The Rough Endoplasmic Reticulum-resident FK506-binding Protein FKBP65 Is a Molecular Chaperone That Interacts with Collagens

Yoshihiro Ishikawa,Janice Vranka,Jackie Wirz,Kazuhiro Nagata,Hans Peter Bächinger

doi:10.1074/jbc.m802535200

Abstract

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and Bächinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.

Highlights

FKBP65 was first identified in mouse NIH3T3 fibroblasts [1], and it consists of four basic FKBP13 domains
We previously observed that FKBP65 can be extracted from the rough endoplasmic reticulum (rER) of chick embryos on a gelatin-Sepharose column [12]
When the gelatin-Sepharose column is eluted with a low pH buffer, the two most abundant proteins are HSP47 and FKBP65

Summary

Introduction

FKBP65 was first identified in mouse NIH3T3 fibroblasts [1], and it consists of four basic FKBP13 domains. The peptidylprolyl cis-trans-isomerase activity of FKBP65 had only a marginal effect on the rate of folding of the collagen triple helix [12, 13], a process that is rate-limited by cis-trans isomerizations [14]. Purified FKBP65 in 50 mM Tris/HCl buffer, pH 7.5, containing 0.2 M NaCl, was mixed with gelatin-Sepharose (Amersham Biosciences) or type I collagen beads for 2 h at 4 °C.

Results

Conclusion