RESUMO
Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.
Assuntos
Colágeno Tipo I/química , Proteínas do Olho/química , Proteoglicanas de Heparan Sulfato/química , Heparina/química , Fatores de Crescimento Neural/química , Peptídeos/química , Serpinas/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/genética , Heparina/metabolismo , Humanos , Camundongos , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Serpinas/genética , Serpinas/metabolismoRESUMO
Collagen is an abundantly distributed extracellular matrix protein in mammalian bodies that maintains structural integrity of the organs and tissues. Besides its function as a structural protein, collagen has various biological functions which regulate cell adhesion, migration and differentiation. In order to develop totally synthetic collagen-surrogates, we recently reported a basic concept for preparing collagen-like triple helical supramolecules based on the self-assembly of staggered trimeric peptides with self-complementary shapes. In this paper, we add one of the specific cellular functions of the native collagen to the collagen-mimetic supramolecule. We synthesized a self-assembling peptide unit containing the integrin-binding sequence Gly-Phe-Hyp-Gly-Glu-Arg. The supramolecule carrying the sequence exhibited significant binding activity to human dermal fibroblasts. The supramolecular structure was found to be essential for function in in vitro cell culture. Cell adhesion was shown to be comparable to that of native collagen, and was further demonstrated to be mediated solely by integrin alpha 2 beta 1. Well-grown focal contacts and stress fibers were observed in cells spread on the supramolecular collagen-mimetic. The results demonstrate the potential of peptide-based artificial collagen as a biomaterial for regulating specific cellular function and fate.
Assuntos
Materiais Biomiméticos/farmacologia , Colágeno/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Integrinas/metabolismo , Peptídeos/síntese química , Peptídeos/farmacologia , Sequência de Aminoácidos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Fibroblastos/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/química , Estrutura Secundária de Proteína , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen-CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen-CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.
