Analysis of tertial structure and function of non-toxic components associating with Clostridium botulinum neurotoxins.

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Clostridium botulinum type A to D hemagglutinin positive progenitor toxins (PTX) consist of three distinct components : neurotoxin (NTX), hemagglutinin (HA), and non-toxic non-HA (NTNH). The HA consists of four subcomponents designated HA1 (〜33kDa), 2 (〜17kDa), 3a (〜22kDa), and 3b (〜53kDa).We have established an easy procedure for purifying type B HA-positive PTX and NTX by employing an affinity gel column-linked lactose. By employing the purified type A to D toxins and their each HA subcomponents prepared as GST-fusion proteins, we have shown that HA, especially HA1 and HA3b, works as an adhesin in the attachment of HA-positive PTXs to the-microvilli of the upper small intestine as well as erythrocytes. Type A (and B) toxin bound to the galactose mainly via HA1, whereas type C (and D) bound to the sialic acid mainly via HA3b. By analyses of primary and tertiary structure of HA1 and HA3b, It became clear that HA1 has two β-trefoil domains like as lectin B-chain of the ricin, whereas HA3b has carbohydrate recognition domain (CRD) of sialo-adhesin family (or Siglec family). In the binding and internalization tests with C HA-positive toxin and human colon carcinoma HT-29 cells, the toxin bound to glycoproteins with high molecular mass, and internalized within 4 min, but not if the cells were pretreated with neuraminidase, indicating that sialic acid of glycoprotein is the receptor for type C toxin and the toxin can internalize quickly into the cells. It was also found that both HA1 and HA3b have adjuvant activity.