Vasostatin‐1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice

Abstract

Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionChromogranin A (ChgA) is a prohormone in neuroendocrine tissues that is a target of pathogenic CD4 + T cells in NOD mice [1,2].This prohormone is an acidic secretory protein found in the granules of neuroendocrine cells, and can be cleaved to form functional proteins, including vasostatin-1 (VS-1, ChgA 1-76), vasostatin-2 (VS-2, ChgA 1-113), and WE-14 [3]. In pancreatic β-cells, VS-1 is a major fragment of ChgA that remains after proteolysis [4][5][6][7] and is the critical peptide for the formation of secretory granules that constitutively store insulin [8].Correspondence: Dr. Bhagirath Singh e-mail: bsingh@uwo.ca BDC2.5 T cells are the most extensively studied T cells of the diabetes-prone NOD mouse. Significantly important findings have been made with T-cell receptor transgenic BDC2.5 NOD mice generated using these T cells [9]. For further functional studies, it is important to evaluate the antigen specificity of these T cells. We have identified the region between residues 29 and 42 of the ChgA molecule to be the antigenic epitope for BDC2.5 T cells in NOD mice [1]. Since this peptide contains cysteine and methionine, oxidation of the −SH and −S-CH 3 side chains of these amino acids could have a significant impact on its antigenic activity [10,11]. Recently, another group [12] reported conflicting results about the antigenicity of ChgA 29-42 peptide and we suggest that it may be due to issues relating to the solubility of the peptide and/or potential oxidation of cysteine and methionine residues. The inhibitory dose of peptide (IC 50 ) for 50% reduction in binding of biotinylated HEL 10-23 to the I-A g7 is shown. Numbers in bold (1 to 9) indicate the binding register. Substitutions of amino acids in the sequence are shown in bold letters.In this study, we have done fine mapping of the VS-1 epitope ChgA 29-42 through amino acid deletion and alanine scanning of the I-A g7 binding register (KCVLEVISD). We characterized the presence of ChgA fragments in the pancreas of diabetes-prone NOD mice. Our findings also indicate the presence of elevated titers of antibody response against the VS-1 epitope ChgA 29-42 in diabetic NOD mice. ResultsChgA 34-42 is a binding register of the ChgA 29-42 epitope for I-A g7Binding of the peptide epitopes to the I-A g7 molecule occurs at P1, P4, P6, P7, and P9 MHC class II anchor positions while P2, P3, P5, and P8 positions act as T-cell receptor contact residues [13]. We generated a library of peptides for binding and T-cell stimulation studies ( Table 1). The binding capacity of the various peptides was determined as a percentage of inhibition of binding for biotinylated hen egg lysozyme (HEL) 10-23 (Bio-HEL) peptide to I-A g7 MHC class II in a competitive assay (Fig. 1B-D). The concentration of the competitor peptide required for 50% inhibition of binding of the labeled HEL peptide indicated as IC 50 (Table 1). The HEL 10-23 and IIP peptides were used as p...

N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes

“…Our functional and structural data presented here led us to conclude that WE14 is an essential part of the epitope for a diverse set of ChgA-specific CD4 + T cells in vivo. Another laboratory has reported that a different unrelated ChgA peptide is the natural epitope for transgenic BDC-2.5 T cells (16,17), but we were not able to reproduce this finding here with the BDC-2.5 hybridoma, perhaps due to sensitivity differences between hybridomas and the transgenic T cells. However, we show here that a set of T-cell hybridomas, including BDC-2.5, whose TCRs contain a variety of Vα, Jα, Vβ, and Jβ segments and CDR3 loops, all respond to the RLGL-WE14 peptide at low concentrations, to the WE14 peptide at high concentrations, and to pancreatic islets from WT ChgA mice, but not ChgA knockout mice.…”

“…3A). They also all responded to high concentrations of the weakly stimulatory natural WE14 peptide, but not to another ChgA peptide (amino acids 11-24, DTKVMKCVLEVISD) that has been suggested by others (16,17) to be the natural ChgA epitope for BDC-2.5 (Fig. 3B).…”

Detection of vasostatin-1-specific CD8+ T cells in non-obese diabetic mice that contribute to diabetes pathogenesis

“…Six-to 8-week-old NOD mice were immunized subcutaneously in the hind footpad under anaesthesia with or without peptide (50 lg) emulsified in 50 ll of incomplete Freund's adjuvant (IFA) [2,3]. The draining lymph nodes were harvested after 10 days and a single-cell suspension was prepared.…”

“…Vasostatin-1 and insulin are both present within the secretory granules of pancreatic islet b cells. We have reported previously that a peptide sequence of ChgA in vasostatin-1 fragment, namely ChgA 29-42, is an antigenic epitope recognized by pathogenic BDC2Á5 CD4 1 T cells in NOD mice [2,3]. Recently a hybrid molecule of insulin and ChgA sequences was reported to stimulate BDC2Á5 T cells [7].…”

“…1 T cells in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D) [1][2][3]. ChgA plays a critical role in T1D development, as NOD mice lacking ChgA are resistant to T1D [4].…”