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Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations. Mutations in genes required for the glycosylation of α-dystroglycan lead to dystroglycanopathies. Here, the authors show that three of these enzymes (ISPD, FKTN and FKRP) work together to attach ribitol phosphate to α-dystroglycan.

Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O -mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure—a ribitol in a phosphodiester linkage—for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains.

Background Oxidized low-density lipoprotein (oxLDL), formed by LDL oxidative modification, drives pathogenesis in cardiovascular diseases and cancers through ER stress, inflammation, and protein aggregation. Stromal-derived factor 2-like 1 (SDF2L1), an ER-resident chaperone, may modulate these processes, but its role in oxLDL-related pathologies remains unclear. Materials and methods We analyzed spatial transcriptomic datasets from human myocardial infarction (MI) and lung cancer tissues. In vitro, we generated stable SDF2L1 knockdown (KD) or overexpression (OE) models in EA.hy926 endothelial and A549 lung epithelial cells, treated with oxLDL (10 μg/mL, 48 h). In vivo, we created global Sdf2l1 knockout ( Sdf2l1 −/− ) mice via CRISPR-Cas9 and administered oxLDL (1.4 mg/kg i.v., 7 days). Assessments included Western blotting for ER stress markers, qPCR for inflammatory genes, PROTEOSTAT assays for protein aggregates, ELISA for O-mannosyltransferase levels, and LC–MS/MS glycoproteomics for O-mannosylation profiles. Results Spatial transcriptomics revealed elevated SDF2L1 in MI ischemic zones and lung cancer tissues, correlating with ATF6 (ER stress) and OLR1 (oxLDL receptor). In cells, both SDF2L1 KD and OE paradoxically exacerbated oxLDL-induced ER stress (elevated p-PERK, p-IRE1α, spliced XBP1, cleaved ATF6), inflammation ( NF-κB , IL-6 , NLRP3 upregulation), and protein aggregation. Sdf2l1 −/− mice showed heightened oxLDL-induced ER stress, inflammatory cytokines ( Il-1β , Il-6 , Tnf ), histopathological changes (mucous degeneration, infiltration), and early disease biomarkers (e.g., Slc7a7 for MI, Kras for lung cancer). Mechanistically, SDF2L1 levels positively regulated O-mannosyltransferase content; Sdf2l1 deficiency reduced these enzymes and altered protein O-mannosylation, increasing TLR4 and MCM8 modifications/protein levels in vivo, while OE decreased them in vitro. Conclusion SDF2L1 exerts a complex modulatory role in oxLDL responses, linking ER stress, protein aggregation, and O-mannosylation to MI and lung cancer. Optimal SDF2L1 levels maintain homeostasis; deviations promote pathology, positioning SDF2L1 as a potential biomarker and therapeutic target for oxLDL-driven diseases.

Cryptococcus neoformans assembles two types of O -linked glycans on its surface proteins, the more abundant major O -glycans that do not contain xylose residues and minor O -glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O -glycans. The human-pathogenic yeast Cryptococcus neoformans assembles two types of O -linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O -glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 ( W SC/ M id2- l ike) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O -mannosylation, respectively. The double deletion of KTR3 and CAP6 ( ktr3 Δ cap6 Δ) completely blocked the mannose addition at the second position of O -glycans, resulting in the accumulation of proteins with O -glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3 Δ cap6 Δ and wml1 Δ wml2 Δ strains. Transcriptome profiling of the ktr3 Δ cap6 Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3 Δ cap6 Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3 Δ cap6 Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3 Δ cap6 Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O -glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans . IMPORTANCE Cryptococcus neoformans assembles two types of O -linked glycans on its surface proteins, the more abundant major O -glycans that do not contain xylose residues and minor O -glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O -glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O -glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3 Δ cap6 Δ double O- glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3 Δ cap6 Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O -glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.

Research background. Glycosyltransferases represent a large and diverse family of enzymes that catalyze the transfer of sugar residues to proteins and lipids, thereby regulating essential cellular processes such as protein quality control and cell wall biosynthesis. In yeast, protein O-mannosyltransferases and other glycosyltransferases are crucial for maintaining cell wall integrity. While the functions of many of these enzymes are well characterized, the role of some of them, such as Gmh5p, remains unknown. This study aims to elucidate the function of Gmh5p, a previously uncharacterized member of the GT34 glycosyltransferase family, in the context of protein and cell wall biosynthesis in Schizosaccharomyces pombe. Experimental approach. To identify proteins and pathways compensating for reduced O-mannosylation, we performed a genetic screening for multi-copy suppressors in a conditional lethal nmt81-oma2+ mutant background. The enzymatic activity of Gmh5p was biochemically characterized, and its functional homology to known mannosyltransferases was assessed through complementation experiments in Saccharomyces cerevisiae. In addition, the N-glycosylation status of model substrates was analyzed in gmh5Δ mutant strains. Results and conclusions. Gmh5p was identified as a suppressor of O-mannosylation defects. Contrary to its predicted function, Gmh5p did not exhibit α-1,2-galactosyltransferase activity but instead showed mannosyltransferase activity. Expression of gmh5+ in S. cerevisiae mnn10 mutants restored hygromycin tolerance to near wild-type levels. Furthermore, N-glycosylation of model substrates was reduced in gmh5Δ mutants. These results demonstrate that Gmh5p is a mannosyltransferase involved in the outer chain elongation of N-linked glycans and functions as a homologue of Mnn10p. Novelty and scientific contribution. This study provides the first functional characterization of Gmh5p as a mannosyltransferase of the GT34 family and demonstrates its role in N-glycan biosynthesis. Our findings expand the current understanding of the diversity and specificity of glycosyltransferases in eukaryotes and highlight their importance in cell wall biology.

Dystroglycanopathies (DGPs) are a group of autosomal recessive neuromuscular diseases with significant clinical and genetic heterogeneity. They originate due to defects in the O-mannosyl glycosylation of α-dystroglycan (α-DG), a prominent linker between the intracellular cytoskeleton and the extracellular matrix (ECM). Fundamentally, such interactions are crucial for the integrity of muscle fibers and neuromuscular synapses, where their defects are mainly associated with muscle and brain dysfunction. To date, biallelic variants in 18 genes have been associated with DGPs, where the underlying cause is still undefined in a significant proportion of patients. Glycosylation of α-DG generates three core motifs where the core M3 is responsible for interaction with the basement membrane. Consistently, all gene defects that corrupt core M3 maturation have been identified as causes of DGPs. POMGNT1 which stimulates the generation of core M1 is also associated with DGPs, as it plays a central role in core M3 processing. Other genes involved in the glycosylation of α-DG seem unrelated to DPGs. The current review illustrates the O -mannosylation pathway of α-DG highlighting the functional properties of related genes and their contribution to the progression of DPGs. Different classes of DPGs are also elaborated characterizing the clinical features of each distinct type and phenotypes associated with each single gene. Finally, current therapeutic approaches with favorable outcomes are addressed. Potential achievements of preclinical and clinical studies would introduce effective curative therapies for this group of disorders in the near future.

Glycosyltransferases that use polyisoprenol-linked donor substrates are categorized in the GT-C superfamily. In eukaryotes, they act in the endoplasmic reticulum (ER) lumen and are involved in N-glycosylation, glypiation, O-mannosylation, and C-mannosylation of proteins. We generated a membrane topology model of C-mannosyltransferases (DPY19 family) that concurred perfectly with the 13 transmembrane domains (TMDs) observed in oligosaccharyltransferases (STT3 family) structures. A multiple alignment of family members from diverse organisms highlighted the presence of only a few conserved amino acids between DPY19s and STT3s. Most of these residues were shown to be essential for DPY19 function and are positioned in luminal loops that showed high conservation within the DPY19 family. Multiple alignments of other eukaryotic GT-C families underlined the presence of similar conserved motifs in luminal loops, in all enzymes of the superfamily. Most GT-C enzymes are proposed to have an uneven number of TDMs with 11 (POMT, TMTC, ALG9, ALG12, PIGB, PIGV, and PIGZ) or 13 (DPY19, STT3, and ALG10) membrane-spanning helices. In contrast, PIGM, ALG3, ALG6, and ALG8 have 12 or 14 TMDs and display a C-terminal dilysine ER-retrieval motif oriented towards the cytoplasm. We propose that all members of the GT-C superfamily are evolutionary related enzymes with preserved membrane topology.

Protein O -mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a β-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general β-trefoil carbohydrate-binding sites (α, β), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous β-trefoil – carbohydrate complexes allows for a functional description of MIR domains in protein O -mannosylation.

Desmocollin‐2 (DSC2) is a desmosomal protein of the cadherin family. Desmosomes are multiprotein complexes, which are involved in cell adhesion of cardiomyocytes and of keratinocytes. The molecular structure of the complete extracellular domain (ECD) of DSC2 was recently described, revealing three disulfide bridges, four N‐glycosylation sites, and four O‐mannosylation sites. However, the functional relevance of these post‐translational modifications for the protein trafficking of DSC2 to the plasma membrane is still unknown. Here, we generated a set of DSC2 mutants, in which we systematically exchanged all N‐glycosylation sites, O‐mannosylation sites, and disulfide bridges within the ECD and investigated the resulting subcellular localization by confocal laser scanning microscopy. Of note, all single and double N‐glycosylation‐ deficient mutants were efficiently incorporated into the plasma membrane, indicating that the absence of these glycosylation sites has a minor effect on the protein trafficking of DSC2. However, the exchange of multiple N‐glycosylation sites resulted in intracellular accumulation. Colocalization analysis using cell compartment trackers revealed that N‐glycosylation‐ deficient DSC2 mutants were retained within the Golgi apparatus. In contrast, elimination of the four O‐mannosylation sites or the disulfide bridges in the ECD has no obvious effect on the intracellular protein processing of DSC2. These experiments underscore the importance of N‐glycosylation at multiple sites of DSC2 for efficient intracellular transport to the plasma membrane. Desmocollin‐2 (DSC2) is a member of the cadherin family. The extracellular domain of DSC2 is known to undergo three types of post‐translational modification. In this study, we demonstrate that in contrast to O‐mannosylation and disulfide bridge modifications, N‐glycosylation of four sites is necessary for efficient incorporation of DSC2 into the plasma membrane.