Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Jul;82(14):4597–4601. doi: 10.1073/pnas.82.14.4597

Cloning and expression of apolipoprotein B, the major protein of low and very low density lipoproteins.

A J Lusis, R West, M Mehrabian, M A Reuben, R C LeBoeuf, J S Kaptein, D F Johnson, V N Schumaker, M P Yuhasz, M C Schotz, et al.
PMCID: PMC390432  PMID: 3860811

Abstract

We report the cloning of cDNAs for rat liver apolipoprotein B (apo B) and the use of the cloned sequences to examine apo B expression at the level of mRNA in rat tissues. Fifteen putative apo B clones were identified by antibody screening of a rat liver cDNA library in the lambda gt11 expression vector. The identity of the clones was confirmed by immunological studies of the fusion protein products. All clones appear to contain sequences found only in apo B PI, the high molecular weight form of rat liver apo B. Blotting studies show that the clones hybridize to a single 20-kilobase liver mRNA species, sufficiently large to encode the entire apo B PI peptide, which is estimated to be 400 kDa in size. Apo B PI mRNA is abundant in liver and present in lower amounts in intestine but is absent in a variety of other tissues examined. This tissue distribution is consistent with that expected from studies on the in vivo synthesis of apo B. One clone, corresponding to a 240-base segment of the apo B PI mRNA, was sequenced and found to exhibit homology with a short region of rat apo E mRNA. Analysis of the secondary structure of the corresponding peptide did not show the preponderance of amphipathic alpha-helical structures characteristic of other apolipoproteins examined thus far.

Full text

PDF
4599

Images in this article

Selected References

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

  1. Bell-Quint J., Forte T., Graham P. Synthesis of two forms of apolipoprotein B by cultured rat hepatocytes. Biochem Biophys Res Commun. 1981 Mar 31;99(2):700–706. doi: 10.1016/0006-291x(81)91800-3. [DOI] [PubMed] [Google Scholar]
  2. Boström K., Wettesten M., Wiklund O., Bondjers G., Lundholm K., Elias P., Norfeldt P. I., Olofsson S. O. Evidence for a structural relationship between apoB75kDa and human plasma apolipoprotein B 100, from translation of human liver mRNA in vitro and immunochemical studies with monoclonal and polyclonal antibodies. Eur J Biochem. 1984 Aug 15;143(1):101–107. doi: 10.1111/j.1432-1033.1984.tb08347.x. [DOI] [PubMed] [Google Scholar]
  3. Brown M. S., Kovanen P. T., Goldstein J. L. Regulation of plasma cholesterol by lipoprotein receptors. Science. 1981 May 8;212(4495):628–635. doi: 10.1126/science.6261329. [DOI] [PubMed] [Google Scholar]
  4. Chen C. H., Aladjem F. Further studies on the subunit structure of human serum low density lipoproteins. Biochem Med. 1978 Apr;19(2):178–187. doi: 10.1016/0006-2944(78)90019-4. [DOI] [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  7. Elovson J., Huang Y. O., Baker N., Kannan R. Apolipoprotein B is structurally and metabolically heterogeneous in the rat. Proc Natl Acad Sci U S A. 1981 Jan;78(1):157–161. doi: 10.1073/pnas.78.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hay R., Getz G. S. Translation in vivo and in vitro of proteins resembling apoproteins of rat plasma very low density lipoprotein. J Lipid Res. 1979 Mar;20(3):334–348. [PubMed] [Google Scholar]
  9. Huang S. S., Lee D. M. A novel method for converting apolipoprotein B, the major protein moiety of human plasma low density lipoproteins, into a water-soluble protein. Biochim Biophys Acta. 1979 Apr 25;577(2):424–441. doi: 10.1016/0005-2795(79)90047-3. [DOI] [PubMed] [Google Scholar]
  10. Innerarity T. L., Mahley R. W. Enhanced binding by cultured human fibroblasts of apo-E-containing lipoproteins as compared with low density lipoproteins. Biochemistry. 1978 Apr 18;17(8):1440–1447. doi: 10.1021/bi00601a013. [DOI] [PubMed] [Google Scholar]
  11. Janero D. R., Lane M. D. Sequential assembly of very low density lipoprotein apolipoproteins, triacylglycerol, and phosphoglycerides by the intact liver cell. J Biol Chem. 1983 Dec 10;258(23):14496–14504. [PubMed] [Google Scholar]
  12. Kane J. P. Apolipoprotein B: structural and metabolic heterogeneity. Annu Rev Physiol. 1983;45:637–650. doi: 10.1146/annurev.ph.45.030183.003225. [DOI] [PubMed] [Google Scholar]
  13. Kane J. P., Hardman D. A., Paulus H. E. Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci U S A. 1980 May;77(5):2465–2469. doi: 10.1073/pnas.77.5.2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kane J. P., Sata T., Hamilton R. L., Havel R. J. Apoprotein composition of very low density lipoproteins of human serum. J Clin Invest. 1975 Dec;56(6):1622–1634. doi: 10.1172/JCI108245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krishnaiah K. V., Walker L. F., Borensztajn J., Schonfeld G., Getz G. S. Apolipoprotein B variant derived from rat intestine. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3806–3810. doi: 10.1073/pnas.77.7.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LeBoeuf R. C., Miller C., Shively J. E., Schumaker V. N., Balla M. A., Lusis A. J. Human apolipoprotein B: partial amino acid sequence. FEBS Lett. 1984 May 7;170(1):105–108. doi: 10.1016/0014-5793(84)81378-2. [DOI] [PubMed] [Google Scholar]
  17. Malloy M. J., Kane J. P., Hardman D. A., Hamilton R. L., Dalal K. B. Normotriglyceridemic abetalipoproteinemia. absence of the B-100 apolipoprotein. J Clin Invest. 1981 May;67(5):1441–1450. doi: 10.1172/JCI110173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marcel Y. L., Hogue M., Theolis R., Jr, Milne R. W. Mapping of antigenic determinants of human apolipoprotein B using monoclonal antibodies against low density lipoproteins. J Biol Chem. 1982 Nov 25;257(22):13165–13168. [PubMed] [Google Scholar]
  19. Olofsson S. O., Boström K., Svanberg U., Bondjers G. Isolation and partial characterization of a polypeptide belonging to apolipoprotein B from low-density lipoproteins of human plasma. Biochemistry. 1980 Mar 18;19(6):1059–1064. doi: 10.1021/bi00547a002. [DOI] [PubMed] [Google Scholar]
  20. Olofsson S. O., Elias P., Boström K., Lundholm K., Norfeldt P. I., Wiklund O., Fager G., Bondjers G. Human liver RNA-programmed in vitro synthesis of a polypeptide related to human apolipoprotein B. FEBS Lett. 1983 May 30;156(1):63–66. doi: 10.1016/0014-5793(83)80249-x. [DOI] [PubMed] [Google Scholar]
  21. Olofsson S. O., Elias P., Boström K., Lundholm K., Norfeldt P. I., Wiklund O., Fager G., Bondjers G. Human liver RNA-programmed in vitro synthesis of a polypeptide related to human apolipoprotein B. FEBS Lett. 1983 May 30;156(1):63–66. doi: 10.1016/0014-5793(83)80249-x. [DOI] [PubMed] [Google Scholar]
  22. Reue K. L., Quon D. H., O'Donnell K. A., Dizikes G. J., Fareed G. C., Lusis A. J. Cloning and regulation of messenger RNA for mouse apolipoprotein E. J Biol Chem. 1984 Feb 25;259(4):2100–2107. [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schwarzbauer J. E., Tamkun J. W., Lemischka I. R., Hynes R. O. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 1983 Dec;35(2 Pt 1):421–431. doi: 10.1016/0092-8674(83)90175-7. [DOI] [PubMed] [Google Scholar]
  25. Smith R., Dawson J. R., Tanford C. The size and number of polypeptide chains in human serum low density lipoprotein. J Biol Chem. 1972 Jun 10;247(11):3376–3381. [PubMed] [Google Scholar]
  26. Socorro L., Camejo G. Preparation and properties of soluble, immunoreactive apoLDL. J Lipid Res. 1979 Jul;20(5):631–638. [PubMed] [Google Scholar]
  27. Socorro L., López F., López A., Camejo G. ApoLDL: evidence for an aggregating system of heterogeneous subunits. J Lipid Res. 1982 Dec;23(9):1283–1291. [PubMed] [Google Scholar]
  28. Sparks C. E., Marsh J. B. Metabolic heterogeneity of apolipoprotein B in the rat. J Lipid Res. 1981 Mar;22(3):519–527. [PubMed] [Google Scholar]
  29. Staden R. An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucleic Acids Res. 1982 May 11;10(9):2951–2961. doi: 10.1093/nar/10.9.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Steele J. C., Jr, Reynolds J. A. Characterization of the apolipoprotein B polypeptide of human plasma low density lipoprotein in detergent and denaturation solutions. J Biol Chem. 1979 Mar 10;254(5):1633–1638. [PubMed] [Google Scholar]
  31. Steele J. C., Jr, Reynolds J. A. Molecular weight and hydrodynamic properties of apolipoprotein B in guanidine hydrochloride and sodium dodecyl sulfate solutions. J Biol Chem. 1979 Mar 10;254(5):1639–1643. [PubMed] [Google Scholar]
  32. Steele J. C., Jr The effect of plasmin on human plasma low density lipoprotein. Thromb Res. 1979;15(3-4):573–579. doi: 10.1016/0049-3848(79)90165-8. [DOI] [PubMed] [Google Scholar]
  33. Tikkanen N. J., Dargar R., Pfleger B., Gonen B., Davie J. M., Schonfeld G. Antigenic mapping of human low density lipoprotein with monoclonal antibodies. J Lipid Res. 1982 Sep;23(7):1032–1038. [PubMed] [Google Scholar]
  34. Tsao B. P., Curtiss L. K., Edgington T. S. Immunochemical heterogeneity of human plasma apolipoprotein B. II. Expression of apolipoprotein B epitopes on native lipoproteins. J Biol Chem. 1982 Dec 25;257(24):15222–15228. [PubMed] [Google Scholar]
  35. Wu A. L., Windmueller H. G. Variant forms of plasma apolipoprotein B. Hepatic and intestinal biosynthesis and heterogeneous metabolism in the rat. J Biol Chem. 1981 Apr 25;256(8):3615–3618. [PubMed] [Google Scholar]
  36. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES