Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen.

K C Ingham,S A Brew,B S Isaacs

doi:10.1016/s0021-9258(18)68828-3

Abstract

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.

Highlights

The fact that theanisotropy response to fragments of fibronectin was qualitatively similar to that of the whole protein argues against this interpretation since selfassociation, totheextentthatithas been characterized, possible that intramolecular associations between domains in fibronectin could affect the environment or conformation of the gelatin-binding domain in such a way as to increase its affinity for the strong site
Triple-helical collagen structures are known to unfold near this temperature and theexistence of such structures could explain a discontinuity in fibronectin binding
Isolated a chains and even CNBr fragments of type ment which appears to contain the strong site but I collagen are capable of reforming triple helical structures, one or more weak sites

Summary

Introduction

The fact that theanisotropy response to fragments of fibronectin was qualitatively similar to that of the whole protein argues against this interpretation since selfassociation, totheextentthatithas been characterized, possible that intramolecular associations between domains in fibronectin could affect the environment or conformation of the gelatin-binding domain in such a way as to increase its affinity for the strong site. Another possibility is that more distant domains of fibronectin have an affinity for collagen which is too weak to be detected independently but which appears to involve heterologous interactions between different contributes to the primary interaction when present as part domains widely separated in the polypeptide chains (14-17).

Results

Conclusion