Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1986 Dec 20;5(13):3495–3507. doi: 10.1002/j.1460-2075.1986.tb04675.x

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

C Cladaras, M Hadzopoulou-Cladaras, R T Nolte, D Atkinson, V I Zannis
PMCID: PMC1167386  PMID: 3030729

Abstract

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.

Full text

PDF
3495

Images in this article

3501Fig. 4.
on p.3501
3505Fig. 6.
on p.3505

Selected References

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

  1. Atkinson D., Small D. M. Recombinant lipoproteins: implications for structure and assembly of native lipoproteins. Annu Rev Biophys Biophys Chem. 1986;15:403–456. doi: 10.1146/annurev.bb.15.060186.002155. [DOI] [PubMed] [Google Scholar]
  2. Beeley J. G. Peptide chain conformation and the glycosylation of glycoproteins. Biochem Biophys Res Commun. 1977 Jun 20;76(4):1051–1055. doi: 10.1016/0006-291x(77)90962-7. [DOI] [PubMed] [Google Scholar]
  3. Beltz G. A., Jacobs K. A., Eickbush T. H., Cherbas P. T., Kafatos F. C. Isolation of multigene families and determination of homologies by filter hybridization methods. Methods Enzymol. 1983;100:266–285. doi: 10.1016/0076-6879(83)00061-0. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Breathnach R., Benoist C., O'Hare K., Gannon F., Chambon P. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4853–4857. doi: 10.1073/pnas.75.10.4853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  7. Carew T. E., Pittman R. C., Marchand E. R., Steinberg D. Measurement in vivo of irreversible degradation of low density lipoprotein in the rabbit aorta. Predominance of intimal degradation. Arteriosclerosis. 1984 May-Jun;4(3):214–224. doi: 10.1161/01.atv.4.3.214. [DOI] [PubMed] [Google Scholar]
  8. Carlsson P., Olofsson S. O., Bondjers G., Darnfors C., Wiklund O., Bjursell G. Molecular cloning of human apolipoprotein B cDNA. Nucleic Acids Res. 1985 Dec 20;13(24):8813–8826. doi: 10.1093/nar/13.24.8813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chapman M. J. Comparative analysis of mammalian plasma lipoproteins. Methods Enzymol. 1986;128:70–143. doi: 10.1016/0076-6879(86)28063-5. [DOI] [PubMed] [Google Scholar]
  10. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  11. Cladaras C., Hadzopoulou-Cladaras M., Avila R., Nussbaum A. L., Nicolosi R., Zannis V. I. Complementary DNA derived structure of the amino-terminal domain of human apolipoprotein B and size of its messenger RNA transcript. Biochemistry. 1986 Sep 23;25(19):5351–5357. doi: 10.1021/bi00367a001. [DOI] [PubMed] [Google Scholar]
  12. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Das H. K., McPherson J., Bruns G. A., Karathanasis S. K., Breslow J. L. Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene. J Biol Chem. 1985 May 25;260(10):6240–6247. [PubMed] [Google Scholar]
  14. De Loof H., Rosseneu M., Brasseur R., Ruysschaert J. M. Use of hydrophobicity profiles to predict receptor binding domains on apolipoprotein E and the low density lipoprotein apolipoprotein B-E receptor. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2295–2299. doi: 10.1073/pnas.83.8.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Deeb S. S., Disteche C., Motulsky A. G., Lebo R. V., Kan Y. W. Chromosomal localization of the human apolipoprotein B gene and detection of homologous RNA in monkey intestine. Proc Natl Acad Sci U S A. 1986 Jan;83(2):419–422. doi: 10.1073/pnas.83.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deeb S. S., Motulsky A. G., Albers J. J. A partial cDNA clone for human apolipoprotein B. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4983–4986. doi: 10.1073/pnas.82.15.4983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Efstratiadis A., Posakony J. W., Maniatis T., Lawn R. M., O'Connell C., Spritz R. A., DeRiel J. K., Forget B. G., Weissman S. M., Slightom J. L. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. doi: 10.1016/0092-8674(80)90429-8. [DOI] [PubMed] [Google Scholar]
  18. Eisenberg D. Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem. 1984;53:595–623. doi: 10.1146/annurev.bi.53.070184.003115. [DOI] [PubMed] [Google Scholar]
  19. Elovson J., Jacobs J. C., Schumaker V. N., Puppione D. L. 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. 1985 Mar 12;24(6):1569–1578. doi: 10.1021/bi00327a042. [DOI] [PubMed] [Google Scholar]
  20. Elshourbagy N. A., Walker D. W., Boguski M. S., Gordon J. I., Taylor J. M. The nucleotide and derived amino acid sequence of human apolipoprotein A-IV mRNA and the close linkage of its gene to the genes of apolipoproteins A-I and C-III. J Biol Chem. 1986 Feb 15;261(5):1998–2002. [PubMed] [Google Scholar]
  21. Gilbert W. Why genes in pieces? Nature. 1978 Feb 9;271(5645):501–501. doi: 10.1038/271501a0. [DOI] [PubMed] [Google Scholar]
  22. Goldstein J. L., Brown M. S. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. doi: 10.1146/annurev.bi.46.070177.004341. [DOI] [PubMed] [Google Scholar]
  23. Hardman D. A., Kane J. P. Isolation and characterization of apolipoprotein B-48. Methods Enzymol. 1986;128:262–272. doi: 10.1016/0076-6879(86)28072-6. [DOI] [PubMed] [Google Scholar]
  24. Huang L. S., Bock S. C., Feinstein S. I., Breslow J. L. Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6825–6829. doi: 10.1073/pnas.82.20.6825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hui D. Y., Harmony J. A., Innerarity T. L., Mahley R. W. Immunoregulatory plasma lipoproteins. Role of apoprotein E and apoprotein B. J Biol Chem. 1980 Dec 25;255(24):11775–11781. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Kannel W. B., Castelli W. P., Gordon T., McNamara P. M. Serum cholesterol, lipoproteins, and the risk of coronary heart disease. The Framingham study. Ann Intern Med. 1971 Jan;74(1):1–12. doi: 10.7326/0003-4819-74-1-1. [DOI] [PubMed] [Google Scholar]
  29. Karathanasis S. K., Zannis V. I., Breslow J. L. Isolation and characterization of the human apolipoprotein A-I gene. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6147–6151. doi: 10.1073/pnas.80.20.6147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Knott T. J., Rall S. C., Jr, Innerarity T. L., Jacobson S. F., Urdea M. S., Levy-Wilson B., Powell L. M., Pease R. J., Eddy R., Nakai H. Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization. Science. 1985 Oct 4;230(4721):37–43. doi: 10.1126/science.2994225. [DOI] [PubMed] [Google Scholar]
  31. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  33. Law S. W., Lackner K. J., Hospattankar A. V., Anchors J. M., Sakaguchi A. Y., Naylor S. L., Brewer H. B., Jr Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8340–8344. doi: 10.1073/pnas.82.24.8340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lawn R. M., Fritsch E. F., Parker R. C., Blake G., Maniatis T. The isolation and characterization of linked delta- and beta-globin genes from a cloned library of human DNA. Cell. 1978 Dec;15(4):1157–1174. doi: 10.1016/0092-8674(78)90043-0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Lee D. C., Rose T. M., Webb N. R., Todaro G. J. Cloning and sequence analysis of a cDNA for rat transforming growth factor-alpha. Nature. 1985 Feb 7;313(6002):489–491. doi: 10.1038/313489a0. [DOI] [PubMed] [Google Scholar]
  37. Lee D. M., Valente A. J., Kuo W. H., Maeda H. Properties of apolipoprotein B in urea and in aqueous buffers. The use of glutathione and nitrogen in its solubilization. Biochim Biophys Acta. 1981 Oct 23;666(1):133–146. doi: 10.1016/0005-2760(81)90099-0. [DOI] [PubMed] [Google Scholar]
  38. Lee P., Breckenridge W. C. Isolation and carbohydrate composition of glycopeptides of human apo low-density lipoprotein from normal and type II hyperlipoproteinemic subjects. Can J Biochem. 1976 Sep;54(9):829–833. doi: 10.1139/o76-119. [DOI] [PubMed] [Google Scholar]
  39. Mahley R. W., Innerarity T. L. Lipoprotein receptors and cholesterol homeostasis. Biochim Biophys Acta. 1983 May 24;737(2):197–222. doi: 10.1016/0304-4157(83)90001-1. [DOI] [PubMed] [Google Scholar]
  40. Mahley R. W., Innerarity T. L., Pitas R. E., Weisgraber K. H., Brown J. H., Gross E. Inhibition of lipoprotein binding to cell surface receptors of fibroblasts following selective modification of arginyl residues in arginine-rich and B apoproteins. J Biol Chem. 1977 Oct 25;252(20):7279–7287. [PubMed] [Google Scholar]
  41. Mahley R. W., Weisgraber K. H., Innerarity T. L. Interaction of plasma lipoproteins containing apolipoproteins B and E with heparin and cell surface receptors. Biochim Biophys Acta. 1979 Oct 26;575(1):81–91. doi: 10.1016/0005-2760(79)90133-4. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mehrabian M., Schumaker V. N., Fareed G. C., West R., Johnson D. F., Kirchgessner T., Lin H. C., Wang X. B., Ma Y. H., Mendiaz E. Human apolipoprotein B: identification of cDNA clones and characterization of mRNA. Nucleic Acids Res. 1985 Oct 11;13(19):6937–6953. doi: 10.1093/nar/13.19.6937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Paik Y. K., Chang D. J., Reardon C. A., Davies G. E., Mahley R. W., Taylor J. M. Nucleotide sequence and structure of the human apolipoprotein E gene. Proc Natl Acad Sci U S A. 1985 May;82(10):3445–3449. doi: 10.1073/pnas.82.10.3445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Protter A. A., Hardman D. A., Schilling J. W., Miller J., Appleby V., Chen G. C., Kirsher S. W., McEnroe G., Kane J. P. Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1467–1471. doi: 10.1073/pnas.83.5.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  49. Pustell J., Kafatos F. C. A convenient and adaptable microcomputer environment for DNA and protein sequence manipulation and analysis. Nucleic Acids Res. 1986 Jan 10;14(1):479–488. doi: 10.1093/nar/14.1.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rall S. C., Jr, Weisgraber K. H., Mahley R. W. Human apolipoprotein E. The complete amino acid sequence. J Biol Chem. 1982 Apr 25;257(8):4171–4178. [PubMed] [Google Scholar]
  51. Ruiz-Opazo N., Weinberger J., Nadal-Ginard B. Comparison of alpha-tropomyosin sequences from smooth and striated muscle. Nature. 1985 May 2;315(6014):67–70. doi: 10.1038/315067a0. [DOI] [PubMed] [Google Scholar]
  52. Schiffer M., Edmundson A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967 Mar;7(2):121–135. doi: 10.1016/S0006-3495(67)86579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Segrest J. P., Jackson R. L., Morrisett J. D., Gotto A. M., Jr A molecular theory of lipid-protein interactions in the plasma lipoproteins. FEBS Lett. 1974 Jan 15;38(3):247–258. doi: 10.1016/0014-5793(74)80064-5. [DOI] [PubMed] [Google Scholar]
  55. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  56. Shoulders C. C., Myant N. B., Sidoli A., Rodriguez J. C., Cortese C., Baralle F. E., Cortese R. Molecular cloning of human LDL apolipoprotein B cDNA. Evidence for more than one gene per haploid genome. Atherosclerosis. 1985 Dec;58(1-3):277–289. doi: 10.1016/0021-9150(85)90073-5. [DOI] [PubMed] [Google Scholar]
  57. Siuta-Mangano P., Howard S. C., Lennarz W. J., Lane M. D. Synthesis, processing, and secretion of apolipoprotein B by the chick liver cell. J Biol Chem. 1982 Apr 25;257(8):4292–4300. [PubMed] [Google Scholar]
  58. Sniderman A., Shapiro S., Marpole D., Skinner B., Teng B., Kwiterovich P. O., Jr Association of coronary atherosclerosis with hyperapobetalipoproteinemia [increased protein but normal cholesterol levels in human plasma low density (beta) lipoproteins]. Proc Natl Acad Sci U S A. 1980 Jan;77(1):604–608. doi: 10.1073/pnas.77.1.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Swaminathan N., Aladjem F. The monosaccharide composition and sequence of the carbohydrate moiety of human serum low density lipoproteins. Biochemistry. 1976 Apr 6;15(7):1516–1522. doi: 10.1021/bi00652a024. [DOI] [PubMed] [Google Scholar]
  60. Wei C. F., Chen S. H., Yang C. Y., Marcel Y. L., Milne R. W., Li W. H., Sparrow J. T., Gotto A. M., Jr, Chan L. 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. 1985 Nov;82(21):7265–7269. doi: 10.1073/pnas.82.21.7265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Weisgraber K. H., Innerarity T. L., Mahley R. W. Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts. J Biol Chem. 1978 Dec 25;253(24):9053–9062. [PubMed] [Google Scholar]
  62. Yamamoto T., Davis C. G., Brown M. S., Schneider W. J., Casey M. L., Goldstein J. L., Russell D. W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell. 1984 Nov;39(1):27–38. doi: 10.1016/0092-8674(84)90188-0. [DOI] [PubMed] [Google Scholar]
  63. Young S. G., Bertics S. J., Scott T. M., Dubois B. W., Curtiss L. K., Witztum J. L. Parallel expression of the MB19 genetic polymorphism in apoprotein B-100 and apoprotein B-48. Evidence that both apoproteins are products of the same gene. J Biol Chem. 1986 Mar 5;261(7):2995–2998. [PubMed] [Google Scholar]
  64. Zannis V. I., Cole F. S., Jackson C. L., Kurnit D. M., Karathanasis S. K. Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages. Biochemistry. 1985 Jul 30;24(16):4450–4455. doi: 10.1021/bi00337a028. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES