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Oligomerization of ß-Dystroglycan in Rabbit Diaphragm and Brain As Revealed by Chemical Crosslinking

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Biochimica et Biophysica Acta 1370Ž. 1998 325±336 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Oligomerization of b-dystroglycan in rabbit diaphragm and brain as revealed by chemical crosslinking Denise M. Finn, Kay Ohlendieck ) Department of Pharmacology, UniÕersity College Dublin, Belfield, Dublin 4, Ireland Received 24 November 1997; accepted 11 December 1997 Abstract The surface component b-dystroglycan is a member of the dystrophin±glycoprotein complex providing a trans-sarco- lemmal linkage between the actin membrane cytoskeleton and the extracellular matrix component laminin-a 2. Although abnormalities in this complex are involved in the pathophysiology of various neuromuscular disorders, little is known about the organization of dystrophin-associated glycoproteins in diaphragm and brain. We therefore investigated the oligomeriza- tion of b-dystroglycan and its connection with the most abundant dystrophin homologues in these two tissues. Employing detergent solubilization and alkaline extraction procedures of native membranes, it was confirmed that b-dystroglycan behaves like an integral surface molecule as predicted by its cDNA sequence. Immunoblot analysis following chemical crosslinking of native membranes showed that b-dystroglycan has a tendency to form high-molecular-mass complexes. Within these crosslinkable complexes, immuno-reactive overlaps were observed between b-dystroglycan, a-dystroglycan, laminin and 427 kDa dystrophin in diaphragm and skeletal muscle. In synaptosomes, the major brain dystrophin isoform Dp116 also exhibited an immuno-reactive overlap with members of the dystroglycan complex. These findings demonstrate that b-dystroglycan does not exist as a monomer in native membranes and imply that certain dystrophin isoforms and dystrophin-associated components interact with this surface protein in diaphragm and brain as has been previously shown for skeletal and heart muscle. q 1998 Elsevier Science B.V. Keywords: Dystroglycan; Dystrophin; Dp-116; Dystrophin±glycoprotein complex; Laminin; Crosslinking 1. Introduction are proposed to provide a trans-sarcolemmal linkage between the basement membrane component laminin- Dystrophin, initially identified as the primary de- a 2 and the actin membrane cytoskeleton in skeletal r wx fect in Duchenne Becker muscular dystrophy 1 , is muscle fibreswx 6±9 . While peripheral a-dystroglycan now well established to be associated with a variety wx of apparent 156 kDa represents a novel non-integrin of surface proteins 2,3 and was shown to exist in class of laminin-binding proteins, 43 kDa b-dys- numerous isoforms in many tissues including brain wx troglycan is an integral dystrophin-associated glyco- 4,5 . Dystrophin-associated components belonging to proteinwx 10 . Both dystroglycan proteins are encoded the highly glycosylated surface dystroglycan-complex by a single gene whose expressed precursor protein product is post-translationally processed into two ) wx Corresponding author. Fax: q353-1-269-2749; E-mail: glycoproteins 11,12 . Molecular studies have shown [email protected] that the extreme COOH-terminus of b-dystroglycan 0005-2736r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S0005-2736Ž. 97 00283-6 326 D.M. Finn, K. OhlendieckrBiochimica et Biophysica Acta 1370() 1998 325±336 constitutes a unique binding site for the second half trophin isoform Dp116 exhibits biochemical proper- of the hinge-4 region and the cysteine-rich domain of ties typical of a membrane cytoskeletal proteinwx 32 . dystrophinwx 13 and that a-dystroglycan can interact Thus, although Dp116 is lacking the N-terminal with the a 2-chain of laminin-2, formerly referred to actin-binding domain of 427 kDa dystrophin, it ap- as merosinwx 11,14,15 . Both proteins co-localize with pears to be a component of the sub-plasma membrane dystrophin to the muscle surfacewx 16±18 , clearly cytoskeleton possibly involved in anchoring proteins co-purify in subcellular fraction studies with the sar- in the periphery of neurons. Another member of the colemma fractionwx 16,19 and contain asparagine-lin- superfamily of spectrin-like components, autosoma- ked oligosaccharideswx 20 . Serinerthreonine-linked lly-encoded utrophin of 395 kDa, has an even broader oligosaccharides on a-sarcoglycan are proposed to distribution in brainwx 33 and may function in the confer a stiff conformation to the peptide core and generation and maintenance of regional substratum- protease resistance to this peripheral cell surface pro- associated membrane specialisations at the blood± teinwx 20 . Although the principal sub-plasmalemmal brain barrierwx 34 . Utrophin was originally described cytoskeleton link to the basement membrane is prob- as a neuromuscular junction-specific dystrophin iso- ably provided by these two tightly associated dystro- formwx 35±37 . Since the utrophin-associated compo- glycans, other sarcolemmal proteins might also be nent a-dystroglycan was found to be a functional involved in this process, i.e., components of the agrin-receptor, the utrophin±glycoprotein complex is sarcoglycan sub-complex ranging in relative molecu- implicated in the anchoring of the nicotinic acetyl- lar mass from apparent 25 kDa to 50 kDawx 2,3 . choline receptor at the neuromuscular junctionw 38± Following the detailed analysis of the spatial con- 40x . Although a- and g-sarcoglycan appear to be figuration of the muscle dystrophin±glycoprotein muscle-specific proteinswx 41 , other dystrophin-asso- complex, it was established that primary or secondary ciated proteins were clearly established to be present defects in the dystrophin±glycoprotein complex lead in neuronal tissueswx 42±45 . In neural retina, the to a variety of neuromuscular disorders, i.e., dystroglycan complex is expressed in the inner limit- DuchennerBecker muscular dystrophy, congenital ing membrane and around blood vessels where it muscular dystrophy and certain forms of limb-girdle co-localises with laminin and utrophinwx 43 . Both muscular dystrophywx 8,9,21±24 . Recently, b-dys- dystroglycans are also present in the outer plexiform troglycan was described as defective in a novel form layer of retina, which is devoid of laminin, but of muscular dystrophywx 25 . However, although a contains dystrophin and utrophinwx 43 . Studies by large number of patients afflicted with Duchenne Mummery et al.wx 42 showed that b-dystroglycan is muscular dystrophy suffer from respiratory complica- enriched in synaptic membranes of adult rat forebrain tionswx 26 , relatively little work has been done on the and that a novel b-dystroglycan-related protein of biochemical and pathophysiological characterization apparent 164 kDa is enriched in the postsynaptic of dystrophin-associated components in the di- density. In addition, analysis of dystrophin-associated aphragm. In addition, the association of brain dys- proteins in rat cerebellum by Tian et al.wx 44 support trophins with other neuronal surface components is the hypothesis that dystrophin interacts with dystro- not well understood and the normal function and glycans in Purkinje neurons. Binding studies with potential involvement of these isoforms in brain dis- purified brain a-dystroglycan established that it can orders is mostly unknown. Since one third of interact with a 2-chain-containing laminin-2wx 44 . In Duchenne patients suffer from non-progressive men- addition, syntrophins were recently shown to co- tal retardation, mutations in brain dystrophins may be localize with various dystrophin isoforms and dystro- the primary or contributing factor leading to these glycans in mouse brainwx 45 . clinical symptomswx 27±29 . Biochemical evidence for a tight association be- Brain dystrophin of 427 kDa has been localised to tween skeletal muscle surface glycoproteins and dys- the postsynaptic densitywx 27 and is present in a wide trophin isoforms exists from chromatographic tech- range of neurons including cerebellar Purkinje cells niques, analytical gradient centrifugation and domain and pyramidal neurons in the cerebral cortexwx 30,31 . binding studieswx 2,3,13,15,20 . Although the dys- Recently, we could show that the major brain dys- trophin±glycoprotein complex can be isolated by D.M. Finn, K. OhlendieckrBiochimica et Biophysica Acta 1370() 1998 325±336 327 laminin affinity columns and labeled laminin binds nize the major brain isoform Dp116, were produced strongly to a-dystroglycan in overlay assayswx 11,15 , as previously described in detailwx 32 . Polyclonal the purified skeletal muscle dystrophin±glycoprotein antibody to crude laminin preparations from base- complex contains no detectable laminin-a 2 chain ment membranes of Englebreth±Holm±SwarmŽ. EHS wx2,3 . This loss in laminin-a 2 may be due to break- mouse sarcoma, monoclonal antibody VC1.1 to the age of weak non-covalent bonds during salt washes HNK-1 antigen, as well as monoclonal antibody SB- to remove actomyosin contaminations from vesicular SP-1 to human erythrocyte spectrin were purchased muscle membrane preparations. Thus, to more di- from Sigma Chemical CompanyŽ. Poole, Dorset, UK . rectly determine the oligomerization of the principal The laminin antibody, although produced against trans-sarcolemma spanning component of the dys- laminin-1Ž. previously named EHS-lamininwx 49 , rec- trophin±glycoprotein complex, b-dystroglycan, and ognizes both laminin-1 and laminin-2Ž previously its association with laminin isoforms and dystrophin, named merosin. wx 49 isoforms.
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