Advertisement

Human Apolipoprotein B

Cloning and Analysis of Liver and Intestinal mRNA and Their Expression in Patients with Abetalipoproteinemia
  • S. W. Law
  • J. C. Monge
  • K. J. Lackner
  • S. Grant
  • K. Higuchi
  • A. V. Hospattanker
  • J. M. Hoeg
  • R. E. Gregg
  • N. Lee
  • A. Sakaguchi
  • S. Naylor
  • H. B. BrewerJr.
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)

Abstract

Human liver apo B-100 has been cloned in plasmid and phage λgt-11 expression vectors and apo B-100 cDNA clones were identified by screening with monospecific anti-apo B antibodies and synthetic oligonucleotides based on peptides isolated and sequenced from apo B-100. Overlapping cDNA clones containing the entire apo B-100 mRNA were isolated and sequenced. All peptides previously isolated from apo B-100 mRNA were identified and their locations defined. Northern blot analysis utilizing radiolabeled cDNA probes revealed that the apoB-100 mRNA is 14.1 kb in size. On the contrary, two distinct molecular species of apo B mRNA are being produced by the small intestine. The larger-molecular-weight species migrate at the same position as liver apo B-100 mRNA. The smaller species is 7.5 kb in size, and most likely codes for apo B-48. Hybridization and nucleic acid sequence studies were carried out to define common domains and apo B-100 mRNA-specific domains. Since apo B is a single-copy gene, we propose a mechanism for the generation of apo B-100 mRNA and apo B-48 mRNA through differential splicing of the precursor apo B RNA transcript. We have also localized the apo B gene to the p23 → pter region of the short arm of chromosome 2 by filter hybridization analysis of human-mouse hybrid cell DNAs. We have also initiated studies on the gene organization and expression of apo B in patients with abetalipoproteinemia. Studies on two unrelated kindreds revealed no major deletions or insertions and that the apo B-100 mRNA and apo B-100 protein are present in the liver cells of abetalipoproteinemia patients.

Keywords

Human Apolipoprotein Southern Blot Hybridization Analysis Partial cDNA Clone Northern Blot Hybridization Analysis Hybrid Cell Clone 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Cardin, A. D., Witt, K. R.. Chao, J.. Margolis. H. S., Donaldson, V. H., and Jackson, R.L., 1984, Degradation of apolipoprotein B-100 of human plasma low density lipoprotein by tissue and plasma kallikreins, J. Biol. Chem. 259:8522–8528.Google Scholar
  2. Carlsson, P., Olofsson, S. O., Bondjers, G., Darnfors, C., Wiklund, O., and Bjursell, G., 1985, Molecular cloning of human apolipoprotein B cDNA, Nucleic Acids Res. 13:8813–8826.PubMedCrossRefGoogle Scholar
  3. Deeb, S. S., Motulsky, A. G.. and Alberts, J. J., 1985, Partial cDNA clone for human apolipoprotein B, Proc. Natl. Acad. Sci. U.S.A. 82:4983–4986.PubMedCrossRefGoogle Scholar
  4. Edge, S. B., Hoeg, J. M., Schneider, P. D., and Brewer, H. B., Jr., 1985, Adult human liver synthesizes apolipoprotein B-100, Arteriosclerosis 3:499–500.Google Scholar
  5. Elovson, J., Jacobs. J. C., Schumaker, V. N., and Puppione, D. L., 1985, Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low-density lipoprotein (apoprotein B-PIII), Biochemistry 24:1569–1578.PubMedCrossRefGoogle Scholar
  6. Goldstein, J. L., and Brown, M. S., 1977, The low-density lipoprotein pathway and its relation to atherosclerosis, Annu. Rev. Biochem. 46:897–930.PubMedCrossRefGoogle Scholar
  7. Green, P. H., Lefkowitch, J. H., Glickman, R. M., Riley, J. W., Quinet, E., and Blum, C. B., 1982, Apolipoprotein localization and quantitation in the human intestine, Gastroenterology 83:1223–1230.PubMedGoogle Scholar
  8. Herbert, P. N., Assmann, G., Gotto, A. M., Jr., and Fredrickson, D. S., 1983, Familial lipoprotein deficiency: Abetalipoproteinemia, hypobetalipoproteinemia, and Tangier disease, in: The Metabolic Basis of Inherited Disease (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown, eds.), McGraw-Hill, New York, pp. 589–621.Google Scholar
  9. Hospattankar, A. V., Fairwell, T., Meng, M., Ronan, R., and Brewer, H. B., Jr., 1986, Identification of sequence homology between human plasma apolipoprotein B-100 and apolipoprotein B-48, J. Biol. Chem. 260:9102–9104.Google Scholar
  10. Jackson, R. L., Morrisett, J. D., and Gotto, A. M., Jr., 1976, Lipoprotein structure and metabolism, Physiol. Rev. 56:259–316.PubMedGoogle Scholar
  11. Kane, J. P., 1983, Apolipoprotein B: Structural and metabolic heterogeneity,Annu. Rev. Physiol.45:637–650.PubMedCrossRefGoogle Scholar
  12. Kane, J. P., 1983, Apolipoprotein B: Structural and metabolic heterogeneity, Annu. Rev. Physiol. 45:637-650. Knott, T. J., Rall, S. C., Innerarity, T. L., Jacobson, S. F., Urdea, M. S., Levy-Wilson, B., Powell, L. M., Pease, R. J., Eddy, R., Nakai, H., Byers, M., Priestly, L. M., Robertson, E., Rall, L. B., Betsholtz, C., Shows, T. B., Mahley, R. W., and Scott, J., 1985, Human apolipoprotein B: Structure of carboxyl-terminal domains, sites of gene expression and chromosomal localization, Science 230:37–43.PubMedCrossRefGoogle Scholar
  13. Law, S. W., Gray, G., and Brewer, H. B., Jr., 1983, cDNA cloning of human apoA-I: Amino acid sequence of preproapoA-I, Biochem. Biophys. Res. Commun. 112:257–264.PubMedCrossRefGoogle Scholar
  14. Law, S. W., Lee, N., Monge, J. C., Sakaguchi, A. Y., Naylor, S. L., and Brewer, H. B., Jr., 1985a, Human apoB-100 gene residues in the 23 region of chromosome 2Biochem. Biophys. Res. Commun. 131:1003–1012.PubMedCrossRefGoogle Scholar
  15. Law, S. W., Lackner, K. J., Hospattankar, A. V., Anchors, J. M., Sakaguchi, A. Y., Naylor, S. L., and Brewer, H. B., Jr., 1985b, Human apolipoprotein B-100: Cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2, Proc. Natl. Acad. Sci. U.S.A. 82:8340–8344.CrossRefGoogle Scholar
  16. LeBoeuf, R. C., Miller, C., Shively, J. E., Schumaker, V. N., Balla, M. A., and Lusis, A. J., 1984, Human apolipoprotein B: Partial amino acid sequence, FEBS Lett. 170:105–108.PubMedCrossRefGoogle Scholar
  17. Lee, D. M., Koren, E., Singh, S., and Mok, T., 1984, Presence of B-100 in rat mesenteric chyle, Biochem. Biophys. Res. Commun. 123:1149–1156.PubMedCrossRefGoogle Scholar
  18. Lusis, A. J., West, R., Mehrabian, M., Reuben, M. A., LeBoeuf, R. C., Kaptein, J. S., Johnson, D. F., Schumaker, V. N., Yuhasz, M. P., Schotz, M. C., and Elovson, J., 1985, Cloning and expression of apolipoprotein B, the major protein of low and very low density lipoproteins, Proc. Natl. Acad. Sci. U.S.A. 82:4596–4601.CrossRefGoogle Scholar
  19. Mehrabian, M., Schumaker, V. N., Fareed, G. C., West, R., Johnson, D. F., Kirchgessner, T., Lin, H. C., Wang, X., Ma, Y., Mendiaz, E., and Lusis, A. J., 1985, Human apolipoprotein B: Identification of cDNA clones and characterization of mRNA, Nucleic Acids Res. 13:6937–6953.PubMedCrossRefGoogle Scholar
  20. Osborne, J. C., Jr., and Brewer, H. B., Jr., 1977, The plasma lipoprotein, Adv. Protein Chem. 31:253–337.PubMedCrossRefGoogle Scholar
  21. Protter, A. A., Hardman, D. A., Schilling, J. W., Miller, J., Appleby, V., Chen, G. C., Kirsher, S. W., McEnroe, G., and Kane, J. P., 1986, Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B, Proc. Natl. Acad. Sci. U.S.A. 83:1467–1471.PubMedCrossRefGoogle Scholar
  22. Salt, H., Wolff, O. H., Lloyd, J. K., Fosbrooke, A. S., Cameron, A. H., and Hubble, D. V., 1960, On having no beta-lipoprotein. A syndrome comprising abeta-lipoproteinemia, acanthocytosis, and steatorrhea, Lancet 2:325–329.PubMedCrossRefGoogle Scholar
  23. Scanu, A. M., and Teng, T., 1979, in: The Biochemistry of Atherosclerosis (A. M. Scanu, ed.), Marcel Dekker, New York, pp. 107–122.Google Scholar
  24. Shoulders, C. C., Myant, N. B., Sidoli, A., Rodriquez, J. C., Cortese, C., Baralle, F. E., and Cortese, R., 1985, Molecular cloning of human LDL apolipoprotein B cDNA, Atherosclerosis 58:277–289.PubMedCrossRefGoogle Scholar
  25. Steele, J. C., Jr., and Reynolds, J. A., 1979, Molecular weight and hydrodynamic properties of apolipoprotein B in guanidine hydrochloride and sodium dodecyl sulfate solution, J. Biol. Chem. 254:1639–1643.PubMedGoogle Scholar
  26. Tertia Belt, K., Carroll, M. C., and Porter, R. R., 1984, The structural basis of the multiple forms of human complement component C4, Cell 36:907–914.CrossRefGoogle Scholar
  27. Wei, C. F., Chen, S. H., Yang, C. Y., Marcel, Y. L., Milne, R. W., Li, W. H., Sparrow, J. T., Gotto, A. M., and Chan, L., 1985, Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxyl-terminal fragment of human apolipoprotein B-100, Proc. Natl. Acad. Sci. U.S.A. 82:7265–7269.PubMedCrossRefGoogle Scholar
  28. Young, S. G., Bertics, S. J., Scott, T. M., Dubois, B. W., Curtiss, L. K., and Witztum, J. L., 1986, Parallel expression of the MB 19 genetic polymorphism in apoprotein B-100 and apoprotein B-48,J. Biol. Chem.261:2995–2998.PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • S. W. Law
    • 1
  • J. C. Monge
    • 1
  • K. J. Lackner
    • 1
  • S. Grant
    • 1
  • K. Higuchi
    • 1
  • A. V. Hospattanker
    • 1
  • J. M. Hoeg
    • 2
  • R. E. Gregg
    • 1
  • N. Lee
    • 1
  • A. Sakaguchi
    • 3
  • S. Naylor
    • 3
  • H. B. BrewerJr.
    • 2
  1. 1.National Heart, Lung, and Blood InstituteNational Institutes of HealthBethesdaUSA
  2. 2.The Molecular Disease Branch. National Heart, Lung, Blood InstituteNational Institutes of HealthBethesdaUSA
  3. 3.Department of Cellular and Structural BiologyUniversity of Texas Health Sciences CenterSan AntonioUSA

Personalised recommendations