Diversity and Relative Levels of Actinidin, Kiwellin, and Thaumatin- Like Allergens in 15 Varieties of Kiwifruit (Actinidia) Ratnasiri Maddumage, † Niels J. Nieuwenhuizen, † Sean M. Bulley, † Janine M. Cooney, ‡ Sol A. Green, † and Ross G. Atkinson* ,† † The New Zealand Institute for Plant & Food Research Limited (PFR), Private Bag 92 169, Auckland, New Zealand ‡ PFR, Ruakura, Private Bag 3123, Hamilton 3240, New Zealand * S Supporting Information ABSTRACT: In the last 30 years the incidence of kiwifruit allergy has increased with the three major allergenic proteins being identified as actinidin, kiwellin, and thaumatin-like protein (TLP). We report wide variation in the levels of actinidin and TLP in 15 kiwifruit varieties from the four most widely cultivated Actinidia species. Acidic and basic isoforms of actinidin were identified in Actinidia deliciosa ‘Hayward’ and Actinidia arguta ‘Hortgem Tahi’, while only a basic isoform of actinidin was identified in Actinidia chinensis ‘Hort16A’. One isoform each of kiwellin and TLP were identified in ripe fruit. The cysteine protease activity of actinidin correlated with protein levels in all species except A. arguta. Protein modeling suggested that modifications to the S2 binding pocket influenced substrate specificity of the A. arguta enzyme. Our results indicate that care is necessary when extrapolating allergenicity results from single varieties to others within the same and between different Actinidia species. KEYWORDS: Actinidia, actinidin, allergen, kiwellin, kiwifruit, thaumatin ■ INTRODUCTION Food allergies affect as many as 8% of young children and 2% of adults in westernized countries, with allergy to any specific fruit generally being found to affect <1% of adults (reviewed in Zuidmeer et al. 1 ). Allergic reactions to kiwifruit (Actinidia spp. Lindl.) have been reported for over 30 years. 2 Clinical symptoms are typically localized to the oral mucosa; however, severe anaphylactic reactions are also observed (reviewed in Lucas et al. 3 ). As kiwifruit’s availability and consumption has increased since the 1970s, an increase in the incidence of kiwifruit allergy has been noted, presumably due to increased contact. 4 This increase is of clinical concern, as kiwifruit allergy has frequently been associated with allergy to other fruit and foods, 5,6 pollen, 7,8 and in latex-fruit syndrome. 9,10 A large number of studies have been conducted on protein extracts from Actinidia deliciosa fruit to identify the IgE-binding components. 7,11-19 A range of IgE-binding proteins have been identified and named by the Allergen Nomenclature Sub- committee of the International Union of Immunological Societies (http://www.allergen.org) including actinidin (Act d 1), a thaumatin-like protein (TLP, Act d 2), a 40 kDa protein (Act d 3), cystatin (Act d 4), kiwellin (Act d 5), pectin methylesterase inhibitor (Act d 6), pectin methylesterase (Act d 7), Bet v1-related proteins (Act d 8), profilin (Act d 9), lipid transfer protein (Act d 10), major latex protein (Act d 11), and chitinase (Act d chitinase). Of these proteins, actinidin, kiwellin, and TLP have received the most scientific attention. The importance of each allergen to allergic patients can vary significantly, for example, no specific IgE recognition was found for Act d 6 when testing 237 patients’ sera. 20 Act d 8 together with Act d 9 are markers for pollen-associated kiwifruit allergy, while Act d 1 is usually linked with kiwifruit monosensitiza- tion. 20 Actinidin is reported to be the major allergen in A. deliciosa fruit extracts in some 13,15,18,21 but not all human popula- tions. 5,12,17 It is recorded as an allergen in other Actinidia species, e.g., Actinidia chinensis (Act c 1), Actinidia arguta (Act a 1), and A. eriantha (Act e 1) in some databases (e.g., www. allergome.org). Actinidin is a member of the papain subfamily of cysteine proteases that includes papain and chymopapain from papaya (Carica papaya), ananain and bromelain from pineapple (Ananas comosus), and aleurain from barley (Hordeum vulgare). Recently, the enzyme’s cysteine protease (CP) activity has been reported to have a beneficial effect on human health by enhancing the gastric digestion of food proteins. 22-24 Actinidin is the most abundant protein in A. deliciosa ‘Hayward’ fruit, but it is not found in the fruit of some A. chinensis cultivars, e.g., gold-fleshed ‘Hort16A’. 25-27 The protein is synthesized as a zymogen and then processed to an active enzyme by removal an endoplasmic reticulum (ER) targeting signal peptide and N- and C-propeptides. The calculated molecular weight is ∼24 kDa; however, the protein has been reported to run on 1-D SDS PAGE from 24 to 30 kDa depending on conditions. In planta, the physiological function of the enzyme is largely unknown, although it has been implicated in pathogen defense 28 and processing of specific proteins, e.g., kiwellin 26 and a class IV chitinase. 29 Kiwellin is specifically recognized by IgE of some patients allergic to kiwifruit. 16,30-32 It is a cysteine-rich protein that has been shown to be a major protein component of the fruit of both A. deliciosa and A. chinensis. It has a calculated molecular Received: October 7, 2012 Revised: December 4, 2012 Accepted: January 4, 2013 Published: January 5, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 728 dx.doi.org/10.1021/jf304289f | J. Agric. Food Chem. 2013, 61, 728-739