Please wait a minute...
浙江大学学报（理学版）
浙江大学学报（理学版）  2020, Vol. 47 Issue (6): 730-742    DOI: 10.3785/j.issn.1008-9497.2020.06.011
海洋科学     
一种新型贝壳基质蛋白的重组表达与功能分析
孙琦, 姜雨婷, 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，浙江 舟山 316022
Recombinant expression and functional analysis of a new type of shell matrix protein
SUN Qi, JIANG Yuting, XU Huanzhi, SHEN Wang, ZHANG Xiaolin, FAN Meihua, LIAO Zhi
Marine Science and Technology College, Zhejiang Ocean University, Zhoushan 316022, Zhejiang Province, China
 全文: PDF(5556 KB)   HTML  
摘要： 贝壳是在贝壳基质蛋白的调控下形成的生物矿化产物，也是生物工程和生物材料学研究的重要对象。贝壳基质蛋白通过调控碳酸钙晶体的成核和生长过程，使贝壳形成有规则的纳米结构并赋予贝壳极强的力学性能。在厚壳贻贝肌棱柱层中鉴定到一种新型贝壳基质蛋白，因其富含谷氨酰胺而被命名为富含谷氨酰胺的贝壳蛋白（Glutamine-rich shell protein，GRSP)，该蛋白序列中含18.2%的谷氨酰胺以及PDZ (Postsynaptic density/Discs large/Zonula occludens)和ZM (ZASP-like Motif)结构域。为了解GRSP在生物矿化过程中的分子机理，在序列分析的基础上，采用密码子优化结合原核重组表达策略，获得重组厚壳贻贝GRSP；进一步利用扫描电镜和傅里叶红外光谱，分析重组GRSP对方解石型以及文石型碳酸钙晶体在形貌和晶型方面的影响；同时，利用沉淀法分析重组GRSP对碳酸钙结晶速度的影响以及与碳酸钙晶体的结合作用。结果表明，重组GRSP可诱导文石型碳酸钙晶体发生形貌变化，并对方解石型碳酸钙晶型产生影响。此外，重组GRSP表现出与碳酸钙结合的活性并能抑制方解石型碳酸钙晶体的结晶速度。上述结果表明，GRSP蛋白可能对贝壳的形成及发育具有重要影响并在贝壳肌棱柱层的形成中发挥重要作用。
关键词： 厚壳贻贝;  贝壳基质蛋白;  生物矿化;  贝壳    
Abstract: It is recognized that mollusk shell is composed mainly by calcium carbonate crystal and produced via mineralization process tightly controlled by shell matrix proteins,as a prominent material for biomaterials and bioengineering study. Shell matrix proteins play an important role in the formation of shell,as well as the regulation of calcium carbonate biomineralization and the promotion of shell growth.A novel Glutamine-rich shell protein (GRSP) with 18.2% Gln has been identified from the myostracum layer of Mytilus coruscus shell previously.A PDZ (postsynaptic density/discs large/ zonula occludes) domain and a ZM (ZASP-like motif) domain have been detected in the sequence of GRSP.However,the roles of GRSP in shell formation are still unclear and no related research has been reported yet.For understanding the mechanism of the GRSP in biomineralization,the GRSP of M.coruscus is recombinant expressed by E.coli BL21 strain with codon optimization.The function of recombinant GRSP is studied and the results showed that,recombinant GRSP can affect the crystal morphology of aragonite calcium carbonate,as well as the polymorph of calcite calcium carbonate.Furthermore,the recombinant GRSP can interact with the calcium carbonate crystal and inhibit the crystalline growth rate of calcite.These results suggest that GRSP may be a biomineralization-related protein in the shell of M.coruscus and play an important role in the formation of the myostracum layer.
Key words: Mytilus coruscus    shell matrix protein    shell    biomineralization
收稿日期: 2019-11-14 出版日期: 2020-11-25
CLC:  Q789  
基金资助: 国家自然科学基金资助项目（31671009）；舟山市科技局项目（2019F12004）.
作者简介: 孙琦(1994—),ORCID：http://orcid.org/0000-0001-5710-7245，女，硕士研究生，主要从事海洋生物活性蛋白质研究，E-mail:1196392377@qq.co；
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
孙琦
姜雨婷
徐焕志
申望
张晓林
范美华
廖智

引用本文:

孙琦, 姜雨婷, 徐焕志, 申望, 张晓林, 范美华, 廖智. 一种新型贝壳基质蛋白的重组表达与功能分析[J]. 浙江大学学报（理学版）, 2020, 47(6): 730-742.

SUN Qi, JIANG Yuting, XU Huanzhi, SHEN Wang, ZHANG Xiaolin, FAN Meihua, LIAO Zhi. Recombinant expression and functional analysis of a new type of shell matrix protein. Journal of Zhejiang University (Science Edition), 2020, 47(6): 730-742.

链接本文:

https://www.zjujournals.com/sci/CN/10.3785/j.issn.1008-9497.2020.06.011    或    https://www.zjujournals.com/sci/CN/Y2020/V47/I6/730

1 KOCOT K M，AGUILERA F，MCDOUGALL C，et al.Sea shell diversity and rapidly evolving secretomes：Insights into the evolution of biomineralization［J］.Front Zool，2016，13：23. DOI：10.1186/s12983-016-0155-z
2 AL OMARI M M，RASHID I S，QINNA N A，et al.Calcium carbonate［J］.Profiles Drug Subst Excip Relat Methodol，2016，41：31-132.
3 CHEN H，QING C，ZHENG J，et al.Synthesis of calcium carbonate using extract components of croaker gill as morphology and polymorph adjust control agent［J］.Materials Science & Engineering C-Materials for Biological Applications，2016，63：485-488. DOI：10.1016/j.msec.2016.03.017
4 GREEN D W，GOTO T K，KIM K S，et al.Calcifying tissue regeneration via biomimetic materials chemistry［J］.Journal of the Royal Society Interface，2014，11（101）：20140537. DOI：10.1098/rsif.2014.0537
5 HARE P E.Amino acids in the proteins from aragonite and calcite in the shells of Mytilus californianus［J］.Science，1963，139（3551）：216-217. DOI：10.1126/science.139.3551.216
6 KAWATA M，AZUMA K，IZAWA H，et al.Biomineralization of calcium phosphate crystals on chitin nanofiber hydrogel for bone regeneration material［J］.Carbohydrate Polymers，2016，136：964-969. DOI：10.1016/j.carbpol.2015.10.009
7 CHOI C S，KIM Y W.A study of the correlation between organic matrics and nanocomposite materials in oyster shell formation［J］.Biomaterials，2000，21（3）：213-222. DOI：10.1016/s0142-9612（99）00120-9
8 ALVARES K.The role of acidic phosphoproteins in biomineralization［J］.Connect Tissue Research，2014，55（1）：34-40. DOI：10.3109/03008207.2013.867336
9 NORIZUKI M，SAMATA T.Distribution and function of the nacrein-related proteins inferred from structural analysis［J］.Marine biotechnology，2008，10（3）：234-241. 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［J］.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［J］. Journal of Zhejiang University（Science Edition），2015，42（3）：339-346. 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［J］.Journal of Proteomics，2015，122：26-40. DOI：10.1016/j.jprot.2015.03.027
12 TE VELTHUIS A J，BAGOWSKI C P.PDZ and LIM domain-encoding genes：Molecular interactions and their role in development［J］.Scientific World Journal，2007，7：1470-1492. DOI：10.1100/tsw. 2007.232
13 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［J］.中国生物化学与分子生物学报，2019，35（10）：1108-1118. SUN Q，JIANG Y T，SHEN W，et al.Recombinant expression and functional analysis of a novel type of shell collagen from Mytilus coruscus［J］.Chinese Journal of Biochemistry and Molecular Biology，2019，35（10）：1108-1118.
14 ZHANG Y，MA Y，YANG M，et al.Expression，purification，and refolding of a recombinant human bone morphogenetic protein 2 in vitro［J］.Protein Expression and Purification，2011，75（2）：155-160. DOI：10.1016/j.pep.2010.07.014
15 YAN Z，JING G，GONG N，et al.N40，a novel nonacidic matrix protein from pearl oyster nacre，facilitates nucleation of aragonite in vitro［J］.Biomacromolecules，2007，8（11）：3597-3601. DOI：10.1021/bm0701494
16 DAVIS K J，DOVE P M，YOREO J J.The role of Mg2+ as an impurity in calcite growth［J］.Science，2000，290：1134-1137. DOI：10.1126/science.290. 5494.1134
17 SHARP P M，LI W H.The codon adaptation index-A measure of directional synonymous codon usage bias，and its potential applications［J］.Nucleic Acids Research，1987，15：1281-1295. US7613662 B2
18 MATTICE W L，RISER J M，CLARK D S.Conformational properties of the complexes formed by proteins and sodium dodecyl sulfate［J］.Biochemistry，1976，15（19）：4264-4272. DOI：10.1021/bi00664a020
19 DAUPHIN Y.Infrared spectra and elemental composition in recent biogenic calcites：Relationships between the υ4 band wavelength and Sr and Mg concentrations［J］.Applied Spectroscopy，1999，53（2）：184-190. DOI：10.1366/0003702991946307
20 SHTUKENBERG A G，WARD M D，KAHR B.Crystal growth with macromolecular additives［J］.Chemical Reviews，2017，117（24）：14042-14090. DOI：10.1021/acs.chemrev.7b00285
21 GAO P，LIAO Z，WANG X X，et al.Layer-by-layer proteomic analysis of Mytilus galloprovincialis shell［J］.PLoS One，2015，10（7）：e0133913. DOI：10.1371/journal.pone.0133913
22 JOUBERT C，PIQUEMAL D，MARIE B，et al.Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell：Focus on biomineralization［J］.BMC Genomics，2010，11：613-620. DOI：10.1186/1471-2164-11-613
23 MANN K，EDSINGER-GONZALES E，MANN M.In-depth proteomic analysis of a mollusc shell：Acid-soluble and acid-insoluble matrix of the limpet Lottia gigantean［J］.Proteome Science，2012，10（1）：28-40. DOI：10.1186/1477-5956-10-28
24 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［J］.中国生物化学与分子生物学报，2018，34（10）：1080-1090. JIANG Y T，SUN Q，LIU H H，et al.The mantle transcriptome and shell proteome of nacre and myostracum from Perna viridis［J］.Chinese Journal of Biochemistry and Molecular Biology，2018，34（10）：1080-1090.
25 ZHANG G，FANG X，GUO X，et al.The oyster genome reveals stress adaptation and complexity of shell formation［J］.Nature，2012，490（7418）：49-54. DOI：10.1165/rcmb.2010-0312OC
26 MARIE B，JOUBERT C，TAYALÉ A，et al.Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell［J］.Proceedings of the National Academy of Sciences，2012，109（51）：20986-20991. DOI：10.1073/pnas.1210552109
27 WANG X，LI L，ZHU Y，et al.Oyster shell proteins originate from multiple organs and their probable transport pathway to the shell formation front［J］.PLoS One，2013，8（6）：e66522. DOI：10.1371/journal.pone.0066522
28 SAMATA T，HAYASHI N，KONO M，et al.A new matrix protein family related to the nacreous layer formation of Pinctada fucata［J］.FEBS Letters，1999，462：225-229. DOI：10.1016/S0014-5793（99）01387-3
29 TSUKAMOTO D，SARASHINA I，ENDO K.Structure and expression of an unusually acidic matrix protein of pearl oyster shells［J］.Biochemical and Biophysical Research Communications，2004，320：1175-1180. DOI：10.1016/j.bbrc.2004.06.072
30 MARIN F，CORSTJENS P，DE GAULEJAC B，et al.Mucins and molluscan clacification：Molecular characterization of mucoperlin，a novel mucin-like protein from the nacreous shell layer of the fan mussel Pinna nobilis （Bivalvia，Pteriomorphia）［J］.Journal of Biological Chemistry，2000，275：20667-20675. DOI：10.1074/jbc.M003006200
31 WRIGHT P E，DYSON H J.Intrinsically unstructured proteins：Reassessing the protein structure-function paradigm［J］.Journal of Molecular Biology，1999，293：321-331.
32 KIM W.Identification of mineral modulation sequences within the nacre-associated oyster shell protein，N16［J］.Crystal growth & design，2004，4：1113-1118. DOI：10.1021/cg049919a
33 MICHENFELDER M，FU G，LAWRENCE C，et al.Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains［J］.Biopolymers，2003，70（4）：522-533. DOI：10.1002/bip.10536
34 KIM I W，MORSE D E，EVANS J S.Molecular characterization of the 30-AA N-terminal mineral interaction domain of the biomineralization protein AP7［J］.Langmuir：The ACS Journal of Surfaces and Colloids，2004，20（26）：11664-11673. DOI：10.1021/la0481400
35 MARCOTTE E M，PELLEGRINI M，YEATES T O，et al.A census of protein repeats［J］.Journal of Molecular Biology，1999，293：151-160. DOI：10.1007/s00535-003-1159-y
36 VERSTREPEN K J，JANSEN A，LEWITTER F，et al.Intragenic tandem repeats generate functional variability［J］.Nature Genetics，2005，37：986-990. DOI：10.1038/ng1618
37 SHENG M，SALA C.PDZ domains and the organization of supramolecular complexes［J］.Annual Review of Neuroscience，2001，24：1-29. DOI：10.1146/annurev.neuro.24.1.1
38 DUNN H A，FERGUSON S S.PDZ protein regulation of G protein-coupled receptor trafficking and signaling pathway［J］.Molecular Pharmacology，2015，88（4）：624-639. DOI：10.1124/mol.115.098509
39 FAULKNER G，PALLAVICINI A，FORMENTIN E，et al.ZASP：A new Z-band alternatively spliced PDZ-motif protein［J］.The Journal of Cell Biology，1999，146（2）：465-475. DOI：10.1083/jcb.146.2.465
40 OTT E B，SAKALIS P A，MARQUES I J，et al.Characterization of the enigma family in zebrafish［J］.Developmental Dynamics，2007，236（11）：3144-3154. DOI：10.1002/dvdy.21346
41 KRCMERY J，CAMARATA T，KULISZ A，et al.Nucleocytoplasmic functions of the PDZ-LIM protein family：New insights into organ development［J］.BioEssays，2010，32（2）：100-108. DOI：10.1002/bies.200900148
42 BAUER K，KRATZER M，OTTE M，et al.Human CLP36，a PDZ-domain and LIM-domain protein，binds to α-actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells［J］.Blood，2000，96（13）：4236-4245. DOI：10. 1016/S0002-9394（99）00202-0
43 POMIES P，MACALMA T，BECKERLE M.C.Purification and characterization of an alpha-actinin-binding PDZ-LIM protein that is up-regulated during muscle differentiation［J］.Journal of Biological Chemistry，1999，274：29242-29250.
44 LIU H，HUANG L，ZHANG Z.et al.LIM mineralization protein-1 inhibits the malignant phenotypes of human osteosarcoma cells［J］.International Journal of Molecular Sciences，2014，15（4）：7037-7048. DOI：10.3390/ijms15047037
45 MORADIAN-OLDAK J，FROLOW F，ADDADI L，et al.Interactions between acidic matrix macromolecules and calcium phosphate ester crystals：Relevance to carbonate apatite formation in biomineralization［J］.Proceedings：Biological Sciences，1992，247（1318）：47-55.
46 FÜREDI M H，MORADIAN O J，WEINER S，et al.Interactions of matrix proteins from mineralized tissues with octacalcium phosphate［J］.Connect Tissue Research，1994，30（4）：251-264. DOI：10.3109/0300 8209409015041
[1] ‘明镇裹;胡华萃;周劲松;翁醒华. rbcL基因在水稻叶绿体DNA基因库克隆中的定位[J]. 浙江大学学报（理学版）, 1996, 23(1): 62-65.