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O-GlcNAc modification of proteins affects volume regulation in Jurkat cells

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Abstract

An increasing amount of recent research has demonstrated that the hexosamine biosynthesis pathway (HBP) plays a significant role in the modulation of intracellular signaling transduction pathways, and affects cellular processes via modification of protein by O-linked β-N-acetylglucosamine (O-GlcNAc). Besides the many known and postulated effects of protein O-GlcNAc modifications, there is little available data on the role of O-GlcNAc in cellular volume regulation. Our objective was to test the effect of increased O-GlcNAc levels on hypotonia-induced volume changes in Jurkat cells. We pretreated Jurkat cells for 1 h with glucosamine (GlcN), PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino-N-phenylcarbamate) an inhibitor of O-GlcNAcase, or a high level of glucose to induce elevated levels of O-GlcNAc. We found that the response of Jurkat cells to hypotonic stress was significantly altered. The hypotonia induced cell-swelling was augmented in both GlcN and PUGNAc-treated cells and, to a lesser extent, in high glucose concentration-treated cells. Evaluated by NMR measurements, GlcN and PUGNAc treatment also significantly reduced intracellular water diffusion. Taken together, increased cell swelling and reduced water diffusion caused by elevated O-GlcNAc show notable analogy to the regulatory volume changes seen by magnetic resonance methods in nervous and other tissues in different pathological states. In conclusion, we demonstrate for the first time that protein O-GlcNAc could modulate cell volume regulation.

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Abbreviations

ADC:

Apparent diffusion coefficient

DPH:

1,6-Diphenyl-1,3,5-hexatriene

EC:

Extracellular compartment

EGTA:

Ethylene glycol tetraacetic acid

ER:

Endoplasmic reticulum

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

Glc:

Glucose

GlcN:

Glucosamine

GlcNAc:

N-Acetyl-glucosamine

HBP:

Hexosamine biosynthesis pathway

Hanks’ BSS:

Hanks’ buffered salt solution

IC:

Intracellular compartment

NMR:

Nuclear magnetic resonance

O-GlcNAc:

O-Linked-N-acetylglucosamine

O-GlcNAcase:

O-GlcNAc hexosaminidase (EC 3.2.1.52)

OGT:

UDP-GlcNAc-polypeptide O-β-N-acetylglucosaminyltransferase (EC2.4.1.94)

PBS:

Phosphate buffered saline

PI:

Propidium Iodide

PUGNAc:

O-(2-Acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate

PVDF:

Poly(vinylidene fluoride)

SDS–PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

UDP-GlcNAc:

UDP-N-acetylglucosamine

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Acknowledgments

We thank Norbert Fülöp for insightful input and Sára Jeges for the support in statistical analysis. We additionally thank Zoltán Berente and the Department of Biochemistry at the University of Pécs for providing the NMR spectrometer and technical expertise with regard to NMR measurements. This work was supported by Hungarian Fund (OTKA 73591 and OTKA 78480) and by the Hungarian Research Council (ETT 401/2006).

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Authors and Affiliations

  1. Department of Laboratory Medicine, Faculty of Medicine, University of Pécs, Ifjúság str. 13, 7624, Pecs, Hungary

    Tamás Nagy, Alfréd Balasa, Dorottya Frank, Orsolya Rideg, Tamás Magyarlaki, Gábor L. Kovács & Attila Miseta

  2. Department of Dentistry, Oral and Maxillofacial Surgery, Faculty of Medicine, University of Pécs, Pecs, Hungary

    Dorottya Frank

  3. Central Laboratory, Kaposi Mór Teaching Hospital, Kaposvár, Hungary

    András Rab

  4. Departments of Medical Informatics and Radiology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

    Gyula Kotek

  5. Department of Radiography, Faculty of Health Sciences, University of Pécs, Pecs, Hungary

    Péter Bogner

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  1. Tamás Nagy
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  2. Alfréd Balasa
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  10. Attila Miseta
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Correspondence to Attila Miseta.

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Nagy, T., Balasa, A., Frank, D. et al. O-GlcNAc modification of proteins affects volume regulation in Jurkat cells. Eur Biophys J 39, 1207–1217 (2010). https://doi.org/10.1007/s00249-009-0573-3

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  • DOI: https://doi.org/10.1007/s00249-009-0573-3

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