AN ESTROGEN-DEPENDENT SHEEP OVIDUCTAL GLYCOPROTEIN HAS GLYCAN LINKAGES TYPICAL OF SIALOMUCINS AND DOES NOT CONTAIN CHITINASE ACTIVITY

The deduced amino acid sequence of an estrogen-dependent sheep oviductal glycoprotein (M(r) 90 000-116 000) revealed the presence of several potential sites for glycan substitution on a protein backbone of M(r) -66 500, and identity with chitinases. In order to further define the nature of the secreted glycoprotein, the objectives of the present study were 1) to devise a method to significantly enrich for the glycoprotein from oviductal secretions, 2) to biochemically characterize the glycoprotein by use of lectin blotting and enzymatic and chemical digestion, and 3) to determine whether unfractionated and enriched fractions containing the glycoprotein have chitinase activity. Oviducts were obtained from ovariectomized ewes treated with estradiol for 6 days and explant-cultured for 24 h. The oviductal glycoprotein was enriched similar to 80-85% from explant culture media by Maackia amurensis agglutinin (MAA) lectin affinity chromatography. Enriched fractions containing the oviductal protein were separated on SDS gels, transferred to polyvinyl difluoride, and probed with digoxigenin-labeled lectins. Lectin blotting revealed that the glycoprotein contained the carbohydrate moieties N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid both in alpha(2,3) and alpha(2,6) linkages, typical of sialomucins. Enzymatic digestion with neuraminidase and N-glycanase indicated that similar to 20% and similar to 6% of the molecular weight of the oviductal glycoprotein can be accounted for by sialic acid and N-linked glycans, respectively. The oviductal glycoprotein was resistent to digestion with O-glycanase alone and chondroitinase ABC, with the latter indicating that it was not a proteoglycan. Treatment with trifluoromethanesulfonic acid resulted in a deglycosylated product of M(r) similar to 66 000 immunoreactive with antibodies to the oviductal glycoprotein. No chitinase activity could be detected for unfractionated culture medium proteins or enriched fractions containing the M(r) 90 000-116 000 oviductal glycoprotein when the substrate methylumbelliferyl chitotriose was used. These data show that 1) MAA lectin chromatography can significantly enrich for the M(r) 90 000-116 DDD glycoprotein from oviductal secretions, 2) the secreted glycoprotein contains saccharide residues typical of sialomucins, and 3) despite primary amino acid sequence identity, the oviductal glycoprotein does not share an enzymatic relationship with chitinases.