Abstract
Successful pregnancy is dependent upon an array of signaling proteins secreted by the trophoblast cells of the placenta. Among these is a group of proteins related to pituitary prolactin, known as the prolactin/growth hormone family. These proteins are expressed at specific times during gestation and synthesized in distinct trophoblast cell types in the rat placenta. We report here that select members of this family, prolactin-like protein (PLP-A), PLP-B, PLP-C, decidual/trophoblast PRP, and placental lactogen I variant, only which are expressed in the spongiotrophoblast, late in rat placental development bear Asn-linked oligosaccharides terminating with NeuAcalpha2,6GalNAcbeta1,4GlcNAcbeta-R. This reflects the concurrent expression of these prolactin/growth hormone family members with the peptide-specific beta1,4GalNAc-transferase and an alpha2,6-sialyltransferase, which can add sialic acid to terminal beta1,4-linked GalNAc. We have determined that at least one of the prolactin-like proteins, PLP-A, is recognized by the protein-specific GalNAc-transferase. The presence of NeuAcalpha2, 6GalNAcbeta1,4GlcNAcbeta-R on only a limited number of glycoproteins synthesized by the spongiotrophoblasts between mid gestation and birth reflects the need for both the GalNAc-transferase and the peptide recognition determinant for efficient addition of GalNAc. Thus, expression of the GalNAc-transferase and specific members of the prolactin/growth hormone family is developmentally regulated in the rat placenta, suggesting a physiological role for the terminal NeuAcalpha2,6GalNAcbeta1,4GlcNAcbeta-R sequence on Asn-linked oligosaccharides of these proteins.
Highlights
The placenta is a complex organ that is essential for successful growth and maturation of the fetus throughout pregnancy [1]
Gestational Development of the Rat Placenta—Extracts of placenta were prepared at various times during gestation and tested for the presence of the glycoprotein hormone-specific GalNAc-transferase and GalNAc-4-sulfotransferase
Since a number of the rat placental glycoproteins that are bound by Wisteria floribunda agglutinin (WFA)-agarose have the same apparent molecular weights as members of the prolactin/growth hormone family and are expressed over the same time frame as the proteinspecific GalNAc-transferase, we examined this family of glycoproteins for the presence of 1,4-linked GalNAc on their Asnlinked oligosaccharides
Summary
Materials—[35S]PAPS was enzymatically synthesized as described previously [34] using 35SO4 from ICN. Reactions consisted of 100 g of postnuclear supernatant protein, 50 mM sodium cacodylate (pH 6.0), 0.5% (w/v) Triton X-100, 50 g of bovine serum albumin, protease inhibitors described above for the GalNAc-transferase assay, 2 M CMP-[3H]NeuAc (2 ϫ 105 cpm), and 20 M GalNAc-Trf in a total volume of 60 l. WGA-Sepharose columns were washed with 25 column volumes of TBS, and bound proteins were eluted with TBS containing 0.5 M GlcNAc. Following removal of GlcNAc by dialysis and lyophilization, the bound glycoproteins were resuspended in 200 l of 20 mM sodium cacodylate (pH 6.0), treated with neuraminidase, and subjected to WFA affinity chromatography as described above. Enzymatic reactions were terminated by the addition of an equal volume of sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.003% bromphenol blue, and 62.5 mM Tris-HCl (pH 6.8)). 35S-Labeled proteins were separated by 10% SDS-PAGE and detected by autoradiography
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