Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1993 Jun;92(3):381–390. doi: 10.1111/j.1365-2249.1993.tb03409.x

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

A M Yamamoto 1, C Mura 1, C De Lemos-Chiarandini 1, R Krishnamoorthy 1, F Alvarez 1
PMCID: PMC1554760  PMID: 7685669

Abstract

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Full text

PDF
381

Images in this article

385Fig. 2
on p.385
386Fig. 4
on p.386
386Fig. 5
on p.386
387Fig. 6
on p.387

Selected References

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

  1. Adelman M. R., Blobel G., Sabatini D. D. An improved cell fractionation procedure for the preparation of rat liver membrane-bound ribosomes. J Cell Biol. 1973 Jan;56(1):191–205. doi: 10.1083/jcb.56.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alvarez F., Bernard O., Homberg J. C., Kreibich G. Anti-liver-kidney microsome antibody recognizes a 50,000 molecular weight protein of the endoplasmic reticulum. J Exp Med. 1985 May 1;161(5):1231–1236. doi: 10.1084/jem.161.5.1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amar-Costesec A., Beaufay H., Wibo M., Thinès-Sempoux D., Feytmans E., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. II. Preparation and composition of the microsomal fraction. J Cell Biol. 1974 Apr;61(1):201–212. doi: 10.1083/jcb.61.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berman P. W., Dowbenko D., Lasky L. A., Simonsen C. C. Detection of antibodies to herpes simplex virus with a continuous cell line expressing cloned glycoprotein D. Science. 1983 Nov 4;222(4623):524–527. doi: 10.1126/science.6312563. [DOI] [PubMed] [Google Scholar]
  5. Brown C. A., Black S. D. Membrane topology of mammalian cytochromes P-450 from liver endoplasmic reticulum. Determination by trypsinolysis of phenobarbital-treated microsomes. J Biol Chem. 1989 Mar 15;264(8):4442–4449. [PubMed] [Google Scholar]
  6. De Lemos-Chiarandini C., Alvarez F., Bernard O., Homberg J. C., Kreibich G. Anti-liver-kidney microsome antibody is a marker for the rat hepatocyte endoplasmic reticulum. Hepatology. 1987 May-Jun;7(3):468–475. doi: 10.1002/hep.1840070310. [DOI] [PubMed] [Google Scholar]
  7. De Lemos-Chiarandini C., Frey A. B., Sabatini D. D., Kreibich G. Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies. J Cell Biol. 1987 Feb;104(2):209–219. doi: 10.1083/jcb.104.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doehmer J., Oesch F. V79 Chinese hamster cells genetically engineered for stable expression of cytochromes P450. Methods Enzymol. 1991;206:117–123. doi: 10.1016/0076-6879(91)06083-f. [DOI] [PubMed] [Google Scholar]
  9. Griffiths G., Brands R., Burke B., Louvard D., Warren G. Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport. J Cell Biol. 1982 Dec;95(3):781–792. doi: 10.1083/jcb.95.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gueguen M., Boniface O., Bernard O., Clerc F., Cartwright T., Alvarez F. Identification of the main epitope on human cytochrome P450 IID6 recognized by anti-liver kidney microsome antibody. J Autoimmun. 1991 Aug;4(4):607–615. doi: 10.1016/0896-8411(91)90180-k. [DOI] [PubMed] [Google Scholar]
  11. Gueguen M., Meunier-Rotival M., Bernard O., Alvarez F. Anti-liver kidney microsome antibody recognizes a cytochrome P450 from the IID subfamily. J Exp Med. 1988 Aug 1;168(2):801–806. doi: 10.1084/jem.168.2.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gueguen M., Yamamoto A. M., Bernard O., Alvarez F. Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1. Biochem Biophys Res Commun. 1989 Mar 15;159(2):542–547. doi: 10.1016/0006-291x(89)90027-2. [DOI] [PubMed] [Google Scholar]
  13. Homberg J. C., Abuaf N., Bernard O., Islam S., Alvarez F., Khalil S. H., Poupon R., Darnis F., Lévy V. G., Grippon P. Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of "autoimmune" hepatitis. Hepatology. 1987 Nov-Dec;7(6):1333–1339. doi: 10.1002/hep.1840070626. [DOI] [PubMed] [Google Scholar]
  14. Hurley W. L., Finkelstein E., Holst B. D. Identification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique. J Immunol Methods. 1985 Dec 17;85(1):195–202. doi: 10.1016/0022-1759(85)90287-x. [DOI] [PubMed] [Google Scholar]
  15. Inamoto T., Brown W. R. IgG is associated with the asialoglycoprotein receptor in the human liver. Hepatology. 1991 Dec;14(6):1070–1075. [PubMed] [Google Scholar]
  16. Jacqz-Aigrain E., Laurent J., Alvarez F. Dextromethorphan phenotypes in paediatric patients with autoimmune hepatitis. Br J Clin Pharmacol. 1990 Jul;30(1):153–154. doi: 10.1111/j.1365-2125.1990.tb03758.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kimura S., Umeno M., Skoda R. C., Meyer U. A., Gonzalez F. J. The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am J Hum Genet. 1989 Dec;45(6):889–904. [PMC free article] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Lenzi M., Bianchi F. B., Cassani F., Pisi E. Liver cell surface expression of the antigen reacting with liver-kidney microsomal antibody (LKM). Clin Exp Immunol. 1984 Jan;55(1):36–40. [PMC free article] [PubMed] [Google Scholar]
  20. Lisanti M. P., Caras I. W., Gilbert T., Hanzel D., Rodriguez-Boulan E. Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7419–7423. doi: 10.1073/pnas.87.19.7419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Loeper J., Descatoire V., Maurice M., Beaune P., Feldmann G., Larrey D., Pessayre D. Presence of functional cytochrome P-450 on isolated rat hepatocyte plasma membrane. Hepatology. 1990 May;11(5):850–858. doi: 10.1002/hep.1840110521. [DOI] [PubMed] [Google Scholar]
  22. Manns M. P., Griffin K. J., Sullivan K. F., Johnson E. F. LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase. J Clin Invest. 1991 Oct;88(4):1370–1378. doi: 10.1172/JCI115443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Manns M. P., Johnson E. F., Griffin K. J., Tan E. M., Sullivan K. F. Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450db1. J Clin Invest. 1989 Mar;83(3):1066–1072. doi: 10.1172/JCI113949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manns M., Zanger U., Gerken G., Sullivan K. F., Meyer zum Büschenfelde K. H., Meyer U. A., Eichelbaum M. Patients with type II autoimmune hepatitis express functionally intact cytochrome P-450 db1 that is inhibited by LKM-1 autoantibodies in vitro but not in vivo. Hepatology. 1990 Jul;12(1):127–132. doi: 10.1002/hep.1840120120. [DOI] [PubMed] [Google Scholar]
  25. Maurice M., Durand-Schneider A. M., Garbarz M., Feldmann G. Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies. Eur J Cell Biol. 1985 Nov;39(1):122–129. [PubMed] [Google Scholar]
  26. McFarlane I. G., McFarlane B. M., Major G. N., Tolley P., Williams R. Identification of the hepatic asialo-glycoprotein receptor (hepatic lectin) as a component of liver specific membrane lipoprotein (LSP). Clin Exp Immunol. 1984 Feb;55(2):347–354. [PMC free article] [PubMed] [Google Scholar]
  27. Monier S., Van Luc P., Kreibich G., Sabatini D. D., Adesnik M. Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane. J Cell Biol. 1988 Aug;107(2):457–470. doi: 10.1083/jcb.107.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ohtani Y., Kakumu S., Fuji A., Murase K. Autoantibodies against liver cell membrane antigens detected by enzyme-linked immunosorbent assay in acute and chronic liver disease. Clin Immunol Immunopathol. 1987 Apr;43(1):9–22. doi: 10.1016/0090-1229(87)90153-x. [DOI] [PubMed] [Google Scholar]
  29. Paradis K., Dib A., Homberg J. C., Bernard O., Alagille D., Alvarez F. Detection of anti-endoplasmic reticulum antibody-positive autoimmune hepatitis in children, using an ELISA technique. J Pediatr Gastroenterol Nutr. 1988 Nov-Dec;7(6):816–822. doi: 10.1097/00005176-198811000-00004. [DOI] [PubMed] [Google Scholar]
  30. Peakman M., Lobo-Yeo A., Mieli-Vergani G., Davies E. T., Mowat A. P., Vergani D. Characterization of anti-liver kidney microsomal antibody in childhood autoimmune chronic active hepatitis: evidence for IgG1 subclass restriction, polyclonality and non cross-reactivity with hepatocyte surface antigens. Clin Exp Immunol. 1987 Sep;69(3):543–549. [PMC free article] [PubMed] [Google Scholar]
  31. Pelloquin F., Lamelin J. P., Lenoir G. M. Human B lymphocytes immortalization by Epstein-Barr virus in the presence of cyclosporin A. In Vitro Cell Dev Biol. 1986 Dec;22(12):689–694. doi: 10.1007/BF02621085. [DOI] [PubMed] [Google Scholar]
  32. Rizzetto M., Swana G., Doniach D. Microsomal antibodies in active chronic hepatitis and other disorders. Clin Exp Immunol. 1973 Nov;15(3):331–344. [PMC free article] [PubMed] [Google Scholar]
  33. Sakaguchi M., Mihara K., Sato R. A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. EMBO J. 1987 Aug;6(8):2425–2431. doi: 10.1002/j.1460-2075.1987.tb02521.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sato T., Sakaguchi M., Mihara K., Omura T. The amino-terminal structures that determine topological orientation of cytochrome P-450 in microsomal membrane. EMBO J. 1990 Aug;9(8):2391–2397. doi: 10.1002/j.1460-2075.1990.tb07414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  36. Swanson N. R., Reed W. D., Yarred L. J., Bartholomaeus W. N., Shilkin K. B., Joske R. A. Autoantibodies to isolated human hepatocyte plasma membranes in liver disease. I. Specificity for chronic active hepatitis. Clin Immunol Immunopathol. 1989 Aug;52(2):291–304. doi: 10.1016/0090-1229(89)90180-3. [DOI] [PubMed] [Google Scholar]
  37. Swanson N. R., Reed W. D., Yarred L. J., Shilkin K. B., Joske R. A. Autoantibodies to isolated human hepatocyte plasma membranes in chronic active hepatitis. II. Specificity of antibodies. Hepatology. 1990 Apr;11(4):613–621. doi: 10.1002/hep.1840110414. [DOI] [PubMed] [Google Scholar]
  38. Szczesna-Skorupa E., Kemper B. NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2. J Cell Biol. 1989 Apr;108(4):1237–1243. doi: 10.1083/jcb.108.4.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Toda G., Ikeda Y., Kashiwagi M., Iwamori M., Oka H. Hepatocyte plasma membrane glycosphingolipid reactive with sera from patients with autoimmune chronic active hepatitis: its identification as sulfatide. Hepatology. 1990 Oct;12(4 Pt 1):664–670. doi: 10.1002/hep.1840120408. [DOI] [PubMed] [Google Scholar]
  40. Waxman D. J., Lapenson D. P., Krishnan M., Bernard O., Kreibich G., Alvarez F. Antibodies to liver/kidney microsome1 in chronic active hepatitis recognize specific forms of hepatic cytochrome P-450. Gastroenterology. 1988 Nov;95(5):1326–1331. doi: 10.1016/0016-5085(88)90368-x. [DOI] [PubMed] [Google Scholar]
  41. Winger L., Winger C., Shastry P., Russell A., Longenecker M. Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4484–4488. doi: 10.1073/pnas.80.14.4484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamamoto A. M., Mura C., Morales M. G., Bernard O., Krishnamoorthy R., Alvarez F. Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies. Clin Exp Immunol. 1992 Feb;87(2):251–255. doi: 10.1111/j.1365-2249.1992.tb02983.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zanger U. M., Hauri H. P., Loeper J., Homberg J. C., Meyer U. A. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8256–8260. doi: 10.1073/pnas.85.21.8256. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES