Design, engineering and production of functional single-chain T cell receptor ligands.
Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen presenting cells (APCs) that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. The peptide binding/T cell recognition domains of rat MHC class II (alpha-1 and beta-1 domains) were expressed as a single exon for structural and functional characterization. These recombinant single-chain T cell receptor ligands (termed 'beta1alpha1' molecules) of approximately 200 amino acid residues were designed using the structural backbone of MHC class II molecules as template, and have been produced in Escherichia coli with and without N-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native MHC class II heterodimer. The proteins exhibited a cooperative two-state thermal folding-unfolding transition. Beta1alpha1 molecules with a covalently linked MBP-72-89 peptide showed increased stability to thermal unfolding relative to the empty beta1alpha1 molecules. This new class of small soluble polypeptide provides a template for designing and refining human homologues useful in detecting and regulating pathogenic T cells.
Burrows
G G
GG
Department of Neurology, Department of Biochemistry and Molecular Biology and Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201, USA.
Chang
J W
JW
Bächinger
H P
HP
Bourdette
D N
DN
Offner
H
H
Vandenbark
A A
AA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D015202:Protein Engineering; D051381:Rats; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000949:Histocompatibility Antigens Class II; D008024:Ligands; D011948:Receptors, Antigen, T-Cell; D004247:DNA