Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide.
Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Lonnie
L
Lybarger
Y Y Lawrence
YY
Yu
Michael J
MJ
Miley
Daved H
DH
Fremont
Nancy
N
Myers
Tina
T
Primeau
Steven M
SM
Truscott
Janet M
JM
Connolly
Ted H
TH
Hansen
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D004527:Egg Proteins / Q000276:immunology / Q000378:metabolism; D006183:H-2 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016056:Immunodominant Epitopes / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010047:Ovalbumin / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004527:Egg Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D016056:Immunodominant Epitopes; C110366:OVA-8; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D010047:Ovalbumin