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. 1987 Dec;84(23):8272–8276. doi: 10.1073/pnas.84.23.8272

Isolation and characterization of cDNAs encoding the heavy chain of human inter-alpha-trypsin inhibitor (I alpha TI): unambiguous evidence for multipolypeptide chain structure of I alpha TI.

J P Salier 1, M Diarra-Mehrpour 1, R Sesboue 1, J Bourguignon 1, R Benarous 1, I Ohkubo 1, S Kurachi 1, K Kurachi 1, J P Martin 1
PMCID: PMC299524  PMID: 2446322

Abstract

Human inter-alpha-trypsin inhibitor (I alpha TI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that I alpha TI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of I alpha TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum I alpha TI. RNA blot analyses of liver RNAs with H- or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000. These results unambiguously establish that I alpha TI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiol-proteinase inhibitors. These data indicate that I alpha TI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.

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Selected References

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

  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bourguignon J., Diarra-Mehrpour M., Sesboü R., Frain M., Sala-Trepat J. M., Martin J. P., Salier J. P. Human inter-alpha-trypsin-inhibitor: characterization and partial nucleotide sequencing of a light chain-encoding cDNA. Biochem Biophys Res Commun. 1985 Sep 30;131(3):1146–1153. doi: 10.1016/0006-291x(85)90210-4. [DOI] [PubMed] [Google Scholar]
  3. Bourguignon J., Vercaigne D., Sesboü R., Martin J. P., Salier J. P. Inter-alpha-trypsin-inhibitor (ITI): two distinct mRNAs in baboon liver argue for a discrete synthesis of ITI and ITI derivatives. FEBS Lett. 1983 Oct 17;162(2):379–383. doi: 10.1016/0014-5793(83)80791-1. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Deininger P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem. 1983 Feb 15;129(1):216–223. doi: 10.1016/0003-2697(83)90072-6. [DOI] [PubMed] [Google Scholar]
  6. Gariépy J., Hodges R. S. Primary sequence analysis and folding behavior of EF hands in relation to the mechanism of action of troponin C and calmodulin. FEBS Lett. 1983 Aug 22;160(1-2):1–6. doi: 10.1016/0014-5793(83)80924-7. [DOI] [PubMed] [Google Scholar]
  7. Higashiyama S., Ohkubo I., Ishiguro H., Kunimatsu M., Sawaki K., Sasaki M. Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain. Biochemistry. 1986 Apr 8;25(7):1669–1675. doi: 10.1021/bi00355a034. [DOI] [PubMed] [Google Scholar]
  8. Hochstrasser K., Schönberger O. L., Rossmanith I., Wachter E. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, V. Attachments of carbohydrates in the human urinary trypsin inhibitor isolated by affinity chromatography. Hoppe Seylers Z Physiol Chem. 1981 Oct;362(10):1357–1362. doi: 10.1515/bchm2.1981.362.2.1357. [DOI] [PubMed] [Google Scholar]
  9. Kastern W., Björck L., Akerström B. Developmental and tissue-specific expression of alpha 1-microglobulin mRNA in the rat. J Biol Chem. 1986 Nov 15;261(32):15070–15074. [PubMed] [Google Scholar]
  10. Kaumeyer J. F., Polazzi J. O., Kotick M. P. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes alpha-1-microglobulin (protein HC). Nucleic Acids Res. 1986 Oct 24;14(20):7839–7850. doi: 10.1093/nar/14.20.7839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lorand L., Credo R. B., Janus T. J. Factor XIII (fibrin-stabilizing factor). Methods Enzymol. 1981;80(Pt 100):333–341. doi: 10.1016/s0076-6879(81)80029-8. [DOI] [PubMed] [Google Scholar]
  12. McKeehan W. L., Sakagami Y., Hoshi H., McKeehan K. A. Two apparent human endothelial cell growth factors from human hepatoma cells are tumor-associated proteinase inhibitors. J Biol Chem. 1986 Apr 25;261(12):5378–5383. [PubMed] [Google Scholar]
  13. Méndez E., Fernández-Luna J. L., Grubb A., Leyva-Cobián F. Human protein HC and its IgA complex are inhibitors of neutrophil chemotaxis. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1472–1475. doi: 10.1073/pnas.83.5.1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ohkubo I., Kurachi K., Takasawa T., Shiokawa H., Sasaki M. Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry. 1984 Nov 20;23(24):5691–5697. doi: 10.1021/bi00319a005. [DOI] [PubMed] [Google Scholar]
  15. Reisinger P., Hochstrasser K., Albrecht G. J., Lempart K., Salier J. P. Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase. Biol Chem Hoppe Seyler. 1985 May;366(5):479–483. doi: 10.1515/bchm3.1985.366.1.479. [DOI] [PubMed] [Google Scholar]
  16. Salier J. P., Martin J. P., Lambin P., McPhee H., Hochstrasser K. Purification of the human serum inter-alpha-trypsin inhibitor by zinc chelate and hydrophobic interaction chromatographies. Anal Biochem. 1980 Dec;109(2):273–283. doi: 10.1016/0003-2697(80)90649-1. [DOI] [PubMed] [Google Scholar]
  17. Salier J. P., Sesboü R., Vercaigne D., Bourguignon J., Martin J. P. Inter-alpha-trypsin inhibitor (ITI): use of new antisera for qualitative studies and discrete quantitation of ITI and its derivatives. Anal Biochem. 1983 Sep;133(2):336–343. doi: 10.1016/0003-2697(83)90093-3. [DOI] [PubMed] [Google Scholar]
  18. Sasagawa T., Ericsson L. H., Walsh K. A., Schreiber W. E., Fischer E. H., Titani K. Complete amino acid sequence of human brain calmodulin. Biochemistry. 1982 May 11;21(10):2565–2569. doi: 10.1021/bi00539a041. [DOI] [PubMed] [Google Scholar]
  19. Takahashi N., Takahashi Y., Putnam F. W. Primary structure of blood coagulation factor XIIIa (fibrinoligase, transglutaminase) from human placenta. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8019–8023. doi: 10.1073/pnas.83.21.8019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wachter E., Hochstrasser K. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, III. Sequence of the two Kunitz-type domains inside the native inter-alpha-trypsin inhibitor, its biological aspects and also of its cleavage products. Hoppe Seylers Z Physiol Chem. 1979 Sep;360(9):1305–1311. doi: 10.1515/bchm2.1979.360.2.1305. [DOI] [PubMed] [Google Scholar]

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