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1.
bioRxiv ; 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-39026824

ABSTRACT

N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher Mr MRS2 bands gel-shifted to lower Mr and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher Mr MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+ under conditions of glucose excess or mitochondrial bioenergetic impairment.

2.
Biochim Biophys Acta Gen Subj ; 1868(10): 130668, 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-38992482

ABSTRACT

Glycosylation of proteins and lipids is of fundamental importance in multicellular eukaryotes. The vast diversity of glycan structures observed is generated in the Golgi apparatus by the concerted activity of >100 distinct enzymes, which include glycosyltransferases and other glycan-modifying enzymes. Well-known for decades, the majority of these enzymes is released from the Golgi apparatus and subsequently secreted into the extracellular space following endoproteolytic cleavage, but the underlying molecular mechanisms and the physiological implications have remained unexplored. This review will summarize our current knowledge of Golgi enzyme proteolysis and secretion and will discuss its conceptual implications for the regulation of cellular glycosylation and the organization of the Golgi apparatus. A particular focus will lie on the intramembrane protease SPPL3, which recently emerged as key protease facilitating Golgi enzyme release and has since been shown to affect a multitude of glycosylation-dependent physiological processes.

3.
J Biol Chem ; : 107584, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39025454

ABSTRACT

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

4.
Article in English | MEDLINE | ID: mdl-39066577

ABSTRACT

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.

5.
Neurol Int ; 16(4): 790-803, 2024 Jul 22.
Article in English | MEDLINE | ID: mdl-39051219

ABSTRACT

Glucocorticoids are used during glioblastoma treatment to prevent the cerebral edema effect surrounding normal brain tissue. The aim of our study was to investigate the long-term effects of multiple administrations of glucocorticoids onto the glycosylated components (proteoglycans and glycosaminoglycans) of normal brain extracellular matrix and the glucocorticoid receptor (GR, Nr3c1) in an experimental model in vivo. Two-month-old male C57Bl/6 mice (n = 90) were injected intraperitoneally with various doses of dexamethasone (DXM) (1; 2.5 mg/kg) for 10 days. The mRNA levels of the GR, proteoglycans core proteins, and heparan sulfate metabolism-involved genes were determined at the 15th, 30th, 60th, and 90th days by a real-time RT-PCR. The glycosaminoglycans content was studied using dot blot and staining with Alcian blue. A DXM treatment increased total GAG content (2-fold), whereas the content of highly sulfated glycosaminoglycans decreased (1.5-2-fold). The mRNA level of the heparan sulfate metabolism-involved gene Hs3St2 increased 5-fold, the mRNA level of Hs6St2 increased6-7-fold, and the mRNA level of proteoglycan aggrecan increased 2-fold. A correlation analysis revealed an association between the mRNA level of the GR and the mRNA level of 8 of the 14 proteoglycans-coding and 4 of the 13 heparan sulfate metabolism-involved genes supporting GR involvement in the DXM regulation of the expression of these genes. In summary, multiple DXM administrations led to an increase in the total GAG content and reorganized the brain extracellular matrix in terms of its glycosylation pattern.

6.
bioRxiv ; 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38948797

ABSTRACT

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

7.
Comput Biol Med ; 179: 108859, 2024 Jul 18.
Article in English | MEDLINE | ID: mdl-39029431

ABSTRACT

O-linked glycosylation is a complex post-translational modification (PTM) in human proteins that plays a critical role in regulating various cellular metabolic and signaling pathways. In contrast to N-linked glycosylation, O-linked glycosylation lacks specific sequence features and maintains an unstable core structure. Identifying O-linked threonine glycosylation sites (OTGs) remains challenging, requiring extensive experimental tests. While bioinformatics tools have emerged for predicting OTGs, their reliance on limited conventional features and absence of well-defined feature selection strategies limit their effectiveness. To address these limitations, we introduced HOTGpred (Human O-linked Threonine Glycosylation predictor), employing a multi-stage feature selection process to identify the optimal feature set for accurately identifying OTGs. Initially, we assessed 25 different feature sets derived from various pretrained protein language model (PLM)-based embeddings and conventional feature descriptors using nine classifiers. Subsequently, we integrated the top five embeddings linearly and determined the most effective scoring function for ranking hybrid features, identifying the optimal feature set through a process of sequential forward search. Among the classifiers, the extreme gradient boosting (XGBT)-based model, using the optimal feature set (HOTGpred), achieved 92.03 % accuracy on the training dataset and 88.25 % on the balanced independent dataset. Notably, HOTGpred significantly outperformed the current state-of-the-art methods on both the balanced and imbalanced independent datasets, demonstrating its superior prediction capabilities. Additionally, SHapley Additive exPlanations (SHAP) and ablation analyses were conducted to identify the features contributing most significantly to HOTGpred. Finally, we developed an easy-to-navigate web server, accessible at https://balalab-skku.org/HOTGpred/, to support glycobiologists in their research on glycosylation structure and function.

8.
J Fungi (Basel) ; 10(7)2024 Jul 20.
Article in English | MEDLINE | ID: mdl-39057387

ABSTRACT

Candidemia is an opportunistic mycosis with high morbidity and mortality rates. Even though Candida albicans is the main causative agent, other Candida species, such as Candida tropicalis, are relevant etiological agents of candidiasis and candidemia. Compared with C. albicans, there is currently limited information about C. tropicalis' biological aspects, including those related to the cell wall and the interaction with the host. Currently, it is known that its cell wall contains O-linked mannans, and the contribution of these structures to cell fitness has previously been addressed using cells subjected to chemical treatments or in mutants where O-linked mannans and other wall components are affected. Here, we generated a C. tropicalis pmt2∆ null mutant, which was affected in the first step of the O-linked mannosylation pathway. The null mutant was viable, contrasting with C. albicans where this gene is essential. The phenotypical characterization showed that O-linked mannans were required for filamentation; proper cell wall integrity and organization; biofilm formation; protein secretion; and adhesion to extracellular matrix components, in particular to fibronectin; and type I and type II collagen. When interacting with human innate immune cells, it was found that this cell wall structure is dispensable for cytokine production, but mutant cells were more phagocytosed by monocyte-derived macrophages. Furthermore, the null mutant cells showed virulence attenuation in Galleria mellonella larvae. Thus, O-linked mannans are minor components of the cell wall that are involved in different aspects of C. tropicalis' biology.

9.
J Biol Chem ; : 107602, 2024 Jul 24.
Article in English | MEDLINE | ID: mdl-39059496

ABSTRACT

Glycosylation is a predominant strategy plants employ to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologues are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

10.
Biomedicines ; 12(7)2024 Jun 28.
Article in English | MEDLINE | ID: mdl-39062019

ABSTRACT

Immune checkpoint proteins have become recent research hotspots for their vital role in maintaining peripheral immune tolerance and suppressing immune response function in a wide range of tumors. Therefore, investigating the immunomodulatory functions of immune checkpoints and their therapeutic potential for clinical use is of paramount importance. The immune checkpoint blockade (ICB) is an important component of cancer immunotherapy, as it targets inhibitory immune signaling transduction with antagonistic antibodies to restore the host immune response. Anti-programmed cell death-1 (PD-1) and anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibodies are two main types of widely used ICBs that drastically improve the survival and prognosis of many patients with cancer. Nevertheless, the response rate of most cancer types remains relatively low due to the drug resistance of ICBs, which calls for an in-depth exploration to improve their efficacy. Accumulating evidence suggests that immune checkpoint proteins are glycosylated in forms of N-glycosylation, core fucosylation, or sialylation, which affect multiple biological functions of proteins such as protein biosynthesis, stability, and interaction. In this review, we give a brief introduction to several immune checkpoints and summarize primary molecular mechanisms that modulate protein stability and immunosuppressive function. In addition, newly developed methods targeting glycosylation on immune checkpoints for detection used to stratify patients, as well as small-molecule agents disrupting receptor-ligand interactions to circumvent drug resistance of traditional ICBs, in order to increase the clinical efficacy of immunotherapy strategies of patients with cancer, are also included to provide new insights into scientific research and clinical treatments.

11.
Biomolecules ; 14(7)2024 Jun 27.
Article in English | MEDLINE | ID: mdl-39062484

ABSTRACT

The endometrium is an important part of women's bodies for menstruation and pregnancy. Various proteins are widely expressed on the surface of endometrial cells, and glycosylation is an important post-translational modification of proteins. Glycosylation modification is closely related not only to endometrial receptivity but also to common diseases related to endometrial receptivity. Glycosylation can improve endometrial receptivity, promote embryo localization and trophoblast cell adhesion and invasion, and contribute to successful implantation. Two diseases related to endometrial receptivity include endometriosis and endometrial cancer. As a common benign disease in women, endometriosis is often accompanied by an increased menstrual volume, prolonged menstrual periods, progressive and aggravated dysmenorrhea, and may be accompanied by infertility. Protein glycosylation modification of the endometrial surface indicates the severity of the disease and may be an important pathogenesis of endometriosis. In cancer, glycosylation modifications on the surface of tumor cells can be a marker to distinguish the type and severity of endometrial cancer. This review highlights the role of protein glycosylation in embryo-maternal endometrial dialogue and explores its potential mechanisms in diseases related to endometrial receptivity, which could provide a new clinical approach for their diagnosis and treatment.


Subject(s)
Endometriosis , Endometrium , Humans , Glycosylation , Female , Endometrium/metabolism , Endometriosis/metabolism , Endometriosis/pathology , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/pathology , Protein Processing, Post-Translational , Embryo Implantation , Pregnancy , Animals
12.
Comput Struct Biotechnol J ; 23: 2580-2594, 2024 Dec.
Article in English | MEDLINE | ID: mdl-39021582

ABSTRACT

Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl-4 hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O-glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs.

13.
Int J Biol Macromol ; : 133943, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39025174

ABSTRACT

Asparagine (Asn, N)-linked glycosylation is an abundant post-translational modification in which Asn, typically in Nglyco-X-S/T; X ≠ P motifs, are modified with N-glycans. It has essential regulatory roles in multicellular organisms. In this study, we systematically investigate the function of three N-glycosylation motifs (Nglyco-A, Nglyco-D and Nglyco-S) previously identified in Phytophthora sojae, through site-directed mutagenesis and functional assays. In P. sojae expressing glycosylation-dead variants pre-PsDMAP1N70A (Nglyco-A motif) or PsADFN64A (Nglyco-D motif), zoospore release or cyst germination is impaired. In particular, the pre-PsDMAP1N70A mutant reduces DNA methylation levels, and the PsADFN64A mutant disrupts the actin forms, which could explain the decrease in pathogenicity after N-glycosylation is destroyed. Similarly, P. sojae expressing PsNRXN132A (Nglyco-S motif) shows increased sensitivity to H2O2 and heat. Through autophagy or 26S proteasome pathway inhibition assays, we found that unglycosylated pre-PsDMAP1N70A and PsADFN64A are degraded via the 26S proteasome pathway, while the autophagy pathway is responsible for PsNRXN132A clearance. These findings demonstrate that glycosylation of these motifs regulates the stability and function of glycoproteins necessary for P. sojae growth, reproduction and pathogenicity, which expands the scope of known N-glycosylation regulatory functions in oomycetes.

14.
J Biol Chem ; : 107583, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39025451

ABSTRACT

Ferroptosis is an iron-dependent cell death mechanism that may be important to prevent tumor formation and useful as a target for new cancer therapies. Transcriptional networks play a crucial role in shaping ferroptosis sensitivity by regulating the expression of transporters, metabolic enzymes, and other proteins. The Cap'n'collar (CNC) protein nuclear factor erythroid 2 like 2 (NFE2L2, also known as NRF2) is a key regulator of ferroptosis in many cells and contexts. Emerging evidence indicates that the related CNC family members BTB and CNC homology 1 (BACH1) and nuclear factor erythroid 2 like 1 (NFE2L1) also have non-redundant roles in ferroptosis regulation. Here, we comprehensively review the role of CNC transcription factors in governing cellular sensitivity to ferroptosis. We describe how CNC family members regulate ferroptosis sensitivity through modulation of iron, lipid, and redox metabolism. We also use examples of ferroptosis regulation by CNC proteins to illustrate the flexible and highly context-dependent nature of the ferroptosis mechanism between cells and conditions.

15.
J Food Sci ; 2024 Jul 09.
Article in English | MEDLINE | ID: mdl-38980988

ABSTRACT

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

17.
Mol Genet Metab ; 142(4): 108509, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38959600

ABSTRACT

OBJECTIVE: Our report describes clinical, genetic, and biochemical features of participants with a molecularly confirmed congenital disorder of glycosylation (CDG) enrolled in the Frontiers in Congenital Disorders of Glycosylation (FCDGC) Natural History cohort at year 5 of the study. METHODS: We enrolled individuals with a known or suspected CDG into the FCDGC Natural History Study, a multicenter prospective and retrospective natural history study of all genetic causes of CDG. We conducted a cross-sectional analysis of baseline study visit data from participants with confirmed CDG who were consented into the FCDGC Natural History Study (5U54NS115198) from October 2019 to November 2023. RESULTS: Three hundred thirty-three subjects consented to the FCDGC Natural History Study. Of these, 280 unique individuals had genetic data available that was consistent with a diagnosis of CDG. These 280 individuals were enrolled into the study between October 8, 2019 and November 29, 2023. One hundred forty-one (50.4%) were female, and 139 (49.6%) were male. Mean and median age at enrollment was 10.1 and 6.5 years, respectively, with a range of 0.22 to 71.4 years. The cohort encompassed individuals with disorders of N-linked protein glycosylation (57%), glycosylphosphatidylinositol anchor disorder (GPI anchor) (15%), disorders of Golgi homeostasis, trafficking and transport (12%), dolichol metabolism disorders (5%), disorders of multiple pathways (6%), and other (5%). The most frequent presenting symptom(s) leading to diagnosis were developmental delay/disability (77%), followed by hypotonia (56%) and feeding difficulties (42%). Mean and median time between first related symptom and diagnosis was 2.7 and 0.8 years, respectively. One hundred percent of individuals in our cohort had developmental differences/disabilities at the time of their baseline visit, followed by 97% with neurologic involvement, 91% with gastrointestinal (GI)/liver involvement, and 88% with musculoskeletal involvement. Severity of disease in individuals was scored on the Nijmegen Progression CDG Rating Scale (NPCRS) with 27% of scores categorized as mild, 44% moderate, and 29% severe. Of the individuals with N-linked protein glycosylation defects, 83% of those with data showed a type 1 pattern on carbohydrate deficient transferrin (CDT) analysis including 82/84 individuals with PMM2-CDG, 6% a type 2 pattern, 1% both type 1 and type 2 pattern and 10% a normal or nonspecific pattern. One hundred percent of individuals with Golgi homeostasis and trafficking defects with data showed a type 2 pattern on CDT analysis, while Golgi transport defect showed a type II pattern 73% of the time, a type 1 pattern for 7%, and 20% had a normal or nonspecific pattern. Most of the variants documented were classified as pathogenic or likely pathogenic using ACMG criteria. For the majority of the variants, the predicted molecular consequence was missense followed by nonsense and splice site, and the majority of the diagnoses are inherited in an autosomal recessive pattern but with disorders of all major nuclear inheritance included. DISCUSSION: The FCDGC Natural History Study serves as an important resource to build future research studies, improve clinical care, and prepare for clinical trial readiness. Herein is the first overview of CDG participants of the FCDGC Natural History Study.

18.
J Proteome Res ; 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-39007905

ABSTRACT

Fcγ-receptors (FcγRs) including FcγRII (CD32) gene family members are expressed on leukocytes, bind the crystallizable fragment (Fc) region of immunoglobulin G (IgG), and bridge humoral and cellular immunity. FcγRIIA and FcγRIIB have opposing roles, with the former responsible for activation and the latter for inhibition of immune cell signaling and effector functions. The extracellular domains of human and murine FcγRIIs share multiple conserved N-glycosylation sites. Understanding the role(s) of FcγRIIA and FcγRIIB glycosylation in autoimmune diseases is precluded by a lack of effective methods to study disease-associated changes in glycosylation. To address this barrier, we developed a method to assess site-specific glycosylation of human FcγRIIA and FcγRIIB, and the mouse ortholog of human FcγRIIB. Among the receptors, conserved glycosylation sites are compared, with the N144/145 site displaying predominantly complex glycans in recombinant FcγRIIs. Differences in sialylation between recombinant human FcγRIIA H/R134 (H/R131) variants at a nearby N145 N-glycosylation site are reported. Further, a potential human FcγRIIA O-glycosylation site, S179 (S212), is reported in recombinant FcγRIIA. The robust method to assess site-specific glycosylation of FcγRIIs reported here, can be utilized to study the potential role of FcγRII family glycosylation in disease. Data are available via ProteomeXchange with identifier PXD049429.

19.
Pharmgenomics Pers Med ; 17: 363-381, 2024.
Article in English | MEDLINE | ID: mdl-38983907

ABSTRACT

Background: Obesity is reaching epidemic proportions in the developed world. The biosynthesis and degradation of human glycoproteins take place at the highest level in the liver. However, the association between glycosylation and the factors affecting obesity and metabolism-associated steatohepatitis (MASH) is still unclear. Materials and Methods: Gene expression data of liver samples from obese patients were retrieved from GSE83452 and GSE89632 databases. Difference analysis and machine learning were used to identify hub genes involved in glycosylation and associated with the response of weight loss treatment. A total of 7 glycosylation-related hub genes were identified and then subjected to correlation analysis, immune cells infiltration analysis and ROC (Receiver Operating Characteristic) analysis. We also evaluated the potential function of 7 hub genes in obesity patients. MASH mice were used to validate the glycosylation-related hub genes. Results: A total of 25 overlapped glycosylation-related genes were identified by DEGs analysis. ACER2, STX17, ARF5, GPC4, ENTPD5, NANP, and DPY19L2 were identified as hub genes. Among these hub genes, ACER2, STX17, ARF5, and ENTPD5 were also differential expressed in MASH patients. ENTPD5 showed increased transcription in obese MASH mice. Conclusion: The current study identified seven glycosylation-related genes, ACER2, STX17, ARF5, GPC4, ENTPD5, NANP, and DPY19L2, that might play key roles in the development of obesity. ENTPD5 might play a key role in the development of MASH. These findings provide fresh perspectives for expanding the investigation of obesity and MASH.

20.
Int J Mol Sci ; 25(13)2024 Jul 08.
Article in English | MEDLINE | ID: mdl-39000590

ABSTRACT

Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift.


Subject(s)
Cysteine , Glycosylation , Cysteine/chemistry , Cysteine/metabolism , Protein Processing, Post-Translational , Glycopeptides/chemistry , Glycopeptides/metabolism , Peptides/chemistry , Peptides/metabolism , Gases/metabolism , Gases/chemistry , Glucose/metabolism , Glucose/chemistry , Proteomics/methods , Tandem Mass Spectrometry/methods
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