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

Endocrine Journal

KUNIO SHIOTA¹  MAYUMI ODA¹  NAKA HATTORI¹  TOMOO NUKADA²  SATOSHI TANAKA¹  TOMOYA OGAWA¹ 

[1] Laboratory of Cellular Biochemistry, Department of Veterinary Medical Sciences/Animal Resource Sciences, University of Tokyo;Laboratory of Synthetic Cellular Chemistry, The Institute of Physical and Chemical Center (RIKEN)

关键词: PL-Im;    N-linked oligosaccharide;    JAK2;    Nb2 lymphoma cell;   

DOI  :  10.1507/endocrj.45.659

学科分类：内分泌与代谢学

来源: Japan Endocrine Society

【 摘 要 】

References(76)Cited-By(2)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 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.

【 授权许可】

Unknown