Abstract
Uteroferrin, a progesterone-induced secretory protein of the pig uterus, can noncovalently associate with additional progesterone-induced glycoproteins (uteroferrin-associated glycoproteins or UfAP) to form a heterodimer. The UfAP were dissociated from uteroferrin by passage through an immunoaffinity column. The flow through material consisted of two immunologically related variants of different size (Mr = 47,000-50,000 and Mr = 39,000-40,000) forms. By using an antiserum to all molecular weight components of the UfAP, it was shown that these glycoproteins were localized in the glandular epithelium of the uterus. Amino acid sequence analysis of the higher molecular weight (Mr = 47,000-50,000) form indicated it had a common amino-terminal sequence which was distinct from that of the lower molecular weight (Mr = 39,000-40,000) form. Endoglycosidase F treatment converted the Mr = 47,000-50,000 form to a common product with Mr = 43,000. Tryptic peptide analysis showed that the Mr = 39,000-40,000 form was closely related in primary sequence to the larger species. When endometrial RNA was translated in vitro, a single major product (Mr = 45,000) was immunoprecipitated by using the UfAP antiserum. These results suggest that the different forms of the UfAP originate from a single precursor by differential glycosylation and peptide cleavage. Endometrial explant cultures released all forms of the glycoproteins. When [32P]orthophosphate was provided, label was incorporated into the 6-position of D-mannosyl residues on the oligosaccharide chains of the UfAP. Therefore the associated glycoproteins have a structural feature normally associated with lysosomal enzymes.
Highlights
Uteroferrin, a progesterone-induced secretory pro- uteroferrin (Uf),’ which is synthesized by the glandular epitein of the pig uterus, cnanoncovalently associatewith thelial cells of the uterine endometrium [1,2,3,4]
[32P]orthophosphatewas provided, label wasincorporated into th6e-position of D-mannOSylresidues on the oligosaccharide chains of the UfAP
In this paper,we have studied the biosynthesis and properties of UfAP in part because it is During pregnancy, pseudopregnancy, or following progesterone treatment, pigs secrete large quantities of proteinaceous material into the uterine tract [1].Among the most abundant of the components in these secretions is a purple a major progesterone-induced component produced in abundance during pregnancy [10] that is taken up by the fetal placental unit and because the functional significance of its association with Uf remains puzzling
Summary
The antigen was removed and the plates were washed with phosphate-buffered saline containing0.1% (w/v) BSA and incubated at room temperature for 1h with serial 2-fold dilutions of anti-UfAP serum. Because of the sloping nature of Translation reactions were incubated for 60 min a t 30 "C and the the protein bands, constituting the UfAP, single-dimension incorporation into radiolabeled UfAP determined by immunoprecip- SDS-PAGE analysis cannot distinguish the MI 47,000 itation followed by 10% SDS-PAGE and autoradiography. 8 column (A); SDS-PAGE of polypeptides in the UfAP after fractionation by reverse phasechromatography ( B ) .A, the gradient was 0-50% (v/v) acetonitrile,0.1% (v/v) trifluoroacetic acid over 40 min. This was followed by a10-min hold in 50% (v/v) acetontrile, 0.1% (v/v) trifluoroacetic acid. Numbers above amino acidsshow residue position relative to NHZ terminus
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