyses revealed that the sialylation of the N- and O-glycans as well as the genotype-dependent N-glycosylation significantly influenced the kinetics and specificity of plasmin-induced cleavage of HRG.

LacdiNAc synthase B4GALNT3 has a unique PA14 domain and suppresses N-glycan capping.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcß1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N-glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galß1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These findings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

Regulation of intracellular activity of N-glycan branching enzymes in mammals.

Most proteins in the secretory pathway are glycosylated, and N-glycans are estimated to be attached to over 7000 proteins in humans. As structural variation of N-glycans critically regulates the functions of a particular glycoprotein, it is pivotal to understand how structural diversity of N-glycans is generated in cells. One of the major factors conferring structural variation of N-glycans is the variable number of N-acetylglucosamine branches. These branch structures are biosynthesized by dedicated glycosyltransferases, including GnT-III (MGAT3), GnT-IVa (MGAT4A), GnT-IVb (MGAT4B), GnT-V (MGAT5), and GnT-IX (GnT-Vb, MGAT5B). In addition, the presence or absence of core modification of N-glycans, namely, core fucose (included as an N-glycan branch in this manuscript), synthesized by FUT8, also confers large structural variation on N-glycans, thereby crucially regulating many protein-protein interactions. Numerous biochemical and medical studies have revealed that these branch structures are involved in a wide range of physiological and pathological processes. However, the mechanisms regulating the activity of the biosynthetic glycosyltransferases are yet to be fully elucidated. In this review, we summarize the previous findings and recent updates regarding regulation of the activity of these N-glycan branching enzymes. We hope that such information will help readers to develop a comprehensive overview of the complex system regulating mammalian N-glycan maturation.

Complex N-glycosylation of mGluR6 is required for trans-synaptic interaction with ELFN adhesion proteins.

Synaptic transmission from retinal photoreceptors to downstream ON-type bipolar cells (BCs) depends on the postsynaptic metabotropic glutamate receptor mGluR6, located at the BC dendritic tips. Glutamate binding to mGluR6 initiates G-protein signaling that ultimately leads to BC depolarization in response to light. The mGluR6 receptor also engages in trans-synaptic interactions with presynaptic ELFN adhesion proteins. The roles of post-translational modifications in mGluR6 trafficking and function are unknown. Treatment with glycosidase enzymes PNGase F and Endo H demonstrated that both endogenous and heterologously expressed mGluR6 contain complex N-glycosylation acquired in the Golgi. Pull-down experiments with ELFN1 and ELFN2 extracellular domains revealed that these proteins interact exclusively with the complex glycosylated form of mGluR6. Mutation of the four predicted N-glycosylation sites, either singly or in combination, revealed that all four sites are glycosylated. Single mutations partially reduced, but did not abolish, surface expression in heterologous cells, while triple mutants had little or no surface expression, indicating that no single glycosylation site is necessary or sufficient for plasma membrane trafficking. Mutation at N445 severely impaired both ELFN1 and ELFN2 binding. All single mutants exhibited dendritic tip enrichment in rod BCs, as did the triple mutant with N445 as the sole N-glycosylation site, demonstrating that glycosylation at N445 is sufficient but not necessary for dendritic tip localization. The quadruple mutant was completely mislocalized. These results reveal a key role for complex N-glycosylation in regulating mGluR6 trafficking and ELFN binding, and by extension, function of the photoreceptor synapses.

The IgG-specific endoglycosidases EndoS and EndoS2 are distinguished by conformation and antibody recognition.

The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation.

Insights into molecular and cellular functions of the Golgi calcium/manganese-proton antiporter TMEM165.

The Golgi compartment performs a number of crucial roles in the cell. However, the exact molecular mechanisms underlying these actions are not fully defined. Pathogenic mutations in genes encoding Golgi proteins may serve as an important source for expanding our knowledge. For instance, mutations in the gene encoding Transmembrane protein 165 (TMEM165) were discovered as a cause of a new type of congenital disorder of glycosylation (CDG). Comprehensive studies of TMEM165 in different model systems, including mammals, yeast, and fish uncovered the new realm of Mn2+ homeostasis regulation. TMEM165 was shown to act as a Ca2+/Mn2+:H+ antiporter in the medial- and trans-Golgi network, pumping the metal ions into the Golgi lumen and protons outside. Disruption of TMEM165 antiporter activity results in defects in N- and O-glycosylation of proteins and glycosylation of lipids. Impaired glycosylation of TMEM165-CDG arises from a lack of Mn2+ within the Golgi. Nevertheless, Mn2+ insufficiency in the Golgi is compensated by the activity of the ATPase SERCA2. TMEM165 turnover has also been found to be regulated by Mn2+ cytosolic concentration. Besides causing CDG, recent investigations have demonstrated the functional involvement of TMEM165 in several other pathologies including cancer and mental health disorders. This systematic review summarizes the available information on TMEM165 molecular structure, cellular function, and its roles in health and disease.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake.

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.