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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2020, Vol. 46 Issue (5): 539-550    DOI: 10.3785/j.issn.1008-9209.2019.12.191
Biological sciences & biotechnology     
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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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     
Received: 19 December 2019      Published: 19 November 2020
CLC:  Q 67  
Corresponding Authors: Zhi LIAO     E-mail: 1315834132@qq.com;liaozhi@zjou.edu.cn
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Yuting JIANG
Qi SUN
Huanzhi XU
Wang SHEN
Xiaolin ZHANG
Meihua FAN
Zhi LIAO
Cite this article:

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


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

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 
Fig. 1 Sequence alignment of the cDNA and the amino acid sequence deduced from the open reading frame of M. coruscus TLPSingle asterisk (*) represents the termination codon; the tailing signal is underlined.
Fig. 2 Phylogenetic tree of M. coruscus TLP with 17 homologous protein sequences from other mollusks constructed by neighbor-joining methodThe maximum sequence difference was set as 0.85; single asterisk (*) represents the M. coruscus TLP.
Fig. 3 Comparison of domain distribution among M. coruscus TLP and homologous protein sequences from other mollusksⅠ. Homologous protein with only one CH domain; Ⅱ. Homologous protein with one CH and one calponin domain; Ⅲ. Homologous protein with one CH and five calponin domains in series; Ⅳ. Homologous protein with one CH and one GAS2 domain.
Fig. 4 Prediction of secondary and tertiary structures of M. coruscus TLPA. Secondary structure of TLP; B. Tertiary structure of TLP (Ⅰ-Ⅵ: α-helix).
Fig. 5 Analysis of codon adaptation index of M. coruscus TLP before and after optimizationA. Before optimization; B. After optimization.
Fig. 6 SDS-PAGE identification of M. coruscus TLP after recombinant expression and purificationM: Protein marker; 1: Recombinant expression product of TLP without induction of isopropyl-β-D-thiogalactoside (IPTG) (negative control); 2: Recombinant expression product of TLP with induction of IPTG for 4 h; 3: Supernatant of the cell lysate from the recombinant expression product of TLP with induction of IPTG; 4: Debris of the cell lysate from the recombinant expression product of TLP with induction of IPTG; 5: Penetrated sample from Ni-NTA column; 6: Eluted sample from Ni-NTA column with 30 mmol/L imidazole; 7: Eluted sample from Ni-NTA column with 200 mmol/L imidazole; 8: Eluted sample from Ni-NTA column with 300 mmol/L imidazole. The target protein band is denoted by an arrow.
Fig. 7 Purification of refolded recombinant TLP （rTLP） by high performance liquid chromatographyThe target protein band is denoted by an arrow.
Fig. 8 Scanning electron microscope (SEM) observation on in vitro crystallization of calcite-type calcium carbonate crystalA. Natural calcite-type calcium carbonate crystal (without protein induction); B. Calcite-type calcium carbonate crystal induced by 50 µg/mL bovine serum albumin (BSA); C. Calcite-type calcium carbonate crystal induced by 10 µg/mL rTLP; D. Calcite-type calcium carbonate crystal induced by 30 µg/mL rTLP; E-F. Calcite-type calcium carbonate crystal induced by 50 µg/mL rTLP. Scale bars: Figs. A, D, and E are 50 µm; Figs. B and C are 100 µm; Fig. F is 30 µm.
Fig. 9 SEM observation on in vitro crystallization of aragonite-type calcium carbonate crystalA. Natural aragonite-type calcium carbonate crystal (without protein induction); B. Aragonite-type calcium carbonate crystal induced by 50 µg/mL BSA; C. Aragonite-type calcium carbonate crystal induced by 10 μg/mL rTLP; D. Aragonite-type calcium carbonate crystal induced by 30 μg/mL rTLP; E-F: Aragonite-type calcium carbonate crystal induced by 50 μg/mL rTLP. Scale bars: Figs. A-E are 40 µm; Fig. F is 20 µm.
Fig. 10 Fourier transform infrared spectra of the calcite- and aragonite-type calcium carbonate crystalsA1-A2: Calcite-type calcium carbonate crystal without or with TLP, respectively, and the arrow represents the aragonite-type characteristic peak; B1-B2: Aragonite-type calcium carbonate crystal without or with TLP, respectively.
Fig. 11 In vitro inhibition of calcium carbonate crystallization by rTLPA. Calcite-type calcium carbonate crystal; B. Aragonite-type calcium carbonate crystal. n=3.
Fig. 12 SDS-PAGE analysis of rTLP precipitated by calcite- and aragonite-type calcium carbonate crystals1: Purified rTLP solution; 2: Supernatant of rTLP incubated with calcite-type calcium carbonate crystal for 2 h; 3: Supernatant of calcite-type calcium carbonate crystal after decalcification by 5% acetic acid; M: Protein marker; 4: Purified rTLP solution; 5: Supernatant of rTLP incubated with aragonite-type calcium carbonate crystal for 2 h; 6: Supernatant of aragonite-type calcium carbonate crystal after decalcification by 5% acetic acid.
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[1] Bao Linfei, Wang Xinxing, He Jianyu, Fan Meihua, Gao Peng, Liao Zhi. Illumina-based transcriptome sequencing of mussel Mytilus coruscus mantle[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(4): 394-406.