I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression

Abstract

The objectives of this work are to characterize the identity of I-domain-antigen conjugate (IDAC) and to evaluate the in vivo efficacy of IDAC in suppressing experimental autoimmune encephalomyelitis (EAE) in mouse model. The hypothesis is that the I-domain delivers PLP(139-151) peptides to antigen-presenting cells (APC) and alters the immune system by simultaneously binding to ICAM-1 and MHC-II, blocking immunological synapse formation. IDAC was synthesized by derivatizing the lysine residues with maleimide groups followed by conjugation with PLP-Cys-OH peptide. Conjugation with PLP peptide does not alter the secondary structure of the protein as determined by CD. IDAC suppresses the progression of EAE, while I-domain and GMB-I-domain could only delay the onset of EAE. As a positive control, Ac-PLP-BPI-NH(2)-2 can effectively suppress the progress of EAE. The number of conjugation sites and the sites of conjugations in IDAC were determined using tryptic digest followed by LC-MS analysis. In conclusion, conjugation of I-domain with an antigenic peptide (PLP) resulted in an active molecule to suppress EAE in vivo.

Figure 1

Schematic representation of a two-step conjugation reaction to prepare IDAC.

Figure 2

Purification and characterization of GMB-I-domain after the reaction of I-domain with GMBS. (A) SEC chromatogram showing the separation between GMB-I-domain and the remaining free GMBS. (B) CD spectra of the parent I-domain (red) and GMB-I-domain (black). (C) Deconvoluted mass spectra of LC ESI-MS analysis of the GMB-I-domain protein and the unmodified I-domain protein (bottom). G, is the number of GMBS molecules conjugated. (D) SDS-PAGE analysis of pure GMB-I-domain protein after staining with Coomassie blue: molecular weight marker (lane 1), the I-domain protein (lane 2), the reaction mixture of I-domain protein and GMBS (lane 3), and GMB-I-domain protein (lane 4).

Figure 3

Purification and characterization of IDAC by SEC, SDS-PAGE, MS, and CD. (A) The SEC chromatogram of IDAC, which is separated from the PLP-Cys peptide. (B) SDS-PAGE analysis of different proteins after staining with Coomassie blue: molecular weight marker (lane 1), the parent I-domain protein (lane 2), the reaction mixture at pH 8.5 to prepare IDAC (lane 3), and the purified IDAC (lane 4). (C) Charge deconvoluted mass spectra of the IDAC after LC desalting. PLP, is the number of PLP molecules conjugated. (D) CD spectra of the parent I-domain (red) and IDAC (black).

Figure 4

Schematic representation of possible hydrolysis products of GMB-I-domain and IDAC.

Figure 5

The X-ray structure of I-domain (PDB code: 1ZON). The residues in red and blue are the respective modified and unmodified lysine sites in IDAC and the residues in green are those located at the MIDAS region. The N- and C-termini are labeled as N- and C-, respectively. The protein images were created using the PyMOL molecular graphics system version 1.4.1 (A) Side view. (B) Top view.

Figure 6

In vivo activity of IDAC, Ac-PLP-BPI-NH₂-2, and PBS in mouse EAE model. After immunization with PLP peptide in CFA, the mice received i.v. injections of 26 nmol/injection/day of the conjugate IDAC on days 4 and 7. For the Ac-PLP-BPI-NH₂-2 treatment group, the mice received i.v. injections of 100 nmol/injection/day of the peptide on days 4, 7, and 10. The control group was treated with PBS on days 4, 7, and 10. Disease progression was evaluated using (A) clinical disease scores, (B) change in body weight, and (C) incidence of disease. The results are expressed as the mean ± S.E. (n ≥ 6).