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 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 Gln substitutions of Asn at putative N-glycosylation sites, were generated via site-directed 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 N-acetylneuraminic 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.
Department of Veterinary Medical Sciences/Animal Resource Sciences, University of Tokyo, Japan.
N
N
Hattori
T
T
Nukada
M
M
Oda
S
S
Tanaka
T
T
Ogawa
K
K
Shiota
D000818:Animals; D019556:COS Cells; D003198:Computer Simulation; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009439:Neuraminidase; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010928:Placental Lactogen / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014415:Tunicamycin
D009844:Oligosaccharides; D011994:Recombinant Proteins; D014415:Tunicamycin; D010928:Placental Lactogen; D009439:Neuraminidase