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1.
Bioprocess Biosyst Eng ; 43(5): 863-875, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-31980903

RESUMEN

O-Glycosylation occurs in recombinant proteins produced by CHO cells, but this phenomenon has not been studied extensively. Here, we report that rituximab is an O-linked N-acetyl-glucosaminylated (O-GlcNAcylated) protein and the production of rituximab is increased by thiamet G, an inhibitor of O-GlcNAcase. The production of rituximab doubled with OGA inhibition and decreased with O-GlcNAc transferase inhibition. O-GlcNAc-specific antibody and metabolic labelling with azidO-GlcNAc confirmed the increased O-GlcNAcylation with thiamet G. Protein mass analysis revealed that serine 7, 12, and 14 of the rituximab light chain were O-GlcNAcylated. S12A mutation of the light chain decreased rituximab stability and failed to increase the production with thiamet G without any significant changes of mRNA level. Cytotoxicity and thermal stability assays confirmed that there were no differences in the biological and physical properties of rituximab produced by thiamet G treatment. Therefore, thiamet G treatment improves the production of rituximab without significantly altering its function.


Asunto(s)
Mutación Missense , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Piranos/farmacología , Rituximab , Tiazoles/farmacología , Sustitución de Aminoácidos , Animales , Células CHO , Cricetulus , Glicosilación/efectos de los fármacos , Humanos , N-Acetilglucosaminiltransferasas/metabolismo , Rituximab/biosíntesis , Rituximab/genética
2.
Mol Cells ; 44(4): 223-232, 2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33935043

RESUMEN

Uniquely expressed in the colon, MS4A12 exhibits store-operated Ca2+ entry (SOCE) activity. However, compared to MS4A1 (CD20), a Ca2+ channel and ideal target for successful leukaemia immunotherapy, MS4A12 has rarely been studied. In this study, we investigated the involvement of MS4A12 in Ca2+ influx and expression changes in MS4A12 in human colonic malignancy. Fluorescence of GCaMP-fused MS4A12 (GCaMP-M12) was evaluated to analyse MS4A12 activity in Ca2+ influx. Plasma membrane expression of GCaMP-M12 was achieved by homo- or hetero-complex formation with no-tagged MS4A12 (nt-M12) or Orai1, respectively. GCaMP-M12 fluorescence in plasma membrane increased only after thapsigargin-induced depletion of endoplasmic reticulum Ca2+ stores, and this fluorescence was inhibited by typical SOCE inhibitors and siRNA for Orai1. Furthermore, GCaMP-MS4A12 and Orai1 co-transfection elicited greater plasma membrane fluorescence than GCaMP-M12 co-transfected with nt-M12. Interestingly, the fluorescence of GCaMP-M12 was decreased by STIM1 over-expression, while increased by siRNA for STIM1 in the presence of thapsigargin and extracellular Ca2+. Moreover, immunoprecipitation assay revealed that Orai1 co-expression decreased protein interactions between MS4A12 and STIM1. In human colon tissue, MS4A12 was expressed in the apical region of the colonic epithelium, although its expression was dramatically decreased in colon cancer tissues. In conclusion, we propose that MS4A12 contributes to SOCE through complex formation with Orai1, but does not cooperate with STIM1. Additionally, we discovered that MS4A12 is expressed in the apical membrane of the colonic epithelium and that its expression is decreased with cancer progression.


Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína ORAI1/metabolismo , Tapsigargina/metabolismo , Fluorescencia , Humanos , Transfección
3.
Cell Calcium ; 92: 102305, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33069962

RESUMEN

CaV1.2 and transient receptor potential canonical channel 3 (TRPC3) are two proteins known to have important roles in pathological cardiac hypertrophy; however, such roles still remain unclear. A better understanding of these roles is important for furthering the clinical understanding of heart failure. We previously reported that Trpc3-knockout (KO) mice are resistant to pathologic hypertrophy and that their CaV1.2 protein expression is reduced. In this study, we aimed to examine the relationship between these two proteins and characterize their role in neonatal cardiomyocytes. We measured CaV1.2 expression in the hearts of wild-type (WT) and Trpc3-/- mice, and examined the effects of Trpc3 knockdown and overexpression in the rat cell line H9c2. We also compared the hypertrophic responses of neonatal cardiomyocytes cultured from Trpc3-/- mice to a representative hypertrophy-causing drug, isoproterenol (ISO), and measured the activity of nuclear factor of activated T cells 3 (NFAT3) in neonatal cardiomyocytes (NCMCs). We inhibited the L-type current with nifedipine, and measured the intracellular calcium concentration using Fura-2 with 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ba2+ influx. When using the Trpc3-mediated Ca2+ influx, both intracellular calcium concentration and calcium influx were reduced in Trpc3-KO myocytes. Not only was the expression of CaV1.2 greatly reduced in Trpc3-KO cardiac lysate, but the size of the CaV1.2 currents in NCMCs was also greatly reduced. When NCMCs were treated with Trpc3 siRNA, it was confirmed that the expression of CaV1.2 and the intracellular nuclear transfer activity of NFAT decreased. In H9c2 cells, the ISO activated- and verapamil inhibited- Ca2+ influxes were dramatically attenuated by Trpc3 siRNA treatment. In addition, it was confirmed that both the expression of CaV1.2 and the size of H9c2 cells were regulated according to the expression and activation level of TRPC3. We found that after stimulation with ISO, cell hypertrophy occurred in WT myocytes, while the increase in size of Trpc3-KO myocytes was greatly reduced. These results suggest that not only the cell hypertrophy process in neonatal cardiac myocytes and H9c2 cells were regulated according to the expression level of CaV1.2, but also that the expression level of CaV1.2 was regulated by TRPC3 through the activation of NFAT.


Asunto(s)
Canales de Calcio Tipo L/metabolismo , Cardiomegalia/metabolismo , Miocitos Cardíacos/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Isoproterenol , Ratones Noqueados , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Canales Catiónicos TRPC/deficiencia
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