EVALUATION OF THE ROLE OF N-LINKED OLIGOSACCHARIDES IN RAT PLACENTAL-LACTOGEN ACTION BY SITE-DIRECTED MUTAGENESIS

To evaluate the role of N-linked oligosaccharides in the molecular act ion of rat placental lactogen (PL), recombinant PL-Im (recPL-Im) and t hree recPL-Im mutants were produced in COS-7 cells. The mutants, carry ing Gin substitutions of Asn at putative N-glycosylation sites, were g enerated via site-directed mutagenesis, i.e. two single mutants (N79Q, N128Q) and one double mutant (N79Q/N128Q). Western blot analysis reve aled 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 dec rease was observed for N79Q/N128Q. PL-Im was therefore N-glycosylated at both Asn(79) and Asn(128). Treatment of the wild type with neuramin idase caused a reduction in molecular mass, indicating that the N-link ed oligosaccharides contained N-acetylneuraminic acids. in the Nb2 cel l bioassay for lactogenic hormones, recPL-Im and its mutants all had g rowth-promoting activity but there was a decline in the growth-stimula ting potency following decreases in N-glycosylation, i.e. the order of relative potencies was the wild type>N128Q>N79Q>N79Q/N128Q, suggestin g that the N-linked oligosaccharides are important in the mitogenic ac tion of the PL-Im. Wild type and all mutants had rat PRL receptor (PRL -R)-binding activity in radioreceptor assays and stimulated JAK2 phosp horylation in Nb2 cells. interestingly however, the binding activity t o PRL-R and phosphorylation of JAK2 was similar in the wild type and m utants, and these results are not in accord with the biological activi ty. In conclusion, the study suggested that PL-Im has two N-linked oli gosaccharides which are involved in its biological activity. The abili ty of PL-Im to bind PRL-R and activate JAK2 appears to be independent of the N-glycosylation.