Identification of fibronectin binding sites in dermatopontin and their biological function
Introduction
Dermatopontin (DP) is an acidic dermal protein that comprises approximately 50 mg/kg wet weight of the dermis [1], [2], [3], [4], [5], [6]. DP interacts with a proteoglycan decorin and regulates collagen fibrillogenesis [7], [8], [9]. The importance of DP in forming the extracellular matrix (ECM) structure was highlighted by analysis of DP knockout mice, where the phenotype demonstrated Ehlers–Danlos syndrome, showing fragile and hyperelastic skin [10], [11]. Then, it was reported that expression of DP is enhanced and sustained in the area of myocardial infarction, suggesting that DP expression is related to the wound healing process [12].
We previously demonstrated that DP is a potent cell adhesion molecule for the epidermal keratinocyte cell line, HaCaT [13]. We also identified the cell surface receptors for DP as α3β1 integrin and syndecan-1. We further showed that the syndecan-1 binding site in DP is DP-4 (PHGQVVVAVRS) [13]. Recently, it was shown that expression of DP in cultured cardiac fibroblasts is enhanced by hypoxia, and that DP promotes adhesion and migration of fibroblasts [14]. Taken together, these reports suggest that DP may have multiple roles in wound healing.
Accordingly, we have found DP in a provisional matrix as well as in both the wound fluid and the serum [15]. The provisional matrix is an initial ECM, which is formed just after wounding, that serves as an early structure to support wound healing [16], [17], [18]. The provisional matrix is composed mainly of fibrin and fibronectin (Fn), and these components serve as an adhesive surface for both fibroblasts and endothelial cells [19], [20]. We have found that DP enhances the interaction between Fn and fibrin, and that DP polymerizes soluble Fn resulting in insoluble Fn fibrils. The Fn fibrils are called activated Fn, and activated Fn enhanced fibroblast adhesion activity [15]. Recently, we reported that DP binds fibrin/fibrinogen and accelerates fibrin formation [21]. The fibrin fibrils formed in the presence of DP demonstrated enhanced endothelial cell adhesion. Thus, DP is a molecule that interacts with other matrix components and can modify their structures and biological activities.
Activated Fn promotes enhanced cell adhesion [22] and has anticancer activity [23], [24], [25]; hence activated Fn may have therapeutic uses. As mentioned above, DP can activate Fn, however, the amount of DP is limited. Therefore, instead of using DP for activating Fn, an active peptide derived from DP would be beneficial since it can be produced chemically and in a large amount. Therefore, we sought to identify the active DP peptide which participates in the interaction with Fn and in the formation of activated Fn.
Section snippets
Materials
DP was purified from newborn calf dermis. Synthetic DP peptides and an anti-DP carboxyl-terminal peptide antibody were produced as reported previously and the sequences of the DP peptides are shown in our previous publication [7]. The sequences of deletion peptides are shown in Table 1. Human plasma Fn, an anti-Fn monoclonal antibody, 70-kDa thermolysin fragments, a recombinant anastellin, and a 120-kDa chymotryptic fragment of Fn were purchased from Sigma (St. Louis, MO). Retronectin, which is
Identification of Fn binding sites on DP
DP bound to immobilized Fn in a dose-dependent manner (Fig. 1A), and the binding profile was identical to that in our previous report [15]. An inhibition study of the interaction between DP and Fn was performed using sixteen sequential peptides that covered the entire DP sequence (Fig. 1B). The DP-4 peptide significantly inhibited DP binding to Fn but the other peptides did not. The direct interaction between DP-4 and Fn was examined using biotinylated DP-4 and a scrambled control DP-4 peptide,
Discussion
In the present study, a functional binding site in DP for Fn was identified as the DP-4 peptide, and the major DP binding site in Fn molecule for DP was identified as the B- and C-strands and their connecting sequence in the III14 domain. The cyclic peptide mimicking the B- and C-strands connecting loop structure enhanced the DP binding activity, suggesting that the non linear structure is important for the interaction between Fn and DP. Further, the DP-4 peptide did not completely inhibit the
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