title: LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin, and is a negative modulator of elastinogenesis.
abstract: Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd=26.47±5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd=24.66±5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.
authors.affiliation: School of Medical Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Sideek
authors.affiliation: School of Medical Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: Clementine
authors.initials: C
authors.lastname: Menz
authors.affiliation: School of Medical Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia.
authors.forename: Mahroo K
authors.initials: MK
authors.lastname: Parsi
authors.affiliation: School of Medical Sciences, University of Adelaide, Adelaide, South Australia 5005, Australia. Electronic address: mark.gibson@adelaide.edu.au.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Gibson
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004547:Elastic Tissue / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D000071838:Fibrillin-1; D000071837:Fibrillins; D005347:Fibroblasts / Q000378:metabolism; D006493:Heparin / Q000235:genetics* / Q000378:metabolism; D006497:Heparan Sulfate / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053779:Latent TGF-beta Binding Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D011485:Protein Binding; D014333:Tropoelastin / Q000235:genetics / Q000378:metabolism*; D054392:Adipokines
chemical_list: D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D006493:Heparin; D006497:Heparan Sulfate; D053779:Latent TGF-beta Binding Proteins; D008840:Microfilament Proteins; D014333:Tropoelastin; C492373:FBLN5 protein, human; C000605722:FBN1 protein, human; D054392:Adipokines; C000605724:Fbn1 protein, mouse; C509035:LTBP2 protein, human
keywords: Fibulin-5; Heparan sulfate; Heparin; LTBP-2; Tropoelastin