Synthetic peptides as putative therapeutic agents in transplantation medicine: application of PEPSCAN to the identification of functional sequences in the extracellular domain of the interleukin-2 receptor beta chain (IL-2Rbeta).
A desired treatment strategy in transplantation medicine is the selective targeting of alloreactive T cells without impairing antileukemic and antiviral activities. One approach is the synthesis of peptides that interfere with the binding of interleukin-2 (IL-2) to its high affinity receptor (IL-2R). This blocks the activation and proliferation of the antigen-activated T cells and the secretion of IL-2. The latter binds to its receptor, via the extracellular domain of the IL-2Rbeta chain, while its cytoplasmic domain is required for intracellular signal transduction. In this study, the PEPSCAN method was applied in order to identify antigenic sequences (epitopes) in the extracellular domain of the IL-2Rbeta. Based on the primary amino acid (aa) sequence of the IL-2Rbeta, a total of 239 overlapping dodecapeptides, spanning the entire sequence of IL-2Rbeta, were synthesized by PEPSCAN and their immunoreactivity was tested by ELISA using monoclonal antibodies (mAbs) specific for IL-2Rbeta such as TU11, Mikbeta1, HuMikbeta1 and TU27. TU11 recognized a linear epitope located in the region 85R-Q(96). None of the 239 synthetic peptides was recognized by TU27. Mikbeta1 (and HuMikbeta1) recognized a discontinuous epitope formed by aa located in the IL-2Rbeta domains L(106) to P(148) and E(170) to A(202). Subsequently, synthetic peptides corresponding to the identified putative epitopic sequences were prepared by solid phase synthesis and their immunogenicity in vivo was assessed by raising polyclonal antibodies. Given that Mikbeta1 and HuMikbeta1 inhibit binding of IL-2 on the IL-2Rbeta, we addressed the question of whether the identified antigenic sequences serve as putative IL-2 binding domains. Synthetic peptides corresponding to these sequences were tested for their ability to compete with IL-2 for binding and, thereby, inhibit IL-2-induced proliferation of mitogen-stimulated human peripheral blood T cells. Sequences 107M-E(118) and 178Y-Q(199) probably represent functional IL-2 binding domains on IL-2Rbeta, since these synthetic peptides significantly inhibited the proliferation of activated T cells and secretion of IL-2.
Laboratory of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, School of Health Sciences, University of Patras, Greece.
H
H
Dionyssopoulou
A
A
Mouzaki
J
J
Slootstra
W
W
Puijk
R
R
Meloen
P
P
Cordopatis
G
G
Sotiropoulou
D000328:Adult; D001665:Binding Sites; D018604:Epitope Mapping / Q000379:methods*; D005544:Forecasting; D006801:Humans; D007166:Immunosuppressive Agents; D007376:Interleukin-2 / Q000378:metabolism*; D008213:Lymphocyte Activation; D008958:Models, Molecular; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D015375:Receptors, Interleukin-2 / Q000276:immunology* / Q000378:metabolism*; D012984:Software
D007166:Immunosuppressive Agents; D007376:Interleukin-2; D009842:Oligopeptides; D010446:Peptide Fragments; D015375:Receptors, Interleukin-2