K. Kudo et al., LOCALIZATION OF CARBOHYDRATE CHAINS OF PIG SPERM LIGAND IN THE GLYCOPROTEIN ZPB OF EGG ZONA-PELLUCIDA, European journal of biochemistry, 252(3), 1998, pp. 492-499
The three glycoproteins of pig zona pellucida (ZPA, ZPB and ZPC) can b
e separated into ZPA and a mixture of ZPB/ZPC by gel-filtration HPLC.
We have shown previously that the neutral complex-type N-linked carboh
ydrate chains obtained from ZPB/ZPC possess sperm-binding activity. In
tact ZPB and ZPC cannot be separated from each other unless acidic N-a
cetyllactosamine regions of their carbohydrate chains are removed by e
ndo-beta-galactosidase digestion. The endo-beta-galactosidase-digested
ZPB retains the sperm-binding activity. Recently, we have reported th
at N-linked carbohydrate chains of N-terminal fragment (residues 137-2
47) obtained from endo-beta-galactosidase-digested ZPB are involved ma
inly in sperm binding [Yonezawa, N., Mitsui, S., Kudo, K. & Nakano, M.
(1997) fur: J. Biochem. 248, 86-92]. In this study, we separated the
intact neutral N-linked chains from the ZPB/ZPC mixture into diantenna
ry chains and triantennary and tetraantennary chains by affinity chrom
atography on Concanavalia ensiformis agglutinin. An in vitro competiti
on assay revealed that triantennary and tetraantennary chains possess
a sperm-binding activity stronger than that of diantennary chains. Thr
ee glycopeptides, having one Asn residue to which the carbohydrate cha
in is linked, were obtained by lysyl endopeptidase digestion of the he
at-solubilized zonae containing intact ZPB and lysyl endopeptidase and
chymotrypsin A digestion of endo-beta-galactosidase-digested ZPB. Fro
m sugar-mapping analysis of the carbohydrate chains from these glycope
ptides and comparison with the carbohydrate structures of the main int
act neutral N-linked chains of ZPB/ZPC, the triantennary and tetraante
nnary chains were shown to be localized mainly at Asn220 of ZPB, and d
iantennary chains were present on all the three potential residues (As
n203, Asn220 and Asn333). These results suggest that the carbohydrate
chains linked to Asn220 of ZPB participate predominantly in sperm-egg
binding.