Tripeptide Containing Selenium with Glutathione Peroxidase Activity†

doi:10.7503/cjcu20160101

Abstract

Abstract:

Six selenium-containing tripeptides(QUW, QWU, WQU, WUQ, UWQ and UQW) were synthesized by solid-phase synthesis technology based on the catalytic triad of native glutathione peroxidase(GPx). The GPx activities and the kinetics of the tripeptide were determined by the enzyme coupling method. The effects of tripeptide on HepG2 cells growth and migration were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, wound assay and Western blot method. The results showed that the GPx activity of the tripeptide with U at the amino terminal was higher than that of U at the middle position or the carboxyl terminal. Among the six tripeptide, the activity of UWQ catalyzed by glutathione(GSH) reduced by hydrogen peroxide(H₂O₂) was higher than those of other tripeptides, and its catalytic mechanism was Ping-Pong mechanism. UWQ can dosedependently discrease migration speed of HepG2 cells to exercise, and reduce the invasion and metastasis of HepG2 cells.

Key words: Gluathione peroxidase, Mimics, Tripeptide containing selenium

CLC Number:

TrendMD:

YIN Juxin, LI Zuhong, MU Ying, LÜ Shaowu, LUO Guimin. Tripeptide Containing Selenium with Glutathione Peroxidase Activity^†[J]. Chem. J. Chinese Universities, 2016, 37(7): 1302.

Figures/Tables 7

Fig.1 Catalytic triad of GPx(PDB data: 1gp1)[3]

Table 1 GPX-like activies of 3P and other GPX mimics

Sepecies	Substrate	Activity/(U·μmol^-1)
WQU	H₂O₂	0.40±0.03
QWU	H₂O₂	0.42±0.04
WUQ	H₂O₂	0.72±0.07
QUW	H₂O₂	0.74±0.05
UQW	H₂O₂	1.12±0.13
UWQ	H₂O₂	2.10±0.11

Fig.2 Double-reciprocal plots for the reduction of H2O2 by GSH catalyzed by UWQ(A) [GSH]/(mmol·L-1): a. 0.5; b.1.0; c. 2.0. (B) [H2O2]/(mmol·L-1): a. 0.2; b. 0.5; c. 1.0.

Table 2 Kinetic parameters of GPx mimics

Sepecies	k_max/min^-1	K_m,GSH/mmol	$K m, H 2 O 2$ /mmol	k_max $K m, GSH - 1$ /(mol^-1·min^-1)	k_max $K m, H 2 O 2 - 1$ /(mol^-1·min^-1)
UWQ	8.83	11.34	8.86	7.79×10²	9.97×1 $02$
UQW	8.46	13.82	8.97	6.12×10²	9.43×10²

Table 2 Kinetic parameters of GPx mimics

Sepecies	k_max/min^-1	K_m,GSH/mmol	$K m, H 2 O 2$ /mmol	k_max $K m, GSH - 1$ /(mol^-1·min^-1)	k_max $K m, H 2 O 2 - 1$ /(mol^-1·min^-1)
UWQ	8.83	11.34	8.86	7.79×10²	9.97×1 $02$
UQW	8.46	13.82	8.97	6.12×10²	9.43×10²

Fig.3 Cell viability of HepG2 cells treated by WUQ for 24 hError bars represent mean±SE for n=8. # p<0.05; ## p<0.01.

Fig.4 Cell migratory ability(A) and relative wound closue(B) of HepG2 cells treated by WUQ for 24 h(A) Conditions, [WUQ]/(mmol·L-1) and time/h: (A1) 0, 0; (A2) 5, 0; (A3) 10, 0; (A4) 15, 0; (A'1) 0, 24; (A'2) 5, 24; (A'3) 10, 24; (A'4) 15, 24. (B) Error bars represent mean±SE for n=5. # p<0.05; ## p<0.01.

Fig.5 Western blot analysis of MMP-9 and AFP protein expression(A) and relative protein expression(B) in HepG2 cells treated by WUQ for 24 h (B) Error bars represent mean±SE for n=3. # p<0.05; ## p<0.01.

References 22

[1]	Flohé L., Günzler W. A., Schock H. H., FEBS Lett., 1973, 32(1), 132—134
[2]	Toppo S., Flohé L., Ursini F., Vanin S., Maiorino M., Biochim. Biophys. Acta,2009, 1790(11), 1486—1500
[3]	Epp O., Ladenstein R., Wendel A., Eur. J. Biochem., 1983, 133(1), 51—69
[4]	Lv S. W., Wang X. G., Mu Y., Zang T. Z., Liu J. Q., Shen J. C., Luo G. M., FEBS J., 2007, 274(15), 3846—3854
[5]	Dong Z. Y., Luo Q., Liu J. Q., Chem. Soc. Rev., 2012, 41, 7890—7908
[6]	Mugesh G., du Mont W. W., Sies H., Chem. Rev., 2001, 101, 2125—2180
[7]	Satheeshkumar K., Mugesh G., Chem. Eur. J., 2011, 17(17), 4849—4857
[8]	Selvakumar K., Shah P., Singh H. B., Chem. Eur. J., 2011, 17(45), 12741—12755
[9]	Chen W. Q., Zheng R. S., Zeng H. M., Zou X. N., Zhang S. W., He J., China Cancer,2015, 24(1), 1—10(陈万青, 郑荣寿, 曾红梅, 邹小农, 张思维, 赫捷. 中国肿瘤, 2015, 24(1), 1—10)
[10]	Koide T., Itoh H., Otaka A.,Yasui H., Kuroda M., Esaki N., Soda K., Fuji N., Chem. Pharm. Bull., 1993, 41(3), 502—506
[11]	Cheng B. P., Li L. S., Zhou R. D., Li L., Zhang H. F., Chem. J. Chinese Universities,2015, 36(5), 872—880(程彪平, 李来生, 周仁丹, 李良, 张宏福. 高等学校化学学报, 2015, 36(5), 872—880)
[12]	Liu Z. J., Hou Y. L., Zhang G. G., Xu N., Mi B., Gong P., Zhao Y. F., Chem. Res. Chinese Universities,2015, 31(4), 235—243
[13]	Wang C., Shen N., Yan G. L.,Sui C. H., Zhuang J. J., Lü S. W., Luo G. M., Mu Y., Int. J. Pept. Res. Ther., 2014, 20(3), 307—324
[14]	Wang W. Q., Shi A. P., Fan Z. M., Chem. Res. Chinese. Universities,2014, 30(2), 108—113
[15]	Lü B. C., Dong S., Xue Q., Zong H., Lü S. W., Luo G. M., Chem. J. Chinese Universities,2013, 34(9), 2146—2151(吕冰聪, 董森, 薛峤, 宗慧, 吕绍武, 罗贵民. 高等学校化学学报, 2013, 34(9), 2146—2151)
[16]	Iwaoka M., Tomoda S., J. Am. Chem. Soc., 1994, 116(6), 2557—2561
[17]	Takebe G., Yarimizu J., Saito Y.,Hayashi T., Nakamura H., Yodoi J., Nagasawa S., Takahashi K., J. Biol. Chem., 2002, 277(43), 41254—41258
[18]	Bell I., Hilvert D., Biochemistry., 1993, 32(50), 13969—13973
[19]	Ren B., Huang W., Akesson B., Ladenstein R., J. Mol. Biol., 1997, 268(5), 869—885
[20]	Lau A. T., Wang Y., Chiu J. F., J. Cell. Biochem., 2008, 104(2), 657—667
[21]	Siegel R., Naishadham D., Jemal A., Ca-Cancer. J. Clin., 2013, 63(1), 11—30
[22]	Marina S. K., Elizaveta S. E., Plos. One., 2013, 8(10), e77469