Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity

Jianya Huan, Roberto Meza-Romero, Jeffery L. Mooney, Yuan K. Chou, David M. Edwards, Cathleen Rich, Jason Link, Arthur Vandenbark, Dennis Bourdette, Hans Peter Bächinger, Gregory G. Burrows

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the 'empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure.

Original languageEnglish (US)
Pages (from-to)2-12
Number of pages11
JournalJournal of Chemical Technology and Biotechnology
Volume80
Issue number1
DOIs
StatePublished - Jan 2005

Fingerprint

T-cells
T-Cell Antigen Receptor
Bioactivity
peptide
Peptides
ligand
mutation
Monomers
Ligands
Mutation
HLA-DR alpha-Chains
aspartic acid
Mutagenesis
Molecules
Autoimmune Experimental Encephalomyelitis
secondary structure
Size exclusion chromatography
Cell proliferation
Dichroism
light scattering

Keywords

  • Autoimmunity
  • Drug design
  • Major histocompatibility complex
  • Protein structure

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)
  • Biotechnology
  • Bioengineering

Cite this

Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity. / Huan, Jianya; Meza-Romero, Roberto; Mooney, Jeffery L.; Chou, Yuan K.; Edwards, David M.; Rich, Cathleen; Link, Jason; Vandenbark, Arthur; Bourdette, Dennis; Bächinger, Hans Peter; Burrows, Gregory G.

In: Journal of Chemical Technology and Biotechnology, Vol. 80, No. 1, 01.2005, p. 2-12.

Research output: Contribution to journalArticle

Huan, Jianya ; Meza-Romero, Roberto ; Mooney, Jeffery L. ; Chou, Yuan K. ; Edwards, David M. ; Rich, Cathleen ; Link, Jason ; Vandenbark, Arthur ; Bourdette, Dennis ; Bächinger, Hans Peter ; Burrows, Gregory G. / Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity. In: Journal of Chemical Technology and Biotechnology. 2005 ; Vol. 80, No. 1. pp. 2-12.
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