Microsoft Word - IAA357BF

The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus. In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I. Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000. The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGX-RSVITXLME. It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic. The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds. Correspondence to: Dr. G.A. Stewart, The Western Australian Research Institute for Child Health, Princess Margaret Hospital, Thomas St., Subiaco, Perth, WA 6008 (Australia) Studies from our laboratory have shown two of the important allergens, designated Derp I and III from the house dust mite, Dermatophagoides pteronyssinus, are proteolytic enzymes [1,2]. These observations suggested other mite-derived enzymes, particularly those involved in mite digestion, might also be allergenic and particular attention was thus directed to a study of amylase. Amylase activity was assayed using a commercial amylase assay and an agarose-starch plate method [3]. Enzyme activity was easily detected in extracts of whole mite and spent growth medium (which was devoid of whole mites) but not in unused growth medium, suggesting it was present in faecal pellets [4]. Solubility data, which showed that the enzyme eluted from dry medium within 40 s after contact with an aqueous solvent, were consistent with this proposal [5]. Amylase was also found in 20 out of 20 extracts of dust obtained from mattresses (geometric mean 1.95; range 0.6–15.3 units/g fine dust) and in 18 out of 20 from lounge room carpets (geometric mean 1.83; range 0.6–4.4 units/g fine dust). Whilst its derivation from mites was not formally proved, it correlated with both mite counts (n = 40; r = 0.35, p < 0.05) and with Derp I concentrations (n = 40; r = 0.41, p < 0.01). Mite amylase was isolated from the growth medium by its capacity to bind to shellfish glycogen [4, 6]. Glycogen was added to medium dissolved in 40% (v/v) ethanol at O°C and the resulting complex, which precipitated under these conditions, was isolated. The glycogen was removed by utilizing the inherent hy-drolytic activity of the mite amylase present and the enzyme further purified by gel filtration and chroma-tofocusing. Mite amylase was shown to be similar to amylases from mammalian sources with regard to apparent molecular weight (56,000 by SDSD ow nl oa de d by : 54 .7 0. 40 .1 1 10 /3 0/ 20 17 1 1: 37 :2 8 P M PAGE) and the presence of free sulphydryl groups, as judged by its binding to an organomercurial affinity chromatog-raphy matrix. It was heterogeneous with regard to charge, with pi in the range 5–7. The N-terminal sequence of the enzyme was shown to be KYXNPHFIGXRSVITXLME and a search of the se358 Lake/Ward/Simpson/Thompson/Stewart Table 1. A summary of the biochemical and physicochemical properties of the major miteallergens ‘ Molecular weight data derived from cDNA studies [1,9],2 gel filtration and SDS-PAGE studies [5], and3 SDS-PAGE studies [4]. Enzyme activity data obtained fromrefs. 2,4, 5.4 Tentative assignmentquence data banks failed to reveal homology with any known protein [4].IgE immunoblot studies were performed using sera from mite-allergic individuals. These studiesshowed the enzyme was allergenic and, in addition, that its expression was dependent on theintegrity of intra-chain disulphide bonds [4]. IgE in sera from both mite-allergic children andadults recognized the enzyme with frequencies of 25 and 46% respectively. These data areconsistent with the concept that several major mite allergens are hydrolytic enzymes involved indigestion [2, 4, 5], and table 1 summarizes their characteristics to date. Amylases from fungaland mammalian sources [7, 8] have also been shown to be allergenic, raising the possibility thatbiochemical activity may be an important sensitizing attribute of such molecules.ReferencesChua, K.Y.; Stewart, G.A.; Thomas, W.R.; Simpson, R.J.; Dil-worth, R.J.; Plozza, T.M.:Sequence analysis of cDNA coding for a major house dust mite allergen, Derp I. Homology withcysteine proteases. J. Exp. Med. 767: 175–182(1988).Stewart, G.A.; Thompson, P.J.; Simpson, R.J.: Protease antigens from house dust mite. Lancet ii:154–155 (1989) and correction: Lancet ii: 462 (1990).Bowman, C.E.; Childs, M.: Polysaccharidases in astigmatid mites (Arthropoda: Acari). Comp.Biochem. Physiol. 72B: 551–557(1982).Lake, F.R.; Stewart, G.A.; Thompson, P.J.; Ward, L.D.; Simpson, R.J.: House dust mite derivedamylase: Allergenicity and physicochemical characterisation. J. Allergy Clin. Immunol., in press.Stewart, G.A.; Lake, F.R.; Thompson, P.J.: Faecally derived hydrolytic enzymes from the housedust mite: Physiochemical characterisation of potential allergens. Int. Arch. Allergy appl.Immun., submitted.Loyter, A.; Schramm, M.: The glycogen-amylase complex as a means of obtaining higly purifiedalpha-amylases. Biochim. Biophys. Acta 65: 200–206 (1962).Flindt, M.L.H.: Allergy to alpha-amylase and papain. Lancet i: 1407–1408(1979).Baur, X.; Fruhmann, G.; Haug, B.; Rasche, B.; Reiher, W.; Weiss, W.: Role of Aspergillusamylase in baker’s asthma. Lancet i: 43 (1986).Chua, K.Y.; Doyle, C.R.; Simpson, R.J.; Turner, K.J.; Stewart, G.A.; Thomas, W.R.: Isolation ofthe major allergen Derp II by IgE plaque immunoassay of a house dust mite cDNA library. Int.Arch. Allergy appl. Immun. 91: 118–123 (1990). Downloadedby: 54.70.40.11-10/30/201711:37:28PM