Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Sep 1;318(Pt 2):591–596. doi: 10.1042/bj3180591

Intracellular single-chain antibody inhibits integrin VLA-4 maturation and function.

Q Yuan 1, K L Strauch 1, R R Lobb 1, M E Hemler 1
PMCID: PMC1217661  PMID: 8809051

Abstract

A single-chain antibody construct was prepared containing the VH and VL regions of anti-(integrin alpha 4) antibody HP1/2, an interchain linker and a KDEL endoplasmic reticulum retention sequence. Intracellular expression of this single-chain antibody caused cell-surface expression of alpha 4 beta 1 integrin to be decreased by 80% on selected RD cells and by 65-100% on selected Jurkat cells, relative to mock transfectants. Immunoprecipitation from single-chain-antibody-transfected cells showed that the single-chain antibody was complexed with the integrin alpha 4 and beta 1 subunits, and the diminished sizes of alpha 4 and beta 1 were consistent with impaired maturation. Furthermore, cell adhesion to alpha 4 beta 1 ligands [VCAM-1 (vascular cell adhesion molecule-1), FN40 (40 kDa chymotryptic fragment of fibronectin) and CS1] was greatly impaired in both RD and Jurkat cells, and cell spreading on immobilized FN40 protein was almost completely eliminated. Thus we conclude that intracellular single-chain antibodies may be used to reduce or eliminate cell-surface expression of a specific integrin, with specific functional consequences. This approach should be generally applicable to other integrin subunits.

Full Text

The Full Text of this article is available as a PDF (471.4 KB).

Selected References

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

  1. Beerli R. R., Wels W., Hynes N. E. Intracellular expression of single chain antibodies reverts ErbB-2 transformation. J Biol Chem. 1994 Sep 30;269(39):23931–23936. [PubMed] [Google Scholar]
  2. Biocca S., Neuberger M. S., Cattaneo A. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 1990 Jan;9(1):101–108. doi: 10.1002/j.1460-2075.1990.tb08085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bretscher M. S. Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1. EMBO J. 1992 Feb;11(2):405–410. doi: 10.1002/j.1460-2075.1992.tb05068.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burkly L. C., Jakubowski A., Newman B. M., Rosa M. D., Chi-Rosso G., Lobb R. R. Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA-4 promotes CD3-dependent T cell proliferation. Eur J Immunol. 1991 Nov;21(11):2871–2875. doi: 10.1002/eji.1830211132. [DOI] [PubMed] [Google Scholar]
  5. Carlson J. R. A new means of inducibly inactivating a cellular protein. Mol Cell Biol. 1988 Jun;8(6):2638–2646. doi: 10.1128/mcb.8.6.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter W. G., Ryan M. C., Gahr P. J. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes. Cell. 1991 May 17;65(4):599–610. doi: 10.1016/0092-8674(91)90092-d. [DOI] [PubMed] [Google Scholar]
  7. Chan B. M., Elices M. J., Murphy E., Hemler M. E. Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on the human B cell line JY. J Biol Chem. 1992 Apr 25;267(12):8366–8370. [PubMed] [Google Scholar]
  8. Chen S. Y., Bagley J., Marasco W. A. Intracellular antibodies as a new class of therapeutic molecules for gene therapy. Hum Gene Ther. 1994 May;5(5):595–601. doi: 10.1089/hum.1994.5.5-595. [DOI] [PubMed] [Google Scholar]
  9. Damle N. K., Klussman K., Leytze G., Aruffo A., Linsley P. S., Ledbetter J. A. Costimulation with integrin ligands intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 augments activation-induced death of antigen-specific CD4+ T lymphocytes. J Immunol. 1993 Sep 1;151(5):2368–2379. [PubMed] [Google Scholar]
  10. Danilenko D. M., Rossitto P. V., Van der Vieren M., Le Trong H., McDonough S. P., Affolter V. K., Moore P. F. A novel canine leukointegrin, alpha d beta 2, is expressed by specific macrophage subpopulations in tissue and a minor CD8+ lymphocyte subpopulation in peripheral blood. J Immunol. 1995 Jul 1;155(1):35–44. [PubMed] [Google Scholar]
  11. Duan L., Zhang H., Oakes J. W., Bagasra O., Pomerantz R. J. Molecular and virological effects of intracellular anti-Rev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains. Hum Gene Ther. 1994 Nov;5(11):1315–1324. doi: 10.1089/hum.1994.5.11-1315. [DOI] [PubMed] [Google Scholar]
  12. Elices M. J., Osborn L., Takada Y., Crouse C., Luhowskyj S., Hemler M. E., Lobb R. R. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell. 1990 Feb 23;60(4):577–584. doi: 10.1016/0092-8674(90)90661-w. [DOI] [PubMed] [Google Scholar]
  13. Fässler R., Pfaff M., Murphy J., Noegel A. A., Johansson S., Timpl R., Albrecht R. Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts. J Cell Biol. 1995 Mar;128(5):979–988. doi: 10.1083/jcb.128.5.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hahn W. C., Menzin E., Saito T., Germain R. N., Bierer B. E. The complete sequences of plasmids pFNeo and pMH-Neo: convenient expression vectors for high-level expression of eukaryotic genes in hematopoietic cell lines. Gene. 1993 May 30;127(2):267–268. doi: 10.1016/0378-1119(93)90731-h. [DOI] [PubMed] [Google Scholar]
  15. Hayashi Y., Iguchi T., Kawashima T., Bao Z. Z., Yacky C., Boettiger D., Horwitz A. F. Repression of integrin beta 1 subunit expression by antisense RNA. Cell Struct Funct. 1991 Jun;16(3):241–249. doi: 10.1247/csf.16.241. [DOI] [PubMed] [Google Scholar]
  16. Hemler M. E., Huang C., Schwarz L. The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight beta subunit. J Biol Chem. 1987 Mar 5;262(7):3300–3309. [PubMed] [Google Scholar]
  17. Hemler M. E., Huang C., Takada Y., Schwarz L., Strominger J. L., Clabby M. L. Characterization of the cell surface heterodimer VLA-4 and related peptides. J Biol Chem. 1987 Aug 25;262(24):11478–11485. [PubMed] [Google Scholar]
  18. Hemler M. E., Ware C. F., Strominger J. L. Characterization of a novel differentiation antigen complex recognize by a monoclonal antibody (A-1A5): unique activation-specific molecular forms on stimulated T cells. J Immunol. 1983 Jul;131(1):334–340. [PubMed] [Google Scholar]
  19. Huston J. S., Levinson D., Mudgett-Hunter M., Tai M. S., Novotný J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. doi: 10.1073/pnas.85.16.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kassner P. D., Hemler M. E. Interchangeable alpha chain cytoplasmic domains play a positive role in control of cell adhesion mediated by VLA-4, a beta 1 integrin. J Exp Med. 1993 Aug 1;178(2):649–660. doi: 10.1084/jem.178.2.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kassner P. D., Kawaguchi S., Hemler M. E. Minimum alpha chain cytoplasmic tail sequence needed to support integrin-mediated adhesion. J Biol Chem. 1994 Aug 5;269(31):19859–19867. [PubMed] [Google Scholar]
  22. Keely P. J., Fong A. M., Zutter M. M., Santoro S. A. Alteration of collagen-dependent adhesion, motility, and morphogenesis by the expression of antisense alpha 2 integrin mRNA in mammary cells. J Cell Sci. 1995 Feb;108(Pt 2):595–607. doi: 10.1242/jcs.108.2.595. [DOI] [PubMed] [Google Scholar]
  23. Koivisto L., Heino J., Häkkinen L., Larjava H. The size of the intracellular beta 1-integrin precursor pool regulates maturation of beta 1-integrin subunit and associated alpha-subunits. Biochem J. 1994 Jun 15;300(Pt 3):771–779. doi: 10.1042/bj3000771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Koopman G., Keehnen R. M., Lindhout E., Newman W., Shimizu Y., van Seventer G. A., de Groot C., Pals S. T. Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J Immunol. 1994 Apr 15;152(8):3760–3767. [PubMed] [Google Scholar]
  25. Lemke H., Hammerling G. J., Hohmann C., Rajewsky K. Hybrid cell lines secreting monoclonal antibody specific for major histocompatibility antigens of the mouse. Nature. 1978 Jan 19;271(5642):249–251. doi: 10.1038/271249a0. [DOI] [PubMed] [Google Scholar]
  26. Lobb R. R., Hemler M. E. The pathophysiologic role of alpha 4 integrins in vivo. J Clin Invest. 1994 Nov;94(5):1722–1728. doi: 10.1172/JCI117519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maciejewski J. P., Weichold F. F., Young N. S., Cara A., Zella D., Reitz M. S., Jr, Gallo R. C. Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro. Nat Med. 1995 Jul;1(7):667–673. doi: 10.1038/nm0795-667. [DOI] [PubMed] [Google Scholar]
  28. Marasco W. A., Haseltine W. A., Chen S. Y. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7889–7893. doi: 10.1073/pnas.90.16.7889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Metzelaar M. J., Wijngaard P. L., Peters P. J., Sixma J. J., Nieuwenhuis H. K., Clevers H. C. CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J Biol Chem. 1991 Feb 15;266(5):3239–3245. [PubMed] [Google Scholar]
  30. Miyake K., Weissman I. L., Greenberger J. S., Kincade P. W. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med. 1991 Mar 1;173(3):599–607. doi: 10.1084/jem.173.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
  32. Nojima Y., Humphries M. J., Mould A. P., Komoriya A., Yamada K. M., Schlossman S. F., Morimoto C. VLA-4 mediates CD3-dependent CD4+ T cell activation via the CS1 alternatively spliced domain of fibronectin. J Exp Med. 1990 Oct 1;172(4):1185–1192. doi: 10.1084/jem.172.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Palmer E. L., Rüegg C., Ferrando R., Pytela R., Sheppard D. Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle. J Cell Biol. 1993 Dec;123(5):1289–1297. doi: 10.1083/jcb.123.5.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Parker C. M., Pujades C., Brenner M. B., Hemler M. E. Alpha 4/180, a novel form of the integrin alpha 4 subunit. J Biol Chem. 1993 Apr 5;268(10):7028–7035. [PubMed] [Google Scholar]
  35. Postigo A. A., Teixidó J., Sánchez-Madrid F. The alpha 4 beta 1/VCAM-1 adhesion pathway in physiology and disease. Res Immunol. 1993 Nov-Dec;144(9):723–762. doi: 10.1016/s0923-2494(93)80057-6. [DOI] [PubMed] [Google Scholar]
  36. Pujades C., Teixidó J., Bazzoni G., Hemler M. E. Integrin alpha 4 cysteines 278 and 717 modulate VLA-4 ligand binding and also contribute to alpha 4/180 formation. Biochem J. 1996 Feb 1;313(Pt 3):899–908. doi: 10.1042/bj3130899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pulido R., Elices M. J., Campanero M. R., Osborn L., Schiffer S., García-Pardo A., Lobb R., Hemler M. E., Sánchez-Madrid F. Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes. J Biol Chem. 1991 Jun 5;266(16):10241–10245. [PubMed] [Google Scholar]
  38. Richardson J. H., Sodroski J. G., Waldmann T. A., Marasco W. A. Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3137–3141. doi: 10.1073/pnas.92.8.3137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Romanic A. M., Madri J. A. The induction of 72-kD gelatinase in T cells upon adhesion to endothelial cells is VCAM-1 dependent. J Cell Biol. 1994 Jun;125(5):1165–1178. doi: 10.1083/jcb.125.5.1165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rosen G. D., Sanes J. R., LaChance R., Cunningham J. M., Roman J., Dean D. C. Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell. 1992 Jun 26;69(7):1107–1119. doi: 10.1016/0092-8674(92)90633-n. [DOI] [PubMed] [Google Scholar]
  41. Ryan D. H., Nuccie B. L., Abboud C. N., Winslow J. M. Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells. J Clin Invest. 1991 Sep;88(3):995–1004. doi: 10.1172/JCI115403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sato T., Tachibana K., Nojima Y., D'Avirro N., Morimoto C. Role of the VLA-4 molecule in T cell costimulation. Identification of the tyrosine phosphorylation pattern induced by the ligation of VLA-4. J Immunol. 1995 Sep 15;155(6):2938–2947. [PubMed] [Google Scholar]
  43. Schreiner C. L., Bauer J. S., Danilov Y. N., Hussein S., Sczekan M. M., Juliano R. L. Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor. J Cell Biol. 1989 Dec;109(6 Pt 1):3157–3167. doi: 10.1083/jcb.109.6.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Smith J. W., Hu D., Satterthwait A., Pinz-Sweeney S., Barbas C. F., 3rd Building synthetic antibodies as adhesive ligands for integrins. J Biol Chem. 1994 Dec 30;269(52):32788–32795. [PubMed] [Google Scholar]
  45. Stephens L. E., Sonne J. E., Fitzgerald M. L., Damsky C. H. Targeted deletion of beta 1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not tissue-specific gene expression. J Cell Biol. 1993 Dec;123(6 Pt 1):1607–1620. doi: 10.1083/jcb.123.6.1607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sánchez-Madrid F., De Landázuri M. O., Morago G., Cebrián M., Acevedo A., Bernabeu C. VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization. Eur J Immunol. 1986 Nov;16(11):1343–1349. doi: 10.1002/eji.1830161106. [DOI] [PubMed] [Google Scholar]
  47. Tavladoraki P., Benvenuto E., Trinca S., De Martinis D., Cattaneo A., Galeffi P. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature. 1993 Dec 2;366(6454):469–472. doi: 10.1038/366469a0. [DOI] [PubMed] [Google Scholar]
  48. Teixidó J., Hemler M. E., Greenberger J. S., Anklesaria P. Role of beta 1 and beta 2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma. J Clin Invest. 1992 Aug;90(2):358–367. doi: 10.1172/JCI115870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Teixidó J., Parker C. M., Kassner P. D., Hemler M. E. Functional and structural analysis of VLA-4 integrin alpha 4 subunit cleavage. J Biol Chem. 1992 Jan 25;267(3):1786–1791. [PubMed] [Google Scholar]
  50. Yang J. T., Rayburn H., Hynes R. O. Embryonic mesodermal defects in alpha 5 integrin-deficient mice. Development. 1993 Dec;119(4):1093–1105. doi: 10.1242/dev.119.4.1093. [DOI] [PubMed] [Google Scholar]
  51. Yurochko A. D., Liu D. Y., Eierman D., Haskill S. Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9034–9038. doi: 10.1073/pnas.89.19.9034. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES