LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation.

Clementine Menz,Julian R J Adams,Zlatko Kopecki,Mark A Gibson,Mohamed A Sideek,Mahroo K Parsi,Allison J Cowin,Christina Lynn Addison

doi:10.1371/journal.pone.0135577

Clementine Menz, Julian R J Adams + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0135577

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Journal: PloS one	Publication Date: Aug 11, 2015
Citations: 17	License type: CC BY 4.0

Affiliation: University of Adelaide, University of South Australia

Abstract

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9–14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.

Highlights

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a member of the fibrillin-LTBP superfamily of extracellular matrix proteins
Full-length recombinant LTBP-2 was tested for binding to a range of growth factors including vascular endothelial growth factor, bone morphogenic proteins (BMPs)-4, BMP-7 and fibroblast growth factor-2 (FGF-2) in an established solid phase binding assay (Fig 2A) [34]
This enabled the Kd for the LTBP-2 / FGF-2 interaction to be calculated by non-linear regression analysis of the curve produced by plotting amount FGF-2 bound versus concentration of FGF-2 added (Fig 3B)

Summary

Introduction

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a member of the fibrillin-LTBP superfamily of extracellular matrix proteins. In addition similar constructs encoding three smaller recombinant fragments spanning the central region of the LTBP-2 molecule were made, each encoding an N-terminal BM40 signal peptide and a C-terminal His6 tag (Fig 1A). A further experiment identified BMP-4 as a false positive, as the BMP-4 antibody showed binding to the LTBP-2 coated wells in the absence of BMP-4 protein (Fig 2B).

Results

Conclusion