Evaluation of the Role of N-linked Oligosaccharides in Rat Placental Lactogen Action by Site-Directed Mutagenesis.

Abstract

Abstract. To evaluate the role of N-linked oligosaccharides in the molecular action of rat placental lactogen (PL), recombinant PL-Im (recPL-Im) and three recPL-Im mutants were produced in COS-7 cells. The mutants, carrying Gin substitutions of Asn at putative N glycosylation sites, were generated via sitedirected mutagenesis, i.e. two single mutants (N79Q, N128Q) and one double mutant (N79Q/N128Q). Western blot analysis revealed that wild type recPL-Im had a molecular mass of 34 kDa , which was reduced to 29 kDa by tunicamycin present during expression. N79Q and N128Q had a lower molecular mass than the wild type, and a further decrease was observed for N79Q/N128Q. PL-Im was therefore N-glycosylated at both Asn79 and Asn128. Treatment of the wild type with neuraminidase caused a reduction in molecular mass, indicating that the N-linked oligosaccharides contained Nacetylneuraminic acids. In the Nb2 cell bioassay for lactogenic hormones, recPL-Im and its mutants all had growth-promoting activity but there was a decline in the growth-stimulating potency following decreases in N-glycosylation, i.e. the order of relative potencies was the wild type>N128Q> N79Q>N79Q/N128Q, suggesting that the N-linked oligosaccharides are important in the mitogenic action of the PL-Im. Wild type and all mutants had rat PRL receptor (PRL-R)-binding activity in radioreceptor assays and stimulated JAK2 phosphorylation in Nb2 cells. Interestingly however, the binding activity to PRL-R and phosphorylation of JAK2 was similar in the wild type and mutants, and these results are not in accord with the biological activity. In conclusion, the study suggested that PL-Im has two N-linked oligosaccharides which are involved in its biological activity. The ability of PL-Im to bind PRL-R and activate JAK2 appears to be independent of the N glycosylation.

PAL31, a Nuclear Protein Required for Progression to the S Phase

“…1 and 2). These data are consistent with our earlier study in which PL-I showed higher proliferating activity than PRL (4).…”

The prolactin and growth hormone families: Pregnancy-specific hormones/cytokines at the maternal-fetal interface

“…It is assumed that PLs activate signal transduction pathways downstream of the PRL-R similar to those activated by PRL. Beyond the abilities of members of the PL-I subfamily to stimulate the Jak-Signal Transducer and Activator of Transcription (STAT) pathway in Nb2 lymphoma cells [95,96] and cultured luteinized granulosa cells [80], information on intracellular signaling pathways triggered by classical members of the placental PRL family is lacking. It seems likely that at least some aspects of PRL signaling are mimicked by PLs; however, whether these uteroplacental ligands reproduce the full spectrum of PRL action or elicit unique responses is unknown.…”

PAL31 Expression in Rat Trophoblast Giant Cells

“…Western blotting of PAL31 and PL-I was performed using standard protocols (20). Cell lysates of proliferating and differentiated Rcho-1 cells were prepared by adding lysis buffer (10 mM phosphate buffer, pH 7.0, containing 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM NaF and protease inhibitors), and incubating for 20 min on ice.…”