FU Berlin Digitale Dissertation
Pamela Tan : Role of tapasin in antigen presentation by MHC class I molecules Die Rolle von Tapasin bei der Antigenpräsentation durch MHC-Klasse-I-Moleküle
|Abstract| |Table of Contents| |More Information|

Abstract Summary An infected cell is recognized by the immune system via pathogen-derived antigenic peptides. The surface presentation of these peptides is achieved by the assembly of a MHC class I-peptide complex in the endoplasmic reticulum (ER). In this PhD thesis tapasin is shown to positively influence peptide loading on MHC class I molecules in quantitative as well as in qualitative terms. Reconstituting the peptide loading defect in the B-lymphoblastoid cell line 721.220 by the expression of tapasin induces the plasma membrane expression of long-lived MHC class I molecules carrying an optimized spectrum of peptides. Tapasin facilitates the selection of peptides in the ER by recruiting the MHC class I molecule into the TAP loading complex. The assembly of two subcomplexes is promoted by tapasin: on one hand calreticulin is recruited to the MHC molecule, and on the other hand the thioreductase ER60 is recruited to TAP through tapasin. On the HLA alleles investigated (HLA-B*4402 anh HLA-B8), the bridging of both subcomplexes by tapasin leads to enhanced binding of optimal length peptides and to the improved formation of Bw4/Bw6 epitopes. Soluble tapasin constructs and mouse tapasin are not able to assemble the complete loading complex consisting of the MHC molecule, calreticulin, ER60 and TAP. In these cases a suboptimal peptide loading is observed, characterized by a reduced half-time of class I molecules on the cell surface and a lower thermostability in lysates. With the findings of this work, we can define functional domains of tapasin. It is shown, that the N-terminal domain of tapasin is essential for peptide loading onto HLA-B8 und HLA-B44 molecules. The deletion of only 19 amino acids at the N-terminus of tapasin abolishes surface expression of these alleles. The transmembrane region of tapasin interacts with TAP. In the presence of membrane-anchored human and murine tapasin the expression of TAP is enhanced by a factor of ten. By contrast, when the transmembrane region is missing, no enhancement of the steady-state levels of TAP were detectable. The importance of the membrane domain of tapasin has also become clear by the fact that a sole amino acid substitution (L410F) in the human protein sequence leads to 70-80% less TAP protein expression. This point mutation putatively destroys a leucin zipper motive which partially abolishes ER retention of tapasin.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title TITEL Die Rolle von Tapasin bei der Antigenpräsentation durch MHC-Klasse-I-Moleküle EINLEITUNG   Das Immunsystem 1 Der Haupthistokompatibitätskomplex (MHC) 3 Chaperone für MHC-Klasse-I-Moleküle 11 Antigenprozessierung und Antigenpräsentation durch MHC-Klasse-I-Moleküle 14 Die Bedeutung der Peptidpräsentation durch MHC-Klasse-I-Moleküle 22 Störungen der Antigenpräsentation durch MHC-Klasse-I-Moleküle 24     FRAGESTELLUNG UND ZIELSETZUNG 27     MATERIAL Geräte 28 Verbrauchsmaterialien 28 Chemikalien 29 DNA 31 Proteine 33 Kits 35 Puffer und Lösungen 36 Zelllinien 42 Mausstämme 42     METHODEN   Molekularbiologische Methoden 43 Proteinchemische Methoden 46 Zellbiologische Methoden 52     ERGEBNISSE   Generierung von 721.220.Tapasin-Transfektanten 57 Der Einfluß von Tapasin auf den Peptidtransport ins ER 60 Tapasin als Chaperon für HC/b2m-Dimere 64 Die Assemblierung des Peptidbeladungskomplexes 67 Der Einfluß von Tapasin auf zelloberflächenexprimierte MHC-Klasse-I-Moleküle 74 Der Einfluß von Tapasin auf die Peptidbeladung von MHC-Klasse-I-Molekülen 79     DISKUSSION 90     ANHANG   Literaturverzeichnis 108 Abkürzungsverzeichnis 121 Danksagungen 122

More Information:
Online available:	http://www.diss.fu-berlin.de/2001/231/indexe.html
Language of PhDThesis:	german
Keywords:	antigen presentation, MHC class I molecule, ER chaperone, peptide loading, peptide editor
DNB-Sachgruppe:	30 Chemie
Date of disputation:	29-Oct-2001
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	PD Dr. Frank Momburg
Second Referee:	Prof. Ralf Erdmann
Contact (Author):	pamela.tan@biomax.de
Contact (Advisor):	f.momburg@dkfz.de
Date created:	28-Nov-2001
Date available:	02-Jan-2002

 

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