Calreticulin Is Expressed on the Cell Surface of Activated Human Peripheral Blood T Lymphocytes in Association with Major Histocompatibility Complex Class I Molecules*
- ‡Laboratory of Molecular Immunology, Institute for Molecular and Cell Biology, University of Porto, 4150 Porto and ‖Department of Hematology, Santo António General Hospital, 4050 Porto, Portugal
Abstract
Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of β2-free MHC class I molecules.
Footnotes
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↵* The American Portuguese Biomedical Research Fund supported this work.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Funded by a Ph.D. fellowship from Fundação para a Ciência e a Technologia (PRAXIS BD 3209/94). To whom correspondence should be addressed: Laboratory of Molecular Immunology, Institute for Molecular and Cell Biology, Rua do Campo Alegre, 823, 4150, Porto, Portugal. Tel.: 351-2-6074956; Fax: 351-2-6098480; E-mail:farosa{at}ibmc.up.pt.
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↵¶ Present address: Ludwig Cancer Research Institute, Norfolk Place, London, W2 1PG UK.
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↵** Funded by a post doctoral fellowship from Fundação para a Ciência e a Technologia (PRAXIS BPD 6010/95).
- Abbreviations:
- ER
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endoplasmic reticulum
- MHC I
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major histocompatibility complex class I
- β2m
-
β 2 microglobulin
- PBL
-
partially purified human peripheral blood T lymphocyte
- Ab
-
antibody
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
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- Received November 18, 1998.
- Revision received March 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
