Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n‐octyl β‐d‐glucoside

Abstract

Dystrophin is purified as a complex with several proteins from the digitonin-solubilized muscle cell membrane. Most of dystrophin-associated proteins (DAPs) are assumed to form a large oligomeric transmembranous glycoprotein complex on the sarcolemma and link dystrophin with a basement membrane protein, laminin. In the present study, we found that the purified dystrophin-DAP complex was dissociated into several groups by n-octyl-P-~-glucoside treatment. In particular, we found that the glycoprotein complex stated above was dissociated into two distinct groups: one composed of 156DAG and 43DAG (A3a) and the other composed of SODAG, 35DAG and A3b. We confirmed by crosslinking and immunoaffinity chromatography that these two groups existed in a complexes. We thus concluded that the glycoprotein complex consists of these two subcomplexes. Furthermore, A3b and 43DAG, which had been formerly treated simply as the 43DAG doublets due to their similar electrophoretic mobilities in SDS/PAGE, were shown to be present in two different subcomplexes. Based on the analyses by two-dimensional gel electrophoresis, peptide mapping and immunoblotting, we concluded that A3b is a novel DAP different from 43DAG. Dystrophin, the protein responsible for Duchenne muscular dystrophy [ l , 21, is purified as a complex with several proteins called dystrophin-associated proteins (DAPs) from digitonin-solubilized rabbit skeletal muscle membrane [3, 41. At least four DAPs, given the symbols 156DAG, SODAG, 43DAG doublets (A3a and A3b) and 35DAG, were shown to exist in the transmembranous glycoprotein complex (GPC) [5, 61. In the structural study of dystrophin or the complex of dystrophin and its associated proteins (dystrophin-DAP complex) by limited calpain digestion [7], we showed that the locus of GPC binding on the dystrophin molecule is confined to the cysteine-rich domain and the first half of the Cterminal domain [6]. Since this locus is known to be the region whose loss is responsible for Duchenne muscular dystrophy [8], this finding was the first experimental evidence showing that the interaction of GPC with dystrophin is essential to prevent muscle degeneration.Among the components of GPC, 156DAG [9, 101 and 43DAG [ l l ] were shown to bind directly to laminin and dystrophin, respectively. However, the molecular organization of GPC has not yet been clarified. Thus, its study is indispensable in order to clarify the molecular basis behind muscular dystrophy. We showed immunochemically that the components of GPC (50DAG and 35DAG) are expressed in striated muscles but not in smooth muscles such as uterus and aorta, whereas 43DAG is rather ubiquitously expressed in various tissues [12, 131. On the other hand, it was reported that in the muscles of patients with severe childhood autosoma1 recessive muscular dystrophy (SCARMD), 5ODAG is lost and 35DAG is reduced, while dystrophin and other DAPs are preserved [ 141. Similar observations were made in the skeletal muscle of dystrophic hamster [15, 161. We also observed that in the skeletal...

Absence of γ‐sarcoglycan (35 DAG) in autosomal recessive muscular dystrophy linked to chromosome 13q12

“…It also appears that the absence of 7-sarcoglycan leads to a reduction of cx-and [~-sarcoglycan suggesting a close association between these three proteins in muscle sarcolemma. The close association of these sarcoglycan proteins has previously been suggested by Yoshida et al [36]. They have shown that n-octyl-13-D-glucoside disrupts the DGC into four groups of proteins: (i) ~-and [3-dystroglycan; (ii) dystrophin; (iii) the syntrophins; and (iv) c~-, 13-and y-sarcoglycan.…”

Early adenovirus-mediated gene transfer effectively prevents muscular dystrophy in alpha-sarcoglycan-deficient mice

“…[1][2][3][4][5][6][7][8] Mutations in four genes encoding sarcoglycan proteins (␣-, ␤-, ␥-and ␦-SG) are responsible for various forms of autosomal recessive limb-girdle muscular dystrophy (LGMD 2D, 2E, 2C and 2F, respectively). [9][10][11][12][13][14][15][16] This heterogeneous group of diseases nevertheless shares a common feature in that a primary deficiency of any of the sarcoglycan proteins leads to the reduction or the absence of all other members of the SG-SSPN complex.…”

Increasing complexity of the dystrophin-associated protein complex.

“…The 43DAG is the same as A3a, as determined by peptide sequence analysis of the purified gel band. A3b is distinct from A3a, as it has a different proteolytic cleavage pattern (14) and is unable to bind dystrophin (16). The 56-kDa amino-terminal core protein once glycosylated at many of the potential O-glycosylation sites gives rise to the fully processed 156DAG (15).…”