On Western blot analysis, two major bands (180 and 135 kDa) and
one minor band (120 kDa) were detected with biotinylated
jacalin
11 in the JBGPs without neuraminidase treatment
(
Fig. 4A ). When the blotted JBGPs with neuraminidase digestion were incubated
with jacalin, a different binding pattern was observed: a 160-kDa band
was detected instead of the 180- and 135-kDa bands, although the
120-kDa band was observed at the same position (
Fig. 4B : JBGPs after
neuraminidase digestion for 17 hours), implying that the 180- and
135-kDa band materials decreased in molecular size to 160- and 120-kDa
bands, respectively, on neuraminidase digestion. These findings showed
that the JBGPs of 180 and 135 kDa are sialo-glycoproteins, whereas that
of 120 kDa is an asialo-glycoprotein. When the JBGPs without
neuraminidase treatment was incubated with the anti-core protein
antibody, 135- and 120-kDa bands intensely and weakly reacted with the
antibody, respectively, whereas the 180-kDa band did not react with it
(Figs. 4C 4D) . In this antibody reaction, the 120-kDa band was more
visible in a blot of a large amount of the JBGPs (
Fig. 4C : 15 μg)
than in one of a small amount (
Fig. 4D : 5 μg). On digestion of the
JBGPs with neuraminidase for 1 hour, the antibody binding to the
135-kDa band decreased in both intensity and molecular size, whereas
that to the 120-kDa band increased
(Fig. 4E) . The 135-kDa band was
faintly and scarcely detected after neuraminidase digestion for 6 and
17 hours, respectively, whereas the 120-kDa band was constantly
detected
(Figs. 4F 4G) . On reaction with the antibody preincubated
with GST–core protein fusion protein, no band was observed for the
JBGPs, with or without neuraminidase digestion (
Fig. 4H : JBGPs without
neuraminidase digestion), implying that both the 135- and 120-kDa bands
were specific for binding to the anti-core protein antibody. These
findings suggested that the terminal sialic acids on the sugar chains
may have caused the different mobilities in the electrophoresed gel of
the glycoproteins in the 135- and 120-kDa bands, the core structures of
which, including the core protein, may be the same. In contrast, the
JBGP of 180 kDa was considered to be a sialo-glycoprotein with a
different core protein, the cDNA of which should be isolated in a
future study.