Placental prolactin-like protein A. Identification and characterization of two major glycoprotein species with antipeptide antibodies.

The purpose of this investigation was to develop immunologic probes to prolactin-like protein A (PLP-A) that could be used to characterize the protein and its distribution in various tissues. Five oligopeptides corresponding to different regions of the predicted PLP-A amino acid sequence (peptides 1-13, 62-76, 101-114, 129-145, and 152-164) were chemically synthesized by solid phase methodology. The peptides were purified to homogeneity by reverse phase high pressure liquid chromatography and coupled to keyhole limpet hemocyanin. The peptide-keyhole limpet hemocyanin conjugates were used to immunize rabbits. Immune responses were monitored by enzyme-linked immunoassay. Reactivity of the antipeptide antisera with placental proteins was determined by immunoblotting and immunoprecipitation analyses. All of the peptides except peptide 1-13 yielded significant immune responses. Antisera to peptides 101-114, 129-145, and 152-164 each specifically recognized proteins of Mr 29,000 and 33,000 from cytosol preparations of rat placental tissue and showed limited or no cross-reactivity with other members of the prolactin-growth hormone family. Three experiments were performed to determine whether the Mr 29,000 and 33,000 species were glycosylated derivatives of an Mr 25,000 precursor. Treatment of placental cytosolic preparations with N-Glycanase prior to immunoblotting resulted in the identification of only an Mr 25,000 species. It was also determined that the Mr 29,000 and 33,000 species specifically bound to concanavalin A. Furthermore, tunicamycin shifted the synthesis of PLP-A by placental explants from the Mr 29,000 and 33,000 forms to the Mr 25,000 species. The Mr 29,000 and 33,000 species were identified in serum obtained from pregnant and fetal rats but not in serum from nonpregnant females or males. We conclude that PLP-A is expressed in rat placenta. An Mr 25,000 precursor (predicted from PLP-A cDNA and these results) is glycosylated to either the Mr 29,000 or 33,000 form, both of which predominate in placenta and in circulation.