牙周致病菌编码的Tannerpin-M对粒细胞释放的丝氨酸蛋白酶的抑制作用

doi:10.3969/j.issn.1674-8115.2024.12.006

摘要/Abstract

摘要：

目的·制备牙周致病相关坦纳菌编码的丝氨酸蛋白酶抑制剂（serine protease inhibitor，Serpin），分析其抑制靶蛋白酶的特异性及其结构特征。方法·通过氨基酸序列分析，选择人类口腔微生物组数据库（eHOMD）中的Serpin，将其转入大肠埃希菌中进行重组表达；采用镍离子亲和层析等方法制备重组蛋白，分析其抑制丝氨酸蛋白酶的特异性，并通过结构生物学手段解析其三维空间结构。结果·在坦纳菌中发现一个新的且活性中心P1残基为甲硫氨酸的Serpin，将其命名为Tannerpin-M，并成功地从大肠埃希菌BL21（DE3）中制备了高纯度的重组蛋白。进一步的活性检测结果表明重组Tannerpin-M可以有效地与粒细胞来源的蛋白酶（人中性粒细胞弹性蛋白酶、组织蛋白酶G和蛋白酶3）、胰弹性蛋白酶以及激肽释放酶1（KLK1）、KLK7等6种蛋白酶形成SDS稳定的共价复合物，其中Tannerpin-M抑制KLK7的二级反应速率常数为4.25×10⁴ L/（mol·s）。解析分辨率为2.4 ?（1 ?=0.1 nm）的Tannerpin-M松弛态构象的晶体结构，结果显示Tannerpin-M具有显著延长的反应中心环，可以发生经典的抑制型Serpin从紧张态向松弛态转变的构象变化。结论·口腔致病菌编码的Tannerpin-M是一个典型的抑制型Serpin，其可以有效抑制粒细胞释放的丝氨酸蛋白酶，提示该口腔致病菌可能以此机制来抵抗人体免疫系统的攻击。

关键词: 丝氨酸蛋白酶抑制剂, 坦纳菌, 原核表达, 晶体结构, 牙周致病菌

Abstract:

Objective ·To prepare a serine protease inhibitor (Serpin) derived from Tannerella which is associated with periodontosis, and analyze its specificity in inhibiting target proteases and its structural characteristics. Methods ·Through amino acid sequence analysis, a Serpin from the human oral microbiome database (eHOMD) was selected and expressed in Escherichia coli. The recombinant protein was purified using methods such as nickel ion affinity chromatography. Its specificity in inhibiting serine proteases was analyzed, followed by an analysis of its three-dimensional spatial structure using structural biology methods. Results ·A novel Serpin, named Tannerpin-M, with methionine as the active center P1 residue, was identified, and a high-purity recombinant protein was successfully prepared from Escherichia coli BL21 (DE3). Further activity testing demonstrated that recombinant Tannerpin-M could effectively form SDS-stable covalent complexes with proteases derived from granulocytes (human neutrophil elastase, cathepsin G, and proteinase 3), as well as with other proteases including kallikrein 1 (KLK1), KLK7, and elastase. Tannerpin-M inhibited KLK7 with a second-order association rate constant of 4.12×10⁴ L/(mol·s). The crystal structure of Tannerpin-M in its relaxed state conformation was resolved at a resolution of 2.4 ? (1 ?=0.1 nm). It revealed that Tannerpin-M possessed a significantly elongated reactive center loop and could undergo the classical conformational transition from a stressed to a relaxed state. Conclusion ·Tannerpin-M, derived from oral pathogenic bacteria, is a typical inhibitory Serpin, and can effectively inhibit the serine protease released by granulocytes, by which it may protect the oral pathogenic bacteria from attacks of the human immune system.

Key words: serine protease inhibitor, Tannerella, prokaryotic expression, crystal structure, periodontal pathogen

中图分类号:

潘子豪, 许佳伟, 周爱武. 牙周致病菌编码的Tannerpin-M对粒细胞释放的丝氨酸蛋白酶的抑制作用[J]. 上海交通大学学报（医学版）, 2024, 44(12): 1536-1544.

PAN Zihao, XU Jiawei, ZHOU Aiwu. Inhibition of Tannerpin-M encoded by periodontal pathogens on serine proteases released by granulocytes[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(12): 1536-1544.

图/表 6

图1 Tannerpin-M与其他相关Serpin的RCL序列对比Note： α1-AT—α1-antitrypsin (Uniprot ID: A0A024R6I7); Tannerpin-M (GenBank: ETK05009.1); Tannerpin (GenBank: PNE27936.1); Miropin (GenBank: TPE18305.1); s1C—strand 1 of β-sheet C.

Fig 1 Comparison of RCL sequences of Tannerpin-M with other related Serpins

图2 重组Tannerpin-M的制备Note： A. Protein expression of Tannerpin-M fusion protein in whole bacterial cell lysate and lysis supernatant. M—marker (Lane 1,8 and 12); Lane 2?4—whole cell lysate of Tannerpin-M expression upon IPTG induction at 0, 6 and 24 h; Lane 5?7—supernatant of Tannerpin-M expression cells upon IPTG induction at 0, 6 and 24 h respectively. B. Enzyme digestion of the fusion protein and purification by reverse nickel ion affinity column (Ni-column). Lane 9—HisTrap-purified SUMO3-Tannerpin-M fusion protein; Lane 10—fusion protein digested with SENP2; Lane 11—flow through of fusion protein digestion from a HisTrap column. C. Gel filtration purification of Tannerpin-M. Lane13?16—fractions of Tannerpin-M from a gel filtration column.

Fig 2 Preparation of recombinant Tannerpin-M

图3 Tannerpin-M抑制丝氨酸蛋白酶的特异性Note： M—marker (Lane 1); Lane 2—Tannerpin-M control (Ctrl). The other lanes are mixed solutions of Tannerpin-M with different serine proteases; Lane 3—FⅡa; Lane 4—HNE; Lane 5—elastase; Lane 6—APC; Lane 7—t-PA; Lane 8—FⅨa; Lane 9—FⅪa; Lane 10—trypsin; Lane 11—CatB; Lane 12—CatG; Lane 13—KLK1; Lane 14—KLK7; Lane 15—PR3.

Fig 3 Inhibition specificity of Tannerpin-M towards serine proteases

图4 动力学检测Tannerpin-M对KLK7的抑制作用Note： The SI value of Tannerpin-M in inhibiting KLK7 was determined by mixing different ratios of Serpin and protease, with the remaining protease activity plotted against the ratio (left). kapp value of Tannerpin-M in inhibiting KLK7 was obtained by plotting the kobs values against the concentrations of Tannerpin-M (right).

Fig 4 Kinetic measurement of the inhibition of Tannerpin-M on KLK7

表1 Tennerpin-M晶体结构信息

Tab 1 Crystallographic statistics of Tannerpin-M structure

Item	Data collection
Beamline	SSRF 18U
Space group	P1211
Cell dimension
a, b, c /Å	46.07, 112.52, 73.62
α, β, γ /(°)	90, 98.81, 90
Wavelength/Å	0.98
Resolution/Å	45.53‒2.4 (2.486‒2.4)
Total No. of observation	54 039 (5 190)
Total No. unique	27 693 (2 698)
R_merge/%	0.066 (0.248)
I/σI	8.81 (3.22)
Completeness/%	95.35 (93.71)
Multiplicity	8.81 (3.22)
R-meas	0.094 (0.351)
CC_1/2	0.993 (0.856)
Refinement
Reflections used in refinement	27 676 (2 697)
Reflections used for R-free	1 369 (140)
Number of non-hydrogen atoms	5 745
Macromolecule	5 714
Ligand	6
Solvent	25
Protein residue	729
R-work	0.221 (0.275)
R-free	0.273 (0.344)
B-factor/Å²	24.78
RMSD bond length/Å	0.002
RMSD bond angle/(°)	0.52
Ramachandran favored/allowed/outlier/%	97.36/2.36/0.28

图5 Tannerpin-M结构模式图Note： A. Crystal structure of cleaved Tannerpin-M. B. Predicted structure of native Tannerpin-M. The central β-sheet A is colored in cyan with the strands traditionally numbered 1?6 from right to left. RCL is colored in red, helix A (hA) is colored in purple, helix F (hF) is colored in orange, and helix D (hD) is colored in gray. P1-P1' residue is shown in sticks.

Fig 5 Structures of Tannerpin-M

参考文献 35

1	LEYS E J, LYONS S R, MOESCHBERGER M L, et al. Association of Bacteroides forsythus and a novel Bacteroides phylotype with periodontitis[J]. J Clin Microbiol, 2002, 40(3): 821-825.
2	FARRELL J J, ZHANG L, ZHOU H, et al. Variations of oral microbiota are associated with pancreatic diseases including pancreatic cancer[J]. Gut, 2012, 61(4): 582-588.
3	KSIAZEK M, MIZGALSKA D, ENGHILD J J, et al. Miropin, a novel bacterial serpin from the periodontopathogen Tannerella forsythia, inhibits a broad range of proteases by using different peptide bonds within the reactive center loop[J]. J Biol Chem, 2015, 290(1): 658-670.
4	LAW R H, ZHANG Q, MCGOWAN S, et al. An overview of the serpin superfamily[J]. Genome Biol, 2006, 7(5): 216.
5	CARRELL R W, EVANS D L, STEIN P E. Mobile reactive centre of serpins and the control of thrombosis[J]. Nature, 1991, 353: 576-578.
6	GETTINS P G. Serpin structure, mechanism, and function[J]. Chem Rev, 2002, 102(12): 4751-4804.
7	HEIT C, JACKSON B C, MCANDREWS M, et al. Update of the human and mouse SERPIN gene superfamily[J]. Hum Genom, 2013, 7: 22.
8	BOUDIER C, BIETH J G. The reaction of serpins with proteinases involves important enthalpy changes[J]. Biochemistry, 2001, 40(33): 9962-9967.
9	HUNTINGTON J A, READ R J, CARRELL R W. Structure of a serpin-protease complex shows inhibition by deformation[J]. Nature, 2000, 407: 923-926.
10	IKEZOE T. Advances in the diagnosis and treatment of disseminated intravascular coagulation in haematological malignancies[J]. Int J Hematol, 2021, 113(1): 34-44.
11	YANG L, MANITHODY C, QURESHI S H, et al. Contribution of exosite occupancy by heparin to the regulation of coagulation proteases by antithrombin[J]. Thromb Haemost, 2010, 103(2): 277-283.
12	MAAS C, DE MAAT S. Therapeutic SERPINs: improving on nature[J]. Front Cardiovasc Med, 2021, 8: 648349.
13	LOMAS D A, LI-EVANS D, FINCH J T, et al. The mechanism of Z α1-antitrypsin accumulation in the liver[J]. Nature, 1992, 357: 605-607.
14	SCHECHTER I, BERGER A. On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain[J]. Biochem Biophys Res Commun, 1968, 32(5): 898-902.
15	SPENCE M A, MORTIMER M D, BUCKLE A M, et al. A comprehensive phylogenetic analysis of the serpin superfamily[J]. Mol Biol Evol, 2021, 38(7): 2915-2929.
16	Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography[J]. Acta Crystallogr D Biol Crystallogr, 1994, 50(pt 5): 760-763.
17	MCCOY A J, GROSSE-KUNSTLEVE R W, ADAMS P D, et al. Phaser crystallographic software[J]. J Appl Crystallogr, 2007, 40(pt 4): 658-674.
18	MURSHUDOV G N, SKUBÁK P, LEBEDEV A A, et al. REFMAC5 for the refinement of macromolecular crystal structures[J]. Acta Crystallogr D Biol Crystallogr, 2011, 67(pt 4): 355-367.
19	EMSLEY P, COWTAN K. Coot: model-building tools for molecular graphics[J]. Acta Crystallogr D Biol Crystallogr, 2004, 60(pt 12 pt 1): 2126-2132.
20	WEI H, CAI H, WU J, et al. Heparin binds lamprey angiotensinogen and promotes thrombin inhibition through a template mechanism[J]. J Biol Chem, 2016, 291(48): 24900-24911.
21	XU J, YE W, YANG T T, et al. DNA accelerates the protease inhibition of a bacterial serpin chloropin[J]. Front Mol Biosci, 2023, 10: 1157186.
22	MAGGIORA L L, SMITH C W, ZHANG Z Y. A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis[J]. J Med Chem, 1992, 35(21): 3727-3730.
23	WEI Z, YAN Y, CARRELL R W, et al. Crystal structure of protein Z-dependent inhibitor complex shows how protein Z functions as a cofactor in the membrane inhibition of factor X[J]. Blood, 2009, 114(17): 3662-3667.
24	IRVING J A, CABRITA L D, ROSSJOHN J, et al. The 1.5 Å crystal structure of a prokaryote serpin: controlling conformational change in a heated environment[J]. Structure, 2003, 11(4): 387-397.
25	IVANOV D, EMONET C, FOATA F, et al. A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases[J]. J Biol Chem, 2006, 281(25): 17246-17252.
26	JUMPER J, EVANS R, PRITZEL A, et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596: 583-589.
27	JIN L, ABRAHAMS J P, SKINNER R, et al. The anticoagulant activation of antithrombin by heparin[J]. Proc Natl Acad Sci USA, 1997, 94(26): 14683-14688.
28	MKAOUAR H, MARIAULE V, RHIMI S, et al. Gut serpinome: emerging evidence in IBD[J]. Int J Mol Sci, 2021, 22(11): 6088.
29	IRVING J A, PIKE R N, DAI W, et al. Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering α₁-antitrypsin to inhibit cathepsin proteases[J]. Biochemistry, 2002, 41(15): 4998-5004.
30	CABRITA L D, IRVING J A, PEARCE M C, et al. Aeropin from the extremophile Pyrobaculum aerophilum bypasses the serpin misfolding trap[J]. J Biol Chem, 2007, 282(37): 26802-26809.
31	TANAKA S, KOGA Y, TAKANO K, et al. Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis[J]. Biochim Biophys Acta, 2011, 1814(2): 299-307.
32	SANRATTANA W, MAAS C, DE MAAT S. SERPINs-from trap to treatment[J]. Front Med (Lausanne), 2019, 6: 25.
33	LAWRENCE D A, OLSON S T, MUHAMMAD S, et al. Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into β-sheet A[J]. J Biol Chem, 2000, 275(8): 5839-5844.
34	OWEN M C, BRENNAN S O, LEWIS J H, et al. Mutation of antitrypsin to antithrombin: α1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder[J]. N Engl J Med, 1983, 309(12): 694-698.
35	GETTINS P G, OLSON S T. Exosite determinants of serpin specificity[J]. J Biol Chem, 2009, 284(31): 20441-20445.

[1]	许佳伟, 周爱武, 杨愈丰. 蓝藻丝氨酸蛋白酶抑制剂arthropin的制备及其靶蛋白酶的筛选[J]. 上海交通大学学报（医学版）, 2023, 43(4): 428-436.
[2]	杨婷婷, 许佳伟, 周爱武, 叶玮. 新型口腔细菌丝氨酸蛋白酶抑制剂Tannerpin的制备与结晶[J]. 上海交通大学学报(医学版), 2021, 41(10): 1297-1302.
[3]	罗冰洁，李盛陶，王宁，高晓冬. ALG3异常的先天性糖基化疾病相关突变蛋白活性的检测[J]. 上海交通大学学报(医学版), 2020, 40(11): 1461-1467.
[4]	吴一凡，宋忠臣，束蓉. Nd: YAG 激光对具核梭杆菌的杀灭作用[J]. 上海交通大学学报（医学版）, 2018, 38(3): 259-.
[5]	付天然，张良. SUMO 化修饰对人源胸腺嘧啶DNA 糖基化酶的结构影响及活性调控[J]. 上海交通大学学报（医学版）, 2018, 38(1): 24-.
[6]	胡淑澄，束蓉，宋忠臣，孙梦君，王依玮. 光化合技术辅助治疗慢性牙周炎的临床效果[J]. 上海交通大学学报（医学版）, 2017, 37(5): 621-.
[7]	章琪，吴佳伟，周爱武. 重组人亲环蛋白CypB的制备及活性鉴定[J]. 上海交通大学学报（医学版）, 2016, 36(12): 1723-.