Novel applications of the complementary peptide molecular recognition approach

Cory Jianke Xian

Previous studies have demonstrated that the complementary peptide (CP) recognition approach can be a successful strategy in studying ligand and receptor interactions. A CP or antisense peptide can be designed in two ways: (1) decoding the complementary or antisense DNA of a ligand; (2) hydropathic anti-complementation through a computer programme from the amino acid sequence of the ligand (CG-CP). Previous work has shown (1) specific binding between a CP and its sense ligand; (2) recognition of the receptor for the ligand by an anti-CP antibody; (3) the idiotype-anti-idiotype relationship between the anti-ligand and anti-CP antibodies; and (4) Prediction and demonstration of previously unknown binding sites or interacting proteins. The work described in this thesis involves the use of the CP approach to novel applications. New Type of Affinity Column. Previous workers have experienced failure or extreme difficulties in various conventional attempts to isolate the 21.5 KD myelin basic protein (MBP) from its other isoforms. Several CPs were constructed according to different methods to 21.5 KD MBP exon II, which is not present in the other isoform of sheep MBP. One of the CPs, CG-CP, was shown to be capable of binding the exon II sense peptide with a high affinity in an ELISA binding assay. A CG-CP column was generated in attempts to isolate the 21.5 KD MBP. Results showed that the column could significantly enrich this isoform from a crude MBP preparation, examined by SDS-PAGE and Western blotting analyses. But there was also some non-specific adsorption of MBP to column matrix, as observed by previous workers with conventional columns. Mimics of Antibody Combining Sites. Previous studies have reported that CPs can be used to generate anti-idiotypic antibodies (anti-ID) (ie., antibodies against an antibody). Several CPs were designed to the anti-human MBP monoclonal antibody (Mab 65) epitope on MBP. ELISA binding assays illustrated specific binding of soluble MBP to the pre-coated CPs (particularly the 3’-5’CP); on the other hand, the soluble CPs did not bind to the pre-coated MBP. An antibody to the 3’-5’CP was generated; but this antibody did not show any anti-ID relation to the Mab 65. Investigation on the ID-anti-ID relationship between the two antibodies raised with the 21.5 KD MBP exon II sense peptide and its 5’-3’CP was also carried out, but results confirmed the finding with the Mab 65 case. T Cell Receptors (TCRs) in Autoimmune Diseases. Experimental autoimmune encephalomyelitis (EAE) is the most widely studied experimental autoimmune disease. A CP (EAE CP) was designed to the EAE epitope (rat MBP 72-82) and it was found that this EAE CP had sequence similarities to TCR, MHC II and LEA molecules in a PIR protein database search. A rabbit antibody to EAE CP was generated and used as an indicator for the interaction between the CP and epitope peptides in an ELISA binding assay, which demonstrated specific and high-affinity binding of EAE CP to the epitope peptides. This anti-EAE CP was also shown to be able to stain rat peripheral blood lymphocytes in an in situ cellular ELISA although not to the EAE specific T cell line in a flow cytometry analysis. A rat TCR. Vp peptide that was used to prevent and treat EAE by others had sequence similarity to the EAE CP, thus the latter was also used in attempts to down-regulate EAE, with results showing that the EAE CP could marginally (but not significantly) delay the onset and reduce the severity of EAE. Sense-antisense complementarity analysis was conducted between interactive yd TCRs and the Adjuvant arthritogenic heat-shock peptides. Results showed that complementaritiescould be observed but tended to be non-specific. Based on the sequence homologies between the receptor complements and ligands, the CP approach was also used to in attempts to predict the antigen(s) possibly involved in multiple sclerosis (MS). CPs to γ TCR sequences from a MS patient and a control designed and used as query sequences to do protein database searches. Some viruses were matched, which had been suggested to be involved in MS by others; but no viruses were consistently matched to all of the MS γ TCR CP sequences. No myelin or other relevant brain proteins were matched. HIV-1 and Receptors. CPs were constructed to HIV-1 gp120 receptor binding region and gp41 fusion peptide, and were found to have sequence similarities respectively to CD4 (a known receptor for HIV) and LFA-1 (suggested to be a possible second receptor by others). Attempts were made to block HIV-1 infectivity with these CP-gp120 and CP-gp41, but with negative results. Specific antibodies to both CPs were also produced but were shown not to be able to stain the human peripheral blood lymphocytes in both in situ cellular ELIS As and How cytometry analyses. The anti-CPgpl20 did not cross-react to the recombinant human CD4. Cytokines and Receptors. CPs to potential receptor binding sites on GM-CSF, IL-3, and IL-5 were designed and were shown to have interesting sequence homologies to their receptor sequences by the BESTFIT sequence analyses. Results with two CPs of GM-CSF (20- 28 and 18-25) indicated that these CPs and an anti-CP-GMCSF18-25 antiserum could not inhibit the GM-CSF binding to GM-CSF high-affinity receptor and that an anti-CP-GMCSF20- 28 antiserum could not stain granulocytes and monocytes. In summary, sense-antisense sequence complementarity (or sequence homology between the sense receptor and the antisense ligand) was observed in the various systems studied. Sense-antisense peptide interaction was demonstrated in several cases examined including (1) EAE epitope peptides and EAE CP; (2) 21.5 KD MBP exon II peptide or whole 21.5 KD MBP and exon II CPs (particularly CG-CP); (3) MBP and the anti-MBP Mab 65 epitope CPs. The interaction between the exon II CG-CP and exon II peptide was successfully utilized to enrich the 21.5 KD MBP from the crude MBP preparation through a CG-CP affinity column. However, no success was achieved in recognizing the receptors for the ligands by using the anti-ligand CP antibodies in the various systems examined (HIV receptor, GMCSF receptor, 21.5 KD MBP interacting protein and EAE specific TCR or MHC II molecules), and in using CPs as biological antagonists (eg., Mab 65-MBP recognition inhibition, EAE prevention, HIV infection inhibition, and cytokine-receptor binding blocking), in contrast to previous workers’ success with peptide hormones. Furthermore, Whitaker’s success in generating anti-ID antibodies by MBP CPs could not be reproduced with two other regions on MBP (exon II and Mab 65 epitope in exon 6). In conclusion, therefore, specific sense-antisense peptide interaction of the CP approach (particularly the CG-CP approach) offers a promising potential for isolating or enriching proteins or peptides; however this CP approach is not universally applicable, especially in designing biological antagonists and generating anti-receptor or anti-ID antibodies with the protein/protein systems of complex structures such as HIV/receptors, cytokines/receptors, epitope/TCR, and MBP/antibodies, in contrast to some peptide hormone/receptor systems.

Novel applications of the complementary peptide molecular recognition approach

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