| Co-Investigator(Kenkyū-buntansha) |
NISHIKAWA Jun Tokyo University of Agriculture and Technology, Agriculture, Assistant Professor, 農学部, 助教授 (30218127)
FUJINAGA Yukako Okayama University, Graduate School of Medicine and Dentisitry, Assistant Researcher, 大学院・医歯学総合研究科, 助手 (60252954)
YOKOTA Kenji Okayama University, Graduate School of Medicine and Dentisitry, Lecturer, 大学院・医歯学総合研究科, 講師 (00243460)
INOUE Kaoru National Institute of Environmental Health Sciences, Visiting Associate, Visiting Associate (40260103)
|
|---|
| Research Abstract |
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.
|
