The effects of post-translational processing on dystroglycan synthesis and trafficking

Dystroglycan is a component of the dystrophin glycoprotein complex that is cleaved into two polypeptides by an unidentified protease. To determine the role of post-translational processing on dystroglycan synthesis and trafficking we expressed the dystroglycan precursor and mutants thereof in a heterologous system. A point mutant in the processing site, S655A, prevented proteolytic cleavage but had no effect upon the surface localisation of dystroglycan. Mutation of two N-linked glycosylation sites that flank the cleavage site inhibited proteolytic processing of the precursor. Furthermore, chemical inhibition of N- and O-linked glycosylation interfered with the processing of the precursor and reduced the levels of dystroglycan at the cell surface. Dystroglycan processing was also inhibited by the proteasome inhibitor lactacystin. N-linked glycosylation is a prerequisite for efficient proteolytic processing and cleavage and glycosylation are dispensable for cell surface targeting of dystroglycan.     

Identification of the beta-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.     

Concerted mutation of Phe residues belonging to the beta-dystroglycan ectodomain strongly inhibits the interaction with alpha-dystroglycan in vitro

The dystroglycan adhesion complex consists of two noncovalently interacting proteins: alpha-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane beta-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of beta-dystroglycan (691-719), and approximately 15 amino acids of alpha-dystroglycan (550-565). Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides beta-dystroglycan (654-750), corresponding to the ectodomain of beta-dystroglycan, and alpha-dystroglycan (485-630), spanning the C-terminal domain of alpha-dystroglycan. By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type alpha-dystroglycan and beta-dystroglycan, and shown the crucial role of two beta-dystroglycan phenylalanines, namely Phe692 and Phe718, for the alpha-beta interaction. Substitution of the alpha-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan-transfected 293-Ebna cells.     

Dystroglycan, a scaffold for the ERK-MAP kinase cascade

Dystroglycan is an important cell adhesion receptor linking the actin cytoskeleton, via utrophin and dystrophin, to laminin in the extracellular matrix. To identify adhesion-related signalling molecules associated with dystroglycan, we conducted a yeast two-hybrid screen and identified mitogen-activated protein (MAP) kinase kinase 2 (MEK2) as a beta-dystroglycan interactor. Pull-down experiments and localization studies substantiated a physiological link between beta-dystroglycan and MEK and localized MEK with dystroglycan in membrane ruffles. Moreover, we also identified active extracellular signal-regulated kinase (ERK), the downstream kinase from MEK, as another interacting partner for beta-dystroglycan and localized both active ERK and dystroglycan to focal adhesions in fibroblast cells. These studies suggest a role for dystroglycan as a multifunctional adaptor or scaffold capable of interacting with components of the ERK-MAP kinase cascade including MEK and ERK. These findings have important implications for our understanding of the role of dystroglycan in normal cellular processes and in disease states such as muscular dystrophy.     

Association of the dystroglycan complex isolated from bovine brain synaptosomes with proteins involved in signal transduction

Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.     

Importance of the propeptide in the biosynthetic maturation of rat cathepsin C

Cathepsin C is a cysteine dipeptidyl-aminopeptidase. Active cathepsin C is found in lysosomes as a 200-kDa multimeric enzyme. Subunits constituting this assembly all arise from the proteolytic cleavage of a single precursor giving rise to three peptides: the propeptide, the alpha- and the beta-chains. Some features of the propeptide such as its length, its high level of glycosylation and its retention in the active lysosomal form of the enzyme suggest an important contribution of the proregion in the transport, maturation and expression of cathepsin C. In order to assess some aspects of this contribution, we transiently expressed mutant molecules of rat cathepsin C either lacking three of the four glycosylation sites, partially deleted in the proregion, or mutated at tryptophan 39 also located in the proregion, and studied their biosynthesis. Our results show that at least one of the three glycosylation sites in the propeptide must be glycosylated in order to obtain targeting and maturation of cathepsin C. We also show that a deletion of 14 amino acids and mutation W39S in the propeptide totally abolishes the biosynthetic processing of the enzyme. These results demonstrate that in addition to its role as a chaperone or in maintaining the latency of the enzymatic activity, the propeptide is required for proper transport and expression of newly synthesized cathepsin C.     

Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine.

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.     

Structural properties of a soluble bioactive precursor for transforming growth factor-alpha

The precursor for transforming growth factor-alpha, proTGF-alpha, is synthesized as an integral membrane glycoprotein with the mature TGF-alpha sequence located in the extracellular domain. Retrovirally transformed rat embryo fibroblasts (FeSV-Fre cells) expressing the endogenous proTGF-alpha gene release and accumulate in the medium mature TGF-alpha as well as a heterogeneous (17-19 kDa) group of soluble, bioactive TGF-alpha precursor forms. These precursors correspond to the heterogeneously glycosylated extracellular domain of proTGF-alpha which is released from the membrane by proteolytic cleavage. They are designated mesoTGF-alpha to denote their intermediate position in the proTGF-alpha processing pathway. The nature of the carbohydrate linked to mesoTGF-alpha has been examined by treatment with glycosidases and the use of metabolic inhibitors of glycosylation. The results indicate that the TGF-alpha precursors from FeSV-Fre cells contain O-linked carbohydrate as well as sialylated N-linked carbohydrate. Heterogeneous N-linked glycosylation of an 11-kDa core polypeptide accounts for the heterogeneous nature of mesoTGF-alpha. MesoTGF-alpha released by cells treated with inhibitors of N-linked carbohydrate processing appears as a 17-kDa species. Treatment with these inhibitors does not alter significantly the production of mesoTGF-alpha or mature TGF-alpha by the cells. However, treatment of cells with an inhibitor of co-translational N-linked glycosylation, tunicamycin, reduces the accumulation of mesoTGF-alpha in the medium and blocks the production of mature TGF-alpha under conditions in which overall protein synthesis is only minimally affected. These findings suggest that the proTGF-alpha processing activity is limiting in FeSV-Fre cells and other transformed cells that accumulate mesoTGF-alpha in the medium and that proTGF-alpha processing depends on a component whose function may require N-linked glycosylation.     

Dystroglycan: a possible mediator for reducing congenital muscular dystrophy?

Alpha-dystroglycan is a highly glycosylated peripheral protein forming a complex with the membrane-spanning beta-dystroglycan and establishing a connection between the extracellular matrix and the cytoskeleton. In skeletal muscle, as part of the larger dystrophin-glycoprotein complex, dystroglycan is believed to be essential for maintaining the structural and functional stability of muscle fibers. Recent work highlights the role of abnormal dystroglycan glycosylation at the basis of glycosyltransferase-deficient congenital muscular dystrophies. Notably, modulation of glycosyltransferase activity can restore alpha-dystroglycan receptor function in these disorders. Moreover, transgenic approaches favoring the interaction between dystroglycan and the extracellular matrix molecules also represent an innovative way to restore skeletal muscle structure. These pioneering approaches might comprise an important first step towards the design of gene-transfer-based strategies for the rescue of congenital muscular dystrophies involving dystroglycan.     

Glycomarkers for muscular dystrophy

During the last 10?years it has become apparent that a significant subset of inherited muscular dystrophy is caused by errors in the glycosylation of α-dystroglycan. Many of these dystrophies are also associated with abnormalities of the central nervous system. Dystroglycan has to be fully glycosylated in order bind to its ligands. To date, six genes have been shown to be essential for functional dystroglycan glycosylation and most, if not all, of these genes act in the formation of O-mannosyl glycans. Genetic heterogeneity indicates that other genes are involved in this pathway. Identification of these additional genes would increase our understanding of this specific and essential glycosylation pathway.     

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.     

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.     

Effect of alternative glycosylation on insulin receptor processing.

The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two beta-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t((1)/(2)) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t((1)/(2)) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.     

Disruption of perlecan binding and matrix assembly by post-translational or genetic disruption of dystroglycan function

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy.     

Intracellular processing of apolipoprotein J precursor to the mature heterodimer.

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.     

In vitro developmental expression of dystroglycan and laminin-alpha2 in human skeletal muscle

The alpha-subunit of dystroglycan, a member of the dystrophin associated protein complex, binds to extracellular laminin-alpha2, while its beta-subunit interacts with cytoskeletal dystrophin. The exact biological role of dystroglycan, especially during human skeletal muscle development, has not been fully explored. Here, we analysed the distribution and expression characteristics of both dystroglycan subunits and laminin-alpha2 in primary human skeletal muscle cells. During development, expression levels of all three proteins increased with differentiation. The proteins were relocated from the sarcoplasm to the sarcolemma. The size of alpha-dystroglycan decreased from 150-220 kDa at the proliferation stage to 100-120 kDa at the late developmental stage. Both alpha- and beta-dystroglycan were involved in forming a complex with their respective partners laminin-alpha2 and dystrophin/utrophin. Our data show that, during development, cells may employ tightly regulated post-translational species-specific modification to produce different isoforms of alpha-dystroglycan to participate in appropriate functions.     

Dystroglycan glycosylation and its role in matrix binding in skeletal muscle

Dystroglycan is an essential component of the dystrophin-glycoprotein complex. Three glycan sequencing studies have identified O-linked mannose chains, including NeuAcalpha 2,3Galbeta 1,4GlcNAcbeta 1,2Manalpha-O, on alpha dystroglycan. Chemical deglycosylation of alpha dystroglycan, antibody blocking studies, and glycan blocking studies all suggest that the O-linked glycans on alpha dystroglycan mediate the binding of extracellular matrix proteins in skeletal muscle. Structural data on laminin G domains and agrin-binding studies also suggest this is the case. Dystroglycan, however, is able to bind proteins via mechanisms that do not involve O-linked glycans. Moreover, laminin and other matrix proteins can bind cell adhesion molecules via their glycan chains. Thus although complex and sometimes not overly convincing, these data suggest that glycosylation plays an important role in dystroglycan binding and function in skeletal muscle.