Pr. Austin et al., EVIDENCE THAT THE A(2) FRAGMENT OF SHIGA-LIKE TOXIN TYPE-I IS REQUIRED FOR HOLOTOXIN INTEGRITY, Infection and immunity, 62(5), 1994, pp. 1768-1775
Escherichia call Shiga-like toxin type I (SLT-I) is a potent cytotoxin
consisting of an enzymatically active A subunit and a pentameric B su
bunit that mediates to?cin binding to susceptible eukaryotic cells. Ev
idence that the carboxy-terminal 38 amino acids of the A subunit are i
nvolved in holotoxin 1A:5B association is presented. We compared the a
bility of purified recombinant SLT-I B subunit (Slt-IB) to combine in
vitro with purified recombinant SLT-I A subunit (Slt-IA; full-length s
ubunit A includes amino acids 1 to 293) and its ability to combine wit
h purified recombinant SLT-I A(1) subunit (Slt-IA(1); truncated subuni
t A includes amino acids 1 to 255). Each mixture was analyzed for biol
ogical and physical evidence of toxin assembly. Although Slt-IA succes
sfully combined with Slt-IB to form a molecular species similar to hol
otoxin that was detectable by nondenaturing polyacrylamide gel electro
phoresis and immunoblotting and yielded a molecule which was cytotoxic
to cultured Vero cells, Slt-IA(1) did not have this ability. Slt-IA(1
) was 36-fold more active than Slt-IA in an in vitro protein synthesis
inhibition assay. These findings suggest that the Slt-IA(2) fragment
is crucial for formation of SLT holotoxin and stabilizes the interacti
on between the A and B subunits.