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Background: Dust mites have been shown to contain a serine protease distinct from the previously reported trypsin and chymotrypsin. The latter enzymes have been shown to be allergens, but the allergenic importance of the former is unknown., Objective: This study was performed to isolate and characterize the novel mite serine protease and determine its allergenicity., Methods: The mite serine protease was isolated from feces-enriched extracts of Dermatophagoides pteronyssinus by ion-exchange chromatography and affinity chromatography, and its physicochemical properties were determined. The allergenicity of the protease was assessed by using the RAST., Results: The protease was enzymatically similar to chymotrypsin and cathepsin G-like enzymes from a variety of sources and was shown to cleave collagen. It had a molecular mass of 23,780 d. N-terminal sequence analysis (18 residues) indicated homology with the mite tryptic allergen, Der p 3, and the chymotryptic allergen, Der p 6. RAST analyses showed that the frequencies of reactivity to the novel allergen and to Der p 1, Der p 2, Der p 3, and Der p 6 were 92%, 97%, 100%, 97%, and 65%, respectively (n = 35). RAST inhibition studies showed some cross-reactivity between the protease and Der p 3 but not Der p 6., Conclusions: A novel mite serine protease was isolated from D. pteronyssinus and found to be a major allergen. This allergen has been tentatively designated Der p 9.

Two isoallergens of Cyn d I were isolated using preparative isoelectric focussing, and were designated Cyn d Ia and b. These isoallergens differ in their pI, molecular weight (Cyn d Ia, 32 kD and Cyn d Ib, 31 kD) and their NH2-terminal sequence. Four monoclonal antibodies (Mabs) were produced using Cyn d Ia as antigen. These Mabs recognized both Cyn d Ia and b. One of the Mabs recognized four different pI forms of Cyn d I on 2D gels. The Mabs also recognized cross-reactive epitopes on proteins from eight other grasses including rye grass, timothy grass, Kentucky bluegrass and Johnson grass.

The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa, have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects. The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a I). This protein has been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N-terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study, IgE binding to Western blots of Pha a I, Lol p I, and Cyn d I was investigated in 24 sera and found to occur in 19/24, 18/24, and 9/24, respectively. IgE binding to all three major allergens, and to both Pha a I and Lol p I, occurred in 8/24 sera. Our findings suggest that while the N-terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia.

Faecally enriched extracts of Dermatophagoides pteronyssinus were shown to contain a trypsin-like enzyme which was allergenic. Chromatofocusing studies revealed the presence of nine major isoforms in D. pteronyssinus, with pI in the range 4 to greater than 8, but only two (range 4-5) in D. farinae. Trypsin isolated from D. pteronyssinus by benzamidine-Sepharose 6B affinity chromatography and gelfiltration was found to be a 31-kDa protein which was enzymatically similar to both invertebrate and vertebrate trypsins. The N-terminal sequence obtained (IVGGEXALAGEXPYQISL) was identical to that reported for the mite allergen Der p III and showed homology with crayfish trypsin and Der f III from D. farinae. Mite trypsin underwent autolysis and the N-terminal sequences of two fragments were found to be ALAGEXPYQI and NNQVXGI respectively. Both showed homology with crayfish trypsin, and the former sequence was identical to residues 7-18 of the native enzyme and Der p III. All isoforms of mite trypsin were showed to be allergenic by radioallergosorbent assay and further studies indicated that the trypsin degradation products were also allergenic. The enzyme was compared with other mite allergens and the rank order of allergenic potency was shown to be: whole mite extract greater than Der p I greater than trypsin. However, all sera from a panel of mite allergic individuals showed IgE reactivity to trypsin, comparable to that seen using whole mite extract and Der p I. These data indicate that mite trypsin is a major allergen corresponding to the previously described allergen, Der p III.

Amylase activity was found in extracts of both Dermatophagoides pteronyssinus whole mite (0.16 U/mg) and spent growth medium (0.01 U/mg) but not in unused growth medium. It was also detected in all extracts of house dust obtained from mattresses (n = 20; geometric mean, 1.95 U/gm) and in 18 extracts of dust obtained from lounge room carpets (n = 20; geometric mean, 0.54 U/gm). Although the origins of amylase in dust are unclear, enzyme activity correlated with mite counts (n = 40; r = 0.35; p less than 0.05) and Der p I concentrations (r = 0.41; p less than 0.01). Mite amylase was purified from spent growth medium by affinity chromatography, gel filtration, and chromatofocusing. It was physicochemically similar to mammalian amylase with regard to molecular weight (60,000), charge heterogeneity (isoelectric point, 5 to 7) and the capacity to bind to an organomercurial affinity matrix. The optimum pH for enzymatic activity was revealed to be 6.4. IgE immunoblot studies demonstrated that the enzyme was allergenic and that its expression was dependent on the integrity of intrachain disulfide bonds. Sera from 25% of mite-allergic children and 46% of mite-allergic adults contained specific IgE to mite amylase. IgE to amylase was associated (p less than 0.01) with increased concentrations of total mite-specific IgE determined with a direct RAST assay.

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

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, KYXNPHFIGXRSVITXLME. 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.

A lambda gt11 library made with cDNA from the house dust mite Dermatophagoides pteronyssinus was screened with human allergic serum by IgE plaque radioimmunoassay. This resulted in the isolation of clones coding for the major allergen Der p II. The cDNA coded for a 129-residue protein of 14,131 daltons with no N-glycosylation sites. No sequence homology with other proteins was evident. The Der p II expressed in Escherichia coli reacted with IgE in 14 of 17 sera from mite-allergic patients giving clonal evidence for its designation as a major allergen. This, along with previous work, has resulted in the cloning of the two major mite allergens.

A major house dust mite allergen, Der p I, was isolated from spent growth medium and physicochemically characterized. These studies show that the allergen is monomeric, contains approximately 216 residues and 4 intra-chain disulphide bonds. The N-terminal amino acid is threonine. Circular dichroism studies show that the allergen contains 10% alpha-helical, 50% beta-pleated sheet and 40% random structures.

A major allergen (Der p1) has been purified to homogeneity from faecal particles from the house dust mite Dermatophagoides pteronyssinus. Reversed-phase microbore HPLC was employed to fractionate and purify a number of tryptic peptides generated from approximately 20 nmol of purified Der p1. N-Terminal amino acid sequence analyses were performed on the intact polypeptide and six tryptic peptides yielding 89 unique assignments; this corresponds to 40% of the molecule. These data are compared with the cDNA-deduced amino acid sequence of the Der p1 allergen. There is extensive similarity between the N-terminal amino acid sequence of Der p1 and the cysteine proteases actinidin and papain. A low Mr protein (approximately 17,000) was resolved from S-carboxymethyl Der p1 by gel-permeation chromatography. Edman degradation of the first 24 residues of this material revealed no similarity with Der p1. It is not clear whether this component is a low Mr disulfide-linked chain derived from Der p1 or represents an unidentified mite component.

A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact residues making up the active site. Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene. These data will now facilitate epitope mapping studies.

A cDNA clone coding for the major house dust mite allergen Der p 1 was isolated from a lambda gt 11 library. Its sequence correlates with known amino acid sequences of Der p 1 and it produces a fusion protein which reacts with rabbit anti-Der p 1 antiserum.

The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa, have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects. The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a 1). This protein has been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N-terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study, IgE binding to Western blots of Pha a I, Lol p I, and Cyn d I was investigated in 24 sera and found to occur in 19/24, 18/24, and &frac924;, respectively. IgE binding to all three major allergens, and to both Pha a I and Lol p I, occurred in &frac824; sera. Our findings suggest that while the N-terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia. [ABSTRACT FROM AUTHOR]