In the present study, we describe a method to specifically isolate intracellular compartments containing endocytosed antigen. We have demonstrated that isolated compartments represent a small proportion of the intracellular material, highly enriched in antigen. Antigen-containing vesicles are specifically sorted from other intracellular compartments, such as endoplasmic reticulum or Golgi apparatus, and from the plasma membrane. They remain functional in vitro since they can be acidified, and the antigen inside has been found to be partially proteolysed. In macrophages, kinetic analysis has revealed that the antigen is first found in compartments of endosomal density, carrying Rab 5 and Rab 7, then in late compartments of lysosomal density, which are rich in proteases. The global protein content of the compartments was mapped by two-dimensional electrophoresis. In B lymphocytes, this method has allowed the isolation of endocytic compartments emerging from receptor-mediated endocytosis of the antigen. After 2 h of chase, the antigen reached vesicles containing large amounts of MHC-class II molecules, invariant chain and human leucocyte antigen-DM, where peptide loading can occur.
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February 1999
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Research Article|
February 08 1999
Purification of intracellular compartments involved in antigen processing: a new method based on magnetic sorting
Laure A. PERRIN-COCON;
Laure A. PERRIN-COCON
1Laboratoire d'Immunochimie (INSERM U238), DBMS/ICH, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France
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Patrice N. MARCHE;
Patrice N. MARCHE
1Laboratoire d'Immunochimie (INSERM U238), DBMS/ICH, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France
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Christian L. VILLIERS
Christian L. VILLIERS
1
1Laboratoire d'Immunochimie (INSERM U238), DBMS/ICH, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France
1To whom correspondence should be addressed (e-mail immuno@dsvgre.cea.fr).
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Publisher: Portland Press Ltd
Received:
August 17 1998
Revision Received:
November 03 1998
Accepted:
November 24 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 338 (1): 123–130.
Article history
Received:
August 17 1998
Revision Received:
November 03 1998
Accepted:
November 24 1998
Citation
Laure A. PERRIN-COCON, Patrice N. MARCHE, Christian L. VILLIERS; Purification of intracellular compartments involved in antigen processing: a new method based on magnetic sorting. Biochem J 15 February 1999; 338 (1): 123–130. doi: https://doi.org/10.1042/bj3380123
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