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• W2137636698 abstract "Dystroglycan is a widely expressed extracellular matrix receptor that plays a critical role in basement membrane formation, epithelial development, and synaptogenesis. Dystroglycan was originally characterized in skeletal muscle as an integral component of the dystrophin glycoprotein complex, which is critical for muscle cell viability. Although the dystroglycan complex has been well characterized in skeletal muscle, there is little information on the structural composition of the dystroglycan complex outside skeletal muscle. Here we have biochemically characterized the dystroglycan complex in lung and kidney. We demonstrate that the presence of sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle. The smooth muscle dystroglycan complex in lung, composed of dystroglycan, dystrophin/utrophin, β-, δ-, ε-sarcoglycan, and sarcospan, can be biochemically separated from epithelial dystroglycan, which is not associated with any of the known sarcoglycans or sarcospan. Similarly, dystroglycan in kidney epithelial cells is not associated with any of the sarcoglycans or sarcospan. Thus, our data demonstrate that there are distinct dystroglycan complexes in non-skeletal muscle organs as follows: one from smooth muscle, which is associated with sarcoglycans forming a similar complex as in skeletal muscle, and one from epithelial cells. Dystroglycan is a widely expressed extracellular matrix receptor that plays a critical role in basement membrane formation, epithelial development, and synaptogenesis. Dystroglycan was originally characterized in skeletal muscle as an integral component of the dystrophin glycoprotein complex, which is critical for muscle cell viability. Although the dystroglycan complex has been well characterized in skeletal muscle, there is little information on the structural composition of the dystroglycan complex outside skeletal muscle. Here we have biochemically characterized the dystroglycan complex in lung and kidney. We demonstrate that the presence of sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle. The smooth muscle dystroglycan complex in lung, composed of dystroglycan, dystrophin/utrophin, β-, δ-, ε-sarcoglycan, and sarcospan, can be biochemically separated from epithelial dystroglycan, which is not associated with any of the known sarcoglycans or sarcospan. Similarly, dystroglycan in kidney epithelial cells is not associated with any of the sarcoglycans or sarcospan. Thus, our data demonstrate that there are distinct dystroglycan complexes in non-skeletal muscle organs as follows: one from smooth muscle, which is associated with sarcoglycans forming a similar complex as in skeletal muscle, and one from epithelial cells. kilobase pair polyacrylamide gel electrophoresis wheat germ agglutinin α-Dystroglycan is a highly glycosylated peripheral membrane protein associated with the membrane-spanning β-dystroglycan. These two proteins were originally isolated from skeletal muscle as components of a large oligomeric complex further comprised of dystrophin, the syntrophin, and sarcoglycan complexes and the recently identified protein sarcospan (1Ervasti J.M. Ohlendieck K. Kahl S.D. Gaver M.G. Campbell K.P. Nature. 1990; 345: 315-319Crossref PubMed Scopus (824) Google Scholar, 2Yoshida M. Ozawa E. J. Biochem. (Tokyo). 1990; 108: 748-752Crossref PubMed Scopus (452) Google Scholar, 3Froehner S.C. Adams M.E. Peters M.F. Gee S.H. Soc. Gen. Physiol. Ser. 1997; 52: 197-207PubMed Google Scholar, 4Lim L.E. Campbell K.P. Curr. Opin. Neurol. 1998; 11: 443-452Crossref PubMed Scopus (125) Google Scholar, 5Crosbie R.H. Heighway J. Venzke D.P. Lee J.C. Campbell K.P. J. Biol. Chem. 1997; 272: 31221-31224Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In skeletal muscle, α-dystroglycan binds to the extracellular matrix component laminin α2-chain (6Sunada Y. Bernier S.M. Kozak C.A. Yamada Y. Campbell K.P. J. Biol. Chem. 1994; 269: 13729-13732Abstract Full Text PDF PubMed Google Scholar), whereas the intracellular domain of β-dystroglycan binds to the cytoskeletal protein dystrophin (7Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Thus, dystroglycan is thought to act as a transmembrane link between the extracellular matrix and the cytoskeleton, and this linkage seems to be crucial for maintaining normal function (4Lim L.E. Campbell K.P. Curr. Opin. Neurol. 1998; 11: 443-452Crossref PubMed Scopus (125) Google Scholar, 8Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1185) Google Scholar, 9Durbeej M. Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1998; 10: 594-601Crossref PubMed Scopus (138) Google Scholar). In addition, a defect in any of the sarcoglycans results in specific loss of the sarcoglycan-sarcospan subcomplex, destabilization of α-dystroglycan, and eventually muscle cell death (4Lim L.E. Campbell K.P. Curr. Opin. Neurol. 1998; 11: 443-452Crossref PubMed Scopus (125) Google Scholar). Many of the dystroglycan-associated proteins found in skeletal and cardiac muscle are also expressed in other tissues. For example, β- and δ-sarcoglycan, sarcospan, and the syntrophins are all at the RNA level expressed in tissues other than skeletal and cardiac muscle (3Froehner S.C. Adams M.E. Peters M.F. Gee S.H. Soc. Gen. Physiol. Ser. 1997; 52: 197-207PubMed Google Scholar, 5Crosbie R.H. Heighway J. Venzke D.P. Lee J.C. Campbell K.P. J. Biol. Chem. 1997; 272: 31221-31224Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 10Lim L.E. Duclos F. Broux O. Bourg N. Sunada Y. Allamand V. Meyer J. Richard I. Moomaw C. Slaughter C. Tomé F.M.S. Fardeau M. Lackson C.E. Beckmann J.S. Campbell K.P. Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (431) Google Scholar, 11Bönnemann C.G. Modi R. Noguchi S. Mizuno Y. Yoshida M. Gussoni E. McNally E.M. Duggan C.A. Hoffman E.P. Ozawa E. Kunkel L.M. Nat. Genet. 1995; 11: 266-273Crossref PubMed Scopus (425) Google Scholar, 12Nigro 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. Puca G.A. Hum. Mol. Genet. 1996; 5: 1179-1186Crossref PubMed Scopus (180) Google Scholar, 13Jung D. Duclos F. Apostol B. Straub V. Lee J.C. Allamand V. Venzke D.P. Sunada Y. Moomaw C.R. Leveille C.J. Slaughter C.A. Crawford T.O. McPherson J.D. Campbell K.P. J. Biol. Chem. 1996; 271: 32321-32329Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In addition, several dystrophin isoforms are ubiquitously expressed, including the autosomal dystrophin homologue utrophin (14Byers T. Lidov H.G. Kunkel L.M. Nat. Genet. 1993; 4: 87-93Crossref PubMed Scopus (252) Google Scholar, 15Schofield J.N. Blake D.J. Simmons C. Morris G.E. Tinsley J.M. Davies K.E. Edwards Y.H. Hum. Mol. Genet. 1994; 8: 1309-1316Google Scholar, 16Durbeej M. Jung D. Hjalt T. Campbell K.P. Ekblom P. Dev. Biol. 1997; 181: 156-167Crossref PubMed Scopus (32) Google Scholar, 17Grady R.M. Merlie J.P. Sanes J.R. J. Cell Biol. 1997; 136: 871-882Crossref PubMed Scopus (195) Google Scholar). By far, dystroglycan is the most widely expressed component. Dystroglycan is expressed at high levels in many cell types and is particularly prominent on the basal side of epithelial cells facing basement membranes (18Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1198) Google Scholar, 19Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Crossref PubMed Scopus (188) Google Scholar, 20Gorecki D.C. Derry J.M.J. Barnard E.A. Hum. Mol. Gen. 1994; 3: 1589-1597Crossref PubMed Scopus (66) Google Scholar, 21Williamson R.A. Henry M.D. Daniels K.J. Hrstka R.F. Lee J.C. Sunada Y. Ibraghimov-Beskrovnaya O. Campbell K.P. Hum. Mol. Genet. 1997; 6: 831-841Crossref PubMed Scopus (450) Google Scholar, 22Blank M. Koulen P. Kroger S. J. Comp. Neurol. 1997; 389: 668-678Crossref PubMed Scopus (42) Google Scholar, 23Durbeej M. Henry M.D. Ferletta M. Campbell K.P. Ekblom P. J. Histochem. Cytochem. 1998; 46: 449-457Crossref PubMed Scopus (155) Google Scholar). Moreover, α-dystroglycan binds laminin-1, agrin, and perlecan (24Gee 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, 25Ruegg M.A. Bixby J.L Trends Neurosci. 1998; 21: 22-27Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 26Gesemannn M. Brancaccio A. Schumacher B. Ruegg M.A. J. Biol. Chem. 1998; 273: 600-605Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 27Peng H.B. Ali A.A. Daggett D.F. Rauvala H. Hassel J.R. Smalheiser N.R. Cell. Adhes. Commun. 1998; 5: 475-489Crossref PubMed Scopus (146) Google Scholar, 28Talts J.F. Zeynep A. Goring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar). β-Dystroglycan, in turn, also binds to the dystrophin isoforms Dp71, Dp116, and Dp260 (7Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 29Saito F. Masaki T. Kamakura K. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Sunada Y. Shimizu T. Matsumura K. J. Biol. Chem. 1999; 274: 8240-8246Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and has been shown to be associated with utrophin (30James M. Man N.T. Wise C.J. Jones G.E. Morris G.E. Cell Motil. Cytoskeleton. 1996; 23: 163-174Crossref Scopus (45) Google Scholar). Thus, different dystroglycan complexes may form in different tissues implying that dystroglycan may have important roles outside skeletal muscle. Indeed, dystroglycan has been implicated as a laminin/agrin receptor involved in epithelial cell development, basement membrane formation, and synaptogenesis (19Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Crossref PubMed Scopus (188) Google Scholar, 21Williamson R.A. Henry M.D. Daniels K.J. Hrstka R.F. Lee J.C. Sunada Y. Ibraghimov-Beskrovnaya O. Campbell K.P. Hum. Mol. Genet. 1997; 6: 831-841Crossref PubMed Scopus (450) Google Scholar, 31Henry M.D. Campbell K.P. Cell. 1998; 95: 859-870Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 32Montanaro F. Gee S.H. Jacobson C. Lindenbaum M.H. Froehner S.C. Carbonetto S. J. Neurosci. 1998; 18: 1250-1260Crossref PubMed Google Scholar, 33Jacobson C. Montanaro F. Lindenbaum M. Carbonetto S. Ferns M. J. Neurosci. 1998; 18: 6340-6348Crossref PubMed Google Scholar). Recently, it was also demonstrated that dystroglycan serves as a receptor for both lymphocytic choriomeningitis virus (34Cao W. Henry M.D. Borrow P. Yamada H. Elder J.H. Ravkov E.V. Nichol S.T. Compans R.W. Campbell K.P. Oldstone M.B. Science. 1998; 11: 2079-2081Crossref Scopus (560) Google Scholar) and Mycobacterium leprae (35Rambukkana A. Yamada H. Zanazzi G. Mathus T. Salzer J.L. Yurchenco P. Campbell K.P. Fischetti V.A. Science. 1998; 22: 2076-2079Crossref Scopus (219) Google Scholar). The roles of the dystroglycan-associated proteins outside skeletal muscle, however, have remained elusive. To elucidate potential functional roles, a first important step includes the determination of the cellular localization of these proteins. Specifically, are the dystroglycan-associated proteins that are present in non-skeletal muscle organs derived from smooth muscle, epithelial cells, or both? ε-Sarcoglycan, a homologue of α-sarcoglycan, was recently identified and shown to be expressed in a wide variety of tissues including epithelial cells (36Ettinger A.J. Feng G. Sanes J.R. J. Biol. Chem. 1997; 272: 32534-32538Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 37McNally E.M. Ly C.T. Kunkel L.M. FEBS Lett. 1998; 422: 27-32Crossref PubMed Scopus (121) Google Scholar). ε-Sarcoglycan has also been identified as an integral component of the smooth muscle sarcoglycan complex 1V. Straub and K. P. Campbell, unpublished data. and could also be part of an epithelial dystroglycan complex, possibly along with β- and δ-sarcoglycan and sarcospan. Although dystroglycan has been shown to play important roles outside skeletal muscle, the dystroglycan complex from other cell types is only partially characterized. In peripheral nerve, the molecular mass of α-dystroglycan is 120 kDa in contrast to 156 kDa in skeletal muscle (38Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Crossref PubMed Scopus (149) Google Scholar, 39Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1119) Google Scholar). The molecular weight of α-dystroglycan in epithelial cells has remained elusive. A 120-kDa band has been identified in lung (18Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1198) Google Scholar), but it is unclear whether the 120-kDa dystroglycan is derived from epithelial cells or smooth muscle. Moreover, α-dystroglycan is believed to act as a laminin-1 receptor involved in the development of kidney epithelial cells (19Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Crossref PubMed Scopus (188) Google Scholar). However, a direct association between kidney α-dystroglycan and laminin-1 has not been demonstrated. Here, we demonstrate that the presence of the sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle within lung, and we show that epithelial dystroglycan is not associated with any of the known sarcoglycans and can be separated from the smooth muscle dystroglycan complex. Dystroglycan, β-sarcoglycan, δ-sarcoglycan, ε-sarcoglycan, and sarcospan are all part of a smooth muscle complex, whereas neither α-, β-, γ-, and δ-sarcoglycan nor sarcospan are expressed in epithelial cells from lung and kidney. ε-Sarcoglycan, on the other hand, is expressed in epithelial cells (36Ettinger A.J. Feng G. Sanes J.R. J. Biol. Chem. 1997; 272: 32534-32538Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) but is not complexed with dystroglycan, as revealed by sucrose gradient fractionation. We have also partially characterized dystroglycan from kidney, an organ mainly composed of epithelial cells. We found that the major part of α-dystroglycan from adult kidney has a molecular mass of 156 kDa, whereas the major part of α-dystroglycan from fetal kidney has a molecular mass of 120 kDa. Both forms were shown to bind laminin-1. Taken together, our results demonstrate a distinct smooth muscle dystroglycan complex and a distinct epithelial dystroglycan complex, and this information will be useful for further investigation of dystroglycan function. New Zealand White rabbits were from Knapp Creek Farms (Amana, IA). Mice (C57BL/10) were bred at the University of Iowa from stocks originally obtained from Jackson Laboratories (Jackson Laboratories, Bar Harbor, ME). F1B control and BIO 14.6 cardiomyopathic hamsters were obtained from BioBreeders (Fitchburg, MA). All animals were kept in the animal care unit of the University of Iowa College of Medicine according to the animal care guidelines. Total RNA from rabbit kidney, lung, and skeletal muscle was extracted using RNAzol B (Tel-Test) according to the manufacturer's specifications. 20 μg of total RNA was electrophoresed on a 1.25% agarose gel containing 5% formaldehyde. Equal loading was verified by ethidium bromide visualization of the RNA, which was subsequently transferred to Hybond N membrane (Amersham Pharmacia Biotech). RNA was cross-linked to the membrane using a Stratagene UV cross-linker. Membranes were then prehybridized and hybridized using standard methods (40Straub V. Duclos F. Venzke D. Lee J.C. Cutshall S. Leveille C.J. Campbell K.P. Am. J. Pathol. 1998; 153: 1623-1630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Hybridization was performed with the following cDNA probes: a 1.1-kb2 cDNA probe representing mouse α-sarcoglycan (41Duclos F. Straub V. Moore S.A. Venzke D.P. Hrstka R.F. Crosbie R.H. Durbeej M. Lebakken C.S. Ettinger A.J. Holt K.H. Lim L.E. Sanes J.R. Davidson B.L. Faulkner J.A. Williamson R. Campbell K.P. J. Cell Biol. 1998; 142: 1461-1471Crossref PubMed Scopus (311) Google Scholar); a 1-kb cDNA probe representing hamster β-sarcoglycan (40Straub V. Duclos F. Venzke D. Lee J.C. Cutshall S. Leveille C.J. Campbell K.P. Am. J. Pathol. 1998; 153: 1623-1630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar); a 1-kb cDNA probe representing hamster γ-sarcoglycan (40Straub V. Duclos F. Venzke D. Lee J.C. Cutshall S. Leveille C.J. Campbell K.P. Am. J. Pathol. 1998; 153: 1623-1630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar); a 1-kb probe representing hamster δ-sarcoglycan (40Straub V. Duclos F. Venzke D. Lee J.C. Cutshall S. Leveille C.J. Campbell K.P. Am. J. Pathol. 1998; 153: 1623-1630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar); a 0.7-kb cDNA probe representing human sarcospan (5Crosbie R.H. Heighway J. Venzke D.P. Lee J.C. Campbell K.P. J. Biol. Chem. 1997; 272: 31221-31224Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar); a rabbit full-length clone of dystroglycan (18Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1198) Google Scholar); a 1-kb cDNA clone corresponding to exon 70 through the beginning of 79 of mouse utrophin; an 850-base pair Eco RI/Xho I fragment derived from EST clone 1149778 corresponding to mouse ε-sarcoglycan (36Ettinger A.J. Feng G. Sanes J.R. J. Biol. Chem. 1997; 272: 32534-32538Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). cDNA inserts were labeled with [α-32P]dCTP to a specific activity of 2 × 108 cpm/μg DNA using Ready Prime kit (Amersham Pharmacia Biotech). Washes were carried out at 65 °C in 1× SSC, 1% SDS initially and then in 0.1× SSC, 0.1% SDS. Membranes were exposed for autoradiography. Mouse monoclonal antibody IIH6 against α-dystroglycan (8Ervasti J.M. Campbell K.P. J. Cell Biol. 1993; 122: 809-823Crossref PubMed Scopus (1185) Google Scholar) and rabbit polyclonal antibodies against α-sarcoglycan (rabbit 98) (42Roberds S.L. Anderson R.D. Ibraghimov-Beskrovnaya O. Campbell K.P. J. Biol. Chem. 1993; 268: 23739-23742Abstract Full Text PDF PubMed Google Scholar), δ-sarcoglycan (rabbit 215) (43Holt K.H. Lim L.E. Straub V. Venzke D. Duclos F. Anderson R.D. Davidson B.L. Campbell K.P. Mol. Cell. 1998; 1: 841-848Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), ε-sarcoglycan (rabbit 232) (41Duclos F. Straub V. Moore S.A. Venzke D.P. Hrstka R.F. Crosbie R.H. Durbeej M. Lebakken C.S. Ettinger A.J. Holt K.H. Lim L.E. Sanes J.R. Davidson B.L. Faulkner J.A. Williamson R. Campbell K.P. J. Cell Biol. 1998; 142: 1461-1471Crossref PubMed Scopus (311) Google Scholar), sarcospan (rabbits 216 and 235) (5Crosbie R.H. Heighway J. Venzke D.P. Lee J.C. Campbell K.P. J. Biol. Chem. 1997; 272: 31221-31224Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar,41Duclos F. Straub V. Moore S.A. Venzke D.P. Hrstka R.F. Crosbie R.H. Durbeej M. Lebakken C.S. Ettinger A.J. Holt K.H. Lim L.E. Sanes J.R. Davidson B.L. Faulkner J.A. Williamson R. Campbell K.P. J. Cell Biol. 1998; 142: 1461-1471Crossref PubMed Scopus (311) Google Scholar), and utrophin (rabbit 56) (44Ohlendieck K. Ervasti J.M. Matsumura K. Kahl S.D. Leveille C.J. Campbell K.P. Neuron. 1991; 7: 499-508Abstract Full Text PDF PubMed Scopus (322) Google Scholar) were previously described. An affinity purified rabbit antibody (rabbit 245) was produced against a COOH-terminal fusion protein of γ-sarcoglycan containing amino acids 167–291. Goat polyclonal antibodies against β-sarcoglycan (goat 26) (41Duclos F. Straub V. Moore S.A. Venzke D.P. Hrstka R.F. Crosbie R.H. Durbeej M. Lebakken C.S. Ettinger A.J. Holt K.H. Lim L.E. Sanes J.R. Davidson B.L. Faulkner J.A. Williamson R. Campbell K.P. J. Cell Biol. 1998; 142: 1461-1471Crossref PubMed Scopus (311) Google Scholar) and goat 20 antiserum (18Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1198) Google Scholar) were described previously. Monoclonal antibodies Ad1/20A6 against α-sarcoglycan, βSarc1/5B1 against β-sarcoglycan, and 35DAG/21B5 against γ-sarcoglycan were generated in collaboration with Louise V. B. Anderson (Newcastle General Hospital, Newcastle upon Tyne, UK). Monoclonal antibody 43 DAG/8D5 against β-dystroglycan was also generated by Louise V. B. Anderson. Polyclonal antibodies against dystroglycan fusion protein B (18Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1198) Google Scholar) were affinity purified from sheep OR12. Sheep OR12 was injected with fusion protein B and boosted with fusion protein D (18Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1198) Google Scholar). Preparation of KCl-washed membranes from adult rabbit kidney, lung, and skeletal muscle were described previously (45Ohlendieck K. Ervasti J.M. Snook J.B. Campbell K.P. J. Cell Biol. 1991; 112: 135-148Crossref PubMed Scopus (241) Google Scholar). Membranes were resolved by SDS-PAGE on 3–12% linear gradients and transferred to nitrocellulose membranes (46Towbin H.T. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44938) Google Scholar). Immunoblot staining was performed as described previously (45Ohlendieck K. Ervasti J.M. Snook J.B. Campbell K.P. J. Cell Biol. 1991; 112: 135-148Crossref PubMed Scopus (241) Google Scholar). Tissues (1–5 g) were solubilized in 100 ml of 50 mm Tris-HCl, pH 7.4, 500 mm NaCl containing 1% digitonin in the presence of pepstatin A (0.6 μg/ml), aprotinin (0.5 μg/ml), leupeptin (0.5 μg/ml), phenylmethylsulfonyl fluoride (0.1 mm), benzamidine (0.75 mm), calpain inhibitor I (5 nm), and calpeptin (5 nm). The solubilized proteins were circulated overnight at 4 °C on an 8-ml wheat germ agglutinin (WGA)-agarose column (Vector Laboratories). The columns were washed with 40 ml of 50 mm Tris-HCl, pH 7.4, 500 mm NaCl containing 0.1% digitonin and eluted with 0.3m N-acetylglucosamine in 50 mmTris-HCl, 500 mm NaCl containing 0.1% digitonin. The WGA eluate was incubated with protein G (Amersham Pharmacia Biotech)-agarose for 1 h, to remove contaminating immunoglobulins. The protein G beads were spun down, and the supernatant was diluted to 100 mm NaCl with 50 mm Tris-HCl, pH 7.4, containing 0.1% digitonin and applied to a DEAE-cellulose column and washed with 50 mm Tris-HCl, pH 7.4, 100 mm NaCl containing 0.1% digitonin. The column was eluted with a gradient of 100–750 mm NaCl buffer containing 50 mmTris-HCl, pH 7.4, and 0.1% digitonin, and 4-ml fractions were collected. The fractions containing dystroglycan were pooled and concentrated to 400 μl using Centricon-30 concentrators (Amicon). The samples were applied to a 5–30% sucrose gradient and centrifuged with a Beckman VTi65.1 vertical rotor at 200,000 × g for 3 h at 4 °C. The gradients were fractionated into 800-μl fractions, which were immunoblotted as described (45Ohlendieck K. Ervasti J.M. Snook J.B. Campbell K.P. J. Cell Biol. 1991; 112: 135-148Crossref PubMed Scopus (241) Google Scholar). Mouse EHS laminin (laminin-1) was generously provided by Dr. Hynda K. Kleinman at the National Institutes of Health. Laminin-1 was biotinylated with NHS-biotin (Vector Laboratories) in a molar ratio of 1:750–1000 in reaction buffer 0.2 m NaHCO3, pH 8.5, containing 0.5 m NaCl. The reaction was quenched, and unbound biotin was removed by dialysis against Tris-buffered saline (50 mm Tris, pH 7.5, 150 mm NaCl). Dystroglycan containing sucrose gradient fractions were separated on 3–12% gradient gels and transferred to nitrocellulose membranes as described (45Ohlendieck K. Ervasti J.M. Snook J.B. Campbell K.P. J. Cell Biol. 1991; 112: 135-148Crossref PubMed Scopus (241) Google Scholar). The blots were blocked in laminin binding buffer (140 mm NaCl, 1 mm MgCl2, 10 mm triethanolamine, pH 7.6) containing 5% non-fat dry milk and subsequently washed for 90 min in laminin binding buffer containing 0.05% Tween 20 (TLBB). Blots were incubated in TLBB containing 3% bovine serum albumin and biotinylated laminin-1 for 8 h and washed in TLBB for 20 min. After incubation with ABC reagents (Vector Laboratories), the blots were washed in TLBB for 20 min and developed in 4-chloro-1-naphthol and H2O2. For immunofluorescence analysis, 8-μm cryosections were prepared from C57BL/10 wild type mouse lungs, kidneys, and skeletal muscle, and F1B and BIO 14.6 hamster lungs, kidneys, and skeletal muscle. All procedures were performed at room temperature. Sections were blocked with 3% bovine serum albumin in phosphate-buffered saline for 30 min and then incubated with the primary antibodies for 90 min or overnight. After washing in phosphate-buffered saline, sections were incubated with Cy3-conjugated secondary antibodies (Vector Laboratories) for 90 min and then mounted with Vectashield mounting medium and observed under a Bio-Rad MRC-600 laser scanning confocal microscope. In order to analyze which proteins are associated with dystroglycan in kidney and lung, we first screened for the presence of sarcoglycan mRNAs in these organs. As expected, the 1.6-kb α-sarcoglycan transcript was only detected in skeletal muscle (Fig. 1). The previously characterized 4.4- and 3.0-kb transcripts representing β- sarcoglycan (10Lim L.E. Duclos F. Broux O. Bourg N. Sunada Y. Allamand V. Meyer J. Richard I. Moomaw C. Slaughter C. Tomé F.M.S. Fardeau M. Lackson C.E. Beckmann J.S. Campbell K.P. Nat. Genet. 1995; 11: 257-265Crossref PubMed Scopus (431) Google Scholar, 11Bönnemann C.G. Modi R. Noguchi S. Mizuno Y. Yoshida M. Gussoni E. McNally E.M. Duggan C.A. Hoffman E.P. Ozawa E. Kunkel L.M. Nat. Genet. 1995; 11: 266-273Crossref PubMed Scopus (425) Google Scholar) were present in both lung and kidney as well as the previously characterized δ-sarcoglycan mRNAs (40Straub V. Duclos F. Venzke D. Lee J.C. Cutshall S. Leveille C.J. Campbell K.P. Am. J. Pathol. 1998; 153: 1623-1630Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) of 9.5, 4.3, 2.3, and 1.4 kb (Fig. 1). In kidney and lung the 9.5-kb δ-sarcoglycan transcript appeared to be the most prominent. γ-Sarcoglycan mRNAs of 2.5 and 1.7 kb were faintly expressed in kidney, moderately in lung, and strongly in skeletal muscle (Fig. 1). Sarcospan mRNA has been shown to be expressed in several organs (5Crosbie R.H. Heighway J. Venzke D.P. Lee J.C. Campbell K.P. J. Biol. Chem. 1997; 272: 31221-31224Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and the 4.5-kb transcript could also be detected in kidney and lung (Fig. 1). In contrast to the sarcoglycan and sarcospan mRNAs, which are all more highly expressed in skeletal muscle than kidney and lung, an equal intensity of the 5.8-kb dystroglycan transcript was detected in the three organs (Fig. 1). The 1.8-kb ε-sarcoglycan mRNA was highly expressed in kidney and lung (Fig. 1). ε-Sarcoglycan was also detected in skeletal muscle but at a lower level. Similarly, the 13-kb utrophin transcript was also detected in kidney and lung and at a lower level in skeletal muscle (Fig. 1). Because it is possible that transcripts are present in cells without being translated, we next analyzed whether the sarcoglycan protein products were expressed in the kidney and lung. KCl-washed membranes from kidney, lung, and skeletal muscle were screened for protein expression. Although the β-, γ-, and δ-sarcoglycan transcripts were readily expressed in the kidney, we were not able to detect their corresponding polypeptides (Fig. 1). In the lung, however, the 43-kDa β-sarcoglycan, the 35-kDa γ-sarcoglycan, and the 35-kDa δ-sarcoglycans were clearly detectable (Fig. 1). Likewise, the 25-kDa sarcospan was not present in the kidney but was present in the lung (Fig. 1). In contrast, dystroglycan with a molecular mass of 156 kDa, the 46-kDa ε-sarcoglycan, and the 395-kDa utrophin were all readily detected in kidney and lung (Fig. 1). Previous reports have shown that full-length dystrophin is present in the smooth muscle of kidney but not epithelial cells (16Durbeej M. Jung D. Hjalt T. Campbell K.P. Ekblom P. Dev. Biol. 1997; 181: 156-167Crossref PubMed Scopus (32) Google Scholar, 47Lidow H.G.W. Kunkel L.M. Lab. Invest. 1998; 78: 1543-1551PubMed Google Scholar). In the lung, dystrophin is exclusively expressed in smooth muscle (48Houzelstein D. Lyons G.E. Chamberlain J.E. Buckingham M.E. J. Cell Biol. 1992; 119: 811-821Crossref PubMed Scopus (45) Google Scholar). The adult lung is mainly composed of epithelial cells and smooth muscle, whereas the kidney is largely composed of epithelial cells. Thus, the expression data suggest that the presence of dystroglycan, β-sarcoglycan, γ/δ-sarcoglycan, ε-sarcoglycan, sarcospan, and utrophin/dystrophin in lung could represent the dystroglycan complex from smooth muscle. Likewise, the presence of dystroglycan, ε-sarcoglycan, and utrophin in the kidney could represent an epithelial complex. To test that hypothesis we used immunofluorescence analysis and sucrose gradient fractionation to determine whether proteins observed by Western blot we" @default.
• W2137636698 created "2016-06-24" @default.
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• W2137636698 modified "2023-10-13" @default.
• W2137636698 title "Biochemical Characterization of the Epithelial Dystroglycan Complex" @default.
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