Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

Abstract

The autocrine͞paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF͞ CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB͞c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.Currently, more than 100 soluble peptide signaling molecules, including peptide hormones, growth factors, and lymphokines, are known. These molecules can be classified into two types: one, like classic hormones, is produced in a specific organ͞cell and exerts a characteristic effect on relatively limited target cells via blood flow (endocrine), and the other is produced in a wide variety of tissues and acts in an autocrine or a paracrine manner with low target specificity. In the latter case, the specificity of the action depends for the most part on spatiotemporarily controlled production of the signaling molecule and the local tissue architecture. The former type of molecules (endocrine factors) are suitable as therapeutic agents as they can be delivered systemically without loss of target specificity. In contrast, autocrine͞paracrine factors may induce diverse responses in various tissues when present in the blood at concentrations higher than a threshold value. Thus, one cannot expect specific therapeutic effects of the factors administered by ordinary methods, though they have many promising biologic activities for clinical applications.

Substrate Recognition by the Collagen-binding Domain ofClostridium histolyticum Class I Collagenase

“…8B). Positive binding of the mutated enzyme to G(POG) 8 peptide was also observed by surface plasmon resonance. In this binding profile (Fig.…”

“…6B). The proteins did not bind to Sepharose beads coupled with any of the following 24-mer peptides; (PG) 12 , (PPPG) 6 , all-D-(PPG) 8 , (APG) 8 , or (PAG) 8 (Fig. 6C, lanes 1-5) or to gelatin-Sepharose beads (Fig.…”

“…For applications, we have shown that the CBD can be used to anchor peptide-signaling molecules to collagen-containing tissues (8). Fusion proteins between growth factors and S2bϩS3 bound not only to insoluble collagen in vitro but to connective tissue in vivo.…”

“…A peptide, G(POG) 8 , was dissolved in 10 mM sodium acetate (pH 6.0), and the solution was incubated at 4°C for 48 h prior to use. The peptide was covalently immobilized to a biosensor chip CM5 through the ␣-amino group following the supplier's protocol.…”

Spatio-Temporal Modification of Collagen Scaffolds Mediated by Triple Helical Propensity

“…Physical interactions, such as hydrogen bonding, hydrophobic, and ionic interactions and biological affinity interactions, provide a much more convenient and natural way of conjugating agents to proteins and other macromolecules as opposed to covalent chemical attachment. 21 For example, biotin-avidin interactions, metal-mediated histidine-NTA interactions, and antigen-antibody binding are all effective protein conjugation tools in biology research. Recent studies on collagens have shown that even at physiological conditions, type I collagen is thermally unstable.…”