Al-bukhari, Talat Abdullah
(2001)
Investigation of the epitope specificities of antibodies to islet β-cell autoantigens.
PhD thesis, University of Nottingham.
Abstract
Glutamic acid decarboxylase-65 (GAD-65) and the tyrosine phosphatase-like protein IA-2 are major targets of autoimmunity in type I diabetes mellitus (type I DM), stiff-man syndrome (SMS) and autoimmune polyendocrine syndrome (APS). In this study, the precise epitopes in GAD-65 of three different mouse monoclonal antibodies (MoAbs) (N-terminal MoAb within amino acid residues 4-17. C-terminal MoAb within amino acid residues 572-585 and GAD-6). human monoclonal antibody (b96.11 huAb) and polyclonal antibodies (SMS patients' sera) were investigated. The precise epitopes in IA-2 of two different MoAbs (76B and 76F) were also investigated. These precise epitope investigations were performed using two different phage-displayed random peptide libraries with different characteristics (T7 phage library gene X C9C and linear 9-mers, and M13 filamentous phage library gene III C7C and linear 12-mers and gene VIII 5C4C4).
Sequencing of N-terminal and C-terminal MoAb reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membranes and in capture ELISA revealed that the most significant motif recognised was P-G-x-x-x-W-S-F and F-L-I-x-E-I/V/L-D-x-L respectively which showed conservative substitutions and may correspond to the position 4-10 amino acids (aa) of GAD-65 (P-G-S-G-F-W-S-F) and to the position 573-581 aa of GAD-65 (F-L-I-E-E-I-E-R-L), respectively. To further define the N-terminal MoAb epitope, sequencing of N-terminal MoAbs reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage Iibrary and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA. revealed a motif of S-T-P which does not correspond to 4-17 aa of GAD-65 and does not overlap with the previous motif of the N-terminal MoAb. i.c. P-G-S-G-F-W-S-F (4-10 aa) of GAD-65. Therefore, the M13 pVIlI 5C4C4 worked with N-terminal MoAb by expressing a relevant sequence for the N-terminal MoAb but which is unlike its epitope in GAD-65. To further define the N-terminal MoAb epitope sequencing of N-terminal MoAb reactive peptides, which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of P-X-X-G which may correspond to 4-7 aa of GAD-65 (P-G-S-G) which overlaps with the previous motif P-G-S-G-F-W-S-F (4-10aa).
Sequencing of GAD-6 MoAb reactive peptides which were obtained from the successful biopanning using T7 gene X C9C phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed two different motifs of R/K-L/ A/I-x-K and M-x-x-A which showed conservative substitutions and may correspond to the position 525-52X and of GAD-65 (R-L-S-K) and to the position 523-526 aa of GAD-65 (M-S-R-L) respectively, which overlap with each other. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using T7 gene X linear 9-mers phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed a motif of R-x-x-K, which may correspond to 525-528 aa of GAD-65 (R-L-S-K) and overlaps with the previous motif of the GAD-6 selected from T7 gene X C9C phage library. To further define the GAD-6 epitope sequencing of GAD-6 reactive peptides which were obtained from the successful biopanning using M13 gene VIII 5C4C4 phage library and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in capture ELISA revealed a motif or M-x-x-A which may correspond to 523-526 aa of GAD-65 (M-S-R-L), and overlaps with the previous motif selected from T7 gene X C9C phage library. Thus, the overall motif or GAD-6 may correspond to 523-528 aa of GAD-65 (M-S-R-L-S-K). To further define the GAD-6 epitope, sequencing or GAD-6 reactive peptides which were obtained from the successful biopanning using MI3 gene III C7C and linear 12-mers phage libraries, did not show a clear motif and did not show reactivity with GAD-6 by capture ELISA. A possible explanation for this is that the peptides which are specific to GAD-6 are not present in these M13 pIII phage libraries.
Sequencing of b96.11 huAb reactive peptides, which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of IV-T/S-A/G/L-T/S-A\L and S-T/S-G/A/L/I which showed conservative substitutions and may correspond to the position 332-336 aa of GAD-65 (V-S-A-T-A) and to the position 338-340 aa of GAD-65 (T-T-V). respectively. Thus the overall motif of b96.11 might correspond to 332-340 aa of GAD-65.
In a capture ELISA system for the detection of GAD-65 specific antibodies, b78 huAb bound slightly better with GAD-6 (rather than N-terminal MoAb ) as the capture MoAb but b96.11 bound much better with GAD-6 as the capture MoAb. This might suggest that GAD-6 does interfere with b78 huAb binding to GAD-65 more than it does with b96.11 huAb. However it must also suggest that the GAD-6 and b78 huAb epitopes are not directly overlapping.
Sequencing of SMS sera reactive peptides which were obtained from the successful biopanning using M13 gene III linear 12-mers phage library and were selected by moderate affinity binding in immnuo-blotting assay using nitro-cellulose membrane revealed two different motifs of L/A-A-x-T/S-R/H/K and of T/S-T-V-I/L-F-E-L/G/I/V/A-H/K-L/G-x-K/R which showed conservative substitutions and may correspond to the position 371-375 aa of GAD-65 (L-L-M-S-R) and to the position 463-472 aa of GAD-65 (T-T-G-F-E-A-H-V-D-K). respectively. as public epitopes of SMS patients' sera.
Sequencing of 76B and 76F MoAbs reactive peptides, which were obtained from the successful biopanning using T7 gene X C9C and M 13 gene III linear 12-mers phage libraries and were selected by high affinity binding in immnuo-blotting assay using nitro-cellulose membrane and in ELISA, revealed that the most significant motif recognised was D-x-K-P-L-S and F-x-Y-Q, respectively which may correspond to the position 477-482 aa of IA-2 (D-Q-K-P-L-S) and to the position 626-629 aa of IA-2 (F-E-Y-Q) respectively.
The studies described in this thesis have shown that the epitope mapping of different antibodies on GAD-65 and IA-2 may help to understand the relationship between antigenicity and structure in these autoantigens which are targets in type I DM and related disorders (e.g. SMS and APS).
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