Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 May 15;316(Pt 1):43–48. doi: 10.1042/bj3160043

Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus.

S Eda 1, Y Suzuki 1, T Kase 1, T Kawai 1, K Ohtani 1, T Sakamoto 1, T Kurimura 1, N Wakamiya 1
PMCID: PMC1217348  PMID: 8645231

Abstract

Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.

Full Text

The Full Text of this article is available as a PDF (381.0 KB).

Selected References

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

  1. Crouch E., Chang D., Rust K., Persson A., Heuser J. Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly. J Biol Chem. 1994 Jun 3;269(22):15808–15813. [PubMed] [Google Scholar]
  2. Drickamer K., Dordal M. S., Reynolds L. Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. J Biol Chem. 1986 May 25;261(15):6878–6887. [PubMed] [Google Scholar]
  3. Erdei A., Reid K. B. Characterization of the human C1q receptor. Behring Inst Mitt. 1989 Jul;(84):216–219. [PubMed] [Google Scholar]
  4. Hartshorn K. L., Crouch E. C., White M. R., Eggleton P., Tauber A. I., Chang D., Sastry K. Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses. J Clin Invest. 1994 Jul;94(1):311–319. doi: 10.1172/JCI117323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hoppe H. J., Barlow P. N., Reid K. B. A parallel three stranded alpha-helical bundle at the nucleation site of collagen triple-helix formation. FEBS Lett. 1994 May 16;344(2-3):191–195. doi: 10.1016/0014-5793(94)00383-1. [DOI] [PubMed] [Google Scholar]
  6. Ikeda K., Sannoh T., Kawasaki N., Kawasaki T., Yamashina I. Serum lectin with known structure activates complement through the classical pathway. J Biol Chem. 1987 Jun 5;262(16):7451–7454. [PubMed] [Google Scholar]
  7. Janeway C. A., Jr The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol Today. 1992 Jan;13(1):11–16. doi: 10.1016/0167-5699(92)90198-G. [DOI] [PubMed] [Google Scholar]
  8. Kawasaki N., Kawasaki T., Yamashina I. Isolation and characterization of a mannan-binding protein from human serum. J Biochem. 1983 Sep;94(3):937–947. doi: 10.1093/oxfordjournals.jbchem.a134437. [DOI] [PubMed] [Google Scholar]
  9. Kawasaki N., Yokota Y., Kawasaki T. Differentiation of conglutination activity and sugar-binding activity of conglutinin after removal of NH2-terminal 54 amino acid residues by endogenous serine protease(s). Arch Biochem Biophys. 1993 Sep;305(2):533–540. doi: 10.1006/abbi.1993.1457. [DOI] [PubMed] [Google Scholar]
  10. Lee Y. M., Leiby K. R., Allar J., Paris K., Lerch B., Okarma T. B. Primary structure of bovine conglutinin, a member of the C-type animal lectin family. J Biol Chem. 1991 Feb 15;266(5):2715–2723. [PubMed] [Google Scholar]
  11. Lim B. L., Wang J. Y., Holmskov U., Hoppe H. J., Reid K. B. Expression of the carbohydrate recognition domain of lung surfactant protein D and demonstration of its binding to lipopolysaccharides of gram-negative bacteria. Biochem Biophys Res Commun. 1994 Aug 15;202(3):1674–1680. doi: 10.1006/bbrc.1994.2127. [DOI] [PubMed] [Google Scholar]
  12. Lu J., Wiedemann H., Holmskov U., Thiel S., Timpl R., Reid K. B. Structural similarity between lung surfactant protein D and conglutinin. Two distinct, C-type lectins containing collagen-like sequences. Eur J Biochem. 1993 Aug 1;215(3):793–799. doi: 10.1111/j.1432-1033.1993.tb18094.x. [DOI] [PubMed] [Google Scholar]
  13. Lu J., Willis A. C., Reid K. B. Purification, characterization and cDNA cloning of human lung surfactant protein D. Biochem J. 1992 Jun 15;284(Pt 3):795–802. doi: 10.1042/bj2840795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Malhotra R., Haurum J. S., Thiel S., Sim R. B. Binding of human collectins (SP-A and MBP) to influenza virus. Biochem J. 1994 Dec 1;304(Pt 2):455–461. doi: 10.1042/bj3040455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Malhotra R., Haurum J., Thiel S., Sim R. B. Interaction of C1q receptor with lung surfactant protein A. Eur J Immunol. 1992 Jun;22(6):1437–1445. doi: 10.1002/eji.1830220616. [DOI] [PubMed] [Google Scholar]
  16. Malhotra R., Thiel S., Reid K. B., Sim R. B. Human leukocyte C1q receptor binds other soluble proteins with collagen domains. J Exp Med. 1990 Sep 1;172(3):955–959. doi: 10.1084/jem.172.3.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ogasawara Y., McCormack F. X., Mason R. J., Voelker D. R. Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction. J Biol Chem. 1994 Nov 25;269(47):29785–29792. [PubMed] [Google Scholar]
  18. Okuno Y., Tanaka K., Baba K., Maeda A., Kunita N., Ueda S. Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system. J Clin Microbiol. 1990 Jun;28(6):1308–1313. doi: 10.1128/jcm.28.6.1308-1313.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Persson A., Chang D., Crouch E. Surfactant protein D is a divalent cation-dependent carbohydrate-binding protein. J Biol Chem. 1990 Apr 5;265(10):5755–5760. [PubMed] [Google Scholar]
  20. Sheriff S., Chang C. Y., Ezekowitz R. A. Human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil. Nat Struct Biol. 1994 Nov;1(11):789–794. doi: 10.1038/nsb1194-789. [DOI] [PubMed] [Google Scholar]
  21. Sim R. B., Reid K. B. C1: molecular interactions with activating systems. Immunol Today. 1991 Sep;12(9):307–311. doi: 10.1016/0167-5699(91)90004-D. [DOI] [PubMed] [Google Scholar]
  22. Spissinger T., Schäfer K. P., Voss T. Assembly of the surfactant protein SP-A. Deletions in the globular domain interfere with the correct folding of the molecule. Eur J Biochem. 1991 Jul 1;199(1):65–71. doi: 10.1111/j.1432-1033.1991.tb16092.x. [DOI] [PubMed] [Google Scholar]
  23. Strang C. J., Slayter H. S., Lachmann P. J., Davis A. E., 3rd Ultrastructure and composition of bovine conglutinin. Biochem J. 1986 Mar 1;234(2):381–389. doi: 10.1042/bj2340381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sumiya M., Super M., Tabona P., Levinsky R. J., Arai T., Turner M. W., Summerfield J. A. Molecular basis of opsonic defect in immunodeficient children. Lancet. 1991 Jun 29;337(8757):1569–1570. doi: 10.1016/0140-6736(91)93263-9. [DOI] [PubMed] [Google Scholar]
  25. Summerfield J. A., Ryder S., Sumiya M., Thursz M., Gorchein A., Monteil M. A., Turner M. W. Mannose binding protein gene mutations associated with unusual and severe infections in adults. Lancet. 1995 Apr 8;345(8954):886–889. doi: 10.1016/s0140-6736(95)90009-8. [DOI] [PubMed] [Google Scholar]
  26. Super M., Thiel S., Lu J., Levinsky R. J., Turner M. W. Association of low levels of mannan-binding protein with a common defect of opsonisation. Lancet. 1989 Nov 25;2(8674):1236–1239. doi: 10.1016/s0140-6736(89)91849-7. [DOI] [PubMed] [Google Scholar]
  27. Suzuki Y., Yin Y., Makino M., Kurimura T., Wakamiya N. Cloning and sequencing of a cDNA coding for bovine conglutinin. Biochem Biophys Res Commun. 1993 Mar 15;191(2):335–342. doi: 10.1006/bbrc.1993.1222. [DOI] [PubMed] [Google Scholar]
  28. Wakamiya N., Okuno Y., Sasao F., Ueda S., Yoshimatsu K., Naiki M., Kurimura T. Isolation and characterization of conglutinin as an influenza A virus inhibitor. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1270–1278. doi: 10.1016/0006-291x(92)90440-v. [DOI] [PubMed] [Google Scholar]
  29. White R. T., Damm D., Miller J., Spratt K., Schilling J., Hawgood S., Benson B., Cordell B. Isolation and characterization of the human pulmonary surfactant apoprotein gene. 1985 Sep 26-Oct 2Nature. 317(6035):361–363. doi: 10.1038/317361a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES