Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Dec 1;288(Pt 2):663–668. doi: 10.1042/bj2880663

Monoclonal antibodies for dystrophin analysis. Epitope mapping and improved binding to SDS-treated muscle sections.

T M Nguyen 1, I B Ginjaar 1, G J van Ommen 1, G E Morris 1
PMCID: PMC1132062  PMID: 1281410

Abstract

A group of 44 monoclonal antibodies (mAbs) raised against the central helical rod (25 mAbs) and C-terminal (19 mAbs) regions of dystrophin were prepared using trpE recombinant fusion proteins as immunogens. Some mAbs cross-react with the structurally related proteins, alpha-actinin and utrophin. Epitope mapping revealed uneven distribution of mAb-binding sites, no mAbs being produced against the C-terminal end of the helical fragment or the cysteine-rich region of the C-terminal dystrophin fragment. The failure of these large regions of the recombinant immunogens to elicit anti-dystrophin antibodies may be because of their inability to fold into the correct dystrophin-like conformation. The mAbs were selected for their ability to recognize 427 kDa dystrophin on Western blots after SDS/PAGE, and/or for immunostaining of the membrane in frozen muscle sections. Although some mAbs obtained by Western-blot screening failed to bind native dystrophin in frozen muscle sections, successful binding could be obtained after SDS or urea treatment of the tissue section to expose the epitopes. This increases the range of mAbs available for detection of dystrophin deletions in muscular dystrophy and evaluation of myoblast therapy.

Full text

PDF
663

Images in this article

664Fig. 1.
on p.664
666Fig. 2.
on p.666
666Fig. 3.
on p.666
666Fig. 5.
on p.666
667Fig. 6.
on p.667

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bar S., Barnea E., Levy Z., Neuman S., Yaffe D., Nudel U. A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution. Biochem J. 1990 Dec 1;272(2):557–560. doi: 10.1042/bj2720557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beggs A. H., Hoffman E. P., Snyder J. R., Arahata K., Specht L., Shapiro F., Angelini C., Sugita H., Kunkel L. M. Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies. Am J Hum Genet. 1991 Jul;49(1):54–67. [PMC free article] [PubMed] [Google Scholar]
  3. Blake D. J., Love D. R., Tinsley J., Morris G. E., Turley H., Gatter K., Dickson G., Edwards Y. H., Davies K. E. Characterization of a 4.8kb transcript from the Duchenne muscular dystrophy locus expressed in Schwannoma cells. Hum Mol Genet. 1992 May;1(2):103–109. doi: 10.1093/hmg/1.2.103. [DOI] [PubMed] [Google Scholar]
  4. Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
  5. Ellis J. M., Man N. T., Morris G. E., Ginjaar I. B., Moorman A. F., van Ommen G. J. Specificity of dystrophin analysis improved with monoclonal antibodies. Lancet. 1990 Oct 6;336(8719):881–882. doi: 10.1016/0140-6736(90)92392-u. [DOI] [PubMed] [Google Scholar]
  6. England S. B., Nicholson L. V., Johnson M. A., Forrest S. M., Love D. R., Zubrzycka-Gaarn E. E., Bulman D. E., Harris J. B., Davies K. E. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature. 1990 Jan 11;343(6254):180–182. doi: 10.1038/343180a0. [DOI] [PubMed] [Google Scholar]
  7. Feener C. A., Koenig M., Kunkel L. M. Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature. 1989 Apr 6;338(6215):509–511. doi: 10.1038/338509a0. [DOI] [PubMed] [Google Scholar]
  8. Ginjaar I. B., Bakker E., den Dunnen J. T., van Paassen M. M., van Ommen G. J., Zubrzycka-Gaarn E., Kloosterman M. D., Wessels A., Moorman A. F. Immunological study of dystrophin in Duchenne fetus. Lancet. 1989 Nov 18;2(8673):1212–1213. doi: 10.1016/s0140-6736(89)91813-8. [DOI] [PubMed] [Google Scholar]
  9. Ginjaar I. B., Bakker E., van Paassen M. M., den Dunnen J. T., Wessels A., Zubrzycka-Gaarn E. E., Moorman A. F., van Ommen G. J. Immunohistochemical studies show truncated dystrophins in the myotubes of three fetuses at risk for Duchenne muscular dystrophy. J Med Genet. 1991 Aug;28(8):505–510. doi: 10.1136/jmg.28.8.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ginjaar I. B., Soffers S., Moorman A. F., Nicholson L. V., Morris G. E., Bakker E., van Haeringen A., van Ommen G. J. Fetal dystrophin to diagnose carrier status. Lancet. 1991 Jul 27;338(8761):258–259. doi: 10.1016/0140-6736(91)90402-b. [DOI] [PubMed] [Google Scholar]
  11. Gussoni E., Pavlath G. K., Lanctot A. M., Sharma K. R., Miller R. G., Steinman L., Blau H. M. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature. 1992 Apr 2;356(6368):435–438. doi: 10.1038/356435a0. [DOI] [PubMed] [Google Scholar]
  12. Helliwell T. R., Ellis J. M., Mountford R. C., Appleton R. E., Morris G. E. A truncated dystrophin lacking the C-terminal domains is localized at the muscle membrane. Am J Hum Genet. 1992 Mar;50(3):508–514. [PMC free article] [PubMed] [Google Scholar]
  13. Hoffman E. P., Brown R. H., Jr, Kunkel L. M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987 Dec 24;51(6):919–928. doi: 10.1016/0092-8674(87)90579-4. [DOI] [PubMed] [Google Scholar]
  14. Hoffman E. P., Fischbeck K. H., Brown R. H., Johnson M., Medori R., Loike J. D., Harris J. B., Waterston R., Brooke M., Specht L. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med. 1988 May 26;318(21):1363–1368. doi: 10.1056/NEJM198805263182104. [DOI] [PubMed] [Google Scholar]
  15. Hoffman E. P., Watkins S. C., Slayter H. S., Kunkel L. M. Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin. J Cell Biol. 1989 Feb;108(2):503–510. doi: 10.1083/jcb.108.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hugnot J. P., Gilgenkrantz H., Vincent N., Chafey P., Morris G. E., Monaco A. P., Berwald-Netter Y., Koulakoff A., Kaplan J. C., Kahn A. Distal transcript of the dystrophin gene initiated from an alternative first exon and encoding a 75-kDa protein widely distributed in nonmuscle tissues. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7506–7510. doi: 10.1073/pnas.89.16.7506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Koenig M., Beggs A. H., Moyer M., Scherpf S., Heindrich K., Bettecken T., Meng G., Müller C. R., Lindlöf M., Kaariainen H. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet. 1989 Oct;45(4):498–506. [PMC free article] [PubMed] [Google Scholar]
  18. Koenig M., Kunkel L. M. Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility. J Biol Chem. 1990 Mar 15;265(8):4560–4566. [PubMed] [Google Scholar]
  19. Koenig M., Monaco A. P., Kunkel L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell. 1988 Apr 22;53(2):219–228. doi: 10.1016/0092-8674(88)90383-2. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lederfein D., Levy Z., Augier N., Mornet D., Morris G., Fuchs O., Yaffe D., Nudel U. A 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5346–5350. doi: 10.1073/pnas.89.12.5346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lemaire C., Heilig R., Mandel J. L. The chicken dystrophin cDNA: striking conservation of the C-terminal coding and 3' untranslated regions between man and chicken. EMBO J. 1988 Dec 20;7(13):4157–4162. doi: 10.1002/j.1460-2075.1988.tb03311.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Love D. R., Hill D. F., Dickson G., Spurr N. K., Byth B. C., Marsden R. F., Walsh F. S., Edwards Y. H., Davies K. E. An autosomal transcript in skeletal muscle with homology to dystrophin. Nature. 1989 May 4;339(6219):55–58. doi: 10.1038/339055a0. [DOI] [PubMed] [Google Scholar]
  24. Morris G. E., Cartwright A. J. Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase. Biochim Biophys Acta. 1990 Jul 6;1039(3):318–322. doi: 10.1016/0167-4838(90)90265-h. [DOI] [PubMed] [Google Scholar]
  25. Nguyen thi Man, Cartwright A. J., Morris G. E., Love D. R., Bloomfield J. F., Davies K. E. Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin. FEBS Lett. 1990 Mar 26;262(2):237–240. doi: 10.1016/0014-5793(90)80199-s. [DOI] [PubMed] [Google Scholar]
  26. Nguyen thi Man, Cartwright A. J., Osborne M., Morris G. E. Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies. Biochim Biophys Acta. 1991 Jan 29;1076(2):245–251. doi: 10.1016/0167-4838(91)90274-4. [DOI] [PubMed] [Google Scholar]
  27. Nguyen T. M., Ellis J. M., Ginjaar I. B., van Paassen M. M., van Ommen G. J., Moorman A. F., Cartwright A. J., Morris G. E. Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin. FEBS Lett. 1990 Oct 15;272(1-2):109–112. doi: 10.1016/0014-5793(90)80460-z. [DOI] [PubMed] [Google Scholar]
  28. Nguyen T. M., Ellis J. M., Love D. R., Davies K. E., Gatter K. C., Dickson G., Morris G. E. Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines. J Cell Biol. 1991 Dec;115(6):1695–1700. doi: 10.1083/jcb.115.6.1695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nicholson L. V., Johnson M. A., Gardner-Medwin D., Bhattacharya S., Harris J. B. Heterogeneity of dystrophin expression in patients with Duchenne and Becker muscular dystrophy. Acta Neuropathol. 1990;80(3):239–250. doi: 10.1007/BF00294640. [DOI] [PubMed] [Google Scholar]
  30. Récan D., Chafey P., Leturcq F., Hugnot J. P., Vincent N., Tomé F., Collin H., Simon D., Czernichow P., Nicholson L. V. Are cysteine-rich and COOH-terminal domains of dystrophin critical for sarcolemmal localization? J Clin Invest. 1992 Feb;89(2):712–716. doi: 10.1172/JCI115640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sedgwick S. G., Nguyen T. M., Ellis J. M., Crowne H., Morris G. E. Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. Nucleic Acids Res. 1991 Nov 11;19(21):5889–5894. doi: 10.1093/nar/19.21.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simmerman H. K., Lovelace D. E., Jones L. R. Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum. Biochim Biophys Acta. 1989 Aug 31;997(3):322–329. doi: 10.1016/0167-4838(89)90203-3. [DOI] [PubMed] [Google Scholar]
  33. Tanaka H., Ishiguro T., Eguchi C., Saito K., Ozawa E. Expression of a dystrophin-related protein associated with the skeletal muscle cell membrane. Histochemistry. 1991;96(1):1–5. doi: 10.1007/BF00266753. [DOI] [PubMed] [Google Scholar]
  34. Walsh F. S., Pizzey J. A., Dickson G. Tissue-specific isoforms of dystrophin. Trends Neurosci. 1989 Jul;12(7):235–238. doi: 10.1016/0166-2236(89)90017-9. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES