June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Mutant LTBP-2 proteins lack secretion ability and fibrillin-1 binding activity
Author Affiliations & Notes
  • Tomoya Akama
    Pharmacology, Kansai Medical University, Moriguchi, Japan
    Tumor Microenvironment, Sanford-Burnham Med Res Inst, La Jolla, CA
  • Yusuke Fujikawa
    Pharmacology, Kansai Medical University, Moriguchi, Japan
  • Tadashi Inoue
    Pharmacology, Kansai Medical University, Moriguchi, Japan
  • Tomoyuki Nakamura
    Pharmacology, Kansai Medical University, Moriguchi, Japan
  • Footnotes
    Commercial Relationships Tomoya Akama, None; Yusuke Fujikawa, None; Tadashi Inoue, None; Tomoyuki Nakamura, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1353. doi:
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    • Get Citation

      Tomoya Akama, Yusuke Fujikawa, Tadashi Inoue, Tomoyuki Nakamura; Mutant LTBP-2 proteins lack secretion ability and fibrillin-1 binding activity. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1353.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Latent TGFβ-binding protein 2 (LTBP-2) is an extracellular matrix protein that belongs to LTBP family proteins. Homozygous mutations on LTBP2, which encodes LTBP-2, have been found on several congenital ocular diseases such as primary congenital glaucoma, Weill-Marchesani syndrome and congenital megalocornea, indicating an important function of LTBP-2 on eye development. Previous reports including ours pointed out direct binding between LTBP-2 and fibrillin-1, an essential extracellular matrix protein for microfibril formation, and suggested involvement of LTBP-2 in microfibril organization. Thus we assumed that mutations found on LTBP2 affect fibrillin-1 binding activity of LTBP-2. In this work, we studied ability of mutant LTBP-2 to bind to fibrillin-1 molecule.

Methods: We constructed expression vectors encoding mutant LTBP-2s and produced the mutants in culture medium of transfected HEK293T cells. These mutant proteins were individually reacted to recombinant fibrillin-1 fragment and subjected to immunoprecipitation to detect specific binding.

Results: By recombinant protein production and immunoprecipitation, we identified that LTBP-2 binds to fibrillin-1 by the latter half of a large domain consists of tandem Ca2+ binding EGF-like repeats. By searching literatures, we obtained four pathogenic mutant LTBP-2s that possess intact fibrillin-1 binding domain, and constructed expression vectors for the mutant proteins. We then transfected each of the expression vectors into HEK293T cells to produce recombinant LTBP-2 in culture medium, however, all the mutant LTBP-2s were not recovered in the medium although all the mutant proteins were detected in cell lysates, indicating the mutant LTBP-2s were produced but not secreted. We further analyzed binding activity of mutant LTBP-2s to fibrillin-1, by recovering the mutant proteins from cell lysates, and found three out of four mutant LTBP-2s did not bind to fibrillin-1.

Conclusions: We found that pathogenic LTBP-2 mutants were secretion-deficient and inactive to fibrillin-1 binding. Because all the tested mutants possess minimal mutations (single amino acid replacement or alteration of C-terminal region), we assume these mutations lead to conformational alteration and result in secretion arrest as well as functional deficiency. These findings may link to explain pathogenensis of ocular diseases caused by LTBP2 mutation.

Keywords: 519 extracellular matrix • 455 ciliary body • 660 proteins encoded by disease genes  

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