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Effect of charge at an amino acid of basic fibroblast growth factor on its mitogenic activity
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Chinese Chemical Letters 21 (2010) 468–471 www.elsevier.com/locate/cclet
Effect of charge at an amino acid of basic fibroblast growth factor on its mitogenic activity Xiao Ping Wu a,*, Xiao Kun Li b a
b
Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China National Engineering Research Center for Gene Medicine, Jinan University, Guangzhou 510632, China Received 26 August 2009
Abstract The amino acid at the 119th position of human basic fibroblast growth factor (hbFGF), lysine (K119), is a critical component for its mitogenic activity. However, little is known about the effects of the characteristics of this residue including charge on the mitogenic activity of hbFGF. Herein, this basic residue was replaced with neutral glutamine residue and acidic glutamic acid residue to construct mutants hbFGFK119Q and hbFGFK119E, respectively. The mutants were produced by BL21(DE3)/pET3c expression system and purified to homogeneity by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. The mitogenic activity analysis showed that neutralization of charge at the 119th residue diminished the mitogenic activity of hbFGF, whereas change of positive charge to negative charge at this residue had no significant effect on its mitogenic activity. Further MAP kinase activation assay revealed that the influence of different charge at the 119th position on the mitogenic activity of hbFGF was related to the signal molecular activation in MAPK pathway. It was deduced that the charge, either positive or negative, at the 119th position of hbFGF is crucial for its full mitogenic activity. # 2009 Xiao Ping Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Human basic fibroblast growth factor; Amino acid charge; Expression; Mitogenic activity; Erk1/2 activation
Basic fibroblast growth factor (bFGF) is a prototype member of fibroblast growth factors (FGFs) which is a large protein family including at least 23 closely related polypeptides [1]. bFGF plays a ubiquitous role in regulating the proliferation, differentiation, migration and survival of a variety of cells by binding and activating FGF receptors (FGFRs) in a heparin sulfate proteoglycan (HSPG)-dependent manner [2]. HSPG binds both FGF and FGFR, not only to facilitate FGF–FGFR dimerization and stabilize FGF against degradation, but also to serve as a FGF storage reservoir [3,4]. It has been shown that HSPG is required for full FGF/FGFR signaling [5,6]. On the basis of site directed mutagenesis, molecular modeling and crystal structure of bFGF, an array of positively charged lysine and arginine is identified as binding sites for HSPG [7–9]. The 119th lysine (K119) of bFGF composed of 146 amino acids is a critical component of the identified HSPG-binding site. To study the effect of charge at the 119th position of hbFGF on its mitogenic activity, here K119 was replaced with neutral glutamine residue and acidic glutamic acid residue to obtain mutants hbFGFK119Q and hbFGFK119E, respectively. The target protein was purified to homogeneity by two purification steps. The results of the mitogenic activity assay showed that the effect of
* Corresponding author. E-mail address: [email protected] (X.P. Wu). 1001-8417/$ – see front matter # 2009 Xiao Ping Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.12.004
X.P. Wu, X.K. Li / Chinese Chemical Letters 21 (2010) 468–471
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hbFGFK119Q on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF, whereas the hbFGFK119E mutant exhibited similar mitogenic activity to wild-type hbFGF. In accordance with the results of cell viability assay, western blotting analysis conducted to monitor the activation of the signal molecular indicated that the level of Erk1/2 activation induced by hbFGFK119E was higher than that observed in hbFGFK119Q treated group and similar to the level induced by wild-type hbFGF. Therefore, the charge at the 119th position of hbFGF might be required for its full mitogenic activity, and the alteration of the mitogenic effect of the mutants was related to the MAPK pathway. 1. Experimental Over-lap polymerase chain reaction (PCR) was applied to amplify two mutant cDNA fragments, which were digested with NdeIand BglII, and then ligated into previously digested pET3c vector to create the corresponding expression vectors, pET3c-hbFGFK119Q and pET3c-hbFGFK119E, respectively. Accuracy of the inserted cDNA was confirmed by automated DNA sequencing conducted by Shanghai Sangon (Shanghai, China). The expression plasmids pET3c-hbFGFK119Q and pET3c-hbFGFK119E were transformed into Escherichia coli strain BL21(DE3), respectively. The transformed cells were grown in Luria broth (LB) medium containing 100 mg/mL ampicillin at 37 8C. When the absorbance at 600 nm of 0.6–0.8 was reached, expression of the mutant proteins was induced by adding isopropyl-b-D-thiogalactoside (IPTG) to a final concentration of 1 mmol/L. After the culture incubated at 37 8C for 4 h, cells were harvested and sonicated in ice-bath. The cell extract was first applied to a CM-Sepharose column equilibrated with PBS (pH 7.4) buffer. Proteins were eluted with 20 mmol/L PBS buffer containing 0.6 mol/L NaCl. Fraction containing the target protein was applied to a Heparin–Sepharose column equilibrated previously with 20 mmol/L PBS buffer containing 0.6 mol/L NaCl. Bound proteins were eluted with 20 mmol/L PBS buffer containing 0.6–2.0 mol/L NaCl. The immunogenic activities of recombinant hbFGF mutants were checked by Western blotting, and the mitogenic activity was determined by cell viability assay. Briefly, Balb/c 3T3 cells were seeded in 96-well plates in Medium 1640 supplemented with 10% fetal bovine serum (FCS) and incubated for 24 h. After starved in Medium 1640 containing 0.4% FCS for 24 h, cells were treated with wild-type hbFGF, hbFGFK119Q and hbFGFK119E at the indicated concentrations and cultured for 48 h. The number of viable cells was determined by the methylthiazoletetrazolium (MTT) method as previously described [10]. The activation of Erk1/2 stimulated by the mutants was further detected. Briefly, Balb/c 3T3 cells were incubated in Medium 1640 containing 10% FCS and starved for 24 h. Wild-type hbFGF, hbFGFK119Q and hbFGFK119E were added to the culture to the final concentration of 25 ng/mL, respectively. After cultured for 10 min, the cells were lysed with RIPA buffer. Protein concentration of the supernatant was determined by the Bio-Rad protein assay method using bovine serum albumin as a standard. Cell lysates with equal amount of protein were run on 12% SDS-PAGE gels, and then transferred onto a polyvinylidene fluoride (PVDF) membrane for Western blotting. 2. Results and discussion The hbFGF mutant cDNA fragments produced by PCR were inserted in pET3c vector to construct the expression plasmids, which were then transformed into BL21(DE3) to obtain the expression strains BL21(DE3)/pET3chbFGFK119Q and BL21(DE3)/pET3c-hbFGFK119E, respectively. IPTG was used to induce the expression of the recombinant hbFGF mutants. The induced proteins corresponding to the predicted sizes appeared in the lanes of induced samples as the arrow indicated (Fig. 1). The recombinant hbFGF mutants were purified from the supernatant of the bacteria lysates by two purification steps: CM-Sepharose and Heparin affinity chromatography. Of note was that during purification of the proteins the concentration of NaCl required to elute the mutant hbFGFK119Q from Heparin–Sepharose resin was close to that for the wild-type protein (1.2 mol/L NaCl), whereas the mutant hbFGFK119E was eluted from immobilized heparin at 0.8 mol/ L NaCl. Detected by SDS–PAGE, the purity of both recombinant mutants determined by optic density scanning was about 95% (Fig. 2A, lanes 3 and 4). Further characterization using Western blotting showed that both of the purified recombinant hbFGF mutants could be recognized by an anti-human hbFGF antibody (Fig. 2B). Bioassay of mitogenicity showed that the mitogenic activity of the recombinant hbFGFK119Q on Balb/c 3T3 cells was lower than that of the wild-type hbFGF at the detected concentrations (Fig. 3). In the light of the results that neutralization of charge at residue K119 diminished the mitogenic activity of hbFGF, one would expect that change of positively charged side chain of K119 to the negatively charged side chain of E119 in hbFGF would strengthen the
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X.P. Wu, X.K. Li / Chinese Chemical Letters 21 (2010) 468–471
Fig. 1. Expression of hbFGFK119Q and hbFGFK119E analyzed by SDS–PAGE. The gel was stained by Coomassie blue G-250. (1) Uninduced BL21(DE3)/pET3c-hbFGFK119Q; (2) induced BL21(DE3)/pET3c-hbFGFK119Q; (3) molecular protein markers; (4) uninduced BL21(DE3)/pET3chbFGFK119E; (5) induced BL21(DE3)/pET3c-hbFGFK119E.
Fig. 2. SDS–PAGE and Western blot analysis of purified hbFGFK119Q and hbFGFK119E. (A) (1) Molecular protein markers; (2) protein sample eluted at 0.8 mol/L NaCl after applied hbFGFK119Q fraction to Heparin affinity column; (3) protein sample eluted at 1.2 mol/L NaCl after applied hbFGFK119Q fraction to Heparin affinity column; (4) protein sample eluted at 0.8 mol/L NaCl after applied hbFGFK119E fraction to Heparin affinity column. (B) Immuno-reactivity of purified hbFGFK119Q and hbFGFK119E with anti-human bFGF antibody. (1) Purified hbFGFK119Q; (2) purified hbFGFK119E.
impact on mitogenic activity of hbFGF. However, this was not the case. The other mutant hbFGFK119E designed to assess the impact of change of positive charge to negative charge at residue K119 displayed no significant change in mitogenic activity compared to wild-type hbFGF (Fig. 3). As the effect of bFGF on cell proliferation was associated with the potential interactions among bFGF, low-affinity binding sites of HSPG, and high-affinity transmembrane receptors, the explanation for the unchangeable mitogenic activity of hbFGFK119E may be that change of partial charge in hbFGF may promote other HSPG-protein–receptors interactions by recruiting other surface-exposed residues. To further clarify the molecular mechanism by which the mitogenic effect of the mutants on Balb/c 3T3 cells changed, and whether the alteration of the mitogenic effect of the mutants is associated with the MAPK pathway, The ability of the mutants and wild-type bFGF to stimulate the activation of two mitogen-activated protein kinases (MAPK), Erk1 and Erk2, was assessed. As shown in Fig. 4, compared to the group treated with wild-type hbFGF, lower level of Erk1/2 activation was observed in the group treated with hbFGFK119Q, and similar activation extent of Erk1/2 was achieved with hbFGFK119E. The results were corresponding with that obtained from MTT assay, suggesting that the alteration of the mitogenic effect of the mutants was associated with the MAPK pathway.
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Fig. 3. Effects of wild-type hbFGF, hbFGFK119Q and hbFGFK119E on the proliferation of Balb/c 3T3 cells. Cells were incubated with samples at the indicated concentrations, and cell proliferation was determined by MTT cell viability assay. Data presented are the mean (SDs) of triplicate tests. (~) p < 0.01 vs. wild-type hbFGF group.
Fig. 4. Western blot analysis of the activation of Erk1/2. Balb/c 3T3 cells were starved and incubated with samples at the concentration of 25 ng/mL for 10 min. Following SDS-PAGE, Western blot analysis was performed using a standard procedure. Relative levels mean density level of pErk1/2 of each sample relative to that of the control. (1) Control; (2) hbFGFK119Q; (3) wild-type hbFGF; (4) hbFGFK119E.
Acknowledgments The work was supported by grants from the Natural Science Foundation of China (No. 30973671), the Natural Science Foundation of Guangdong Province of China (No. 9151064001000031), and Guangdong Provincial Key Discipline in Biochemistry and Molecular biology. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
A. Beenken, M. Mohammadi, Nat. Rev. Drug Discov. 8 (2009) 235. L. Dailey, D. Ambrosetti, A. Mansukhani, et al. Cytokine Growth Factor Rev. 16 (2005) 233. M. Mohammadi, S.K. Olsen, O.A. Ibrahimi, Cytokine Growth Factor Rev. 16 (2005) 107. N.J. Harmer, Biochem. Soc. Trans. 34 (2006) 442. K. Forsten-Williams, C.L. Chu, M. Fannon, et al. Ann. Biomed. Eng. 36 (2008) 2134. U. Ha¨cker, K. Nybakken, N. Perrimon, Nat. Rev. Mol. Cell. Biol. 6 (2005) 530. L.D. Thompson, M.W. Pantoliano, B.A. Springer, Biochemistry 33 (1994) 3831. W.F. Heath, A.S. Cantrell, N.G. Mayne, et al. Biochemistry 30 (1991) 5608. L.Y. Li, M. Safran, D. Aviezer, et al. Biochemistry 33 (1994) 10999. X. Li, L. Jiang, Y. Wang, et al. Cancer Lett. 256 (2007) 29.
Chinese Chemical Letters 21 (2010) 468–471 www.elsevier.com/locate/cclet
Effect of charge at an amino acid of basic fibroblast growth factor on its mitogenic activity Xiao Ping Wu a,*, Xiao Kun Li b a
b
Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China National Engineering Research Center for Gene Medicine, Jinan University, Guangzhou 510632, China Received 26 August 2009
Abstract The amino acid at the 119th position of human basic fibroblast growth factor (hbFGF), lysine (K119), is a critical component for its mitogenic activity. However, little is known about the effects of the characteristics of this residue including charge on the mitogenic activity of hbFGF. Herein, this basic residue was replaced with neutral glutamine residue and acidic glutamic acid residue to construct mutants hbFGFK119Q and hbFGFK119E, respectively. The mutants were produced by BL21(DE3)/pET3c expression system and purified to homogeneity by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. The mitogenic activity analysis showed that neutralization of charge at the 119th residue diminished the mitogenic activity of hbFGF, whereas change of positive charge to negative charge at this residue had no significant effect on its mitogenic activity. Further MAP kinase activation assay revealed that the influence of different charge at the 119th position on the mitogenic activity of hbFGF was related to the signal molecular activation in MAPK pathway. It was deduced that the charge, either positive or negative, at the 119th position of hbFGF is crucial for its full mitogenic activity. # 2009 Xiao Ping Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Human basic fibroblast growth factor; Amino acid charge; Expression; Mitogenic activity; Erk1/2 activation
Basic fibroblast growth factor (bFGF) is a prototype member of fibroblast growth factors (FGFs) which is a large protein family including at least 23 closely related polypeptides [1]. bFGF plays a ubiquitous role in regulating the proliferation, differentiation, migration and survival of a variety of cells by binding and activating FGF receptors (FGFRs) in a heparin sulfate proteoglycan (HSPG)-dependent manner [2]. HSPG binds both FGF and FGFR, not only to facilitate FGF–FGFR dimerization and stabilize FGF against degradation, but also to serve as a FGF storage reservoir [3,4]. It has been shown that HSPG is required for full FGF/FGFR signaling [5,6]. On the basis of site directed mutagenesis, molecular modeling and crystal structure of bFGF, an array of positively charged lysine and arginine is identified as binding sites for HSPG [7–9]. The 119th lysine (K119) of bFGF composed of 146 amino acids is a critical component of the identified HSPG-binding site. To study the effect of charge at the 119th position of hbFGF on its mitogenic activity, here K119 was replaced with neutral glutamine residue and acidic glutamic acid residue to obtain mutants hbFGFK119Q and hbFGFK119E, respectively. The target protein was purified to homogeneity by two purification steps. The results of the mitogenic activity assay showed that the effect of
* Corresponding author. E-mail address: [email protected] (X.P. Wu). 1001-8417/$ – see front matter # 2009 Xiao Ping Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2009.12.004
X.P. Wu, X.K. Li / Chinese Chemical Letters 21 (2010) 468–471
469
hbFGFK119Q on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF, whereas the hbFGFK119E mutant exhibited similar mitogenic activity to wild-type hbFGF. In accordance with the results of cell viability assay, western blotting analysis conducted to monitor the activation of the signal molecular indicated that the level of Erk1/2 activation induced by hbFGFK119E was higher than that observed in hbFGFK119Q treated group and similar to the level induced by wild-type hbFGF. Therefore, the charge at the 119th position of hbFGF might be required for its full mitogenic activity, and the alteration of the mitogenic effect of the mutants was related to the MAPK pathway. 1. Experimental Over-lap polymerase chain reaction (PCR) was applied to amplify two mutant cDNA fragments, which were digested with NdeIand BglII, and then ligated into previously digested pET3c vector to create the corresponding expression vectors, pET3c-hbFGFK119Q and pET3c-hbFGFK119E, respectively. Accuracy of the inserted cDNA was confirmed by automated DNA sequencing conducted by Shanghai Sangon (Shanghai, China). The expression plasmids pET3c-hbFGFK119Q and pET3c-hbFGFK119E were transformed into Escherichia coli strain BL21(DE3), respectively. The transformed cells were grown in Luria broth (LB) medium containing 100 mg/mL ampicillin at 37 8C. When the absorbance at 600 nm of 0.6–0.8 was reached, expression of the mutant proteins was induced by adding isopropyl-b-D-thiogalactoside (IPTG) to a final concentration of 1 mmol/L. After the culture incubated at 37 8C for 4 h, cells were harvested and sonicated in ice-bath. The cell extract was first applied to a CM-Sepharose column equilibrated with PBS (pH 7.4) buffer. Proteins were eluted with 20 mmol/L PBS buffer containing 0.6 mol/L NaCl. Fraction containing the target protein was applied to a Heparin–Sepharose column equilibrated previously with 20 mmol/L PBS buffer containing 0.6 mol/L NaCl. Bound proteins were eluted with 20 mmol/L PBS buffer containing 0.6–2.0 mol/L NaCl. The immunogenic activities of recombinant hbFGF mutants were checked by Western blotting, and the mitogenic activity was determined by cell viability assay. Briefly, Balb/c 3T3 cells were seeded in 96-well plates in Medium 1640 supplemented with 10% fetal bovine serum (FCS) and incubated for 24 h. After starved in Medium 1640 containing 0.4% FCS for 24 h, cells were treated with wild-type hbFGF, hbFGFK119Q and hbFGFK119E at the indicated concentrations and cultured for 48 h. The number of viable cells was determined by the methylthiazoletetrazolium (MTT) method as previously described [10]. The activation of Erk1/2 stimulated by the mutants was further detected. Briefly, Balb/c 3T3 cells were incubated in Medium 1640 containing 10% FCS and starved for 24 h. Wild-type hbFGF, hbFGFK119Q and hbFGFK119E were added to the culture to the final concentration of 25 ng/mL, respectively. After cultured for 10 min, the cells were lysed with RIPA buffer. Protein concentration of the supernatant was determined by the Bio-Rad protein assay method using bovine serum albumin as a standard. Cell lysates with equal amount of protein were run on 12% SDS-PAGE gels, and then transferred onto a polyvinylidene fluoride (PVDF) membrane for Western blotting. 2. Results and discussion The hbFGF mutant cDNA fragments produced by PCR were inserted in pET3c vector to construct the expression plasmids, which were then transformed into BL21(DE3) to obtain the expression strains BL21(DE3)/pET3chbFGFK119Q and BL21(DE3)/pET3c-hbFGFK119E, respectively. IPTG was used to induce the expression of the recombinant hbFGF mutants. The induced proteins corresponding to the predicted sizes appeared in the lanes of induced samples as the arrow indicated (Fig. 1). The recombinant hbFGF mutants were purified from the supernatant of the bacteria lysates by two purification steps: CM-Sepharose and Heparin affinity chromatography. Of note was that during purification of the proteins the concentration of NaCl required to elute the mutant hbFGFK119Q from Heparin–Sepharose resin was close to that for the wild-type protein (1.2 mol/L NaCl), whereas the mutant hbFGFK119E was eluted from immobilized heparin at 0.8 mol/ L NaCl. Detected by SDS–PAGE, the purity of both recombinant mutants determined by optic density scanning was about 95% (Fig. 2A, lanes 3 and 4). Further characterization using Western blotting showed that both of the purified recombinant hbFGF mutants could be recognized by an anti-human hbFGF antibody (Fig. 2B). Bioassay of mitogenicity showed that the mitogenic activity of the recombinant hbFGFK119Q on Balb/c 3T3 cells was lower than that of the wild-type hbFGF at the detected concentrations (Fig. 3). In the light of the results that neutralization of charge at residue K119 diminished the mitogenic activity of hbFGF, one would expect that change of positively charged side chain of K119 to the negatively charged side chain of E119 in hbFGF would strengthen the
470
X.P. Wu, X.K. Li / Chinese Chemical Letters 21 (2010) 468–471
Fig. 1. Expression of hbFGFK119Q and hbFGFK119E analyzed by SDS–PAGE. The gel was stained by Coomassie blue G-250. (1) Uninduced BL21(DE3)/pET3c-hbFGFK119Q; (2) induced BL21(DE3)/pET3c-hbFGFK119Q; (3) molecular protein markers; (4) uninduced BL21(DE3)/pET3chbFGFK119E; (5) induced BL21(DE3)/pET3c-hbFGFK119E.
Fig. 2. SDS–PAGE and Western blot analysis of purified hbFGFK119Q and hbFGFK119E. (A) (1) Molecular protein markers; (2) protein sample eluted at 0.8 mol/L NaCl after applied hbFGFK119Q fraction to Heparin affinity column; (3) protein sample eluted at 1.2 mol/L NaCl after applied hbFGFK119Q fraction to Heparin affinity column; (4) protein sample eluted at 0.8 mol/L NaCl after applied hbFGFK119E fraction to Heparin affinity column. (B) Immuno-reactivity of purified hbFGFK119Q and hbFGFK119E with anti-human bFGF antibody. (1) Purified hbFGFK119Q; (2) purified hbFGFK119E.
impact on mitogenic activity of hbFGF. However, this was not the case. The other mutant hbFGFK119E designed to assess the impact of change of positive charge to negative charge at residue K119 displayed no significant change in mitogenic activity compared to wild-type hbFGF (Fig. 3). As the effect of bFGF on cell proliferation was associated with the potential interactions among bFGF, low-affinity binding sites of HSPG, and high-affinity transmembrane receptors, the explanation for the unchangeable mitogenic activity of hbFGFK119E may be that change of partial charge in hbFGF may promote other HSPG-protein–receptors interactions by recruiting other surface-exposed residues. To further clarify the molecular mechanism by which the mitogenic effect of the mutants on Balb/c 3T3 cells changed, and whether the alteration of the mitogenic effect of the mutants is associated with the MAPK pathway, The ability of the mutants and wild-type bFGF to stimulate the activation of two mitogen-activated protein kinases (MAPK), Erk1 and Erk2, was assessed. As shown in Fig. 4, compared to the group treated with wild-type hbFGF, lower level of Erk1/2 activation was observed in the group treated with hbFGFK119Q, and similar activation extent of Erk1/2 was achieved with hbFGFK119E. The results were corresponding with that obtained from MTT assay, suggesting that the alteration of the mitogenic effect of the mutants was associated with the MAPK pathway.
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Fig. 3. Effects of wild-type hbFGF, hbFGFK119Q and hbFGFK119E on the proliferation of Balb/c 3T3 cells. Cells were incubated with samples at the indicated concentrations, and cell proliferation was determined by MTT cell viability assay. Data presented are the mean (SDs) of triplicate tests. (~) p < 0.01 vs. wild-type hbFGF group.
Fig. 4. Western blot analysis of the activation of Erk1/2. Balb/c 3T3 cells were starved and incubated with samples at the concentration of 25 ng/mL for 10 min. Following SDS-PAGE, Western blot analysis was performed using a standard procedure. Relative levels mean density level of pErk1/2 of each sample relative to that of the control. (1) Control; (2) hbFGFK119Q; (3) wild-type hbFGF; (4) hbFGFK119E.
Acknowledgments The work was supported by grants from the Natural Science Foundation of China (No. 30973671), the Natural Science Foundation of Guangdong Province of China (No. 9151064001000031), and Guangdong Provincial Key Discipline in Biochemistry and Molecular biology. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
A. Beenken, M. Mohammadi, Nat. Rev. Drug Discov. 8 (2009) 235. L. Dailey, D. Ambrosetti, A. Mansukhani, et al. Cytokine Growth Factor Rev. 16 (2005) 233. M. Mohammadi, S.K. Olsen, O.A. Ibrahimi, Cytokine Growth Factor Rev. 16 (2005) 107. N.J. Harmer, Biochem. Soc. Trans. 34 (2006) 442. K. Forsten-Williams, C.L. Chu, M. Fannon, et al. Ann. Biomed. Eng. 36 (2008) 2134. U. Ha¨cker, K. Nybakken, N. Perrimon, Nat. Rev. Mol. Cell. Biol. 6 (2005) 530. L.D. Thompson, M.W. Pantoliano, B.A. Springer, Biochemistry 33 (1994) 3831. W.F. Heath, A.S. Cantrell, N.G. Mayne, et al. Biochemistry 30 (1991) 5608. L.Y. Li, M. Safran, D. Aviezer, et al. Biochemistry 33 (1994) 10999. X. Li, L. Jiang, Y. Wang, et al. Cancer Lett. 256 (2007) 29.