FGF9 monomer/dimer equilibrium regulates. extracellular matrix affinity and tissue diffusion

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1 SUPPLEMENTARY INFORMATGION FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion Masayo Harada, Hirotaka Murakami, Akihiko Okawa, Noriaki Okimoto, Shuichi Hiraoka, Taka Nakahara, Ryogo Akasaka, Yo-ichi Shiraishi, Noriyuki Futatsugi, Yoko Mizutani-Koseki, Atsushi Kuroiwa, Mikako Shirouzu, Shigeyuki Yokoyama, Makoto Taiji, Sachiko Iseki, David M. Ornitz and Haruhiko Koseki CONTENTS Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Supplementary Methods Supplementary Reference 1

2 Supplementary Figure 1 a fb Fgf9 +/+ E18.5 pb b fb Fgf9 Eks/Eks pb c fb HE pb d fb HE pb Kossa Kossa Supplementary Figure 1 Premature fusion of coronal suture in Fgf9 Eks/Eks fetuses. Hemaotxylin and eosin staining (a, b) and von Kossa staining (c, d) in the coronal suture of Fgf9 +/+ and Fgf9 Eks/Eks fetuses at E18.5. fb, frontal bone; pb, parietal bone. Scale bars, 100 µm. 2

3 Supplementary Figure 2 a E. Coli Extraction 25% ammonium sulfate supernatant 50% ammonium sulfate precipitate b kda FGF9 WT FGF9 Eks Desalting by dialysis TOYOPEARL AF-Heparin HC-650M Desalting by dialysis TOYOPEARL CM-650M 6 TOYOPEARL HW-50F Supplementary Figure 2 Purification of FGF9 WT and FGF9 Eks proteins. (a) Outline of the procedure used for purification of FGF9 WT and FGF9 Eks proteins. (b) SDS-PAGE of purified FGF9 WT and FGF9 Eks. Both FGF9 WT and FGF9 Eks are detected with a molecular mass of approximately 20 kda. 3

4 Supplementary Figure 3 E10.5 E11.5 Fgf9 +/+ Fgf9 Eks/Eks Fgf9 +/+ a b i j Fgf9 Eks/Eks c Fgf9 Fgf9 Fgf9 Fgf9 d k l e Fgfr1 f Fgfr1 m Fgfr1 n Fgfr1 g Fgfr2 h Fgfr2 Fgfr2 Fgfr2 o p Fgfr3 Fgfr3 Fgfr3 Fgfr3 Supplementary Figure 3 Fgf9 is expressed in myoblasts, whereas Fgfrs are expressed in the mesenchymal cells in the prospective elbow joint. In situ detection of Fgf9 (a, b, i, j), Fgfr1(c, d, k, l), Fgfr2 (e, f, m, n) and Fgfr3 (g, h, o, p) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. Scale bars, 100 µm. 4

5 Supplementary Figure 4 a One soaked bead 100 ng b FGF9 WT FGF9 Eks FGF9 WT FGF9 Eks FGF9 WT FGF9 Eks (h) 20k Da 20k Da Supplementary Figure 4 Equal loading of FGF9 WT and FGF9 Eks in the AffiGel-Blue beads and the equal diffusion rate of FGF9 WT and FGF9 Eks from the beads. (a) The holding capacity of the AffiGel-Blue beads for FGF9 WT or FGF9 Eks was determined by soaking the beads in an FGF9 WT or FGF9 Eks solution (500 µg/ml) and subsequent FGF9 detection by western blotting analysis. Lanes 1-6, western blotting of one bead per lane which had been soaked in FGF9 WT or FGF9 Eks solution. Lanes 7, 8, recombinant FGF9 WT and FGF9 Eks (100 ng). (b) The diffusion rate of FGF9 WT and FGF9 Eks from the Affigel-Blue beads was determined by soaking the FGF9 WT -or FGF9 Eks -beads in PBS for indicated time at 37 C and subsequent detection of retaining FGF9 in the beads by western blotting analysis. 5

6 Supplementary Figure 5 Bsr I exon 3 Fgf9 +/+ Fgf9 Eks/Eks 42 bp P(Fgf9 Eks/+ ) C57BL/6J DBA/2J JF1/Ms 147 bp 189 bp F1(Fgf9 Eks/+ ) Fgf9 Eks/Eks Fgf9 Eks/+ Fgf9 +/+ 189 bp 147 bp Supplementary Figure 5 Genotyping of Fgf9 Eks mutation. A 189-bp genomic PCR fragment including the nucleotide substitution of the Fgf9 gene in the Eks mutants is not digested by the BsrI restriction enzyme, whereas PCR fragments from +/+, C57BL/6, DBA/2J and JF1/Ms mice are digested. 6

7 Supplementary Table 1 Supplementary Table1 Kinetic parameters for the interactions of FGF9 WT and FGF9 Eks with heparin k a (M -1 s -1 ) k d (s -1 ) K D (M) FGF9 WT / heparin FGF9 Eks / heparin P 3.85 x x x x x x x x x x x x x 10-8 The ka, kd, and KD values were calculated from the sensorgrams using five concentrations of analyte (FGF9 WT or FGF9 Eks ) in three independent experiments (mean ± s.e.m.). P values represent the significance of differences between FGF9 WT /heparin and FGF9 Eks /heparin (two-tailed Student s t-test). 7

8 Supplementary Table 2 Supplementary Table 2 Inter-monomer hydrogen bonds in FGF9 WT homodimer, FGF9 WT/Eks heterodimer and FGF9 Eks homodimer determined by MD simulations FGF9 WT (a) - FGF9 WT (b) FGF9 Eks (a) - FGF9 WT (b) FGF9 Eks (a) - FGF9 Eks (b) Inter-monomer hydrogen bonds N143 - Y67 N143 - R69 N143 - Y145 Y67 - N143 R69 - N143 Y145 - N143 R62 - D193 R64 - D193 D193 - R62 D193 - R64 T143 - Y67 Y67 - N143 R69 - N143 Y145 - N143 R62 - D193 R64 - D193 D193 - R62 D193 - R64 T143 - Y145 Y145 - T143 R62 - D193 R64 - D193 D193 - R62 D193 - R64 Amino acid residues contributing to hydrogen bond formation involved in dimerization are indicated using the single-letter amino acid code and residue number. 8

9 Supplementary Table 3 Supplementary Table 3 Primers for genotyping of the Fgf9 Eks allele Fwd: 5 -CACAGGAATGTGTGTTCAGA-3 Rev: 5 -GGTCCACTGGTCTAGGTAAA-3 9

10 SUPPLEMENTARY METHODS FGF9 WT and FGF9 Eks expression and purification. Purified FGF9 WT and FGF9 Eks were produced essentially according to a procedure previously described (Koyama et al., 2001), except for production of the recombinant proteins in E. coli. Using RT-PCR, we generated cdna fragments which encode the initiation Met and the downstream peptide sequence between residues 35 and 208 in FGF9 WT and FGF9 Eks. The first 33 N-terminal residues were not included since they are implicated only in secretion. Each fragment was inserted between the NdeI and HindIII sites in the pcoldiv bacterial expression vector (Takara). The resulting plasmid was introduced into the E.coli BL21 strain, and production of the recombinant protein was induced in SB medium supplemented with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 24 hours at 15. The bacteria were harvested and subsequently disrupted by lysozyme treatment and sonication. After removal of cell debris by centrifugation, FGF9 WT and FGF9 Eks were recovered in the supernatant by fractionation with 25% saturation of ammonium sulfate, and subsequently by precipitating that supernatant with 50% ammonium sulfate. The precipitates were solubilized in 25 mm ammonium acetate buffer (ph 5.5) and dialyzed against 25 mm ammonium acetate buffer (ph 5.5) containing 1% ammonium sulfate. We further separated the obtained fraction, as shown in Figure S2A, through a series of chromatography columns: TOYOPEARL AF-Heparin HC- 650M column (TOSOH), TOYOPEARL CM-650M column (TOSOH), TOYOPEARL HW-50F column (TOSOH). We performed these chromatography steps using the buffer system previously described mg of highly purified FGF9 WT and 100 mg of FGF9 Eks 10

11 were obtained from 20 L bacterial culture by this method (Supplementary Fig. 1b). SUPPLEMENTARY REFERENCE 1. Koyama, N. et al. Improved preparation and crystallization of 25 kda human fibroblast growth factor-9. Biotechnol. Appl. Biochem. 33, (2001). 11