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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2020, Vol. 46 Issue (5): 539-550    DOI: 10.3785/j.issn.1008-9209.2019.12.191
生物科学与技术     
厚壳贻贝转凝蛋白类贝壳基质蛋白的重组表达与功能分析
姜雨婷(),孙琦,徐焕志,申望,张晓林,范美华,廖智()
浙江海洋大学海洋科学与技术学院，海洋生物蛋白质工程研究室，浙江 舟山 316022
Recombinant expression and functional analysis of transgelin-like protein from the shell of Mytilus coruscus
Yuting JIANG(),Qi SUN,Huanzhi XU,Wang SHEN,Xiaolin ZHANG,Meihua FAN,Zhi LIAO()
Laboratory of Marine Biology Protein Engineering, College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, Zhejiang, China
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摘要：

为探明从厚壳贻贝（Mytilus coruscus）贝壳肌棱柱层中鉴定的一种新型贝壳基质蛋白——转凝蛋白类蛋白（transgelin-like protein, TLP）在贝壳形成过程中可能的分子机制，在序列分析基础上，对厚壳贻贝TLP进行原核重组表达及其表达产物的纯化，并分析其对方解石型和文石型碳酸钙晶体形貌的诱导作用及对晶型的影响，同时，分析其对2种晶型碳酸钙晶体结晶速度的抑制及结合作用。序列分析结果显示，厚壳贻贝TLP含有一段钙调理蛋白同源（calponin homology, CH）结构域，其三级结构以α-螺旋结构域为主。功能分析结果表明，重组厚壳贻贝TLP能诱导方解石型和文石型碳酸钙晶体在形貌上产生变化，其中：对方解石型碳酸钙晶体的晶型具有向文石型转化的作用，但对文石型碳酸钙晶体的晶型无影响。重组TLP对方解石型碳酸钙晶体的结晶速度具有明显的抑制作用，而对文石型碳酸钙晶体的结晶速度在蛋白质量浓度较高时具有促进作用。此外，重组TLP具有结合方解石型碳酸钙晶体的作用，但与文石型碳酸钙晶体无明显结合。以上结果表明，TLP对贝壳的形成具有影响并可能在贝壳肌棱柱层的形成中起到了重要作用。
关键词： 转凝蛋白类蛋白;  钙调理蛋白同源结构域;  贝壳基质蛋白;  生物矿化    
Abstract:

Transgelin-like protein (TLP) is a novel shell matrix protein identified previously from the myostracum layer of Mytilus coruscus shell. For exploring its function in the shell formation of mussel, the TLP was recombinantly expressed by Escherichia coli expression system based on the sequence analysis and codon optimization. The functions of recombinant TLP (rTLP) on calcite- and aragonite-type calcium carbonate crystals were then investigated, including morphology, polymorph, crystallization rate, and binding ability of calcium carbonate crystals. Sequence analysis showed that the TLP contained a calponin homology (CH) domain, and the spatial structure of TLP predicted by SWISS-MODEL presented a conformation formed predominately by α-helices. Functional analyses showed that the rTLP had significant effects on the morphological change of aragonite-type calcium carbonate crystal and polymorph change of calcite-type calcium carbonate crystal, suggesting the function of this protein in the transformation of calcite to aragonite. In addition, the rTLP showed inhibition of calcite-type calcium carbonate crystallization rate in vitro, and promotion of aragonite-type calcium carbonate crystallization rate under high protein concentration. Moreover, the rTLP presented binding abilities to the calcite-type calcium carbonate crystal rather than the aragonite-type calcium carbonate crystal. Considering the myostracum layer is composed of aragonite-type calcium carbonate crystal, we speculate that the TLP may play important roles in the shell biomineralization and the formation of myostracum layer.
Key words: transgelin-like protein    calponin homology domain    shell matrix protein    biomineralization
收稿日期: 2019-12-19 出版日期: 2020-11-19
CLC:  Q 67  
基金资助: 国家自然科学基金(31671009);浙江省舟山市科技局项目(2019F12004)
通讯作者: 廖智     E-mail: 1315834132@qq.com;liaozhi@zjou.edu.cn
作者简介: 姜雨婷（https://orcid.org/0000-0002-8921-9775），E-mail：1315834132@qq.com
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引用本文:

姜雨婷,孙琦,徐焕志,申望,张晓林,范美华,廖智. 厚壳贻贝转凝蛋白类贝壳基质蛋白的重组表达与功能分析[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(5): 539-550.

Yuting JIANG,Qi SUN,Huanzhi XU,Wang SHEN,Xiaolin ZHANG,Meihua FAN,Zhi LIAO. Recombinant expression and functional analysis of transgelin-like protein from the shell of Mytilus coruscus. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(5): 539-550.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.12.191    或    http://www.zjujournals.com/agr/CN/Y2020/V46/I5/539

图1  厚壳贻贝TLP的cDNA序列及其由开放阅读框推导的氨基酸序列比对*代表终止密码子；下划线代表加尾信号。
图2  厚壳贻贝TLP与17条来自其他软体动物且一致性在88%以上的同源蛋白序列基于邻接法构建的系统进化树最大序列差异设置为0.85；*代表厚壳贻贝的TLP。
图3  厚壳贻贝TLP与来自其他软体动物的同源蛋白序列的结构域分布比较Ⅰ.仅含1个CH结构域的同源蛋白；Ⅱ.含1个CH和1个钙调理蛋白结构域的同源蛋白；Ⅲ.含1个CH和5个串联钙调理蛋白结构域的同源蛋白；Ⅳ.含1个CH和1个GAS2结构域的同源蛋白。
图4  厚壳贻贝TLP的二级和三级结构预测A. TLP的二级结构；B. TLP的三级结构（Ⅰ～Ⅵ：α-螺旋）。
图5  厚壳贻贝TLP在优化前后的密码子适应指数分析A.优化前；B.优化后。
图6  厚壳贻贝TLP重组表达和纯化后的SDS-PAGE鉴定M：蛋白质分子质量标志物；1：未经异丙基-β-D-硫代半乳糖苷（IPTG）诱导的重组菌表达产物（阴性对照）；2：经IPTG诱导4 h后的重组菌表达产物；3：经IPTG诱导表达后重组菌菌体经裂解离心后的上清液部分；4：经IPTG诱导表达后重组菌菌体经裂解离心后的沉淀部分； 5：表达产物经镍柱分离的穿透峰部分；6：表达产物经镍柱分离及30 mmol/L咪唑溶液洗脱后的产物；7：表达产物经镍柱分离及200 mmol/L咪唑溶液洗脱后的产物；8：表达产物经镍柱分离及300 mmol/L咪唑溶液洗脱后的产物。箭头代表目标条带。
图7  重组TLP复性后的高效液相色谱法纯化箭头指示目标条带。
图8  方解石型碳酸钙晶体的扫描电子显微镜观察A.天然方解石型碳酸钙晶体；B.牛血清白蛋白（50 µg/mL）作用下的方解石型碳酸钙晶体；C. 10 μg/mL重组TLP作用下的方解石型碳酸钙晶体；D. 30 µg/mL重组TLP作用下的方解石型碳酸钙晶体；E. 50 μg/mL重组TLP作用下的方解石型碳酸钙晶体；F. 50 μg/mL重组TLP作用下的方解石型碳酸钙晶体放大图。A、D～E：标尺为 50 µm；B～C：标尺为100 µm；F：标尺为30 µm。
图9  文石型碳酸钙晶体的扫描电子显微镜观察A.天然文石型碳酸钙晶体；B.牛血清白蛋白（50 µg/mL）作用下的文石型碳酸钙晶体；C. 10 μg/mL重组TLP作用下的文石型碳酸钙晶体；D. 30 μg/mL重组TLP作用下的文石型碳酸钙晶体；E. 50 μg/mL重组TLP作用下的文石型碳酸钙晶体；F. 50 μg/mL 重组TLP作用下的文石型碳酸钙晶体放大图。A～E：标尺为40 µm；F：标尺为20 µm。
图10  方解石型和文石型碳酸钙晶体的傅里叶变换红外光谱图A1～A2：方解石型碳酸钙晶体在加入50 µg/mL重组TLP前后的比较，箭头所示为文石型特征峰；B1～B2：文石型碳酸钙晶体在加入50 µg/mL重组TLP前后的比较。
图11  重组TLP对碳酸钙晶体结晶速度的抑制作用A.方解石型碳酸钙晶体；B.文石型碳酸钙晶体。n=3。
图12  重组TLP与方解石型及文石型碳酸钙晶体结合的SDS-PAGE分析1：重组TLP溶液；2：重组TLP与方解石型碳酸钙晶体孵育2 h并离心后的上清液；3：重组TLP与方解石型碳酸钙晶体孵育并离心后的沉淀进一步经5%乙酸脱钙并离心后的上清液；M：蛋白质分子质量标志物；4：重组TLP溶液；5：重组TLP与文石型碳酸钙晶体孵育2 h并离心后的上清液；6：重组TLP与文石型碳酸钙晶体孵育并离心后的沉淀进一步经5%乙酸脱钙并离心后的上清液。
1 SONG X R, LIU Z Q, WANG L L, et al. Recent advances of shell matrix proteins and cellular orchestration in marine molluscan shell biomineralization. Frontiers in Marine Science, 2019,6:41. DOI:10.3389/fmars.2019.00041
doi: 10.3389/fmars.2019.00041
2 TAYLOR J D, REID D G. Shell microstructure and mineralogy of the Littorinidae: ecological and evolutionary significance. Hydrobiologia, 1990,193(1):199-215.
3 OCHIAI E. Biomineralization: principles and applications in bioinorganic chemistry-Ⅴ. Journal of Chemical Education, 1991,68(8):627-637. DOI:10.1021/ed068p627
doi: 10.1021/ed068p627
4 KOCOT K M, AGUILERA F, MCDOUGALL C, et al. Sea shell diversity and rapidly evolving secretomes: insights into the evolution of biomineralization. Frontiers in Zoology, 2016,13(1):23-32. DOI:10.1186/s12983-016-0155-z
doi: 10.1186/s12983-016-0155-z
5 TALHAM D R. Biomineralization: principles and concepts in bioinorganic materials chemistry Stephen Mann. Crystal Growth & Design, 2002,2(6):675-676. DOI:10.1021/cg020033l
doi: 10.1021/cg020033l
6 CARIOLOU M A, MORSE D E. Purification and characterization of calcium-binding conchiolin shell peptides from the mollusc, Haliotis rufescens, as a function of development. Journal of Comparative Physiology B: Biochemical Systemic and Environmental Physiology, 1988,157(6):717-729.
7 ADDADI L, WEINER S. Biomineralization: a pavement of pearl. Nature, 1997,389(6654):912-915.
8 ALVARES K. The role of acidic phosphoproteins in biomineralization. Connect Tissue Research, 2014,55(1):34-40. DOI:10.3109/03008207.2013.867336
doi: 10.3109/03008207.2013.867336
9 NORIZUKI M, SAMATA T. Distribution and function of the nacrein-related proteins inferred from structural analysis. Marine Biotechnology, 2008,10(3):234-241. DOI:10.1007/s10126-007-9061-x
doi: 10.1007/s10126-007-9061-x
10 赵鲁苹,徐焕志,陈东,等.厚壳贻贝贝壳的微结构及光谱分析. 浙江大学学报(理学版),2015,42(3):339-346. DOI:10.3785/j.issn.1008-9497.2015.03.018
ZHAO L P, XU H Z, CHEN D, et al. Microstructure and spectral analysis of Mytilus coruscus shell. Journal of Zhejiang University (Science Edition), 2015,42(3):339-346. (in Chinese with English abstract)
doi: 10.3785/j.issn.1008-9497.2015.03.018
11 LIAO Z, BAO L F, FAN M H, et al. In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell. Journal of Proteomics, 2015,122:26-40. DOI:10.1016/j.jprot.2015.03.027
doi: 10.1016/j.jprot
12 GAO P, LIAO Z, WANG X X, et al. Layer-by-layer proteomic analysis of Mytilus galloprovincialis shell. PLoS ONE, 2015,10(7):e0133913. DOI:10.1371/journal.pone.0133913
doi: 10.1371/journal.pone.0133913
13 ZHANG G F, FANG X D, GUO X M, et al. The oyster genome reveals stress adaptation and complexity of shell formation. Nature, 2012,490(7418):49-54. DOI:10.1038/nature11413
doi: 10.1038/nature11413
14 姜雨婷,孙琦,刘宏汉,等.翡翠贻贝外套膜转录组及贝壳珍珠质层和肌棱柱层蛋白质组分析. 中国生物化学与分子生物学报,2018,34(10):1080-1090. DOI:10.13865/j.cnki.cjbmb.2018.10.09
JIANG Y T, SUN Q, LIU H H, et al. The mantle transcriptome and shell proteome of nacre and myostracum from Perna viridis. Chinese Journal of Biochemistry and Molecular Biology, 2018,34(10):1080-1090. (in Chinese with English abstract)
doi: 10.13865/j.cnki.cjbmb.2018.10.09
15 DVORAKOVA M, NENUTIL R, BOUCHAL P. Trans-gelins, cytoskeletal proteins implicated in different aspects of cancer development. Expert Review of Proteomics, 2014,11(2):149-165. DOI:10.1586/14789450.2014.860358
doi: 10.1586/14789450.2014.860358
16 RIVERO F, CVRCKOVA F. Origins and evolution of the actin cytoskeleton. Advances in Experimental Medicine and Biology, 2007,607:97-110. DOI:10.1007/978-0-387-74021-8_8
doi: 10.1007/978-0-387-74021-8_8
17 PLAZYO O, LIU R, HOSSAIN M M, et al. Deletion of calponin 2 attenuates the development of calcific aortic valve disease in ApoE－/－mice. Journal of Molecular and Cellular Cardiology, 2018,121:233-241. DOI:10.1016/j.yjmcc.2018.07.249
doi: 10.1016/j.yjmcc
18 JIN J P, ZHANG Z, BAUTISTA J. Isoform diversity, regulation, and functional adaptation of troponin and calponin. Critical Reviews in Eukaryotic Gene Expression, 2008,18(2):93-124. DOI:10.1615/CritRevEukarGeneExpr.v18.i2.10
doi: 10.1615/CritRevEukarGeneExpr.v18.i2.10
19 LIU R, JIN J P. Calponin isoforms CNN1, CNN2 and CNN3: regulators for actin cytoskeleton functions in smooth muscle and non-muscle cells. Gene, 2016,585(1):143-153. DOI:10.1016/j.gene.2016.02.040
doi: 10.1016/j.gene.2016.02.040
20 STRADAL T, KRANEWITTER W, WINDER S J, et al. CH domains revisited. FEBS Letters, 1998,431(2):134-137.
21 BASHOUR A M, FULLERTON A T, HART M J, et al. IQGAP1, a Rac- and Cdc42-binding protein, directly binds and cross-links microfilaments. Journal of Cell Biology, 1997,137(7):1555-1566.
22 BRILL S, LI S, LYMAN C W, et al. The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases. Molecular and Cellular Biology, 1996,16(9):4869-4878.
23 HART M J, CALLOW M G, SOUZA B, et al. IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs. The EMBO Journal, 1996,15(12):2997-3005.
24 FUKATA M, KURODA S, FUJII K, et al. Regulation of cross-linking of actin filament by IQGAP1, a target for Cdc42. Journal of Biological Chemistry, 1997,272(47):29579-29583. DOI:10.1074/jbc.272.47.29579
doi: 10.1074/jbc.272.47.29579
25 孙琦,姜雨婷,申望,等.厚壳贻贝一种新型贝壳胶原蛋白的重组表达与功能分析. 中国生物化学与分子生物学报,2019,35(10):1108-1118. DOI:10.13865/j.cnki.cjbmb.2019.10.10
SUN Q, JIANG Y T, SHEN W, et al. Recombinant expression and functional analysis of a novel type of shell collagen from Mytilus coruscus. Chinese Journal of Biochemistry and Molecular Biology, 2019,35(10):1108-1118. (in Chinese with English abstract)
doi: 10.13865/j.cnki.cjbmb.2019.10.10
26 SHTUKENBERG A G, WARD M D, KAHR B, et al. Crystal growth with macromolecular additives. Chemical Reviews, 2017,117(24):14042-14090. DOI:10.1021/acs.chemrev.7b00285
doi: 10.1021/acs.chemrev
27 MATSUSHIRO A, MIYASHITA T, MIYAMOTO H, et al. Presence of protein complex is prerequisite for aragonite crystallization in the nacreous layer. Marine Biotechnology, 2003,5(1):37-44. DOI:10.1007/s10126-002-0048-3
doi: 10.1007/s10126-002-0048-3
28 BRAMHAM J, HODGKISON J L, SMITH B O, et al. Solution structure of the calponin CH domain and fitting to the 3D-helical reconstruction of F-actin: calponin. Structure, 2002,10(2):249-258. DOI:10.1016/S0969-2126(02)00703-7
doi: 10.1016/S0969-2126(02)00703-7
29 SHARP P M, LI W. The codon adaptation index: a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Research, 1987,15(3):1281-1295.
30 ALBECK S, ADDADI L, WEINER S, et al. Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins. Connective Tissue Research, 1996,35(1/2/3/4):365-370. DOI:10.3109/03008209609029213
doi: 10.3109/03008209609029213
31 GAJJERAMAN S, NARAYANAN K, HAO J J, et al. Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite. Journal of Biological Chemistry, 2007,282(2):1193-1204. DOI:10.1074/jbc.M604732200
doi: 10.1074/jbc.M6047
32 CENKER C, BOMANS P H, FRIEDRICH H, et al. Peptide nanotube formation: a crystal growth process. Soft Matter, 2012,8(28):7463-7470. DOI:10.1039/c2sm25671a
doi: 10.1039/c2sm25671a
33 NAGASAWA H. Macromolecules in biominerals of aquatic organisms. Thalassas: An International Journal of Marine Sciences, 2004,20(1):15-24.
34 YANG L, MA X M, ZHANG X Y, et al. Cooperativity between bovine serum albumin (BSA) and crystal line of calcium carbonate in distilled water. Journal of the Chinese Chemical Society, 2002,49(1):45-50. DOI:10.1002/jccs.20020008
doi: 10.1002/jccs.2002
35 GOODMAN A, GOODE B L, MATSUDAIRA P, et al. The Saccharomyces cerevisiae calponin/transgelin homolog Scpl functions with fimbrin to regulate stability and organization of the actin cytoskeleton. Molecular Biology of the Cell, 2003,14(7):2617-2629. DOI:10.1091/mbc.E03-01-0028
doi: 10.1091/mbc.E03-01-0028
36 MARIE B, RAMOS-SILVA P, MARIN F, et al. Proteomics of CaCO3 biomineral-associated proteins: how to properly address their analysis. Proteomics, 2013,13(21):3109-3116. DOI:10.1002/pmic.201300162
doi: 10.1002/pmic.201300162
37 LIAO Z, JIANG Y T, SUN Q, et al. Microstructure and in-depth proteomic analysis of Perna viridis shell. PLoS ONE, 2019,14(7):e0219699. DOI:10.1371/journal.pone.0219699
doi: 10.1371/journal.pone.0219699
38 LIANG J, XU G R, XIE J, et al. Dual roles of the lysine-rich matrix protein (KRMP)-3 in shell formation of pearl oyster, Pinctada fucata. PLoS ONE, 2015,10(7):e0131868. DOI:10.1371/journal.pone.0131868
doi: 10.1371/journal.pone.0131868
39 MA Z J, HUANG J, SUN J, et al. A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata. Journal of Biological Chemistry, 2007,282(32):23253-23263. DOI:10.1074/jbc.M700001200
doi: 10.1074/jbc.M700001200
40 MARIE B, ZANELLACLEON I, CORNEILLAT M, et al. Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus. FEBS Journal, 2011,278(12):2117-2130. DOI:10.1111/j.1742-4658.2011.08129.x
doi: 10.1111/j.1742-4658.2011.08129.x
41 YAN Z G, JING G, GONG N P, et al. N40, a novel nonacidic matrix protein from pearl oyster nacre, facilitates nucleation of aragonite in vitro. Biomacromolecules, 2007,8(11):3597-3601. DOI:10.1021/bm0701494
doi: 10.1021/bm0701494
42 YAN Y, YANG D, YANG X, et al. A novel matrix protein, PfY2, functions as a crucial macromolecule during shell formation. Scientific Reports, 2017,7(1):6021. DOI:10.1038/s41598-017-06375-w
doi: 10.1038/s41598-017-06375-w
43 TRECCANI L, MANN K, HEINEMANN F, et al. Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals. Biophysical Journal, 2006,91(7):2601-2608. DOI:10.1529/biophysj.106.086108
doi: 10.1529/biophysj.106.086108
44 KONO M, HAYASHI N, SAMATA T. Molecular mechanism of the nacreous layer formation in Pinctada maxima. Biochemical and Biophysics Research Communication, 2000,269(1):213-218. DOI:10.1006/bbrc.2000.2274
doi: 10.1006/bbrc.2000.2274
45 LEESMILLER J P, HEELEY D H, SMILLIE L B, et al. Isolation and characterization of an abundant and novel 22-kDa protein (SM22) from chicken gizzard smooth muscle. Journal of Biological Chemistry, 1987,262(7):2988-2993.
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