Matriglycan maintains t-tubule structural integrity in cardiac muscle.

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.

Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

Polymerizing laminins in development, health, and disease.

Polymerizing laminins are multi-domain basement membrane (BM) glycoproteins that self-assemble into cell-anchored planar lattices to establish the initial BM scaffold. Nidogens, collagen-IV and proteoglycans then bind to the scaffold at different domain loci to create a mature BM. The LN domains of adjacent laminins bind to each other to form a polymer node, while the LG domains attach to cytoskeletal-anchoring integrins and dystroglycan, as well as to sulfatides and heparan sulfates. The polymer node, the repeating unit of the polymer scaffold, is organized into a near-symmetrical triskelion. The structure, recently solved by cryo-electron microscopy in combination with AlphaFold2 modeling and biochemical studies, reveals how the LN surface residues interact with each other and how mutations cause failures of self-assembly in an emerging group of diseases, the LN-lamininopathies, that include LAMA2-related dystrophy and Pierson syndrome.

Dual FKRP/FST gene therapy normalizes ambulation, increases strength, decreases pathology, and amplifies gene expression in LGMDR9 mice.

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRPP448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes.

Protein tyrosine phosphatase 69D is a substrate of protein O-mannosyltransferases 1-2 that is required for the wiring of sensory axons in Drosophila.

Mutations in protein O-mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Protein tyrosine phosphatase 69D (PTP69D) as a gene interacting with POMTs in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked mannose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs. These modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of receptor-type protein tyrosine phosphatase functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

Dystroglycan-HSPG interactions provide synaptic plasticity and specificity.

AIM: This study examined the roles of the laminin and proteoglycan receptor dystroglycan (DG) in extracellular matrix stabilization and cellular mechanosensory processes conveyed through communication between the extracellular matrix (ECM) and cytoskeleton facilitated by DG. Specific functional attributes of HS-proteoglycans (HSPGs) are conveyed through interactions with DG and provide synaptic specificity through diverse interactions with an extensive range of cell attachment and adaptor proteins which convey synaptic plasticity. HSPG-DG interactions are important in phototransduction and neurotransduction and facilitate retinal bipolar-photoreceptor neuronal signaling in vision. Besides synaptic stabilization, HSPG-DG interactions also stabilize basement membranes and the ECM and have specific roles in the assembly and function of the neuromuscular junction. This provides neuromuscular control of muscle systems that control conscious body movement as well as essential autonomic control of diaphragm, intercostal and abdominal muscles and muscle systems in the face, mouth and pharynx which assist in breathing processes. DG is thus a multifunctional cell regulatory glycoprotein receptor and regulates a diverse range of biological and physiological processes throughout the human body. The unique glycosylation of the αDG domain is responsible for its diverse interactions with ECM components in cell-ECM signaling. Cytoskeletal cell regulatory switches assembled by the ßDG domain in its role as a nuclear scaffolding protein respond to such ECM cues to regulate cellular behavior and tissue homeostasis thus DG has fascinating and diverse roles in health and disease.

Analysis of the GFP-labelled ß-dystroglycan interactome in HEK-293 transfected cells reveals novel intracellular networks.

Dystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane ß-DG whose, cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. ß-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases. To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with ß-DG, HEK-293 cells were transiently transfected with a plasmid carrying the ß-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing ß-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG. Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis led to the identification of an interactome formed by 313 exclusive protein matches for ß-DG binding. A series of already known ß-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel ß-DG interactors and their related networks, were identified in diverse subcellular compartments, such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamina-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein, whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with ß-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires ß-DG for a proper nuclear localization. These results expand the role of ß-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy.

The extracellular matrix protein agrin is essential for epicardial epithelial-to-mesenchymal transition during heart development.

During heart development, epicardial cells residing within the outer layer undergo epithelial-mesenchymal transition (EMT) and migrate into the underlying myocardium to support organ growth and morphogenesis. Disruption of epicardial EMT results in embryonic lethality, yet its regulation is poorly understood. Here, we report epicardial EMT within the mesothelial layer of the mouse embryonic heart at ultra-high resolution using scanning electron microscopy combined with immunofluorescence analyses. We identified morphologically active EMT regions that associated with key components of the extracellular matrix, including the basement membrane-associated proteoglycan agrin. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by downregulation of Wilms' tumor 1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing ß-catenin and promoting pFAK localization at focal adhesions, and promoted the aggregation of dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan in vivo, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the extracellular matrix in heart development and implicate agrin as a crucial regulator of epicardial EMT.

Validation of a novel western blot assay to monitor patterns and levels of alpha dystroglycan in skeletal muscle of patients with limb girdle muscular dystrophies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

The many roles of dystroglycan in nervous system development and function: Dystroglycan and neural circuit development: Dystroglycan and neural circuit development.

The glycoprotein dystroglycan was first identified in muscle, where it functions as part of the dystrophin glycoprotein complex to connect the extracellular matrix to the actin cytoskeleton. Mutations in genes involved in the glycosylation of dystroglycan cause a form of congenital muscular dystrophy termed dystroglycanopathy. In addition to its well-defined role in regulating muscle integrity, dystroglycan is essential for proper central and peripheral nervous system development. Patients with dystroglycanopathy can present with a wide range of neurological perturbations, but unraveling the complex role of Dag1 in the nervous system has proven to be a challenge. Over the past two decades, animal models of dystroglycanopathy have been an invaluable resource that has allowed researchers to elucidate dystroglycan's many roles in neural circuit development. In this review, we summarize the pathways involved in dystroglycan's glycosylation and its known interacting proteins, and discuss how it regulates neuronal migration, axon guidance, synapse formation, and its role in non-neuronal cells.

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation.

How extracellular matrix contributes to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. We found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell-autonomous control of F-actin fiber orientation relies on the preceding BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a crucial organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic α-Dystroglycan O-Mannosylated Core M1 Glycoconjugates In situ.

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.

An atypical expression of core α-Dystroglycan and Laminin-α2 in skin fibroblasts of patients with congenital muscular dystrophies.

BACKGROUND: Congenital muscular dystrophies (CMDs) result from genetically inherited defects in the biosynthesis and/or the posttranslational modification (glycosylation) of laminin-α2 and α-dystroglycan (α-DG), respectively. The interaction between both proteins is responsible for the stability and integrity of the muscle cell. We aimed to study the expression profiles of both proteins in two classes of CMDs. SUBJECTS AND METHODS: Whole-exome sequencing (WES) was done for four patients with neuromuscular manifestations. The expression of core α-DG and laminin-α2 subunit in skin fibroblasts and MCF-7 cells was assessed by western blot. RESULTS: WES revealed two cases with nonsense mutations; c.2938G > T and c.4348 C > T, in LAMA2 encodes laminin-α2. It revealed also two cases with mutations in POMGNT1 encode protein O-mannose beta-1,2-N-acetylglucosaminyltransferase mutations. One patient had a missense mutation c.1325G > A, and the other had a synonymous variant c.636 C > T. Immunodetection of core α-DG in skin fibroblasts revealed the expression of truncated forms of core α-DG accompanied by reduced expression of laminin-α2 in POMGNT1-CMD patients and one patient with LAMA2-CMD. One patient with LAMA2-CMD had overexpression of laminin-α2 and expression of a low level of an abnormal form of increased molecular weight core α-DG. MCF-7 cells showed truncated forms of core α-CDG with an absent laminin-α2. CONCLUSION: A correlation between the expression pattern/level of core α-DG and laminin-α2 could be found in patients with different types of CMD.

Defective perlecan-associated basement membrane regeneration and altered modulation of transforming growth factor beta in corneal fibrosis.

In the cornea, the epithelial basement membrane (EBM) and corneal endothelial Descemet's basement membrane (DBM) critically regulate the localization, availability and, therefore, the functions of transforming growth factor (TGF)ß1, TGFß2, and platelet-derived growth factors (PDGF) that modulate myofibroblast development. Defective regeneration of the EBM, and notably diminished perlecan incorporation, occurs via several mechanisms and results in excessive and prolonged penetration of pro-fibrotic growth factors into the stroma. These growth factors drive mature myofibroblast development from both corneal fibroblasts and bone marrow-derived fibrocytes, and then the persistence of these myofibroblasts and the disordered collagens and other matrix materials they produce to generate stromal scarring fibrosis. Corneal stromal fibrosis often resolves completely if the inciting factor is removed and the BM regenerates. Similar defects in BM regeneration are likely associated with the development of fibrosis in other organs where perlecan has a critical role in the modulation of signaling by TGFß1 and TGFß2. Other BM components, such as collagen type IV and collagen type XIII, are also critical regulators of TGF beta (and other growth factors) in the cornea and other organs. After injury, BM components are dynamically secreted and assembled through the cooperation of neighboring cells-for example, the epithelial cells and keratocytes for the corneal EBM and corneal endothelial cells and keratocytes for the corneal DBM. One of the most critical functions of these reassembled BMs in all organs is to modulate the pro-fibrotic effects of TGFßs, PDGFs and other growth factors between tissues that comprise the organ.

The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi.

The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency.

Laminin α2 controls mouse and human stem cell behaviour during midbrain dopaminergic neuron development.

Development of the central nervous system requires coordination of the proliferation and differentiation of neural stem cells. Here, we show that laminin alpha 2 (lm-α2) is a component of the midbrain dopaminergic neuron (mDA) progenitor niche in the ventral midbrain (VM) and identify a concentration-dependent role for laminin α2ß1γ1 (lm211) in regulating mDA progenitor proliferation and survival via a distinct set of receptors. At high concentrations, lm211-rich environments maintain mDA progenitors in a proliferative state via integrins α6ß1 and α7ß1, whereas low concentrations of lm211 support mDA lineage survival via dystroglycan receptors. We confirmed our findings in vivo, demonstrating that the VM was smaller in the absence of lm-α2, with increased apoptosis; furthermore, the progenitor pool was depleted through premature differentiation, resulting in fewer mDA neurons. Examination of mDA neuron subtype composition showed a reduction in later-born mDA neurons of the ventral tegmental area, which control a range of cognitive behaviours. Our results identify a novel role for laminin in neural development and provide a possible mechanism for autism-like behaviours and the brainstem hypoplasia seen in some individuals with mutations of LAMA2.

Identification of potential plasma markers for hepatitis B virus-related chronic hepatitis and liver fibrosis/cirrhosis.

The aim of this study was to identify potential plasma biomarkers for hepatitis B virus (HBV)-related liver diseases. High-throughput transcriptome sequencing analysis was performed on five patients with chronic hepatitis B (CHB), five patients with HBV-associated liver fibrosis/liver cirrhosis (LF/LC), and four healthy participants. By short time-series expression miner and functional analysis, aquaporin 1 (AQP1), dystroglycan 1 (DAG1), and hemoglobin subunit beta (HBB) were identified as potential biomarkers. Immunohistochemical analysis revealed that the expression levels of AQP1, DAG1, and HBB were upregulated in the three groups. Subsequent enzyme-linked immunosorbent assay tests on the training cohort (n = 150) indicated that the plasma levels of AQP1 and DAG1 were highest in LF/LC patients, followed by those in CHB patients, and the lowest in healthy controls. APAD model, a diagnostic panel incorporating age, platelet, AQP1, and DAG1 levels, exhibited the strongest stratification ability to distinguish LF/LC patients from CHB patients, and to differentiate CHB patients from healthy controls. Furthermore, the diagnostic accuracies of the biomarkers and APAD model were verified in an independent cohort consisting of 230 participants. In conclusion, both AQP1 and DAG1 have good diagnostic values and APAD model greatly enhances the diagnostic accuracy for HBV-related hepatic diseases.

TRAPPC11-related muscular dystrophy with hypoglycosylation of alpha-dystroglycan in skeletal muscle and brain.

AIMS: TRAPPC11, a subunit of the transport protein particle (TRAPP) complex, is important for complex integrity and anterograde membrane transport from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment. Several individuals with TRAPPC11 mutations have been reported with muscle weakness and other features including brain, liver, skeletal and eye involvement. A detailed analysis of brain and muscle pathology will further our understanding of the presentation and aetiology of TRAPPC11 disease. METHODS: We describe five cases of early-onset TRAPPC11-related muscular dystrophy with a systematic review of muscle pathology in all five individuals, post-mortem brain pathology findings in one and membrane trafficking assays in another. RESULTS: All affected individuals presented in infancy with muscle weakness, motor delay and elevated serum creatine kinase (CK). Additional features included cataracts, liver disease, intellectual disability, cardiomyopathy, movement disorder and structural brain abnormalities. Muscle pathology in all five revealed dystrophic changes, universal hypoglycosylation of alpha-dystroglycan and variably reduced dystrophin-associated complex proteins. Membrane trafficking assays showed defective Golgi trafficking in one individual. Neuropathological examination of one individual revealed cerebellar atrophy, granule cell hypoplasia, Purkinje cell (PC) loss, degeneration and dendrite dystrophy, reduced alpha-dystroglycan (IIH6) expression in PC and dentate neurones and absence of neuronal migration defects. CONCLUSIONS: This report suggests that recessive mutations in TRAPPC11 are linked to muscular dystrophies with hypoglycosylation of alpha-dystroglycan. The structural cerebellar involvement that we document for the first time resembles the neuropathology reported in N-linked congenital disorders of glycosylation (CDG) such as PMM2-CDG, suggesting defects in multiple glycosylation pathways in this condition.

Involvement of abnormal dystroglycan expression and matriglycan levels in cancer pathogenesis.

Dystroglycan (DG) is a glycoprotein composed of two subunits that remain non-covalently bound at the plasma membrane: α-DG, which is extracellular and heavily O-mannosyl glycosylated, and ß-DG, an integral transmembrane polypeptide. α-DG is involved in the maintenance of tissue integrity and function in the adult, providing an O-glycosylation-dependent link for cells to their extracellular matrix. ß-DG in turn contacts the cytoskeleton via dystrophin and participates in a variety of pathways transmitting extracellular signals to the nucleus. Increasing evidence exists of a pivotal role of DG in the modulation of normal cellular proliferation. In this context, deficiencies in DG glycosylation levels, in particular those affecting the so-called matriglycan structure, have been found in an ample variety of human tumors and cancer-derived cell lines. This occurs together with an underexpression of the DAG1 mRNA and/or its α-DG (core) polypeptide product or, more frequently, with a downregulation of ß-DG protein levels. These changes are in general accompanied in tumor cells by a low expression of genes involved in the last steps of the α-DG O-mannosyl glycosylation pathway, namely POMT1/2, POMGNT2, CRPPA, B4GAT1 and LARGE1/2. On the other hand, a series of other genes acting earlier in this pathway are overexpressed in tumor cells, namely DOLK, DPM1/2/3, POMGNT1, B3GALNT2, POMK and FKTN, hence exerting instead a pro-oncogenic role. Finally, downregulation of ß-DG, altered ß-DG processing and/or impaired ß-DG nuclear levels are increasingly found in human tumors and cell lines. It follows that DG itself, particular genes/proteins involved in its glycosylation and/or their interactors in the cell could be useful as biomarkers of certain types of human cancer, and/or as molecular targets of new therapies addressing these neoplasms.

The role of protein glycosylation in muscle diseases.

INTRODUCTION: As a post-translational modification, glycosylation plays vital role in regulating the folding and function of proteins necessary for many biological processes. Unlike glycation, glycosylation is an enzymatic process; glycosyltransferases transfer sugars to proteins, forming glycosidic bonds with amino acid residues on proteins. Changes that interfere with the enzymatic reaction and result in abnormal glycosylation can spatio-temporally affect the balance of glycosylation, leading to disease states. Muscle diseases have been associated with dysfunctional protein glycosylation, and many studies have focused on the pathophysiology underlying this association. This review aims to summarize the research progress on protein glycosylation in the pathogenesis of muscle diseases and provides new insight into the muscle research field. METHODS: Literatures were reviewed comparatively and data were organized to find information about protein glycosylation and its role in muscle disease. RESULTS: Protein glycosylation modification is closely related to the occurrence of muscle diseases. α-DG is a key protein in the study of inherited muscle diseases and has a wide range of glycosylation, including O-linked glycosylation and N-linked glycosylation. Besides, O-GlcNAc glycosylation is an important mechanism of protein glycosylation, helping maintaining the structure and function of skeletal muscle and participating in multiple biological processes. Protein glycosylation is also connected to muscle disease and neurodegenerative diseases, especially Alzheimer's disease. CONCLUSIONS: Taken together, better understanding of protein glycosylation and its implication in muscle disease would help provide new perspectives in the prevention and treatment measures for human muscle diseases.