Characterization of the allergenic potential of proteins: an assessment of the kiwifruit allergen actinidin.

Assessment of the potential allergenicity (IgE-inducing properties) of novel proteins is an important challenge in the overall safety assessment of foods. Resistance to digestion with pepsin is commonly measured to characterize allergenicity, although the association is not absolute. We have previously shown that specific IgE antibody production induced by systemic [intraperitoneal (i.p.)] exposure of BALB/c strain mice to a range of proteins correlates with allergenic potential for known allergens. The purpose of the present study was to explore further the utility of these approaches using the food allergen, actinidin. Recently, kiwifruit has become an important allergenic foodstuff, coincident with its increased consumption, particularly as a weaning food. The ability of the kiwifruit allergen actinidin to stimulate antibody responses has been compared with the reference allergen ovalbumin, and with the non-allergen bovine haemoglobin. Haemoglobin was rapidly digested by pepsin whereas actinidin was resistant unless subjected to prior chemical reduction (reflecting intracellular digestion conditions). Haemoglobin stimulated detectable IgG antibody production at relatively high doses (10%), but failed to provoke detectable IgE. In contrast, actinidin was both immunogenic and allergenic at relatively low doses (0.25% to 1%). Vigorous IgG and IgG1 antibody and high titre IgE antibody responses were recorded, similar to those provoked by ovalbumin. Thus, actinidin displays a marked ability to provoke IgE, consistent with allergenic potential. These data provide further encouragement that in tandem with analysis of pepsin stability, the induction of IgE after systemic exposure of BALB/c strain mice provides a useful approach for the prospective identification of protein allergens.

Relationship between protein digestibility and allergenicity: comparisons of pepsin and cathepsin.

An association between protein allergenicity and resistance to pepsin digestion in the gastrointestinal tract has been proposed. However, although widely accepted, such an association is inconsistent with known labile allergens and resistant nonallergens. Given the central role of antigen presenting cells, and in particular dendritic cells (DC), in the development of allergic responses, the stability of allergens to intracellular processing may be more relevant than resistance to extracellular pepsin digestion. We have characterised the expression by DC of cathepsins (proteolytic enzymes), and compared the proteolytic activity of the most highly expressed cathepsin with pepsin for a range of 9 allergens and 4 putative nonallergens. Cathepsin expression in bone marrow-derived DC (BM-DC) derived from BALB/c strain mice was characterised by flow cytometry; cathepsins D, E and S were identified, with cathepsin D being the most highly expressed. Digestion studies revealed that the majority of allergens (5/9) were pepsin resistant, whereas non-allergens (3/4) were labile. If the generation of pepsin-resistant fragments was considered as a feature of allergenicity, this increased to 7/9 allergens and 4/4 nonallergens. In contrast, most of the proteins examined were resistant to cathepsin digestion, with significant digestion recorded for only 2/9 allergens and 2/4 non-allergens. Chemical reduction (to mimic intracellular reducing conditions) increased the susceptibility of proteins to digestion by cathepsins, but did not improve discrimination between allergens and nonallergens on this basis. These data confirm that there is a general relationship between resistance to digestion with pepsin and allergenicity. The relationship is not absolute, but the information gained from this characteristic does provide useful information in a weight of evidence approach for allergenicity assessment. The most abundant cathepsin detected in antigen processing BM-DC, cathepsin D, is not an appropriate substitute for pepsin. The hypothesis that pepsin stability may be a surrogate for stability to digestion within DC may still hold true, but consideration of a single enzyme in this context is possibly an oversimplification.

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature.

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögren's syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log(10) odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.