葉 強(qiáng),金曉琴,劉偉娜,韓鳳琴,康 振,楊 莉

(浙江師范大學(xué) 化學(xué)與生命科學(xué)學(xué)院,浙江 金華 321004)

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植物蛋白質(zhì)N-糖基化修飾研究進(jìn)展*

葉 強(qiáng),金曉琴,劉偉娜,韓鳳琴,康 振,楊 莉

(浙江師范大學(xué) 化學(xué)與生命科學(xué)學(xué)院,浙江 金華 321004)

N-糖基化與植物蛋白質(zhì)正確折疊、細(xì)胞凋亡、器官發(fā)育及信號轉(zhuǎn)導(dǎo)等生物學(xué)功能密切相關(guān).主要對植物蛋白N-糖基化的結(jié)構(gòu)、生物合成、加工修飾、相關(guān)酶生物學(xué)功能，以及糖蛋白的分離鑒定方法等進(jìn)行了綜述,并探討了植物糖基化蛋白功能研究的應(yīng)用前景及存在的問題.

植物蛋白質(zhì)；N-糖基化修飾；糖苷合成；生物學(xué)功能

真核生物細(xì)胞內(nèi)的多肽及蛋白質(zhì)分子經(jīng)核糖體合成后大多需翻譯后修飾,如泛素化、磷?；?、糖基化等,確保蛋白質(zhì)正常行使其生物學(xué)功能[1].其中,糖基化是真核生物體內(nèi)常見的蛋白翻譯后修飾,糖蛋白占細(xì)胞蛋白質(zhì)的50%以上,參與細(xì)胞識別、分化、發(fā)育、信號轉(zhuǎn)導(dǎo)和免疫應(yīng)答等多個重要的生命過程[2-3].

蛋白質(zhì)的糖基化修飾是指糖鏈與蛋白質(zhì)上特定氨基酸殘基共價結(jié)合的過程.根據(jù)連接方式,主要分為N-糖基化、O-糖基化、C-糖基化及糖基磷脂酰肌醇錨定連接4種類型[3-5].N-糖基化是指內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum,ER)上糖基轉(zhuǎn)移酶催化轉(zhuǎn)移至新生肽Asn-X-Ser/Thr(X是除脯氨酸Pro外的任一氨基酸；Asn為天冬酰胺；Ser為絲氨酸；Thr為蘇氨酸)基序的Asn殘基,是蛋白質(zhì)糖基化修飾的重要形式,胞外分泌蛋白、膜整合蛋白及構(gòu)成內(nèi)膜系統(tǒng)的可溶性駐留蛋白大多經(jīng)N-糖基化修飾.酵母、哺乳動物和細(xì)菌中相關(guān)糖蛋白的鑒定為植物N-糖基化蛋白功能分析奠定了基礎(chǔ)，但植物蛋白N-糖基化研究尚處于起步階段.蛋白質(zhì)的N-糖基化主要包括糖鏈的生物合成、糖鏈的轉(zhuǎn)移及糖鏈的進(jìn)一步加工.本文主要從植物蛋白的N-糖鏈結(jié)構(gòu)、N-糖基化過程及其生物學(xué)功能等方面進(jìn)行了綜述.

1 植物蛋白N-糖鏈的基本結(jié)構(gòu)

植物N-糖蛋白的糖鏈包含1個核心五糖,根據(jù)其結(jié)構(gòu)可分為4種類型(見圖1)：1)寡甘露糖型：最簡單的N-糖鏈,僅含核心五糖結(jié)構(gòu)；2)高甘露糖型：具有5個及以上的甘露糖(mannose,Man)殘基；3)復(fù)雜型：糖鏈除含3個Man核心及與Man連接的N-乙酰葡糖胺糖基(GlcNAc)外,還包含唾液酸及其衍生物；4)雜合型：具有復(fù)雜型和高甘露糖型2類糖鏈結(jié)構(gòu)元件[6-7].

(a)寡甘露糖型；(b)高甘露糖型；(c)復(fù)雜型；(d)雜合型.虛框內(nèi)為N-糖鏈核心結(jié)構(gòu)圖1 植物糖蛋白N-糖鏈的4種基本結(jié)構(gòu)

圖2 脂連寡糖(LLO)的生物合成

2 植物蛋白質(zhì)的N-糖基化修飾

蛋白質(zhì)的N-糖基化修飾主要包括糖鏈的生物合成、糖鏈轉(zhuǎn)移至新合成蛋白，以及蛋白糖鏈的進(jìn)一步加工.

2.1N-糖鏈的生物合成

蛋白質(zhì)的N-糖基化修飾主要發(fā)生于內(nèi)質(zhì)網(wǎng)(ER)和高爾基體(Golgi)上,糖鏈與肽鏈的生物合成同步進(jìn)行,動物與植物N-糖鏈的生物合成基本一致[8].糖鏈與新生肽結(jié)合前以脂連寡糖(lipid-linked oligosaccharide,LLO)形式存在[9-10],LLO的生物合成主要包括4個步驟(見圖2)：1)面向胞質(zhì)一側(cè)的ER膜上,2分子GlcNAc經(jīng)GlcNAc-1-磷酸轉(zhuǎn)移酶(GlcNAc-1-P transferase,GPT,如ALG7與ALG13/14)催化與二磷酸-多萜醇(Dol-PP)結(jié)合生成GlcNAc2-PP-Dol；2)5分子Man在糖基轉(zhuǎn)移酶(glycosyltransferase,GT,如ALG1,ALG2與ALG11)作用下依次與GlcNAc結(jié)合,生成具有2個分枝的核心五糖；3)核心五糖經(jīng)翻轉(zhuǎn)酶催化進(jìn)入ER內(nèi)腔；4)在ER內(nèi)腔,核心五糖經(jīng)特定GT(ALG3,ALG6,ALG8-ALG10及ALG12)催化添加4分子Man及3分子葡萄糖(glucose,Glc),生成包含14個糖基的LLO前體[3-4,11].在進(jìn)入ER腔前,LLO糖鏈合成時糖基供體為鳥嘌呤核苷二磷酸(UDP)-甘露糖與尿嘧啶核苷二磷酸(GDP)-N-乙酰葡糖胺；LLO翻轉(zhuǎn)進(jìn)入ER內(nèi)腔后,糖基供體則為多萜醇-葡萄糖與多萜醇-甘露糖.

2.2 蛋白質(zhì)N-糖基化修飾

糖鏈LLO生物合成的同時,新合成的蛋白質(zhì)也經(jīng)易位子進(jìn)入ER腔[3].LLO在寡聚糖轉(zhuǎn)移酶(oligosaccharyltransferase,OST)作用下轉(zhuǎn)移至新生肽Asn-X-Ser/Thr基序的Asn殘基[9-10].LLO轉(zhuǎn)移至新生肽后,新生肽上的LLO將繼續(xù)被修飾,主要包括以下2個過程：

1)鈣聯(lián)蛋白-鈣網(wǎng)蛋白(calnexin-calreticulin cycle,CNX-CRT)循環(huán)

如圖3所示：轉(zhuǎn)移至新合成蛋白的LLO分別經(jīng)α-葡糖苷酶Ⅰ(α-glucosidase Ⅰ,GCSⅠ/GI)與α-葡糖苷酶Ⅱ(GCSⅡ/GⅡ)水解末端2個Glc殘基,生成的寡糖結(jié)構(gòu)Glc1Man9GlcNAc2被CNX或(和)CRT識別并結(jié)合；糖鏈上最后一個Glc殘基經(jīng)GCSⅡ水解后(結(jié)構(gòu)為Man9GlcNAc2),糖蛋白脫離CNX-CRT循環(huán),轉(zhuǎn)運(yùn)至Golgi進(jìn)一步修飾[12-14].若蛋白質(zhì)未正確折疊,糖鏈會經(jīng)UDP-Glc：糖蛋白糖基轉(zhuǎn)移酶(UDP-glucose:glycoprotein-glucosyltransferase,UGGT)再次糖基化,重新進(jìn)入CNX-CRT循環(huán),或者直接進(jìn)入蛋白降解程序[15].

圖3 鈣聯(lián)蛋白-鈣網(wǎng)蛋白(CNX-CRT)循環(huán)

2)蛋白糖鏈在Golgi上的再加工

糖蛋白在ER完成修飾后,通過COPⅡ型膜泡運(yùn)輸至Golgi再加工[6,11].首先,Man9GlcNAc2或Man8GlcNAc2經(jīng)α-甘露糖苷酶Ⅰ(Golgi-α-ManⅠ,MNS1/2)水解糖鏈上3～4分子Man殘基；接著,N-乙酰氨基葡萄糖轉(zhuǎn)移酶Ⅰ(glucosamine-phosphateN-acetyltransferase Ⅰ,GlcNAcT或GnTI)催化向糖鏈添加1分子GlcNAc殘基,生成GlcNAcMan5GlcNAc2(見圖4)；最后,在XylT,FucT和GalT等酶的作用下加工生成復(fù)雜型或雜合型糖鏈[16-17].

圖4 復(fù)雜型N-糖鏈在高爾基體上的再加工

3 蛋白質(zhì)N-糖基化修飾的相關(guān)酶及其生物學(xué)功能

3.1 蛋白質(zhì)N-糖基化修飾相關(guān)酶

植物中蛋白質(zhì)保守的N-糖基化修飾進(jìn)程主要是由糖基轉(zhuǎn)移酶(glycosyltransferase,GT)、α-葡糖苷酶(α-glucosidase,GCS)及甘露糖苷酶(mannosidase,MNS)完成.

1)GT：主要負(fù)責(zé)LLO的生物合成.根據(jù)糖基供體,GT可分為Leloir與non-Leloir 2種類型.Leloir型GT以糖核苷酸GDP-Man及UDP-GlcNAc為糖基供體,LLO從胞質(zhì)一側(cè)翻轉(zhuǎn)至ER內(nèi)腔前所涉及的糖基轉(zhuǎn)移酶多為該類型[18].non-Leloir型GT則以磷酸酯連接的糖Dol-Glc與Dol-Man為糖基供體[19].

2)GCS：在CNX-CRT循環(huán)中起重要作用的是GCSⅠ與GCSⅡ.GCSⅠ與OST復(fù)合物緊密相連,為Ⅱ型膜蛋白,水解LLO末端第一個Glc殘基[15].GCSⅡ由α亞基(功能域)與β亞基(定位域)構(gòu)成,在N-糖苷合成早期敲除α亞基后糖鏈末端多出1～2個Glc,N-糖鏈不能進(jìn)行后續(xù)修飾[14,20].目前尚未發(fā)現(xiàn)能夠替代GCSⅡ功能的酶.

3)MNS：由ER型(α-ManⅠ,MNS3)及Golgi型(α-ManⅠ,MNS1/2)組成,負(fù)責(zé)去除糖鏈上的Man殘基(見圖4).糖蛋白離開CNX-CRT循環(huán)后,其糖鏈可被MNS3水解生成Man8GlcNAc2,再進(jìn)入Golgi；也可不經(jīng)水解,直接進(jìn)入Golgi[21-22].MNS1-3與ER執(zhí)行錯誤折疊糖蛋白降解(endoplastic reticulum-associated degradation machinery,ERAD)途徑密切相關(guān),但具體機(jī)制尚未明確[17,23].

3.2 蛋白質(zhì)N-糖基化修飾在植物中的生物學(xué)功能

真核生物蛋白質(zhì)的N-糖苷具有幫助蛋白質(zhì)正確折疊、輔助蛋白質(zhì)功能發(fā)揮、抑制或延緩蛋白質(zhì)降解等作用[7].

1)N-糖苷幫助蛋白質(zhì)正確折疊.

蛋白質(zhì)的N-糖基化修飾過程中,CNX-CRT能夠?qū)Ｒ蛔R別糖蛋白上的GlcMan9GlcNAc2結(jié)構(gòu)[14],與未折疊糖蛋白結(jié)合,避免折疊中間體及錯誤折疊蛋白從ER逃逸[22,24-25].錯誤折疊且無法修復(fù)的糖蛋白進(jìn)入ERAD途徑,由ERAD復(fù)合物運(yùn)輸至胞質(zhì)溶膠,經(jīng)糖基肽酶水解去除糖鏈后,蛋白進(jìn)入26S蛋白酶體降解[15,26-27].

2)N-糖基化修飾蛋白與細(xì)胞凋亡密切相關(guān).

錯誤折疊的糖蛋白滯留于ER,激發(fā)未折疊蛋白應(yīng)答反應(yīng)(unfold protein response,UPR).Iwata等[28]發(fā)現(xiàn)衣霉素(Tunicamycin,GT抑制劑)能夠誘導(dǎo)煙草懸浮細(xì)胞產(chǎn)生UPR反應(yīng).DAD1(OST復(fù)合物亞基之一)作為抗細(xì)胞凋亡因子,缺失引起DGL1(OST復(fù)合物亞基之一)快速降解,引發(fā)細(xì)胞凋亡；超表達(dá)則保護(hù)原生質(zhì)體免受紫外線引起的DNA斷裂和細(xì)胞損傷[29-31].

3)N-糖基化修飾蛋白影響細(xì)胞壁的組成與含量.

植物細(xì)胞壁主要由纖維素、半纖維素、果膠質(zhì)等多糖組成.N-糖基化受阻時,植物細(xì)胞壁成分與含量將發(fā)生改變.例如：DGL1能夠影響細(xì)胞壁多糖的形成[32-34]；突變體gcs1與rsw3的細(xì)胞壁纖維素含量顯著降低[35]；Mns1-3也觀察到細(xì)胞壁不均勻等現(xiàn)象[36].

4)N-糖基化修飾蛋白對根發(fā)育的影響.

VTC1編碼GDP-Man焦磷酸化酶,合成GDP-Man,VTC1缺失會引起根生長的不可逆抑制[37].文獻(xiàn)[38]發(fā)現(xiàn)擬南芥突變體cgl1對鹽脅迫敏感,根生長受抑制,根尖形態(tài)發(fā)生異常.此外,SWP1(OST復(fù)合物亞基之一)突變體將抑制側(cè)根發(fā)生與伸長[39]；Osdgl1的根細(xì)胞體積變小,部分根細(xì)胞死亡[40]；AtMns1-3也觀察到根變短的現(xiàn)象[21,36].

5)N-糖苷對果實(shí)發(fā)育的影響.

在果實(shí)的成熟過程中,N-聚糖大量積累,因此，N-糖基化也能夠影響植物果實(shí)的發(fā)育.大量的實(shí)驗(yàn)證明,N-聚糖的增加能夠促進(jìn)果實(shí)轉(zhuǎn)色并引起乙烯含量的增加,而對N-糖基化修飾過程進(jìn)行抑制后果實(shí)延遲成熟.例如，對β-D-乙酰氨基己糖苷酶的酶活進(jìn)行抑制能夠延長果實(shí)的貨架期[41-43].

此外,蛋白質(zhì)的N-糖基化修飾在細(xì)胞分化、免疫、信號轉(zhuǎn)導(dǎo)及激素調(diào)控等多個重要的生命進(jìn)程都起到十分重要的作用[44-45].

4 N-糖蛋白的富集分離和鑒定方法

目前，有關(guān)植物蛋白的N-糖基化修飾研究較少,對于植物特定發(fā)育時期哪些糖蛋白發(fā)生N-糖基化修飾及N-糖基化位點(diǎn)等仍然知之甚少.植物體內(nèi)糖蛋白上糖鏈的合成與修飾十分復(fù)雜,無固定模板與結(jié)構(gòu),且N-糖基化修飾的蛋白豐度遠(yuǎn)遠(yuǎn)低于未經(jīng)N-糖基化修飾的蛋白.因此，富集分離N-糖苷、N-糖肽與N-糖蛋白十分困難[46-47].

近年來，隨著蛋白質(zhì)組學(xué)的飛速發(fā)展,應(yīng)用蛋白質(zhì)組學(xué)全面分析植物N-糖蛋白質(zhì),大大加快了糖蛋白質(zhì)組的研究.目前最常用的N-糖蛋白組研究主要包括富集分離、酶解消化和鑒定3個步驟：首先應(yīng)用刀豆蛋白A凝集素[47]、麥胚凝集素[48]及小扁豆凝集素[49]等凝集素與N-糖鏈特有的結(jié)構(gòu)共價結(jié)合,將N-糖蛋白從眾多蛋白中分離出來；分離出的糖蛋白再經(jīng)PNGase F等糖苷酶處理,N-糖肽與N-糖鏈相連處由天冬酰胺轉(zhuǎn)化為天冬氨酸,N-糖肽分子量發(fā)生改變,再采用質(zhì)譜技術(shù)分析N-糖肽序列及N-糖蛋白的糖基化位點(diǎn).文獻(xiàn)[50]采用刀豆蛋白A凝集素層析結(jié)合二維液相色譜富集分離糖蛋白,經(jīng)胰蛋白酶消化后進(jìn)行液相色譜串聯(lián)基質(zhì)輔助激光解吸附質(zhì)譜技術(shù)(LC-MALDI-MS/MS)分析,共鑒定出133個糖蛋白,并預(yù)測了其中大部分糖蛋白的糖基化位點(diǎn).文獻(xiàn)[51]采用類似方法，從二穗短柄草中鑒定出46個N-糖蛋白,以及47個糖基化位點(diǎn).此外,Silva-Sanchez等[52]應(yīng)用糖蛋白特異熒光染料 ProQ 染色2D蛋白膠,從玉米胚乳己糖缺失突變體mn1及野生型中分離出45個差異糖蛋白.

5 展 望

隨著糖生物學(xué)研究的迅速發(fā)展,糖蛋白已成為生物化學(xué)研究的熱點(diǎn)和前沿,在糖蛋白的結(jié)構(gòu)、生物合成、代謝及其生理作用等方面已取得了不少的成果,但仍有很多糖蛋白的結(jié)構(gòu)和功能未知.由于糖的合成無固定模板和糖結(jié)構(gòu)與功能的不對應(yīng)性,糖蛋白的鑒定、功能與開發(fā)利用研究相對困難,尤其是植物糖蛋白的研究落后于動物、酵母等的相關(guān)研究[25,45].

隨著多種植物基因組測序的完成,以及大量新技術(shù)、新方法及新軟件的開發(fā)利用,加速了植物中糖蛋白的分離及結(jié)構(gòu)與生物學(xué)功能的鑒定.但是,一個蛋白質(zhì)可能有幾十種甚至上百種不同的多聚糖修飾基團(tuán)，而且蛋白糖基化修飾在細(xì)胞中豐度也較低,如何明確這些糖蛋白的生物學(xué)功能仍面臨巨大的挑戰(zhàn).此外,糖蛋白藥物對人類健康的重要性已開始受到重視,已上市的醫(yī)藥蛋白中70%以上為糖蛋白[53-54].應(yīng)用植物作為生物反應(yīng)器大量生產(chǎn)藥用糖蛋白，將是今后的研究方向之一.為確保糖蛋白藥物的安全性、均一性和藥效,研究植物糖蛋白的糖基化結(jié)構(gòu)與生物活性,優(yōu)化蛋白糖基化修飾的方法與條件,對于開發(fā)利用植物生產(chǎn)藥用重組糖蛋白也是必不可少的.

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(責(zé)任編輯 薛 榮)

Research progress onN-glycosylation proteins in plants

YE Qiang,JIN Xiaoqin,LIU Weina,HAN Fengqin,KANG Zhen,YANG Li

(CollegeofChemistryandLifeSciences,ZhejiangNormalUniversity,Jinhua321004,China)

N-glycosylation was closely related to correctly folded proteins,apoptosis,organ developments and signal transductions.It was reviewed the structures,biosynthesis,modifications and functions ofN-glycosylation,methods for isolation and identification of glycoprotein in plants.The application perspectives and potential problems were also discussed.

plant proteins; glycosylation;N-glycan synthesis; biological functions

10.16218/j.issn.1001-5051.2016.01.015

??2015-04-20；

2015-05-18

浙江省自然科學(xué)基金資助項(xiàng)目(LY14C150001)；浙江省大學(xué)生新苗人才計(jì)劃項(xiàng)目(2104R404024)

葉 強(qiáng)(1992-),男,浙江金華人,碩士研究生.研究方向：生物化學(xué)與分子生物學(xué).通信作者：楊 莉.E-mail:yangli@zjnu.cn

Q51

A

1001-5051(2016)01-080-07