Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly
Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues including the vision. the collagen acknowledgement by PEDF Elf1 is definitely sequence- and conformation-specific and the high affinity binding motif is definitely KG(14) reported that PEDF inhibits VEGF-induced angiogenesis by regulating intracellular proteolysis of VEGF-receptor-1 (VEGFR-1) and inhibiting VEGFR-1 phosphorylation. Becerra SR141716 and SR141716 co-workers (15) reported the lipase-linked cell membrane SR141716 protein is definitely a neurotrophic receptor of PEDF. Involvement of the 37/67-kDa laminin receptor in the transmission transduction of PEDF has also been reported (16). Konson (17) further reported the serine phosphorylations of PEDF by protein kinase A and casein kinase 2 enhanced the binding of PEDF to 37/67-kDa laminin receptor. Beccera and co-workers (18) recently recognized ATP synthase F1 as an angiogenic PEDF receptor. The cells PEDF is not likely to be a soluble element but is rather deposited in extracellular matrices (ECMs) (19). PEDF was shown to associate with ECM components such as collagen heparan sulfate hyaluronan and chondroitin sulfate A-C (1 20 Although the native PEDF has an (19) showed that the tissue PEDF is deposited along the collagen fibers in the blood vessel-poor regions of the tissue from their immunohistochemical observations. These findings motivated us to investigate further details of the PEDF-collagen interaction to clarify the importance of this property. Collagen is the predominant ECM protein playing important roles in cell adhesion migration and differentiation. This protein is characterized by a unique triple helical structure constituted by long tandem repeats of Gly-triplets in which the and positions are frequently occupied by Pro and 4-hydroxyproline (Hyp (O)) residues. To date 28 types of collagen have been identified and classified into several groups according to the higher order structures. The polypeptide chain (called α-chain) compositions of collagen are variable; among the fibril-forming collagens collagen I is a heterotrimer consisting of two α1-chains and an α2-chain whereas collagens II and III are homotrimers. Self-trimerizing collagen-mimetic peptides have been utilized in the identification of biologically active sequences in collagen triple helices such as integrin α2β1-binding sequence GFOGER (26) and heparin/HSPG-binding sequence KGHR (27). HSPGs SR141716 are proteoglycans bearing heparan sulfate chains and have a wide range of biological functions and activities. Heparan sulfate which is a structural analog of heparin consists of sulfated d-glucuronic acid/l-iduronic acid and glucosamine disaccharide repeats (28). HSPGs interact with collagen I via heparan sulfate chains (29). Furthermore heparin/HSPG-binding collagen-like peptide was reported to inhibit vascular endothelial cell tube formation (27). This result suggested that HSPGs have pro-angiogenic roles. In this study we aimed to elucidate collagen recognition by PEDF using synthetic collagen-like peptides and to shed light on the functional relationship between PEDF and ECM components during angiogenesis. EXPERIMENTAL PROCEDURES Prediction of PEDF-binding Sequences An original text search program was written in Perl CGI script (Perl 5.8.1) and run on an Apache 1.3.33 HTML server. Using this program we extracted 9-10 amino acid peptides that contain both Arg and Lys residues from the sequences of SR141716 human α1(I)(776-1012) and α2(I)(776-1012) corresponding to the fragments. Peptides Peptide chains were constructed manually on the basis of a standard Fmoc (expressing glutathione is the change in absorbance at 313 nm from time 0 to 120 min; μ+ is the typical Δof wells including collagen and PEDF or heparin and μ? may be the SR141716 normal modification of absorbance from the collagen just in charge wells on a single dish. Obtained data had been analyzed using Source edition 7.5 data analysis software (OriginLab Northampton MA) and 50% inhibitory concentration (IC50) values were estimated through the midpoint from the inhibition curves. Enzyme-linked Immunosorbent Assay (ELISA) Wells of 96-well plates (Nunc Kamstrup Denmark) had been covered with 50 μl of 10 μg/ml.