Abstract
Latent transforming growth factor (TGF) beta-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFbeta. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit "exquisite specificities," a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.
Highlights
Fibrillin microfibrils are ubiquitous structural elements in the connective tissue
Latent transforming growth factor (TGF)-binding protein (LTBP)3-1 associates with fibrillin microfibrils in the perichondrium and in osteoblast cultures (7, 8), and LTBP-1 and LTBP-4 interact with fibrillin (9)
Extracellular Matrix Incorporation of LTBPs Requires Fibrillin-1—Dermal fibroblasts established from neonatal wild type, fibrillin-1 (Fbn1) null, and fibrillin-2 (Fbn2) null mice were examined by immunofluorescence, Western blotting, and quantitative real-time PCR, to determine the status of LTBPs in the absence of fibrillin-1
Summary
Recombinant Proteins—Construction and purification of recombinant fibrillin-1 polypeptides rF23 (10) and rF31 (26) were previously described. New recombinant fibrillin-1 polypeptides were generated with specific mutations designed within the context of rF23 Generation of these new expression constructs is described in the following paragraphs. All fibrillin-1 recombinant polypeptides were harvested from the media of stably transfected 293/EBNA cells and were purified using chelating chromatography, followed by molecular sieve chromatography, as we have described previously (9). For construction of rF80 (deletion of Hybrid1), pCEPSP-rF23 was used as a template along with primers pCEP-5Ј and JE3cb1-AS to generate a NheI-BbsI PCR product. Histagged human fibulin-4 was purified using nickel-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) followed by molecular sieve chromatography on a Superose 12 column (HR16/50) equilibrated in 2 M urea, 50 mM Tris-HCl, pH 8.0
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