Both WFIKKN1 and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11

Abstract

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP, a follistatin, an immunoglobulin, two Kunitz-type protease inhibitor domains, and an NTR domain. Recent experiments have shown that WFIKKN2 protein binds mature GDF8/myostatin and myostatin propeptide and inhibits the biological activity of myostatin (Hill, J. J., Qiu, Y., Hewick, R. M., and Wolfman, N. M. (2003) Mol. Endocrinol. 17, 1144-1154). Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.

FIGURE 1.

Characterization of human myostatin-propeptide expressed in E. coli. A, SDS-PAGE of refolded recombinant myostatin propeptide. Lane 1, reduced myostatin propeptide; lane 2, reduced proteins of the low molecular weight calibration kit; lane 3, nonreduced myostatin propeptide. B, far-ultraviolet circular dichroism spectra of recombinant myostatin propeptide. Spectra were recorded in 10 mm Tris-HCl, pH 8.0, at 25 °C using 0.1 mg/ml protein. The solid line indicates the spectrum of the recombinant protein, and the dashed line indicates the CDPro-predicted spectrum of a protein consisting of 0.202 regular β-strand, 0.113 distorted β-strand, 0.065 regular helix, and 0.088 distorted helix, 0.217 turn, and 0.315 unordered structure. C, temperature dependence of the CD spectra of myostatin propeptide. Changes in the CD of the protein were monitored at 203 nm during the course of heating from 25 to 90 °C at a heating rate of 60 °C/h. D, melting temperature was determined by derivative processing of changes in CD (of C) using the spectra analysis program for WINDOWS 95/NT version 1.53.04 (Jasco).

FIGURE 2.

Characterization of the interaction of WFIKKN1 and WFIKKN2 with GDF8 and GDF11 by surface plasmon resonance assays. Sensorgrams of the interactions are as follows. A, WFIKKN1 (25, 50, 100, 150, 200, and 400 nm) with myostatin; B, WFIKKN1 (12.5, 25, 37.5, 50, 100, and 150 nm) with GDF11; C, WFIKKN2 (2.5, 5, 10, 20, and 40 nm) with myostatin; D, WFIKKN2 (3, 6, 12.5, 25, and 50 nm) with GDF11. Various concentrations of WFIKKN1 or WFIKKN2 in 20 mm HEPES buffer, pH 7.5, containing 150 mm NaCl, 5 mm EDTA, 0.005% Tween 20 were injected over myostatin or GDF11 immobilized on CM5 sensorchips. For each type of experiment, one set of representative data of three parallels are shown.