Conformational epitopes recognized by protective anti-neisserial surface protein a antibodies
Conformational epitopes recognized by protective anti-neisserial surface protein a antibodies
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DOI：
10.1128/iai.71.12.6844-6849.2003
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发表时间：
2003-12-01
影响因子：
3.1
通讯作者：
Granoff, DM
中科院分区：
文献类型：
--
作者：
Hou, VC;Moe, GR;Granoff, DM
NspA是一种保守的膜蛋白，在小鼠中激发针对脑膜炎奈瑟菌的保护性抗体应答。最近的晶体学研究表明，NspA在洗涤剂中重构时采用八链β-桶结构。为了确定保护性鼠抗NspA抗体识别的含NspA表位的区段，我们研究了两种杀菌性和保护性抗NspA单克隆抗体（MAb）AL 12和14 C7的结合。两种MAb均不与对应于NspA的整个成熟序列的重叠合成肽（10聚体、12聚体和环状12聚体）或变性重组NspA（rNspA）结合，尽管与蛋白质的结合可以通过在脂质体中重折叠来恢复。基于两种单克隆抗体结合大肠杆菌微囊泡的能力，从一组rNspA变体通过位点特异性诱变产生，单克隆抗体和NspA之间的最重要的接触似乎位于环3的LGG段内。环2的构象似乎也是一个重要的决定因素，因为该片段中残基的特定组合导致抗体结合的丧失。因此，两种抗NspA MAb识别由NspA的三维结构中的环2和3的紧密接近引起的不连续构象表位。数据表明，使用rNspA的最佳免疫原性疫苗将需要允许蛋白质正确折叠的制剂。
NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-stranded beta-barrell structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein.