Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Abstract

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type-1 diabetes (T1D) and stiff-person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ-aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme-linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N-terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14-23 and 91-99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side-chain functionality, rather than amino acid side-chain specificity. Additionally, results suggested that non-contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide-based therapeutics against SPS.

Peptides, Antibodies, Peptide Antibodies and More

“…This technique is still the major method for MAb production, as described by Köhler and Milstein, even though minor modifications may be introduced, mainly in the screening phase, where an assay system identical to the end stage use should be used to avoid selection of clones with aberrant properties [94]. Upon successful screening and cloning, highly specific MAbs are obtained and the epitopes of these can be characterised with high precision, as illustrated by recent studies of MAbs to the glutamate decarboxylase isoforms 65 and 67, which were shown to recognise linear epitopes located in flexible structures [47,95]. Similar results have been obtained with many other MAbs, which verifies the stringent requirements for an optimal antibody-antigen interaction [33,46,96,97].…”

“…Amino acid composition Hydrophilic aas, charged aas, Pro, Gly (represented in loops) [33,138] Peptide length 8-25 aa [46,130,[132][133][134] Peptide structure Linear, flexible, cyclic, loops, turns, helices [25,86,87,[93][94][95] Protein target Accessible epitope, areas of high conservation, areas of hypervariability, N/C-termini, post-translational modifications [128,129,138] Following peptide synthesis, the peptides are conjugated to carriers to ensure immune responses with high antibody titres, since many peptides are not immunogenic by themselves. Traditional carriers include bovine serum albumin, keyhole limpet hemocyanin, and ovalbumin [45,128,[139][140][141].…”

Fine Mapping of Glutamate Decarboxylase 65 Epitopes Reveals Dependency on Hydrophobic Amino Acids for Specific Interactions

“…The majority of epitopes are composed of 8–12 aas [1,2,3,4,5,6]. Except from antibodies recognizing modified epitopes, are epitopes seldom shorter than 6 aas, as antibody-antigen interactions depend on several aas for a stable interaction [7,8,9].…”

Enhanced GAD65 production in plants using the MagnICON transient expression system: Optimization of upstream production and downstream processing

“…Notably, when the GAD67/65 fusion protein was expressed in plants it was localized predominantly in the cytosol . A truncated version of GAD65mut would be preferable to a fusion protein for diagnostic and therapeutic applications to avoid cross‐reaction with autoantibodies against GAD67, which are not produced in diabetes but are relevant in other autoimmune diseases such as stiff‐person syndrome .…”

“…We previously described the cytosolic targeting of GAD65 in plants by replacing the N‐terminus of the protein with the homologous region of GAD67, the hydrophilic isoform of GAD . However, the N‐terminal region of GAD67 contains epitopes representing stiff‐person syndrome, another autoimmune disease , and this could interfere with the diagnosis and treatment of T1D. We therefore evaluated three N‐terminal deletions of GAD65mut as a strategy to prevent its association with membranes in plant cells.…”