Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption
Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption
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DOI：
10.1111/1348-0421.12565
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发表时间：
2018-02-01
影响因子：
2.6
通讯作者：
Fujinaga, Yukako
中科院分区：
文献类型：
--
作者：
Amatsu, Sho;Matsumura, Takuhiro;Fujinaga, Yukako
血凝素（HA）是肉毒杆菌神经毒素（BoNT）复合物的成分之一，它通过至少两种特定机制促进BoNT通过肠上皮的吸收：碳水化合物结合的细胞表面附着和e -钙粘蛋白结合的上皮屏障破坏。已知透明质酸形成三臂结构，其中三个原聚体各有三个碳水化合物结合位点和一个e-钙粘蛋白结合位点。三臂形式的HA被认为同时与这些配体结合。在本研究中，我们研究了透明质酸的多价效应如何影响其屏障破坏活性。我们制备了B型全长HA（三臂型）和mini-HA，这是一个缺失三聚体形成结构域的突变体。尺寸排除色谱分析表明，mini-HA以二聚体（双臂形式）和单体（单臂形式）存在，然后分离。我们检测了透明质酸对屏障破坏活性、e -钙粘蛋白结合活性和基底外侧细胞表面附着活性的多价效应。我们的研究结果表明，HA最初通过碳水化合物结合附着在Caco-2细胞的基表面，然后移动到细胞外侧表面，在那里HA破坏上皮屏障。结果表明，HA的多价效应增强了Caco-2细胞的屏障破坏活性。我们发现，HA的多价效应增强了基底细胞对固定E-cadherin的表面附着和结合能力。以上结果表明，至少这两种由HA多价效应诱导的因素导致了屏障破坏活性的增强。
Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity.