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Monoclonal Antibodies to Human Thioredoxin Reductase
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Medical Nobel Institute for Biochemistry, Department of Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0001-5082-6423
1998 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 249, no 1, 86-89 p.Article in journal (Refereed) Published
Abstract [en]

The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH is an electron donor for ribonucleotide reductase but has also been implicated in other cellular events, including secretion, growth promotion, regulation of transcription factors, protection against oxidative stress, and apoptosis. Mammalian TrxR is a dimeric flavoprotein with 58 kDa subunits each with a catalytically active selenocysteine residue. To study the function and expression of TrxR, we have produced and characterized, for the first time, monoclonal antibodies against human TrxR. Native placenta TrxR was used for immunization of BALB/c mice, followed by hybridization, cloning, and establishment of hybridomas producing specific antibodies against human TrxR. Three clones of IgG1,κ subclass, termed anti-TrxR1, anti-TrxR2, and anti-TrxR3, were studied in detail. The isoelectric points (pIs) of the mAbs were 6.5, 6.0, and 6.5, respectively. The affinities (Ka) of the mAbs were 2 × 108M−1.Inhibition ELISA using biotin-labeled versus nonconjugated mAb IgG revealed that all three mAbs recognized one immunodominant epitope. Western blot analysis showed that the antibodies specifically bound to a 58 kDa protein, representing the subunit of TrxR. A Trx-dependent insulin reduction assay was used for analysis of enzymatic activity and the antibodies neutralized the reductase activity.

Place, publisher, year, edition, pages
1998. Vol. 249, no 1, 86-89 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79884DOI: 10.1006/bbrc.1998.9053OAI: oai:DiVA.org:liu-79884DiVA: diva2:544465
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Thioredoxin system in normal and transformed human cells
Open this publication in new window or tab >>Thioredoxin system in normal and transformed human cells
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Thioredoxin (Trx) and thioredoxin reductase (TrxR), together with NADPH, constitute the Trx system, a major antioxidant entity that helps maintain a reducing environment within living cells. Trx designates a family of proteins that are related on the basis of structure and function. Human full-length Trx is a 12 kDa redox-active protein that contains the evolutionarily conserved active site sequence Cys-Gly Pro-Cys. Truncated Trx is a shorter, 10 kDa form of human Trx that shows complete homology to the N-terminal 80 or 84 amino acids of 12 kDa Trx. Truncated Trx displays no reducing activity, even though it contains an intact active site. Human TrxR is a homodimeric, FAD-containing, selenoprotein that reduces oxidized Trx back to the enzymatically active form by consuming NADPH. Expression of human Trx and TrxR is induced by a variety of stressors. The Trx system functions directly and indirectly in important biological features such as DNA synthesis, gene expression, co-cytokine activity, chemokine properties, scavenging reactive oxygen intermediates, apoptosis, and growth regulation.

Aims: The overall objective of this research was to examine the regulation and biological role of the Trx system in normal and transformed cells, and a possible connection with tumorigenesis. The specific aims were as follows: i) to study the presence and function of full-length and truncated Trx in normal and transformed human cells; ii) to assess the expression and function of TrxR; iii) to investigate the functional importance of truncated Trx; iv) to examine the regulatory mechanisms behind secretion of Trx, truncated Trx, and TrxR; v) to study full-length secreted Trx in human plasma.

Methods: Using Köhler and Milstein hybridoma technology, monoclonal antibodies against full-length and truncated Trx, as well as TrxR, were developed and applied in various immunological methods to detect the proteins in and secreted from normal and transformed human cells.

Results: Using selective monoclonal antibodies distinct intracellular localization of full-length and truncated Trx was shown. The latter was present in lower amounts, primarily associated with the plasma membrane; only small amounts of the 12 kDa were present on the plasma membrane. Moreover, we found that human TrxR was secreted from normal and transformed cells via a Golgi-dependent pathway. Using sensitive EUSPOT, TrxR release was quantified at the single-cell level and found to be inducible. TrxR was detected in plasma from healthy blood donors, and this protein was overexpressed in transformed cells, compared to their normal counterparts. Truncated 10 kDa Trx was present in and secreted from normal and transformed cells. Treatment of normal peripheral blood monocytes with LPS, IL-1, IFN-γ, PMA, ionomycin, and the thiol oxidant diarnide induced secretion of Trx. Trx was found within platelets, and liberation of this Trx was induced by diamide, implying a novel mechanism for release of Trx.

Conclusions: Monoclonal antibodies were generated against full-length and truncated Trx as well as TrxR and proved to be useful tools for investigating these proteins. Full-length and truncated Trx were found to have different cellular functions, suggesting that C-temrinal truncation regulates the activity of Trx. Secretion of TrxR and the presence of thls enzyme in plasma imply that the Trx system has both intracellular and extracellular functions. The presence of Trx in platelets indicates participation of the protein in coagulation and possibly also in the effects of platelets that maintain the reducing capacity of human plasma.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 642
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25707 (URN)10083 (Local ID)91-7219-743-9 (ISBN)10083 (Archive number)10083 (OAI)
Public defence
2000-10-13, Berzeliussalen, Hälsouniversitetet, Linköping, 09:30 (Swedish)
Opponent
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Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-09-22Bibliographically approved

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Söderberg, AnitaRosén, Anders

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