Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary

Kim, Dae Jung; Park, Chaewon; Byambaragchaa, Munkhzaya; Kim, Shin‐Kwon; Lee, Bae-Ik; Hwang, Hyung-Kyu; Myeong, Jeong‐In; Hong, Sungwon; Kang, Myung‐Hwa; Min, Kwan-Sik

doi:10.1016/j.dib.2016.05.069

Cited by 11 publications

(21 citation statements)

References 4 publications

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“…A homogeneous time-resolved fluorescence (HTRF) cAMP assay kit was purchased from Cisbio (Codolet, France). Monoclonal antibodies (5A11, 11A8, and 14F5) and rec-eelLH from CHO-K1 cells were produced in our lab, as previously reported [30]. Rec-eelLH produced using a baculovirus expression system was kindly donated by Dr. Sun-Me Hong (Institute of Gyeongbuk Marine Bio-Industry).…”

Section: Methods Materialsmentioning

confidence: 99%

“…Culture media were collected on day 7 after transfection; supernatants were collected and frozen at -80 °C. The concentration of rec-eelLH was analyzed using an enzyme-linked immunosorbent assay (ELISA), previously developed in our laboratory [30].…”

Section: Site-directed Mutagenesis Of Activation and Inactivation Sitesmentioning

confidence: 99%

“…Rec-eelLH was quantified using a sandwich ELISA performed in plates coated with the monoclonal antibody, 5A11, as described previously [30]. A volume of 100µL of rec-eelLH sample was added to the wells and then incubated for 1 h at room temperature (RT).…”

Section: Elisa Analysis Of Rec-eellh Proteinmentioning

confidence: 99%

“…Absorbance at 450 nm was measured in each well using a microplate reader (Cytation 3). cAMP analysis by homogeneous time-resolved fluorescence (HTRF) cAMP accumulation in CHO-K1 cells expressing eelLHR-WT and eelLHR mutants was measured using cAMP Dynamic 2 assay kits (Cisbio Bioassays, Codolet, France), as described previously [30]. Briefly cells transfected with eelLHR-WT and eelLHR mutants were added at 10,000 cells per well into a 384well plate 48 h after transfection.…”

Section: Elisa Analysis Of Rec-eellh Proteinmentioning

confidence: 99%

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Characterization of constitutive activation and inactivation mutants in conserved regions of the eel luteinizing hormone receptor

Byambaragchaa¹,

Kim²,

Kim³

et al. 2019

Preprint

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Background: We analyzed signal transduction of three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel luteinizing hormone receptor (LHR), known to be naturally occurring in humans as LHR. The objective of the present study is to assess the functional effects of these mutations in signal transduction. Methods: Site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation, stimulated by recombinant eelLH (rec-eelLH), was measured by homogeneous time-resolved fluorescence (HTRF) assays. Results: The cAMP response in cells expressing the wild-type eel LHR (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in the basal cAMP response, respectively. However, their maximal responses to agonist were approximately 73, 53, and 92%, respectively, of the maximal response of the LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP concentration in the absence of ligand. The inactivating mutations did not completely impair signal transduction. The maximal responses of the inactivating mutants, D383N and Y546F, were 32% and 24% of the LHR-WT, respectively. Conclusion: We report here the first characterization of activating and inactivating mutations in eelLHR. These results provide important data on the signal transduction of constitutively active and inactive eelLHR mutants. Keywords: eel LHR, constitutively activating/inactivating mutation

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Section: Methods Materialsmentioning

confidence: 99%

Section: Site-directed Mutagenesis Of Activation and Inactivation Sitesmentioning

confidence: 99%

Section: Elisa Analysis Of Rec-eellh Proteinmentioning

confidence: 99%

Section: Elisa Analysis Of Rec-eellh Proteinmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of constitutive activation and inactivation mutants in conserved regions of the eel luteinizing hormone receptor

Byambaragchaa¹,

Kim²,

Kim³

et al. 2019

Preprint

Self Cite

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show abstract

“…Rec-eelLH was quantified using a double-sandwich ELISA performed in plates coated with the monoclonal antibody, 5A11, which is directed against the -subunit of eelLH as described previously [30]. The standard of rec-eelLH was produced from E. coli and purified by performing Ni-NTA Sepharose column chromatography with 250 mM imidazole.…”

Section: Elisa Analysis Of Rec-eellh Proteinmentioning

confidence: 99%

Mutations in Conserved Regions of Eel Luteinizing Hormone Receptor Result in Constitutive Activation or Inactivation

Byambaragchaa¹,

Kim²,

Kim³

et al. 2019

Preprint

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Luteinizing hormone receptor (LHR) is a member of the seven-transmembrane (TM) receptor family. Several mutations in LHR have been identified in many mammalian species, leading to either constitutive activation or inactivation of the receptor. Mutations in highly conserved regions of the TM domain have been reported. In this study, we analyzed signal transduction by three constitutively active mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of eelLHR known as naturally occurring in human LHR . To directly assess the functional effects of these mutations, site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation stimulated by recombinant eelLH (rec-eelLH) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The cAMP response in cells expressing eelLHR wild-type (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response, respectively. However, their maximal responses were approximately 73, 53, and 92%, respectively, of the maximal response of LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP production in the absence of agonist. The maximal responses of the inactivating mutants, D383N and Y546F, were 32 and 24% of LHR-WT, respectively. However, the inactivating mutations did not completely impair signal transduction. Thus, we report here the first characterization of activating and inactivating mutations in eelLHR and we show that these mutations have similar effects as those reported for mammalian LHRs. Moreover, eelLHR with activating mutations showed constitutive cAMP responses. These results provide important data on the signal transduction of constitutively active and inactive LHR mutants. Further studies should aim to identify the mechanism responsible for the significant increase in basal cAMP response in the constitutively activated eelLHR mutants.

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Heterologous Production and Glycosylation of Japanese Eel Follitropin Using Silkworm

Hong

Choi

et al. 2019

Biotechnol Bioproc E

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Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary

Cited by 11 publications

References 4 publications

Characterization of constitutive activation and inactivation mutants in conserved regions of the eel luteinizing hormone receptor

Characterization of constitutive activation and inactivation mutants in conserved regions of the eel luteinizing hormone receptor

Mutations in Conserved Regions of Eel Luteinizing Hormone Receptor Result in Constitutive Activation or Inactivation

Heterologous Production and Glycosylation of Japanese Eel Follitropin Using Silkworm

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