Characterization of δ-Sarcoglycan, a Novel Component of the Oligomeric Sarcoglycan Complex Involved in Limb-Girdle Muscular Dystrophy (1996)

The sarcoglycan complex is known to be involved in limb-girdle muscular dystrophy (LGMD) and is composed of at least three proteins: α-, β-, and γ-sarcoglycan. δ-Sarcoglycan has now been identified as a second 35-kDa sarcolemmal transmembrane glycoprotein that shares high homology with γ-sarcoglycan and is expressed mainly in skeletal and cardiac muscle. Biochemical analysis has demonstrated that γ- and δ-sarcoglycan are separate entities within the sarcoglycan complex and that all four sarcoglycans exist in the complex on a stoichiometrically equal basis. Immunohistochemical analysis of skeletal muscle biopsies from patients with LGMD2C, LGMD2D, and LGMD2E demonstrated a reduction of the entire sarcoglycan complex in these muscular dystrophies. Furthermore, we have mapped the human δ-sarcoglycan gene to chromosome 5q33-q34 in a region overlapping the recently linked autosomal recessive LGMD2F locus. The sarcoglycan complex is known to be involved in limb-girdle muscular dystrophy (LGMD) and is composed of at least three proteins: α-, β-, and γ-sarcoglycan. δ-Sarcoglycan has now been identified as a second 35-kDa sarcolemmal transmembrane glycoprotein that shares high homology with γ-sarcoglycan and is expressed mainly in skeletal and cardiac muscle. Biochemical analysis has demonstrated that γ- and δ-sarcoglycan are separate entities within the sarcoglycan complex and that all four sarcoglycans exist in the complex on a stoichiometrically equal basis. Immunohistochemical analysis of skeletal muscle biopsies from patients with LGMD2C, LGMD2D, and LGMD2E demonstrated a reduction of the entire sarcoglycan complex in these muscular dystrophies. Furthermore, we have mapped the human δ-sarcoglycan gene to chromosome 5q33-q34 in a region overlapping the recently linked autosomal recessive LGMD2F locus. INTRODUCTIONThe dystrophin-glycoprotein complex (DGC) 1The abbreviations used are: DGCdystrophin-glycoprotein complexDMDDuchenne muscular dystrophyLGMDlimb-girdle muscular dystrophyBSAbovine serum albuminPCRpolymerase chain reactionPBSphosphate-buffered salineFITCfluoroisothiocyanatePAGEpolyacrylamide gel electrophoresisHPLChigh pressure liquid chromatography. (1Campbell K.P. Kahl S.D. Nature. 1989; 338: 259-262Crossref PubMed Scopus (602) Google Scholar, 2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 4Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1099) Google Scholar) in skeletal muscle is a complex of sarcolemmal proteins and glycoproteins. It is composed of dystrophin, a cytoskeletal actin-binding protein (5Hoffman E.P. Brown Jr., R.H. Kunkel L.M. Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3602) Google Scholar, 6Koenig M. Monaco A.P. Kunkel L.M. Cell. 1988; 53: 219-228Abstract Full Text PDF PubMed Scopus (1255) Google Scholar, 7Hemmings L. Kuhlman P.A. Critchley D.R. J. Cell Biol. 1992; 116: 1369-1380Crossref PubMed Scopus (154) Google Scholar); the syntrophins, a 59-kDa triplet of intracellular proteins that bind the C-terminal domain of dystrophin (8Adams M.E. Butler M.H. Dwyer T.M. Peters M.F. Murnane A.A. Froehner S.C. Neuron. 1993; 11: 531-540Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 9Yang B. Ibraghimov-Beskrovnaya O. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1994; 269: 6040-6044Abstract Full Text PDF PubMed Google Scholar, 10Yang B. Jung D. Rafael J.A. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 4975-4978Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Ahn A.H. Kunkel L.M. J. Cell. Biol. 1995; 128: 363-371Crossref PubMed Scopus (189) Google Scholar, 12Suzuki A. Yoshida M. Ozawa E. J. Cell. Biol. 1995; 128: 373-381Crossref PubMed Scopus (151) Google Scholar); α-dystroglycan, a 156-kDa extracellular proteoglycan that binds the G domain of laminin (13Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1182) Google Scholar, 14Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1159) Google Scholar, 15Gee S.H. Blacher R.W. Douville P.J. Provost P.R. Yurchenco P.D. Carbonetto S. J. Biol. Chem. 1993; 268: 14972-14980Abstract Full Text PDF PubMed Google Scholar); β-dystroglycan, a 43-kDa transmembrane glycoprotein (2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 13Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1182) Google Scholar) that binds the cysteine-rich region of dystrophin (16Jung D. Yang B. Meyer J. Chamberlain J.A. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 17Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Crossref PubMed Scopus (219) Google Scholar); α-, β-, and γ-sarcoglycan, transmembrane glycoproteins of 50, 43, and 35 kDa, respectively (18Roberds S.L. Anderson R.D. Ibraghimov-Beskrovnaya O. Campbell K.P. J. Biol. Chem. 1993; 268: 23739-23742Abstract Full Text PDF PubMed Google Scholar, 19Roberds S.L. et al.Cell. 1994; 78: 625-633Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 20McNally E.M. Yoshida M. Mizuno Y. Ozawa E. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9690-9694Crossref PubMed Scopus (90) Google Scholar, 21Lim L.E. Duclos F. Broux O. et al.Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (430) Google Scholar, 22Bönnemann C.G. et al.Nature Genet. 1995; 11: 266-273Crossref PubMed Scopus (423) Google Scholar, 23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar, 24Jung D. et al.FEBS Lett. 1996; 381: 15-20Crossref PubMed Scopus (52) Google Scholar); and a 25-kDa transmembrane protein (1Campbell K.P. Kahl S.D. Nature. 1989; 338: 259-262Crossref PubMed Scopus (602) Google Scholar, 2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 4Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1099) Google Scholar). Recent experiments have demonstrated the existence of two complexes within the DGC (24Jung D. et al.FEBS Lett. 1996; 381: 15-20Crossref PubMed Scopus (52) Google Scholar, 25Yoshida M. Suzuki A. Yamamoto H. Mizuno Y. Ozawa E. Eur. J. Biochem. 1994; 222: 1055-1061Crossref PubMed Scopus (190) Google Scholar): the dystroglycan complex, composed of α- and β-dystroglycan, and the sarcoglycan complex, consisting of α-, β-, and γ-sarcoglycan.Defects in DGC components lead to muscle fiber necrosis, the major pathological event in muscular dystrophies (26Campbell K.P. Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (753) Google Scholar). In Duchenne muscular dystrophy (DMD), mutations in the dystrophin gene cause the loss of dystrophin and a reduction of the dystrophin-associated proteins (2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 5Hoffman E.P. Brown Jr., R.H. Kunkel L.M. Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3602) Google Scholar). One form of congenital muscular dystrophy has recently been characterized as being caused by mutations in the laminin α2-chain gene (27Helbling-Leclerc A. et al.Nat. Genet. 1995; 11: 216-218Crossref PubMed Scopus (560) Google Scholar, 28Nissinen M. et al.Am. J. Hum. Genet. 1996; 58: 1177-1184PubMed Google Scholar). Limb-girdle muscular dystrophy (LGMD) represents a clinically and genetically heterogeneous class of disorders (29Stevenson A.C. Ann. Eugen. 1953; 18: 50-91Crossref PubMed Scopus (8) Google Scholar, 30Walton J.N. Nattrass F.J. Brain. 1954; 77: 169-231Crossref PubMed Scopus (285) Google Scholar). They are inherited as either autosomal dominant or recessive traits. An autosomal dominant form, LGMD1A, was mapped to 5q31-q33 (31Speer M.C. et al.Am. J. Hum. Genet. 1992; 50: 1211-1217PubMed Google Scholar, 32Yamaoka L.Y. et al.Neuromusc. Disord. 1994; 4: 471-475Abstract Full Text PDF PubMed Scopus (28) Google Scholar), while six genes involved in the autosomal recessive forms were mapped to 15q15.1 (LGMD2A) (33Beckmann J.S. et al.C. R. Acad. Sci. (Paris). 1991; 312: 141-148PubMed Google Scholar), 2p16-p13 (LGMD2B) (34Bashir R. Strachan T. Keers S. Stephenson A. Mahjneh I. Marconi G. Nashef L. Bushby K.M. Hum. Mol. Genet. 1994; 3: 455-457Crossref PubMed Scopus (182) Google Scholar), 13q12 (LGMD2C) (23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar, 35Ben Othmane K. et al.Nat. Genet. 1992; 2: 315-317Crossref PubMed Scopus (173) Google Scholar, 36Azibi K. et al.Hum. Mol. Genet. 1993; 2: 1423-1428Crossref PubMed Scopus (106) Google Scholar), 17q12-q21.33 (LGMD2D) (19Roberds S.L. et al.Cell. 1994; 78: 625-633Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 20McNally E.M. Yoshida M. Mizuno Y. Ozawa E. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9690-9694Crossref PubMed Scopus (90) Google Scholar), 4q12 (LGMD2E) (21Lim L.E. Duclos F. Broux O. et al.Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (430) Google Scholar, 22Bönnemann C.G. et al.Nature Genet. 1995; 11: 266-273Crossref PubMed Scopus (423) Google Scholar), and most recently 5q33-q34 (LGMD2F) (37Passos-Bueno M.R. Moreira E.S. Vainzof M. Marie S.K. Zatz M. Hum. Mol. Genet. 1996; 5: 815-820Crossref PubMed Scopus (95) Google Scholar). Patients with LGMD2C, −2D, and −2E have a deficiency of components of the sarcoglycan complex resulting from mutations in the genes encoding γ-, α-, and β-sarcoglycan, respectively (19Roberds S.L. et al.Cell. 1994; 78: 625-633Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 21Lim L.E. Duclos F. Broux O. et al.Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (430) Google Scholar, 22Bönnemann C.G. et al.Nature Genet. 1995; 11: 266-273Crossref PubMed Scopus (423) Google Scholar, 23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar, 38Matsumura K. Tomé F.M. Collin H. Azibi K. Chaouch M. Kaplan J.-C. Fardeau M. Campbell K.P. Nature. 1992; 359: 320-322Crossref PubMed Scopus (232) Google Scholar, 39Piccolo F. et al.Nat. Genet. 1995; 10: 243-245Crossref PubMed Scopus (8) Google Scholar, 40Passos-Bueno M.R. et al.Hum. Mol. Genet. 1995; 4: 1163-1167Crossref PubMed Scopus (66) Google Scholar). The gene responsible for LGMD2A has been identified as the muscle-specific calpain (41Richard I. et al.Cell. 1995; 81: 27-40Abstract Full Text PDF PubMed Scopus (858) Google Scholar), whereas the genes responsible for LGMD1A, −2B, and −2F are still unknown.Here, we first describe the cloning of a cDNA encoding a fourth sarcoglycan protein (δ-sarcoglycan), a novel 35-kDa component of the dystrophin-glycoprotein complex. Human δ-sarcoglycan is a transmembrane glycoprotein that is mainly expressed in skeletal and cardiac muscle and shares about 60% protein sequence identity with the recently cloned γ-sarcoglycan. We demonstrate that γ- and δ-sarcoglycan are separate entities within the DGC, despite their similar molecular weights and protein sequences. δ-Sarcoglycan is not an isoform of γ-sarcoglycan. We also show that α-, β-, γ-, and δ-sarcoglycan are equal in the sarcoglycan complex on a stoichiometric basis. We demonstrate that δ-sarcoglycan is reduced along with α-, β-, and γ-sarcoglycan in LGMD2C, −2D, and −2E. Therefore, a common feature of LGMD2C, −2D, and −2E is a specific loss of the entire sarcoglycan complex from the sarcolemma membrane. Finally, we map the human δ-sarcoglycan gene to chromosome 5q33-34 within the recently reported disease interval for LGMD2F (5q33-34) (37Passos-Bueno M.R. Moreira E.S. Vainzof M. Marie S.K. Zatz M. Hum. Mol. Genet. 1996; 5: 815-820Crossref PubMed Scopus (95) Google Scholar).DISCUSSIONThe different components of the DGC were initially characterized based on their electrophoretic mobilities (1Campbell K.P. Kahl S.D. Nature. 1989; 338: 259-262Crossref PubMed Scopus (602) Google Scholar, 2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 4Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1099) Google Scholar). One component, identified as a 35-kDa protein band has recently been cloned and named γ-sarcoglycan (23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar). However, we demonstrated here by direct protein sequencing and molecular cloning that the 35-kDa protein band contained a second protein distinct from γ-sarcoglycan and was encoded by a different gene. The identity of this protein, named δ-sarcoglycan, was characterized using a specific antibody. In particular, δ-sarcoglycan copurified with the DGC and sarcoglycan complexes and colocalized with the other components of the DGC to the sarcolemma. The identification of this novel component of the sarcoglycan complex allowed us to update the molecular organization of the dystrophin-glycoprotein complex (Fig. 7).The predicted molecular mass of δ-sarcoglycan is 29 kDa, whereas its apparent molecular mass is 35 kDa. This discrepancy is mainly due to N-glycosylation of δ-sarcoglycan. Interestingly, the structure of δ-sarcoglycan is similar to β- and γ-sarcoglycan, with a short N-terminal intracellular domain and a large glycosylated C-terminal extracellular domain. Furthermore, δ- and γ-sarcoglycan share about 60% identity in primary structure, suggesting that these two proteins may have a similar function within the sarcoglycan complex. The structural homology as well as the similarity in molecular weight between γ- and δ-sarcoglycan have rendered these proteins indistinguishable by Western blot analysis. However, we demonstrated here that deglycosylation of purified DGC allows the separation of γ- and δ-sarcoglycan. Furthermore, the predicted isoelectric point (pI) of δ-sarcoglycan is 9.48, whereas γ-sarcoglycan displays a pI of 5.0 (23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar). Yamamoto et al. (51Yamamoto H. Hagiwara Y. Mizuno Y. Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1993; 114: 132-139Crossref PubMed Scopus (66) Google Scholar) have shown that the 35-kDa band from purified DGC is resolved on two-dimensional SDS-PAGE into two distinct spots, one being identified as γ-sarcoglycan with an acid pI and the second as an uncharacterized more basic protein. We observed that this more basic protein is specifically detected with the anti-δ-sarcoglycan antibodies. 2D. Jung, unpublished results. Therefore, two-dimensional SDS-PAGE gel analysis of purified DGC may also allow the separation of γ-sarcoglycan from δ-sarcoglycan.δ-Sarcoglycan, similarly to α- and γ-sarcoglycan, is strongly expressed in skeletal and cardiac muscle, whereas the β-sarcoglycan transcript is ubiquitously expressed. Furthermore, several transcripts were detected for δ-sarcoglycan, suggesting the existence of alternatively spliced forms. However, two peptides from the 35-kDa protein band were not found in γ- or δ-sarcoglycan. Using the TBLASTN search program, we identified an EST from mouse embryonic skeletal muscle containing one of these peptides. This EST encodes a C-terminal isoform of δ-sarcoglycan. Further work is currently in progress to characterize this isoform in human skeletal muscle.Immunostaining of skeletal muscle biopsies from patients with LGMD2C, −2D, or −2E demonstrated a reduction of all the sarcoglycan proteins (α, β, γ, and δ) at the sarcolemma, while the other components of the DGC were preserved. Therefore, a common feature of LGMD2C, −2D, and −2E is a specific absence of the sarcoglycan complex at the sarcolemma. This finding demonstrated that all sarcoglycans are required to maintain the integrity of the complex. We have previously demonstrated that in normal muscle the sarcoglycan proteins are tightly associated (24Jung D. et al.FEBS Lett. 1996; 381: 15-20Crossref PubMed Scopus (52) Google Scholar). This tight association may confer a high stability to the complex. The loss of only one sarcoglycan may render the complex more vulnerable to degradation and result in a destabilization of the complex during the process of its translocation to the sarcolemma. Such a mechanism has already been described for the cystic fibrosis transmembrane conductance regulator (52Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1215) Google Scholar). The mechanism by which the absence of the sarcoglycan complex from the membrane leads to a dystrophic phenotype is currently unknown. It is possible that the sarcoglycan complex contributes to the DGC function, which is believed to link the extracellular matrix and the cytoskeleton. Absence of this functional link may result in sarcolemmal instability and greater susceptibility to stress-induced damage, as it has been suggested for Duchenne muscular dystrophy (26Campbell K.P. Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (753) Google Scholar).So far, LGMD2C, −2D, and −2E have been characterized by primary defects in γ-, α-, and β-sarcoglycan, respectively (19Roberds S.L. et al.Cell. 1994; 78: 625-633Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 21Lim L.E. Duclos F. Broux O. et al.Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (430) Google Scholar, 22Bönnemann C.G. et al.Nature Genet. 1995; 11: 266-273Crossref PubMed Scopus (423) Google Scholar, 23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar, 38Matsumura K. Tomé F.M. Collin H. Azibi K. Chaouch M. Kaplan J.-C. Fardeau M. Campbell K.P. Nature. 1992; 359: 320-322Crossref PubMed Scopus (232) Google Scholar, 39Piccolo F. et al.Nat. Genet. 1995; 10: 243-245Crossref PubMed Scopus (8) Google Scholar), leading to the absence of the entire sarcoglycan complex. Since, the four sarcoglycan proteins are equal on a stoichiometric basis within the complex and that each protein is required to maintain the integrity of the complex, one may expect that a defect in δ-sarcoglycan also leads to the disruption of the sarcoglycan complex resulting in a LGMD phenotype. Interestingly, the δ-sarcoglycan gene has been mapped to chromosome 5q33-34, which overlaps with the region recently linked to a new form of autosomal recessive LGMD, LGMD2F (37Passos-Bueno M.R. Moreira E.S. Vainzof M. Marie S.K. Zatz M. Hum. Mol. Genet. 1996; 5: 815-820Crossref PubMed Scopus (95) Google Scholar).Note Added in ProofRecently, two papers have been published which report the cloning of δ-sarcoglycan (Nigro, V., Piluso, G., Belsito, A., Politano, L., Puca, A. A., Papparella, S., Rossi, E., Viglietto, G., Esposito, M. G., Abbondanza, C., Medici, N., Molinari, A. M., Nigro, G., and Puca, G. A. (1996) Hum. Mol. Genet. 5, 1179-1186) and the demonstration that a mutation in δ-sarcoglycan causes LGMD2F (Nigro, V., de Sá Moreira, E., Piluso, G., Vainzof, M., Belsito, A., Politano, L., Puca, A. A., Passos-Bueno, M. R., and Zatz, M. (1996) Nat. Genet. 14, 195-198). Interestingly, the reported sequence of δ-sarcoglycan differs in the C-terminal region from our δ-sarcoglycan sequence suggesting the existence of two isoforms of δ-sarcoglycan (δ1 and δ2) in skeletal muscle. INTRODUCTIONThe dystrophin-glycoprotein complex (DGC) 1The abbreviations used are: DGCdystrophin-glycoprotein complexDMDDuchenne muscular dystrophyLGMDlimb-girdle muscular dystrophyBSAbovine serum albuminPCRpolymerase chain reactionPBSphosphate-buffered salineFITCfluoroisothiocyanatePAGEpolyacrylamide gel electrophoresisHPLChigh pressure liquid chromatography. (1Campbell K.P. Kahl S.D. Nature. 1989; 338: 259-262Crossref PubMed Scopus (602) Google Scholar, 2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 4Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1099) Google Scholar) in skeletal muscle is a complex of sarcolemmal proteins and glycoproteins. It is composed of dystrophin, a cytoskeletal actin-binding protein (5Hoffman E.P. Brown Jr., R.H. Kunkel L.M. Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3602) Google Scholar, 6Koenig M. Monaco A.P. Kunkel L.M. Cell. 1988; 53: 219-228Abstract Full Text PDF PubMed Scopus (1255) Google Scholar, 7Hemmings L. Kuhlman P.A. Critchley D.R. J. Cell Biol. 1992; 116: 1369-1380Crossref PubMed Scopus (154) Google Scholar); the syntrophins, a 59-kDa triplet of intracellular proteins that bind the C-terminal domain of dystrophin (8Adams M.E. Butler M.H. Dwyer T.M. Peters M.F. Murnane A.A. Froehner S.C. Neuron. 1993; 11: 531-540Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 9Yang B. Ibraghimov-Beskrovnaya O. Moomaw C.R. Slaughter C.A. Campbell K.P. J. Biol. Chem. 1994; 269: 6040-6044Abstract Full Text PDF PubMed Google Scholar, 10Yang B. Jung D. Rafael J.A. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 4975-4978Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Ahn A.H. Kunkel L.M. J. Cell. Biol. 1995; 128: 363-371Crossref PubMed Scopus (189) Google Scholar, 12Suzuki A. Yoshida M. Ozawa E. J. Cell. Biol. 1995; 128: 373-381Crossref PubMed Scopus (151) Google Scholar); α-dystroglycan, a 156-kDa extracellular proteoglycan that binds the G domain of laminin (13Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1182) Google Scholar, 14Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1159) Google Scholar, 15Gee S.H. Blacher R.W. Douville P.J. Provost P.R. Yurchenco P.D. Carbonetto S. J. Biol. Chem. 1993; 268: 14972-14980Abstract Full Text PDF PubMed Google Scholar); β-dystroglycan, a 43-kDa transmembrane glycoprotein (2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 13Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1182) Google Scholar) that binds the cysteine-rich region of dystrophin (16Jung D. Yang B. Meyer J. Chamberlain J.A. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 17Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Crossref PubMed Scopus (219) Google Scholar); α-, β-, and γ-sarcoglycan, transmembrane glycoproteins of 50, 43, and 35 kDa, respectively (18Roberds S.L. Anderson R.D. Ibraghimov-Beskrovnaya O. Campbell K.P. J. Biol. Chem. 1993; 268: 23739-23742Abstract Full Text PDF PubMed Google Scholar, 19Roberds S.L. et al.Cell. 1994; 78: 625-633Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 20McNally E.M. Yoshida M. Mizuno Y. Ozawa E. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9690-9694Crossref PubMed Scopus (90) Google Scholar, 21Lim L.E. Duclos F. Broux O. et al.Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (430) Google Scholar, 22Bönnemann C.G. et al.Nature Genet. 1995; 11: 266-273Crossref PubMed Scopus (423) Google Scholar, 23Noguchi S. et al.Science. 1995; 270: 819-822Crossref PubMed Scopus (475) Google Scholar, 24Jung D. et al.FEBS Lett. 1996; 381: 15-20Crossref PubMed Scopus (52) Google Scholar); and a 25-kDa transmembrane protein (1Campbell K.P. Kahl S.D. Nature. 1989; 338: 259-262Crossref PubMed Scopus (602) Google Scholar, 2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 3Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (448) Google Scholar, 4Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1099) Google Scholar). Recent experiments have demonstrated the existence of two complexes within the DGC (24Jung D. et al.FEBS Lett. 1996; 381: 15-20Crossref PubMed Scopus (52) Google Scholar, 25Yoshida M. Suzuki A. Yamamoto H. Mizuno Y. Ozawa E. Eur. J. Biochem. 1994; 222: 1055-1061Crossref PubMed Scopus (190) Google Scholar): the dystroglycan complex, composed of α- and β-dystroglycan, and the sarcoglycan complex, consisting of α-, β-, and γ-sarcoglycan.Defects in DGC components lead to muscle fiber necrosis, the major pathological event in muscular dystrophies (26Campbell K.P. Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (753) Google Scholar). In Duchenne muscular dystrophy (DMD), mutations in the dystrophin gene cause the loss of dystrophin and a reduction of the dystrophin-associated proteins (2Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-331Crossref PubMed Scopus (807) Google Scholar, 5Hoffman E.P. Brown Jr., R.H. Kunkel L.M. 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The gene responsible for LGMD2A has been identified as the muscle-specific calpain (41Richard I. et al.Cell. 1995; 81: 27-40Abstract Full Text PDF PubMed Scopus (858) Google Scholar), whereas the genes responsible for LGMD1A, −2B, and −2F are still unknown.Here, we first describe the cloning of a cDNA encoding a fourth sarcoglycan protein (δ-sarcoglycan), a novel 35-kDa component of the dystrophin-glycoprotein complex. Human δ-sarcoglycan is a transmembrane glycoprotein that is mainly expressed in skeletal and cardiac muscle and shares about 60% protein sequence identity with the recently cloned γ-sarcoglycan. We demonstrate that γ- and δ-sarcoglycan are separate entities within the DGC, despite their similar molecular weights and protein sequences. δ-Sarcoglycan is not an isoform of γ-sarcoglycan. We also show that α-, β-, γ-, and δ-sarcoglycan are equal in the sarcoglycan complex on a stoichiometric basis. We demonstrate that δ-sarcoglycan is reduced along with α-, β-, and γ-sarcoglycan in LGMD2C, −2D, and −2E. Therefore, a common feature of LGMD2C, −2D, and −2E is a specific loss of the entire sarcoglycan complex from the sarcolemma membrane. Finally, we map the human δ-sarcoglycan gene to chromosome 5q33-34 within the recently reported disease interval for LGMD2F (5q33-34) (37Passos-Bueno M.R. Moreira E.S. Vainzof M. Marie S.K. Zatz M. Hum. Mol. Genet. 1996; 5: 815-820Crossref PubMed Scopus (95) Google Scholar).