甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白結(jié)構(gòu)與功能研究進(jìn)展

鄭征帆，呂艷杰，寧黔冀

( 河南師范大學(xué) 生命科學(xué)學(xué)院，河南 新鄉(xiāng) 453007 )

甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白結(jié)構(gòu)與功能研究進(jìn)展

鄭征帆，呂艷杰，寧黔冀

( 河南師范大學(xué) 生命科學(xué)學(xué)院，河南 新鄉(xiāng) 453007 )

甲殼動(dòng)物；幾丁質(zhì)結(jié)合蛋白；結(jié)構(gòu)；功能

由于堅(jiān)硬表皮的限制，甲殼動(dòng)物必須經(jīng)過蛻皮才能生長。蛻皮是一個(gè)周期性的動(dòng)態(tài)過程，包括舊表皮降解和新表皮的形成。作為表皮組成成分的幾丁質(zhì)是和蛋白質(zhì)以結(jié)合的形式存在，此蛋白即為幾丁質(zhì)結(jié)合蛋白(CBPs)，是甲殼動(dòng)物表皮中重要的結(jié)構(gòu)性蛋白，該類蛋白一般含有特征性的結(jié)構(gòu)域，由蛻皮激素的靶組織——表皮上皮細(xì)胞按照一定的周期和步驟表達(dá)合成。幾丁質(zhì)結(jié)合蛋白與幾丁質(zhì)構(gòu)成的復(fù)合物不僅為鈣化作用提供有機(jī)支架[1]，而且參與控制鈣化的程度，因此，幾丁質(zhì)結(jié)合蛋白被認(rèn)為在表皮的形成、維護(hù)以及功能的調(diào)節(jié)中起重要作用。近年來，傳統(tǒng)的分離純化技術(shù)、尤其是高通量測序技術(shù)的應(yīng)用，大大加快了甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白新基因發(fā)現(xiàn)的進(jìn)程，本文將在結(jié)構(gòu)和功能等方面介紹此類蛋白的研究進(jìn)展。

1 甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白的結(jié)構(gòu)

甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白分別含有特征性的保守基序，包括RR基序、富含半胱氨酸的幾丁質(zhì)結(jié)合結(jié)構(gòu)域、postmolt-18基序、crust-18基序等。

1.1 RR基序

對(duì)昆蟲的研究表明，RR基序是結(jié)構(gòu)性表皮蛋白中分布最廣、研究最為深入的基序[2-5]，首先在煙草天蛾(Manducasexta)幼蟲的表皮發(fā)現(xiàn)[6-7]。離體研究顯示，該基序能結(jié)合幾丁質(zhì)，覆蓋35個(gè)氨基酸殘基，以發(fā)現(xiàn)者Rebers和Riddiford的名字命名為Rebers-Riddiford共識(shí)序列，簡稱RR基序。首先鑒定出的是RR-1基序[Gx8Gx6YxAxExGYx7Px2P](X代表任意氨基酸，阿拉伯?dāng)?shù)字代表任意氨基酸的數(shù)目，后同)，之后又發(fā)現(xiàn)了兩個(gè)與之相似的變異型——RR-2基序[Gx8Gx6YxAx4GFNAVV]和RR-3基序[APVx2VxTxYHAODxLGOxSFGHx4OxRxEx2DAAGNKxGx8Gx6YxAxExGYx7Px2P][8]。RR-2基序N端前4個(gè)不連續(xù)的保守氨基酸與RR-1基序一致，而C端的GFNAVV代替了RR-1基序C端富含脯氨酸的序列。含RR-1和RR-2的幾丁質(zhì)結(jié)合蛋白在NCBI中標(biāo)注為pfam00379家族[9]。根據(jù)隱馬爾可夫模型發(fā)展的工具可在線提交幾丁質(zhì)結(jié)合蛋白的蛋白序列，更客觀便捷地區(qū)分含RR-1或RR-2基序[10]。研究表明，含RR-1的幾丁質(zhì)結(jié)合蛋白主要來自內(nèi)表皮，而含RR-2的幾丁質(zhì)結(jié)合蛋白主要來自外表皮[8]。RR-3基序覆蓋75個(gè)氨基酸殘基，C端包含RR-1基序，目前發(fā)現(xiàn)的含RR-3的幾丁質(zhì)結(jié)合蛋白種類較少，特征不夠明確。

對(duì)甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白的研究遠(yuǎn)遜于昆蟲，早期通常采用直接分離純化的方法。Andersen[11]采取尿素三氟乙酸提取、離子交換層析和雙向凝膠電泳等方法，從北方長額蝦(Pandalusborealis)的表皮中分離純化出一些蛋白，氨基酸組分分析表明，蛋白富含甘氨酸和丙氨酸，且大部分蛋白酸性氨基酸的含量大于15%。蛋白質(zhì)序列的獲得依賴于Edman降解、質(zhì)譜、酶解等方法的建立，先后獲得了北方長額蝦表皮蛋白Hla序列[12]、美洲螯龍蝦(Homarusamericanus)未鈣化表皮中的6個(gè)幾丁質(zhì)結(jié)合蛋白序列等，對(duì)后者分析發(fā)現(xiàn)，這些蛋白的pI≈4，Mr≈12 ku，其中，5個(gè)蛋白僅有幾個(gè)殘基不一致，而另一個(gè)蛋白序列則有一半以上的差異[13]；從黃道蟹(Cancerpagurus)未鈣化表皮中分離純化出5個(gè)幾丁質(zhì)結(jié)合蛋白，pI為3.5～10， Mr為20～43 ku[14]。這些結(jié)果反映了種內(nèi)以及種間幾丁質(zhì)結(jié)合蛋白的多樣性，也預(yù)示了這類蛋白功能的復(fù)雜性。

高通量測序等技術(shù)的應(yīng)用大大加快了甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白的研究進(jìn)程，越來越多的編碼幾丁質(zhì)結(jié)合蛋白的新基因被發(fā)現(xiàn)，如日本囊對(duì)蝦(Marsupenaeusjaponicus)的DD9A、DD9B[15]、DD5[16]和crustocalcin[17]，克氏原螯蝦(Procambarusclarkii)的CAP-1、CAP-2[18-20]、Casp-2[21]和SCBP-1[22]，藍(lán)蟹(Callinectessapidus)的CsAMP8.1、CsAMP6.0、CsCP8.2、CsCP8.5[23]和CsAMP9.3、CsAMP16.5、CsAMP13.4、CsAMP16.3、CsCP14.1、CsCP6.1[24]等，研究發(fā)現(xiàn)，除crustocalcin和CsCP6.1包含RR基序的部分序列外，上述其他幾丁質(zhì)結(jié)合蛋白基因均包含完整的RR-1基序。對(duì)克氏原螯蝦表皮SCBP-1基因分析表明，其cDNA的1～26位為5′UTR區(qū)，第27～71位編碼以起始密碼子ATG為開端的信號(hào)肽序列，覆蓋15個(gè)氨基酸殘基，幾丁質(zhì)結(jié)合結(jié)構(gòu)域——RR-1基序從第81位氨基酸開始至第115位氨基酸結(jié)束，覆蓋35個(gè)氨基酸殘基，SCBP-1共包含155個(gè)氨基酸殘基，以TAA為終止密碼子，3′UTR區(qū)有多聚腺苷酸信號(hào)AATAAA[22]。藍(lán)蟹CsCP6.1的信號(hào)肽和幾丁質(zhì)結(jié)合結(jié)構(gòu)域之間還具有跨膜區(qū)[25]。大部分幾丁質(zhì)結(jié)合蛋白的RR-1基序以單一非重復(fù)形式出現(xiàn)，但也有串聯(lián)重復(fù)形式，日本囊對(duì)蝦DD5蛋白在1116個(gè)氨基酸殘基的ORF中，有100個(gè)氨基酸左右的串聯(lián)重復(fù)單元，每個(gè)單元均包含RR-1基序[16]，這種串聯(lián)重復(fù)RR模式可能參與表皮中幾丁質(zhì)纖維的交叉連接，并且這些重復(fù)區(qū)整齊的大小可能反映了幾丁質(zhì)纖維有規(guī)律的空間排布。RR-2基序目前僅見于紅螯螯蝦(Cheraxquadricarinatus)轉(zhuǎn)錄物組中[26]。有關(guān)RR-3基序的報(bào)道也較少，僅在美洲螯龍蝦HaCP18.8[8]和藍(lán)蟹CsAMP/CP13.7[27]中有分布，其中，HaCP18.8在鈣化表皮表達(dá)，CsAMP/CP13.7在滑動(dòng)關(guān)節(jié)膜表皮和鈣化表皮都有表達(dá)。

1.2 其他基序

cys-CBD是富含半胱氨酸的幾丁質(zhì)結(jié)合結(jié)構(gòu)域，分為cys-CBD1和cys-CBD2。cys-CBD1只在真菌中有報(bào)道[28]，cys-CBD2在昆蟲的表皮蛋白和圍食膜蛋白中有分布[29]，其蛋白質(zhì)一級(jí)結(jié)構(gòu)中的6個(gè)cys構(gòu)成3個(gè)二硫鍵。截至目前，尚未在甲殼動(dòng)物表皮分離出含有cys-CBD2的幾丁質(zhì)結(jié)合蛋白，但在紅螯螯蝦轉(zhuǎn)錄物組中發(fā)現(xiàn)了許多具有cys-CBD2的重疊克隆群[19]，且紅螯螯蝦胃石蛋白GAP-65具有cys-CBD2[30-31]，該基序在甲殼動(dòng)物表皮中的分布有待進(jìn)一步研究。

postmolt-18基序[VxDTPEVAAAKAAFxAAY]，覆蓋18個(gè)氨基酸殘基，含此基序的表皮蛋白主要在蛻皮后期表達(dá)，所以稱為postmolt-18基序[24]。從美洲螯龍蝦鈣化表皮中分離純化出的HaCP18.8和HaCP20.2[8]、黃道蟹鈣化表皮中分離純化出的CpCP14.99和CpCP18.76[14]蛋白、藍(lán)蟹中克隆的CsCP15.0、CsCP19.0[24]以及紅螯螯蝦轉(zhuǎn)錄物組cq post-molt protein 1[32]均含有postmolt-18基序，其中藍(lán)蟹CsCP15.0是個(gè)例外，在蛻皮前和蛻皮后都有表達(dá)。

crust-18基序{x[L/V][I/V]GPSGIV[T/S]x[D/N]GxN[I/V]Q[V/L]}，同樣覆蓋18個(gè)氨基酸殘基，此基序只在甲殼動(dòng)物表皮蛋白中發(fā)現(xiàn)，故稱crust-18基序[14]。如9個(gè)源自美洲螯龍蝦鈣化表皮中的幾丁質(zhì)結(jié)合蛋白[24]，黃道蟹鈣化表皮中的CpCP 4.34、CpCP 4.59、CpCP 4.63、CpCP 4.66、CpCP 4.98、CpCP 11.58、CpCP 12.43、CpCP 12.46[14]以及紅螯螯蝦的兩個(gè)轉(zhuǎn)錄物組cq crustacean CP 1和cq crustacean CP 2[32]均含有crust-18基序。crust-18基序一般以2倍或4倍的串聯(lián)重復(fù)模式出現(xiàn)，推測可能與幾丁質(zhì)結(jié)合蛋白特殊折疊的空間構(gòu)象有關(guān)。

2 甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白的功能

2.1 結(jié)合幾丁質(zhì)

幾丁質(zhì)結(jié)合蛋白最基本的功能是結(jié)合幾丁質(zhì)，形成鈣沉積的網(wǎng)架，通常采用離體的評(píng)價(jià)方法[22]。先后從克氏原螯蝦表皮中分離純化獲得的2種幾丁質(zhì)結(jié)合蛋白——CAP-1[18]和CAP-2[20]均有較強(qiáng)的幾丁質(zhì)結(jié)合能力。高通量測序技術(shù)的應(yīng)用加快了新的幾丁質(zhì)結(jié)合蛋白基因發(fā)現(xiàn)進(jìn)程，多采用大腸桿菌(Escherichiacoli)原核表達(dá)，直接評(píng)價(jià)重組蛋白的結(jié)合能力，如克氏原螯蝦Casp-2的重組蛋白顯示出弱的幾丁質(zhì)結(jié)合能力，與前期研究時(shí)較易分離相一致[21]。由于缺乏翻譯后修飾或限于原核的分段表達(dá)等諸多原因，結(jié)合力的強(qiáng)弱并不完全與直接提取幾丁質(zhì)結(jié)合蛋白的難易程度一致，克氏原螯蝦重組SCBP-1體外試驗(yàn)僅顯示弱的幾丁質(zhì)結(jié)合能力，這與分離純化時(shí)該蛋白強(qiáng)的幾丁質(zhì)結(jié)合能力明顯不同[22]。

2.2 控制鈣化

甲殼動(dòng)物起到支持和防衛(wèi)作用的堅(jiān)硬表皮除了內(nèi)/外表皮的結(jié)構(gòu)，還與鈣化有關(guān)[33-34]，控制鈣化是表皮幾丁質(zhì)結(jié)合蛋白的另一個(gè)重要功能，常規(guī)的評(píng)價(jià)方法是離體條件下過飽和CaCO3溶液中幾丁質(zhì)結(jié)合蛋白對(duì)CaCO3沉淀的抑制效應(yīng)[18,35-37]。分離純化的克氏原螯蝦CAP-2[20]、Casp-2[21]和重組的日本囊對(duì)蝦crustocalcin[17]均表現(xiàn)出對(duì)CaCO3沉淀的抑制，表明此類幾丁質(zhì)結(jié)合蛋白具有結(jié)合Ca2+的能力。構(gòu)效關(guān)系的研究表明， 幾丁質(zhì)結(jié)合蛋白分子中絲氨酸的磷酸化與抑制活性有關(guān)，克氏原螯蝦CAP-1第70位絲氨酸磷酸化后，對(duì)CaCO3沉淀的抑制活性顯著提高[19]。

幾丁質(zhì)結(jié)合蛋白控制鈣化的分子機(jī)制是目前研究的熱點(diǎn)問題。Inoue等[20]利用同位素標(biāo)記技術(shù)，研究克氏原螯蝦重組CAP-1和CAP-2與45Ca2+的結(jié)合作用，結(jié)果表明此類幾丁質(zhì)結(jié)合蛋白可直接結(jié)合45Ca2+，推測幾丁質(zhì)結(jié)合蛋白可引發(fā)晶核的形成。對(duì)日本囊對(duì)蝦的1種幾丁質(zhì)結(jié)合蛋白crustocalcin的研究表明，分段表達(dá)的N端、中間和C端重組蛋白，只有中間肽段重組蛋白結(jié)合45Ca2+[17]，推測幾丁質(zhì)結(jié)合蛋白行使成核劑功能時(shí)具有特異的Ca2+結(jié)合位點(diǎn)；Endo等[38]分段表達(dá)crustocalcin蛋白，將獲得的重組蛋白Fr2a(包含crustocalcin第76～210位氨基酸)和Fr3(包含crustocalcin第197～321位氨基酸)附著于直鏈淀粉珠表面，在含鈣溶液中孵育，發(fā)現(xiàn)其表面有CaCO3晶體顆粒形成，且相同條件下Fr3珠比Fr2a珠表面形成的晶體顆粒數(shù)多，進(jìn)一步說明了不同位點(diǎn)結(jié)合CaCO3的差異。甲殼動(dòng)物表皮幾丁質(zhì)結(jié)合蛋白不僅通過引發(fā)成核來促進(jìn)CaCO3晶體的形成，對(duì)晶型也有一定影響。Hennig等[39]利用鼠婦(Porcellioscaber)胸骨腹片提取的幾丁質(zhì)結(jié)合蛋白，體外模擬CaCO3在表皮的沉淀，SEM觀察顯示，與陰性對(duì)照蛋白相比，CaCO3在提取的幾丁質(zhì)結(jié)合蛋白中沉淀的形狀與在體CaCO3球型沉淀輻射狀結(jié)構(gòu)十分相似。

2.3 與蛻皮周期具有相關(guān)性

在甲殼動(dòng)物周期性蛻皮過程中，表皮結(jié)構(gòu)也不斷地變化，其相對(duì)穩(wěn)定時(shí)期——間期表皮基本結(jié)構(gòu)由外到內(nèi)分別是上表皮、外表皮、內(nèi)表皮、膜狀層和上皮細(xì)胞層，這種變化受蛻皮激素的調(diào)節(jié)，合成幾丁質(zhì)結(jié)合蛋白的表皮上皮細(xì)胞是蛻皮激素的靶組織[32,40]，理論上，幾丁質(zhì)結(jié)合蛋白的表達(dá)應(yīng)該與蛻皮周期密切相關(guān)。據(jù)報(bào)道，藍(lán)蟹、日本囊對(duì)蝦和克氏原螯蝦的相關(guān)基因在蛻皮前期呈高表達(dá)，而有些基因則在蛻皮后期表達(dá)[32]。

有學(xué)者利用高通量測序技術(shù)得到的大量數(shù)據(jù)建立模型，研究表明，克氏原螯蝦含有RR基序的重疊克隆群在蛻皮前期D2、D4階段有高峰[41]。上述基因表達(dá)的時(shí)序性差異可能與其編碼的蛋白在表皮的分布有關(guān)，甲殼動(dòng)物在蛻去舊表皮之前，新的上表皮和外表皮已經(jīng)形成，在蛻皮后期，內(nèi)表皮形成及表皮鈣化[42-43]，所以，蛻皮前期高表達(dá)的幾丁質(zhì)結(jié)合蛋白可能分布在外表皮，而蛻皮后期高表達(dá)的幾丁質(zhì)結(jié)合蛋白可能分布于內(nèi)表皮[44]。

甲殼動(dòng)物幾丁質(zhì)結(jié)合蛋白的時(shí)序性表達(dá)對(duì)于表皮結(jié)構(gòu)的完整性可能至關(guān)重要，如果其中某一幾丁質(zhì)結(jié)合蛋白異?？赡軙?huì)不同程度地影響動(dòng)物蛻皮周期的進(jìn)程甚至引起死亡。運(yùn)用RNAi技術(shù)沉默紅螯螯蝦Cq-M13的表達(dá)，試驗(yàn)組蛻皮前期的天數(shù)比對(duì)照組延長了兩倍[45]；沉默藍(lán)蟹CsEarlyCP1的表達(dá)導(dǎo)致蛻皮前死亡率升高，免疫組化結(jié)果顯示，背部表皮CsEarlyCP1的含量減少[46]。有關(guān)幾丁質(zhì)結(jié)合蛋白對(duì)甲殼動(dòng)物表皮完整性的研究盡管目前尚未見報(bào)道，但對(duì)昆蟲的觀察表明，幾丁質(zhì)結(jié)合蛋白的缺失導(dǎo)致表皮超微結(jié)構(gòu)異常，影響表皮的完整性。赤擬谷盜(Triboliumcastaneum)幼蟲的TcCPR18和TcCPR27基因沉默，成蟲表皮出現(xiàn)褶皺、變短[47]，其中，TcCPR27缺陷個(gè)體的內(nèi)表皮中，幾丁質(zhì)—蛋白質(zhì)水平片層和垂直孔道具有異常的電子透明， TcCPR18缺陷個(gè)體的鞘翅前表皮具有無組織的片層和孔道[48-50]。

3 展 望

幾丁質(zhì)結(jié)合蛋白作為表皮中的結(jié)構(gòu)性蛋白，其表達(dá)特點(diǎn)關(guān)乎表皮形成及結(jié)構(gòu)的完整性以及各種生理功能的發(fā)揮，進(jìn)而影響甲殼動(dòng)物的生長、防御、免疫等各方面。雖然相當(dāng)數(shù)量的表皮幾丁質(zhì)結(jié)合蛋白或基因相繼被發(fā)現(xiàn)，但其確切的功能卻知之甚少，如同一種幾丁質(zhì)結(jié)合蛋白在表皮不同鈣化部位以及表皮各層(主要是外表皮和內(nèi)表皮)的分布特點(diǎn)，不同幾丁質(zhì)結(jié)合蛋白對(duì)表皮完整性和蛻皮周期的影響等。另外，對(duì)于幾丁質(zhì)結(jié)合蛋白的探究需進(jìn)一步拓寬研究的物種范圍，探討此類基因表達(dá)的調(diào)控途徑及其功能誘導(dǎo)與阻遏等，為最終闡明蛻皮周期的分子機(jī)理奠定基礎(chǔ)。

[1] Glazer L,Tom M,Weil S,et al.Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith [J].Journal of Experimental Biology, 2013, 216(10):1898-1904.

[2] Togawa T,Dunn W A,Emmons A C,et al.Developmental expression patterns of cuticular protein genes with the R&R Consensus fromAnophelesgambiae[J].Insect Biochemistry and Molecular Biology, 2008, 38(5):508-519.

[3] Charles J P.The regulation of expression of insect cuticle protein genes[J].Insect Biochemistry and Molecular Biology, 2010, 40(3):205-213.

[4] Willis J H.Structural cuticular proteins from arthropods: annotation, nomenclature, and sequence characteristics in the genomics era[J].Insect Biochemistry and Molecular Biology, 2010, 40(3):189-204.

[5] Zhu K Y,Merzendorfer H,Zhang W,et al.Biosynthesis, turnover, and functions of chitin in insects[J]. Annual Review of Entomology,2016,61(1):177-196.

[6] Rebers J E,Riddiford L M.Structure and expression of aManducasextalarval cuticle gene homologous toDrosophilacuticle genes[J].Journal of Molecular Biology,1988,203(2):411-423.

[7] Rebers J E, Willis J H.A conserved domain in arthropod cuticular proteins binds chitin[J].Insect Biochemistry and Molecular Biology, 2001,31(11):1083-1093.

[8] Andersen S O.Studies on proteins in post-ecdysial nymphal cuticle of locust,Locustamigratoria, and cockroach,Blaberuscraniifer[J].Insect Biochemistry and Molecular Biology, 2000, 30(7):569-577.

[9] Tetreau G,Dittmer N T,Cao X,et al.Analysis of chitin-binding proteins fromManducasextaprovides new insights into evolution of peritrophin A-type chitin-binding domains in insects[J].Insect Biochemistry and Molecular Biology,2015,62(11):127-141.

[10] Ioannidou Z S,Theodoropoulou M C,Papandreou N C,et al.CutProtFam-Pred: detection and classification of putative structural cuticular proteins from sequence alone, based on profile Hidden Markov Models[J]. Insect Biochemistry and Molecular Biology,2014,52(5):51-59.

[11] Andersen S O. Cuticular proteins from the shrimp,Pandalusborealis[J].Comparative Biochemistry and Physiology Part B,1991,99(2):453-458.

[12] Jacobsen S L,Andersen S O,Hojrup P.Amino acid sequence determination of a protein purified from the shell of the shrimp,Pandalusborealis[J].Comparative Biochemistry and Physiology Part B,1994,109(2/3):209-217.

[13] Andersen S O.Characterization of proteins from arthrodial membranes of the lobster,Homarusamericanus[J].Comparative Biochemistry and Physiology Part A,1998,121(4):375-383.

[14] Andersen S O.Exoskeletal proteins from the crab,Cancerpugurus[J].Comparative Biochemistry and Physiology Part A,1999,123(2):203-211.

[15] Watanabe T,Persson P,Endo H, et al.Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustaceanPenaeusjaponicus[J].Comparative Biochemistry and Physiology Part B,2000,125(1):127-136.

[16] Ikeya T,Persson P,Kono M,et al.The DD5 gene of the decapods crustaceanPenaeusjaponicasencodes a putative exoskeletal protein with a novel tandem repeat structure[J].Comparative Biochemistry and Physiology Part B,2001,128(3):379-388.

[17] Endo H, Persson P, Watanabe T.Molecular cloning of the crustacean DD4 cDNA encoding a Ca2+-binding protein[J].Biochemical and Biophysical Research Communications, 2000,276(1):286-291.

[18] Inoue H,Ozaki N,Nagasawa H.Purification and structural determination of a phosphorylated peptide with anticalcification and chitin-binding activities in the exoskeleton of the crayfish,Procambarusclarkii[J].Bioscience, Biotechnology and Biochemistry,2001,65(8):1840-1848.

[19] Inoue H,Ohira T,Ozaki N,et al.Cloning and Expression of a cDNA encoding a matrix peptide associated with calcification in the exoskeleton of the crayfish[J].Comparative Biochemistry and Physiology Part B,2003,136(4):755-765.

[20] Inoue H,Ohira T,Ozaki N,et al.A novel calcium-binding peptide from the cuticle of the crayfish,Procambarusclarkii[J].Biochemical and Biophysical Research Communications,2004,318(3):649-654.

[21] Inoue H,Yuasa-Hashimoto N,Suzuki M,et al.Structure determination and functional analysis of a soluble matrix protein associated with calcification of exoskeleton of the crayfish,Procambarusclarkii[J].Bioscience, Biotechnology and Biochemistry,2008,72(10): 2697-2707.

[22] Suzuki M,Sugisaka-Nobayshi A,Kogure T,et al.Structural and functional analyses of a strong chitin-binding protein-1 (SCBP-1) from the exoskeleton of the crayfishProcambarusclarkii[J].Bioscience, Biotechnology and Biochemistry,2013,77(2):361-368.

[23] Wynn A,Shafer T H.Four differentially expressed cDNAs inCallinectessapiduscontaining Rebers-Riddiford consensus sequence[J].Comparative Biochemistry and Physiology Part B, 2005,141(3):294-306.

[24] Faircloth L M,Shafer T H.Differential expression of eight transcripts and their roles in the cuticle of the blue crab,Callinectessapidus[J].Comparative Biochemistry and Physiology Part B, 2007, 146(3):370-383.

[25] Kuballa A V,Merritt D J,Elizur A.Gene expression profiling of cuticular proteins across the moult cycle of the crabPortunuspelagicus[J].Bmc Biology,2007,5(1):1-26.

[26] Abehsera S,Glazer L,Tynyakov J,et al.Binary gene expression patterning of the molt cycle: the case of chitin metabolism[J].PloS One,2015,10(6):e0130787.

[27] Shafer T H,McCartney M A,Faircloth L M.Identifying exoskeleton proteins in the blue crab from an expressed sequence tag (EST) library[J].Integrative and Comparative Biology,2006,46(6):978-990.

[28] Wright H T,Sandrasegaram G,Wright C S.Evolution of a family of N-acetylglucosamine binding proteins containing the disulfide-rich domain of wheat germ agglutinin[J].Journal of Molecular Evolution,1991,33(3):283-294.

[29] Jasrapuria S,Arakane Y,Osman G,et al.Genes encoding proteins with peritrophin A-type chitin-binding domains inTriboliumcastaneum, are grouped into three distinct families based on phylogeny, expression and function[J].Insect Biochemistry and Molecular Biology, 2010,40(3):214-227.

[30] Shechter A,Glazer L,Cheled S,et al.A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix[J]. Proceedings of the National Academy of Sciences of the USA,2008,105(20):7129-7134.

[31] Glazer L,Sagi A.On the involvement of proteins in the assembly of the crayfish gastrolith extracellular matrix[J].Invertebrate Reproduction and Development,2012,56(1):57-65.

[32] Roer R,Abehsera S,Sagi A.Exoskeletons across the pancrustacea: comparative morphology, physiology, biochemistry and genetics[J].Integrative and Comparative Biology,2015,55(5):771-791.

[33] Luquet G.Biomineralizations insights and prospects[J].Zookeys,2012(176):103-121.

[34] Dillaman R,Hequembourg S,Gay M.Early pattern of calcification in the dorsal carapace of the blue crab,Callinectessapidus[J].Journal of Morphology,2005,263(3):356-374.

[35] Suzuki M,Kogure T,Sakuda S,et al.Identification of ligament intra-crystalline peptide (LICP) from the hinge ligament of the bivalve,Pinctadafucata[J].Marine Biotechnology,2015,17(2):153-161.

[36] Liang J,Xu G,Xie J,et al.Dual roles of the lysine-rich matrix protein (KRMP)-3 in shell formation of pearl oyster,Pinctadafucata[J].PloS One,2015,10(7):e0131868.

[37] Dhami N K,Reddy M S,Mukherjee A.Synergistic role of bacterial urease and carbonic anhydrase in carbonate mineralization[J].Applied Biochemistry and Biotechnology,2014,172(5):2552-2561.

[38] Endo H,Takagi Y,Ozaki N,et al.A crustacean Ca2+-binding protein with a glutamate-rich sequence promotes CaCO3crystallization[J].Biochemical Journal,2004,384(1):159-167.

[39] Hennig S,Hild S,Fabritius H O,et al.Influence of near-physiological salines and organic matrix proteins from amorphous CaCO3deposits ofPorcellioscaberon in vitro CaCO3precipitation[J].Crystal Growth and Design,2012,12(2):646-655.

[40] Roer R,Dillaman R.The structure and calcification of the crustacean cuticle[J]. Integrative and Comparative Biology,1984,24(4):893-909.

[41] Tom M,Manfrin C,Chung S J,et al.Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfishProcambarusclarkiiduring premolt[J].Journal of Experimental Biology,2014,217(23):4193-4202.

[42] Kuballa A V,Elizur A.Differential expression profiling of components associated with exoskeletal hardening in crustaceans[J].Bmc Genomics,2008,9(1):575-589.

[43] Kuballa A V,Holton T A,Paterson B,et al.Moult cycle specific differential gene expression profiling of the crabPortunuspelagicus[J].Bmc Genomics,2011,12(1):147-165.

[44] Nagasawa H.The crustacean cuticle: structure, composition and mineralization[J].Frontiers in Bioscience,2012,4(1):711-720.

[45] Tynyakov J,Bentov S,Abehsera S,et al.A novel chitin binding crayfish molar tooth protein with elasticity properties[J].PloS One, 2015, 10(5):e0127871.

[46] Jenkins J.Investigating the roles of cuticular proteins of the blue crab,Callinectessapidususing RNA interference[D].Wilmington:University of North Carolina,2010.

[47] Arakane Y,Lomakin J,Gehrke S H,et al.Formation of rigid, non-flight forewings (elytra) of a beetle requires two major cuticular proteins[J].PloS Genetics, 2012, 8(4):e1002682.

[48] Noh M Y,Kramer K J,Muthurkrishnan S,et al.Two major cuticular proteins are required for assembly of horizontal laminae and vertical pore canals in rigid cuticle ofTriboliumcastaneum[J].Insect Biochemistry and Molecular Biology,2014,53(4):22-29.

[49] Mun S, Noh M Y, Dittmer N T, et al.Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt[J].Scientific Reports,2015(5):10484.

[50] Noh M Y,Muthukrishnan S,Kramer K J,et al.TriboliumcastaneumRR-1 cuticular protein TcCPR4 is required for formation of pore canals in rigid cuticle[J].PloS Genetics,2015,11(2):e1004963.

ResearchProgressonStructureandFunctionofCrustaceanCuticularChitin-bindingProteins:aReview

ZHENG Zhengfan, Lü Yanjie, NING Qianji

( College of Life Science, Henan Normal University, Xinxiang 453007, China )

crustacean; chitin-binding protein; structure; function

10.16378/j.cnki.1003-1111.2017.04.023

S917

C

1003-1111(2017)04-0538-05

2016-08-05；

2016-12-20.

鄭征帆(1990-)，女，碩士研究生；研究方向：甲殼動(dòng)物生長發(fā)育的體液調(diào)節(jié). E-mail: zhengzhengfan@163.com.通訊作者：寧黔冀(1964-)，女，教授，博士；研究方向：甲殼動(dòng)物激素調(diào)控. E-mail: nqjnqj1964@163.com.