Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Sep 15;98(6):1313–1323. doi: 10.1172/JCI118918

Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

L Lavaissiere 1, S Jia 1, M Nishiyama 1, S de la Monte 1, A M Stern 1, J R Wands 1, P A Friedman 1
PMCID: PMC507557  PMID: 8823296

Abstract

To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.

Full Text

The Full Text of this article is available as a PDF (634.5 KB).

Selected References

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

  1. Bullerdiek J., Takla G., Bartnitzke S., Brandt G., Chilla R., Haubrich J. Relationship of cytogenetic subtypes of salivary gland pleomorphic adenomas with patient age and histologic type. Cancer. 1989 Aug 15;64(4):876–880. doi: 10.1002/1097-0142(19890815)64:4<876::aid-cncr2820640419>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  2. Coffman C. R., Skoglund P., Harris W. A., Kintner C. R. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell. 1993 May 21;73(4):659–671. doi: 10.1016/0092-8674(93)90247-n. [DOI] [PubMed] [Google Scholar]
  3. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Diederich R. J., Matsuno K., Hing H., Artavanis-Tsakonas S. Cytosolic interaction between deltex and Notch ankyrin repeats implicates deltex in the Notch signaling pathway. Development. 1994 Mar;120(3):473–481. doi: 10.1242/dev.120.3.473. [DOI] [PubMed] [Google Scholar]
  5. Ellisen L. W., Bird J., West D. C., Soreng A. L., Reynolds T. C., Smith S. D., Sklar J. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell. 1991 Aug 23;66(4):649–661. doi: 10.1016/0092-8674(91)90111-b. [DOI] [PubMed] [Google Scholar]
  6. Fortini M. E., Rebay I., Caron L. A., Artavanis-Tsakonas S. An activated Notch receptor blocks cell-fate commitment in the developing Drosophila eye. Nature. 1993 Oct 7;365(6446):555–557. doi: 10.1038/365555a0. [DOI] [PubMed] [Google Scholar]
  7. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  8. Frohlich M., Motté P., Galvin K., Takahashi H., Wands J., Ozturk M. Enhanced expression of the protein kinase substrate p36 in human hepatocellular carcinoma. Mol Cell Biol. 1990 Jun;10(6):3216–3223. doi: 10.1128/mcb.10.6.3216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gronke R. S., VanDusen W. J., Garsky V. M., Jacobs J. W., Sardana M. K., Stern A. M., Friedman P. A. Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX. Proc Natl Acad Sci U S A. 1989 May;86(10):3609–3613. doi: 10.1073/pnas.86.10.3609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gronke R. S., Welsch D. J., VanDusen W. J., Garsky V. M., Sardana M. K., Stern A. M., Friedman P. A. Partial purification and characterization of bovine liver aspartyl beta-hydroxylase. J Biol Chem. 1990 May 25;265(15):8558–8565. [PubMed] [Google Scholar]
  11. Hartmann E., Rapoport T. A., Lodish H. F. Predicting the orientation of eukaryotic membrane-spanning proteins. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5786–5790. doi: 10.1073/pnas.86.15.5786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. He L., Isselbacher K. J., Wands J. R., Goodman H. M., Shih C., Quaroni A. Establishment and characterization of a new human hepatocellular carcinoma cell line. In Vitro. 1984 Jun;20(6):493–504. doi: 10.1007/BF02619623. [DOI] [PubMed] [Google Scholar]
  13. Itin C., Kappeler F., Linstedt A. D., Hauri H. P. A novel endocytosis signal related to the KKXX ER-retrieval signal. EMBO J. 1995 May 15;14(10):2250–2256. doi: 10.1002/j.1460-2075.1995.tb07219.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jhappan C., Gallahan D., Stahle C., Chu E., Smith G. H., Merlino G., Callahan R. Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands. Genes Dev. 1992 Mar;6(3):345–355. doi: 10.1101/gad.6.3.345. [DOI] [PubMed] [Google Scholar]
  15. Jia S., McGinnis K., VanDusen W. J., Burke C. J., Kuo A., Griffin P. R., Sardana M. K., Elliston K. O., Stern A. M., Friedman P. A. A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7227–7231. doi: 10.1073/pnas.91.15.7227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jia S., VanDusen W. J., Diehl R. E., Kohl N. E., Dixon R. A., Elliston K. O., Stern A. M., Friedman P. A. cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase. J Biol Chem. 1992 Jul 15;267(20):14322–14327. [PubMed] [Google Scholar]
  17. Kopan R., Nye J. S., Weintraub H. The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development. 1994 Sep;120(9):2385–2396. doi: 10.1242/dev.120.9.2385. [DOI] [PubMed] [Google Scholar]
  18. Kopan R., Schroeter E. H., Weintraub H., Nye J. S. Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1683–1688. doi: 10.1073/pnas.93.4.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Korioth F., Gieffers C., Frey J. Cloning and characterization of the human gene encoding aspartyl beta-hydroxylase. Gene. 1994 Dec 15;150(2):395–399. doi: 10.1016/0378-1119(94)90460-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Lieber T., Kidd S., Alcamo E., Corbin V., Young M. W. Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 1993 Oct;7(10):1949–1965. doi: 10.1101/gad.7.10.1949. [DOI] [PubMed] [Google Scholar]
  23. Lyman D., Young M. W. Further evidence for function of the Drosophila Notch protein as a transmembrane receptor. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10395–10399. doi: 10.1073/pnas.90.21.10395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manfioletti G., Brancolini C., Avanzi G., Schneider C. The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol. 1993 Aug;13(8):4976–4985. doi: 10.1128/mcb.13.8.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Monkovic D. D., VanDusen W. J., Petroski C. J., Garsky V. M., Sardana M. K., Zavodszky P., Stern A. M., Friedman P. A. Invertebrate aspartyl/asparaginyl beta-hydroxylase: potential modification of endogenous epidermal growth factor-like modules. Biochem Biophys Res Commun. 1992 Nov 30;189(1):233–241. doi: 10.1016/0006-291x(92)91549-6. [DOI] [PubMed] [Google Scholar]
  26. Myllylä R., Günzler V., Kivirikko K. I., Kaska D. D. Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases. Biochem J. 1992 Sep 15;286(Pt 3):923–927. doi: 10.1042/bj2860923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nye J. S., Kopan R., Axel R. An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells. Development. 1994 Sep;120(9):2421–2430. doi: 10.1242/dev.120.9.2421. [DOI] [PubMed] [Google Scholar]
  28. Pear W. S., Aster J. C., Scott M. L., Hasserjian R. P., Soffer B., Sklar J., Baltimore D. Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J Exp Med. 1996 May 1;183(5):2283–2291. doi: 10.1084/jem.183.5.2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pinkel D., Landegent J., Collins C., Fuscoe J., Segraves R., Lucas J., Gray J. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9138–9142. doi: 10.1073/pnas.85.23.9138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rebay I., Fehon R. G., Artavanis-Tsakonas S. Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell. 1993 Jul 30;74(2):319–329. doi: 10.1016/0092-8674(93)90423-n. [DOI] [PubMed] [Google Scholar]
  31. Roehl H., Kimble J. Control of cell fate in C. elegans by a GLP-1 peptide consisting primarily of ankyrin repeats. Nature. 1993 Aug 12;364(6438):632–635. doi: 10.1038/364632a0. [DOI] [PubMed] [Google Scholar]
  32. Schutze M. P., Peterson P. A., Jackson M. R. An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum. EMBO J. 1994 Apr 1;13(7):1696–1705. doi: 10.1002/j.1460-2075.1994.tb06434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shouval D., Eilat D., Carlson R. I., Adler R., Livni N., Wands J. R. Human hepatoma-associated cell surface antigen: identification and characterization by means of monoclonal antibodies. Hepatology. 1985 May-Jun;5(3):347–356. doi: 10.1002/hep.1840050302. [DOI] [PubMed] [Google Scholar]
  34. Stenflo J., Lundwall A., Dahlbäck B. beta-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S. Proc Natl Acad Sci U S A. 1987 Jan;84(2):368–372. doi: 10.1073/pnas.84.2.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stenflo J. Structure-function relationships of epidermal growth factor modules in vitamin K-dependent clotting factors. Blood. 1991 Oct 1;78(7):1637–1651. [PubMed] [Google Scholar]
  36. Stitt T. N., Conn G., Gore M., Lai C., Bruno J., Radziejewski C., Mattsson K., Fisher J., Gies D. R., Jones P. F. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell. 1995 Feb 24;80(4):661–670. doi: 10.1016/0092-8674(95)90520-0. [DOI] [PubMed] [Google Scholar]
  37. Struhl G., Fitzgerald K., Greenwald I. Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell. 1993 Jul 30;74(2):331–345. doi: 10.1016/0092-8674(93)90424-o. [DOI] [PubMed] [Google Scholar]
  38. Takahashi H., Ozturk M., Wilson B., Maki A., Ozawa K., Koizumi M., Endo K., Strauss W., Shouval D., Wands J. In vivo expression of two novel tumor-associated antigens and their use in immunolocalization of human hepatocellular carcinoma. Hepatology. 1989 Apr;9(4):625–634. doi: 10.1002/hep.1840090419. [DOI] [PubMed] [Google Scholar]
  39. Varnum B. C., Young C., Elliott G., Garcia A., Bartley T. D., Fridell Y. W., Hunt R. W., Trail G., Clogston C., Toso R. J. Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6. Nature. 1995 Feb 16;373(6515):623–626. doi: 10.1038/373623a0. [DOI] [PubMed] [Google Scholar]
  40. Wang Q. P., VanDusen W. J., Petroski C. J., Garsky V. M., Stern A. M., Friedman P. A. Bovine liver aspartyl beta-hydroxylase. Purification and characterization. J Biol Chem. 1991 Jul 25;266(21):14004–14010. [PubMed] [Google Scholar]
  41. Wilson B., Ozturk M., Takahashi H., Motté P., Kew M., Isselbacher K. J., Wands J. R. Cell-surface changes associated with transformation of human hepatocytes to the malignant phenotype. Proc Natl Acad Sci U S A. 1988 May;85(9):3140–3144. doi: 10.1073/pnas.85.9.3140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Witsell D. L., Weissler M. C., Livanos E., Bova-Hill C., Gilmer T. M. Unique chromosomal rearrangement and mucin production in a novel salivary myoepithelial cell strain. Arch Otolaryngol Head Neck Surg. 1993 Oct;119(10):1151–1157. doi: 10.1001/archotol.1993.01880220107013. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES