甘露聚糖结合凝集素与相关丝氨酸蛋白酶相互作用的初步研究

发布时间：2018-06-09 19:53

本文选题：甘露聚糖结合凝集素 + 胶原样区　；参考：《第一军医大学》2007年博士论文

【摘要】： 天然免疫是机体识别病原微生物，抵御病原体感染的第一道防线。甘露聚糖结合凝集素(mannan-binding lectin，MBL)是天然免疫系统中的关键分子，为肝细胞分泌的血浆蛋白，属C型凝集素超家族中胶凝素(collectins)家族成员。MBL籍其模式识别作用选择性识别多种病原体的糖结构，然后通过激活补体凝集素途径而发挥溶破和间接调理功能，并能与吞噬细胞胶凝素受体(即C1qR)结合而启动调理吞噬，还可介导MBL依赖的细胞介导的细胞毒作用。而且，MBL也是一个重要的粘膜表面防御分子。成熟MBL肽链自N端至C端依次有4个结构域：富含Cys的N端区、胶原样区(collagen-like region，CLR)、颈区和C端糖识别域(carbohydrate-recognition domain，CRD)。完整MBL分子是同质三肽链结构单位的寡聚体，多至六聚体。只有高寡聚体MBL分子才具有生物学活性。CRD是MBL分子的识别功能区，能选择性识别多种病原体的糖结构；而CLR是其效应功能区，MBL激活补体和结合胶凝素受体的功能定位于此区。MBL分子通过其CLR与MBL相关丝氨酸蛋白酶(MBL associated serine proteases，MASP1、MASP2)结合形成复合物，在其CRD识别、结合病原微生物的糖结构后，可活化MASPs酶原，从而激活补体凝集素途径而发挥天然抗感染免疫效应。 MASPs肽链由6个功能区组成，从N端至C端依次为CUB区(complement subcomponent C1r／C1s-like domain)、EGF区(epidermal growth factor-likedomain)、CUB区、补体调控蛋白(complement control proteins，CCP)区、CCP区和丝氨酸蛋白酶(serine proteases，SP)区。初步研究表明，N端的3个结构域即2个CUB区和1个EGF区是MASPs与MBL-CLR结合的区域，能以Ca~(2+)依赖方式与MBL-CLR相互作用。然而，这种相互作用的详细情况、特别是MBL-CLR中MASPs结合位点尚不明了。已在MBL基因编码CLR的第一外显子发现3个点突变CGT52TGT、GGC54GAC和GGA57GAA，而人群中这些点突变的基因频率极高，可引起血清MBL水平低下及活性降低从而导致调理吞噬作用缺损。MBL缺损主要表现为各种病原体的反复急慢性感染，甚至HBV、HIV等特殊病原体的感染及其发展转归也与MBL缺损有关。MBL基因突变还与自身免疫病如(SLE、RA、IgA肾炎及皮肌炎等)密切相关。然而，MBL基因突变引起免疫缺损的发病机理迄今未明。本实验研究首先采用原核表达系统分别表达了MBL-CLR和MASPs蛋白，利用哺乳细胞表达了突变MBL蛋白，然后建立MBL与MASPs结合的体系，合成一系列CLR短肽来阻断这种结合，并探索突变MBL蛋白与MASPs的相互作用，以阐述MBL分子CLR精细的结构-功能关系。其意义在于：从一个方面揭示MBL介导天然免疫防御的机理；有助于阐明MBL基因突变引起调理吞噬缺损的机制。一、人MBL-CLR蛋白的原核表达及鉴定使用Primer premier5.0软件，设计并合成引物，以含有汉族人野生型MBL全长编码区cDNA的质粒pGEM-MBL为模板，PCR扩增出长度约180bp的人MBL-CLR基因片段，将其克隆至pET32a原核表达载体中，构建的重组表达载体经BamHⅠ和HindⅢ酶切后出现约5900bp和180bp的片段，经测序鉴定序列正确后，导入大肠杆菌BL21(DE3)中，以IPTG诱导重组pET32a-MBL-CLR质粒表达Trx-CLR融合蛋白。对以可溶性表达的Trx-CLR融合蛋白经Ni+-NTA agarose层析柱纯化，获得了纯化的融合蛋白。纯化蛋白经SDS-PAGE电泳出现Mr约为30000、60000和120000的3条带，Western blot分析表明，3条蛋白带均可与抗His抗体起反应，3条蛋白带对应于融合蛋白的单体和寡聚体。ELISA证实，纯化蛋白能与鼠抗重组人MBL蛋白抗体结合。二、MASP1、MASP2 N端和C端蛋白的原核表达及鉴定使用Primer premier5.0软件设计并合成引物，分别以含人MASP1、MASP2全长编码区cDNA的pGEM-MASP1、pGEM-MASP2质粒为模板，分别扩增出长度约860bp和840bp的人MASP1和MAS2的N端区基因片段，分别将其克隆至pGEX-4T-1原核表达载体中，经双酶切和测序鉴定序列正确后，导入大肠杆菌，以IPTG分别诱导重组pGEX-4T-MASP1-N和pGEX-4T-MASP2-N质粒表达GST-MASP1-N、GST-MASP2-N融合蛋白。对以包涵体形式存在的GST-MASP1-N和GST-MASP2-N融合蛋白经8mol／L尿素变性、梯度复性，获得结构折叠正确的重组蛋白，经GSTrap层析柱纯化后，获得了纯化的融合蛋白。经ELISA和SDS-PAGE鉴定，证实为GST-MASP1-N、GST-MASP2-N融合蛋白。同样构建了pET17b-MASP1-C和pET17b-MASP2-C原核表达载体，诱导表达了MASP1-C和MASP2-C蛋白，对以包涵体形式表达的蛋白进行变性复性后经Ni~+-NTA agarose层析柱纯化，获得了纯化的MASP1-C和MASP2-C融合蛋白。三、定位MBL-CLR的MASPs结合位点建立MASPs与野生型MBL蛋白和MBL-CLR蛋白结合的反应体系，设计合成一系列CLR短肽对MBL与MASPs的结合进行抑制或阻断实验。从MBL-CLR蛋白阻断野生型MBL与MASPs结合反应的结果来看，MBL-CLR可有效地抑制MASP1-N、MASP2-N蛋白与野生型MBL的结合，其50％抑制浓度分别为0.3g／L和0.15g／L；MBL-CLR也能很好地与MASP1-N和MASP2-N蛋白结合，进一步证明MASPs确实是与MBL-CLR结合。 MBL-CLR区由59个氨基酸残基组成，由19个规律的Gly-X-Y胶原三联体重复顺序构成，其中有一处Gly-Gln断裂。根据CLR的序列及特点先后设计了9条肽进行合成。其中1～4号肽每个肽约22个氨基酸残基，覆盖了CLR全长59个氨基酸，并有部分序列重叠；5～8号肽主要针对点突变位点设计，，5号肽为野生型，6、7、8号肽分别是含32Cys、34Asp、37Glu残基的肽。对于MASP1-N、MASP2-N蛋白与重组MBL野生型MBL蛋白、MBL-CLR蛋白的结合，3号和5号肽有一定抑制作用，抑制率在25％-45％之间，2号肽有较好抑制活性，抑制率可达50％以上，而1、4、6、7、8号肽抑制作用不明显。根据这些结果，以2号肽序列为主，考虑其与3号和5号肽的重叠部分，重新设计合成了9号肽，并将序列中2个脯氨酸残基(P)改为羟基脯氨酸(O)：GLRGLQGPOGKLGPOG。抑制实验发现，9号肽能有效抑制MASP1-N、MASP2-N蛋白与重组野生型MBL蛋白、MBL-CLR蛋白结合，抑制率达70％以上。9号肽序列位于MBL-CLR中45～60位氨基酸残基，正巧是Gly-Gln断裂之后的16个残基。从哺乳动物CLR的序列来看，9号肽氨基酸序列是相对保守的，可能各种属动物的MBL均通过这一区域结合MASPs。当然，这一结论尚需进一步验证。此外，9号肽与MASPs结合的亲和力仍然偏低，尚需在今后的研究中进一步提高短肽的亲和力。同样也利用MBL-CLR能与U937细胞胶凝素受体的作用建立了细胞ELISA的体系，从MBL-CLR与U937细胞的结合试验来看，CLR确实具有胶凝素类似的作用能够和U937细胞上的胶凝素受体结合。但从合成肽抑制试验来看，没有肽段起到有效的抑制作用，这表明CLR与U937细胞的胶凝素的结合位点并不同于MASPs的结合位点，而真正的结合位点仍需进一步研究。四、突变型MBL蛋白与MASPs的结合作用 MASP1-N和MASP2-N蛋白不仅能与重组野生型MBL蛋白结合也能与32Cys、34Asp、37Glu突变型MBL蛋白结合，然而，在同样的包被浓度下，与野生型MBL蛋白的结合力高于其它3种突变蛋白。在同等条件下，32Cys突变型蛋白与MASP1-N、MASP2-N蛋白的结合力最低，与34Asp突变型蛋白的结合力次之，与37Glu突变蛋白的结合力相对较高。此外，9号合成肽可以抑制MASP1-N和MASP2-N蛋白与3种突变型MBL蛋白结合。结果表明，MBL-CLR的这3个点突变与MBL的MASPs结合位点不重叠，虽然对MBL与MASPs的结合有一定影响，但推测是MBL点突变导致MBL寡聚化程度降低而影响其MASPs结合亲和力，而非突变产物的直接效应。
[Abstract]:Natural immunity is the first defense against pathogen infection , which is a key molecule in the innate immune system . The mannan - binding lectin is a key molecule in the innate immune system .

There are four domains from the N - terminal to the C - terminus of the mature peptide chain : the N - terminal region rich in Cys , the collagen - like region ( CLR ) , the neck region and the C - terminal sugar recognition domain ( CRD ) . The intact mbl molecule is an oligomer of the homogeneous tripeptide chain structure unit , which is a poly - to - hexamer . Only the high oligomeric mbl molecule has biological activity . the crd is the recognition functional region of the mbl molecule , which can selectively recognize the sugar structure of a plurality of pathogens ;
In this region , the function of the binding lectin receptor is localized in the region . The binding of the binding agent and the binding of the binding lectin receptor in the region is localized in this region .

The MASPs peptide chain consists of six functional zones , from the N - terminal to the C - terminus , the CUB region , the epidermal growth factor - like domain , the CUB region , the complement regulatory protein ( CCP ) region , the CCP region and the serine protease ( SP ) region .

Three point mutations CGT52TGT , GGC54GAC and GGA57GAA have been found in the first exon encoding the CLR gene . The gene frequency of these mutations in the population is extremely high , which can lead to low levels of serum levels and decreased activity .

In this study , we used prokaryotic expression system to express the protein of the protein , the expression of the mutein , the expression of the mutein in the mammalian cell , and then establish a system of binding to MASPs , synthesize a series of CLR short peptides to block the binding , and explore the structure - functional relationship between the mutein and MASPs .
It is helpful to elucidate the mechanism of modulating the phagocytosis of the defect caused by the gene mutation of the mbl .

Prokaryotic Expression and Identification of One - and One - Man - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1 - 1

The recombinant expression vector was cloned into the prokaryotic expression vector of pET32a . The recombinant expression vector was cloned into the prokaryotic expression vector pET32a . The recombinant expression vector was cloned into the prokaryotic expression vector of pET32a . After sequencing , the recombinant expression vector was cloned into E . coli BL21 ( DE3 ) .

Prokaryotic expression and identification of two , MASP1 , MASP2 N - terminal and C - terminal proteins

The expression vector of GST - MASP1 - N and GST - MASP2 - N was amplified by ELISA and SDS - PAGE .

III . Binding site of MASPs to locate the mbl - CLR

The binding of MASP1 - N , MASP2 - N protein to wild - type mbl was inhibited and the 50 % inhibition concentration was 0.3g / L and 0.15g / L , respectively .
The binding of MASP1 - N and MASP2 - N proteins is also well demonstrated by the binding of the MASPs to the MASP1 - N and MASP2 - N proteins .

According to the sequence and characteristics of the CLR , 9 peptides were synthesized .
No . 9 peptide has a certain inhibitory effect on the binding of MASP1 - N , MASP2 - N protein and recombinant wild - type mbl protein , and the inhibition rate can reach more than 50 % .

In the same way , the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the binding sites of the masPs and the binding sites of the binding sites of the MASPs are different , and the true binding sites still need further study .

Study on the Binding Effect of Mutant Protein - protein and MASPs

The binding force of MASP1 - N and MASP2 - N protein was higher than that of the other three mutant proteins .
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【引证文献】