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(guò)蛻皮才能生長(zhǎng)。蛻皮是一個(gè)周期性的動(dòng)態(tài)過(guò)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(lái)，傳統(tǒng)的分離純化技術(shù)、尤其是高通量測(cè)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(chóng)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(chóng)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(jiǎn)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(lái)自內(nèi)表皮，而含RR-2的幾丁質(zhì)結(jié)合蛋白主要來(lái)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(chóng)，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(zhǎng)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(zhǎng)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(cè)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(lái)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(kāi)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(kāi)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(jiàn)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(chóng)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(cè)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(cè)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(guò)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(wèn)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(cè)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(cè)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(shuō)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(guò)引發(fā)成核來(lái)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(guò)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(cè)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(zhǎng)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(jiàn)報(bào)道，但對(duì)昆蟲(chóng)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(chóng)的TcCPR18和TcCPR27基因沉默，成蟲(chóng)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(wú)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(zhǎng)、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)。

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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(zhǎng)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.