When crude extracts of Spirometra mansoni plerocercoid
(sparganum) were analysed by SDS–polyacrylamide gel electrophoresis
(PAGE)/immunoblot using patients' sera, IgE antibodies
reacted specifically with 21, 27 and 53 kDa proteins.
The 21 and 27 kDa proteins have been previously characterized as
cysteine proteases. In this study, the 53 kDa protein
was confirmed, by immunoprecipitation, to induce a specific IgE
response. The protein was purified by affinity chromatography using an
IgG1
(κ2) type mAb. The protein was partially sensitive to
peptide-N4-(N-acetyl-β-glucosaminyl)asparagine
amidase F (endo F) digestion. It exhibited an endoproteinase activity in
a
thiol-dependent manner preferentially degrading
benzoyloxycarboxyl-phenylalanyl-arginyl-4-methoxy-β-naphthylamide
(Z-phe-arg-MNA) of a panel of substrates tested. This endoprotease activity
was
maximal at pH 6·5 and in 0·1 M sodium phosphate.
The proteolytic activity was inhibited by 10−5ML-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
(E-64) and
1 mM iodoacetamide (IAA), and potentiated by dithiothreitol
(DTT, 5 mM).