Evaluation of the role of N-linked oligosaccharides in rat placental lactogen action by site-directed mutagenesis

Naka Hattori, Tomoo Nukada, Mayumi Oda, Satoshi Tanaka, Tomoya Ogawa, Kunio Shiota

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish
Pages (from-to)659-674
Number of pages16
JournalEndocrine Journal
Volume45
Issue number5
Publication statusPublished - 1998 Oct
Externally publishedYes

Fingerprint

Placental Lactogen
Site-Directed Mutagenesis
Oligosaccharides
Prolactin Receptors
Glycosylation
Phosphorylation
Sialic Acids
Tunicamycin
Radioligand Assay
COS Cells
Neuraminidase
Biological Assay
Western Blotting
Hormones
Growth

Keywords

  • JAK2
  • N-linked oligosaccharide
  • Nb2 lymphoma cell
  • PL-Im

ASJC Scopus subject areas

  • Endocrinology

Cite this

Hattori, N., Nukada, T., Oda, M., Tanaka, S., Ogawa, T., & Shiota, K. (1998). Evaluation of the role of N-linked oligosaccharides in rat placental lactogen action by site-directed mutagenesis. Endocrine Journal, 45(5), 659-674.

Evaluation of the role of N-linked oligosaccharides in rat placental lactogen action by site-directed mutagenesis. / Hattori, Naka; Nukada, Tomoo; Oda, Mayumi; Tanaka, Satoshi; Ogawa, Tomoya; Shiota, Kunio.

In: Endocrine Journal, Vol. 45, No. 5, 10.1998, p. 659-674.

Research output: Contribution to journalArticle

Hattori, N, Nukada, T, Oda, M, Tanaka, S, Ogawa, T & Shiota, K 1998, 'Evaluation of the role of N-linked oligosaccharides in rat placental lactogen action by site-directed mutagenesis', Endocrine Journal, vol. 45, no. 5, pp. 659-674.
Hattori, Naka ; Nukada, Tomoo ; Oda, Mayumi ; Tanaka, Satoshi ; Ogawa, Tomoya ; Shiota, Kunio. / Evaluation of the role of N-linked oligosaccharides in rat placental lactogen action by site-directed mutagenesis. In: Endocrine Journal. 1998 ; Vol. 45, No. 5. pp. 659-674.
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