Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA.
Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA.
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DOI：
10.1186/1475-2875-7-104
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发表时间：
2008-06-06
期刊：
影响因子：
3
通讯作者：
Salanti A
中科院分区：
文献类型：
--
作者：
Resende M;Nielsen MA;Dahlbäck M;Ditlev SB;Andersen P;Sander AF;Ndam NT;Theander TG;Salanti A
妊娠期疟疾是由恶性疟原虫感染的红细胞结合胎盘受体硫酸软骨素A （CSA）引起的。这导致寄生虫在胎盘中积累，对母亲和未出生的孩子造成严重的临床后果。妇女对胎盘疟疾产生抗药性，因为获得了专门针对在胎盘中结合的受感染红细胞表面的抗体。VAR2CSA最有可能是寄生虫编码的蛋白，介导与胎盘受体CSA的结合。几个结构域已被证明在体外结合CSA；很明显，基于var2csa的疫苗不能适应所有的CSA结合域和血清变体。因此，在整个VAR2CSA分子中确定最小配体结合区是当务之急。为了确定VAR2CSA的最小csa结合区/肽，构建了基于整个VAR2CSA编码区的噬菌体展示库。该文库在固定的CSA和表达CSA的细胞上进行筛选，产生有限数量的CSA结合噬菌体。对这些肽的抗体进行了亲和纯化，并测试了对csa结合感染红细胞的反应性。最常见的噬菌体表达了先前定义为CSA结合的VAR2CSA部分的肽。此外，大多数结合区映射到VAR2CSA的表面暴露部分。用合成肽证实了DBL2X肽与CSA的结合。针对csa结合的DBL2X肽的抗体与感染红细胞表面发生反应，表明感染红细胞上的天然VAR2CSA抗体可接近该表位。定义了VAR2CSA对多种CSA具有亲和力的短连续区域。许多这些区域定位于CSA结合区域和这些区域的表面暴露区域，并且与DBL2中肽序列相对应的合成肽被证明与CSA结合而不是与CSC结合。这些表位中的一些可能参与了原生寄生虫CSA的粘附。然而，针对单个表位的抗体不能抑制寄生虫的粘附。本研究支持噬菌体展示技术作为鉴定大型蛋白（如VAR2CSA）的csa结合区域的技术。
Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule. To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes. The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes. Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.
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