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Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro

Authors:
Jana Pytelková at The Czech Academy of Sciences
Jana Pytelková
Martin Lepsik at Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry
Martin Lepsik
Miloslav Sanda at Max Planck Institute for Heart and Lung Research
Miloslav Sanda
Pavel Talacko at Charles University in Prague
Pavel Talacko
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Abstract and Figures

Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite. A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found. We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.
Distribution of α-amylase and protease activities in the whole body extract and faecal extract of A. siro (A.s.) and D. farinae (D.f.). The α-amylase activities were assayed at the respective pH optima with RBB-starch as a substrate. The protease activities were assayed with azocasein as a substrate; the contribution of cysteine proteases (dashed) was determined as the part of protease activity inhibited by E-64. The specific activities (units per mg protein) are normalized to the maximum value measured for α-amylases and proteases, respectively; mean values ± SE are given.
Distribution of α-amylase and protease activities in the whole body extract and faecal extract of A. siro (A.s.) and D. farinae (D.f.). The α-amylase activities were assayed at the respective pH optima with RBB-starch as a substrate. The protease activities were assayed with azocasein as a substrate; the contribution of cysteine proteases (dashed) was determined as the part of protease activity inhibited by E-64. The specific activities (units per mg protein) are normalized to the maximum value measured for α-amylases and proteases, respectively; mean values ± SE are given.
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Purification and IgE reactivity of Aca s 4. The whole body extracts (20 μg) of A. siro (A.s WB) and D. farinae (D.f. WB) and the purified Aca s 4 (2.5 μg) were resolved by SDS-PAGE. Left-hand panel: A gel stained for protein with Coomassie blue. Right-hand panel: Western blot probed with pooled sera from mite-allergic patients sensitive to Dermatophagoides spp. and with anti-IgE antibodies and developed by chemiluminescence. For immunostaining inhibition (inhib.), the pooled sera were preincubated with purified Aca s 4. The arrows mark the position of Aca s 4 (~56 kDa) with the N-terminal sequence determined by Edman sequencing. Molecular mass standards are indicated.
Purification and IgE reactivity of Aca s 4. The whole body extracts (20 μg) of A. siro (A.s WB) and D. farinae (D.f. WB) and the purified Aca s 4 (2.5 μg) were resolved by SDS-PAGE. Left-hand panel: A gel stained for protein with Coomassie blue. Right-hand panel: Western blot probed with pooled sera from mite-allergic patients sensitive to Dermatophagoides spp. and with anti-IgE antibodies and developed by chemiluminescence. For immunostaining inhibition (inhib.), the pooled sera were preincubated with purified Aca s 4. The arrows mark the position of Aca s 4 (~56 kDa) with the N-terminal sequence determined by Edman sequencing. Molecular mass standards are indicated.
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Multiple sequence alignment of Aca s 4 with other α-amylases of mite origin and human α-amylase. Aca s 4: Acarus siro (GenBank: ABL09312); Tyr p 4: Tyrophagus putrescentiae (GenBank: ABM53754); Blo t 4: Blomia tropicalis (GenBank: AAQ24543) [12]; Eur m 4: Euroglyphus maynei (GenBank: AAD38943) [13]; Der p 4: Dermatophagoides pteronyssinus (GenBank: AAD38942) [13]; HPA: human pancreatic α-amylase (GenBank: AAH07060). The sequence similarities to Aca s 4 are 74%, 70%, 64%, 66%, and 50%, respectively. Full-length sequences of mature proteins are aligned. Amino acids identical to those of Aca s 4 are shaded. In the Aca s 4 sequence, the N-terminal sequence determined by Edman sequencing (dotted underline) and fragments determined by LC-MS/MS analysis (solid underline) are indicated. Positions of catalytic residues (@) and residues binding the Cl- (#) and the Ca2+ (*) are marked. N-glycosylation signals are double underlined (Aca s 4 and Blo t 4 have no predicted N-glycosylation sites). Note the Asn295Ser mutation located in the Cl- -binding site of mite α-amylases (compared to HPA and other animal α-amylases). Cys residues forming four conserved disulfide bridges (‡) and one specific disulfide (+) in the Aca s 4 molecule are marked.
Multiple sequence alignment of Aca s 4 with other α-amylases of mite origin and human α-amylase. Aca s 4: Acarus siro (GenBank: ABL09312); Tyr p 4: Tyrophagus putrescentiae (GenBank: ABM53754); Blo t 4: Blomia tropicalis (GenBank: AAQ24543) [12]; Eur m 4: Euroglyphus maynei (GenBank: AAD38943) [13]; Der p 4: Dermatophagoides pteronyssinus (GenBank: AAD38942) [13]; HPA: human pancreatic α-amylase (GenBank: AAH07060). The sequence similarities to Aca s 4 are 74%, 70%, 64%, 66%, and 50%, respectively. Full-length sequences of mature proteins are aligned. Amino acids identical to those of Aca s 4 are shaded. In the Aca s 4 sequence, the N-terminal sequence determined by Edman sequencing (dotted underline) and fragments determined by LC-MS/MS analysis (solid underline) are indicated. Positions of catalytic residues (@) and residues binding the Cl- (#) and the Ca2+ (*) are marked. N-glycosylation signals are double underlined (Aca s 4 and Blo t 4 have no predicted N-glycosylation sites). Note the Asn295Ser mutation located in the Cl- -binding site of mite α-amylases (compared to HPA and other animal α-amylases). Cys residues forming four conserved disulfide bridges (‡) and one specific disulfide (+) in the Aca s 4 molecule are marked.
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Enzymatic properties of purified Aca s 4. (a) pH profile determined with RBB-starch as a substrate. Mean values ± SE are normalized to the maximum value. (b) Activation effect of NaCl on amylolytic activity of Aca s 4 (solid line) and porcine pancreatic α-amylase (PPA) (dashed line) measured with the RBB-starch assay. Mean values ± SE are expressed as a percentage of activity relative to the control without NaCl (100%). (c) Inhibition profile of Aca s 4 determined by a library of PAMIs with the general structure Ac-XHWYYRCW-NH2; the X position contains one of the 20 naturally occurring amino acids as indicated. The inhibition sensitivity of the purified Aca s 4 (hatched) is compared to that of whole body extracts of A. siro (gray) and D. farinae (black). Mean values ± SE are expressed as the percentage of amylolytic activity (RBB-starch assay) relative to the uninhibited control (100%).
Enzymatic properties of purified Aca s 4. (a) pH profile determined with RBB-starch as a substrate. Mean values ± SE are normalized to the maximum value. (b) Activation effect of NaCl on amylolytic activity of Aca s 4 (solid line) and porcine pancreatic α-amylase (PPA) (dashed line) measured with the RBB-starch assay. Mean values ± SE are expressed as a percentage of activity relative to the control without NaCl (100%). (c) Inhibition profile of Aca s 4 determined by a library of PAMIs with the general structure Ac-XHWYYRCW-NH2; the X position contains one of the 20 naturally occurring amino acids as indicated. The inhibition sensitivity of the purified Aca s 4 (hatched) is compared to that of whole body extracts of A. siro (gray) and D. farinae (black). Mean values ± SE are expressed as the percentage of amylolytic activity (RBB-starch assay) relative to the uninhibited control (100%).
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Spatial model of Aca s 4 built by homology modeling and simulations. (a) The Aca s 4 structure (in ribbon representation) is composed of the consensus α-amylase domains A (green), B (red), and C (brown). Three catalytic residues (D196, E232, D297) in the active site are highlighted (orange ball-and-stick). The calcium ion is depicted as a magenta sphere. The chloride binding site (blue sticks) is composed of the conserved binding residues R194 and R334 and the residue S295, which is specific for mite α-amylases; the chloride ion is shown as a light blue sphere. The disulfide bridges are represented by yellow sticks. (b) A superposition of Cα traces of Aca s 4 (magenta) with HPA (cyan; PDB: 2CPU) used as a template for Aca s 4 modeling. The disulfide bridges are represented by yellow (Aca s 4) and orange (HPA) sticks; non-conserved disulfides are marked by asterisks. (c) The surface model of Aca s 4; the right-hand view is in the same orientation as in (a). The molecule is colored red for residues that are identical for Aca s 4 and Der p 4.
Spatial model of Aca s 4 built by homology modeling and simulations. (a) The Aca s 4 structure (in ribbon representation) is composed of the consensus α-amylase domains A (green), B (red), and C (brown). Three catalytic residues (D196, E232, D297) in the active site are highlighted (orange ball-and-stick). The calcium ion is depicted as a magenta sphere. The chloride binding site (blue sticks) is composed of the conserved binding residues R194 and R334 and the residue S295, which is specific for mite α-amylases; the chloride ion is shown as a light blue sphere. The disulfide bridges are represented by yellow sticks. (b) A superposition of Cα traces of Aca s 4 (magenta) with HPA (cyan; PDB: 2CPU) used as a template for Aca s 4 modeling. The disulfide bridges are represented by yellow (Aca s 4) and orange (HPA) sticks; non-conserved disulfides are marked by asterisks. (c) The surface model of Aca s 4; the right-hand view is in the same orientation as in (a). The molecule is colored red for residues that are identical for Aca s 4 and Der p 4.
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Article
  • Jan 2002
Human pancreatic R-amylase (HPA) is a member of the R-amylase family involved in the degradation of starch. Some members of this family, including HPA, require chloride for maximal activity. To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced. Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes. X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes. However, the introduction of these mutations did alter the kinetic properties of the enzyme. Mutations to residue R195 resulted in a 20 -450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH. Interestingly, replacement of R337 with a nonbasic amino acid resulted in an R-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity. We propose that the chloride is required to increase the pKa of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue.
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Allergenicity and Physicochemical Characterization of House Dust Mite Derived Amylase
Article
  • Jan 1991
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.
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Combined effect of antifeedant α-amylase inhibitor and predator Cheyletus malaccensis in controlling the stored mite pest Acarus siro
Article
  • Aug 2006
  • PHYSIOL ENTOMOL
Acarus siro is the most abundant and frequent mite to infest stored-food products, causing allergies and transmitting mycotoxin producing fungi. The predatory mite Cheyletus malaccensis is a candidate species in the biocontrol programme for this pest. In vitro, the α-amylase inhibitor acarbose is effective against the α-amylase of A. siro but not against that of C. malaccensis. In vivo, the impact of acarbose on a population of A. siro is investigated along with the interaction with the predator. Various densities of adult parthenogenetic females of C. malaccensis are reared on A. siro feeding on either a control diet or a diet containing different acarbose concentrations. The combined action of both factors significantly improved the final biocontrol efficiency with C. malaccensis, compensating for the lower energetic content of the prey on acarbose by increasing the number of prey caught. Acarbose had no negative effects on the longevity and the length of oviposition period of C. malaccensis but partially reduced its fecundity. The results are discussed in the context of the integrated control of stored-product mite pests.
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The importance of starch and sucrose digestion in nutritive biology of synanthropic acaridid mites: α-Amylases and α-glucosidases are suitable targets for inhibitor-based strategies of mite control
Article
  • Jul 2009
  • ARCH INSECT BIOCHEM
The adaptation of nine species of mites that infest stored products for starch utilization was tested by (1) enzymatic analysis using feces and whole mite extracts, (2) biotests, and (3) inhibition experiments. Acarus siro, Aleuroglyphus ovatus, and Tyroborus lini were associated with the starch-type substrates and maltose, with higher enzymatic activities observed in whole mite extracts. Lepidoglyphus destructor was associated with the same substrates but had higher activities in feces. Dermatophagoides farinae, Chortoglyphus arcuatus, and Caloglyphus redickorzevi were associated with sucrose. Tyrophagus putrescentiae and Carpoglyphus lactis had low or intermediate enzymatic activity on the tested substrates. Biotests on starch additive diets showed accelerated growth of species associated with the starch-type substrates. The inhibitor acarbose suppressed starch hydrolysis and growth of the mites. We suggest that the species with higher starch hydrolytic activity in feces were more tolerant to acarbose, and alpha-amylase and alpha-glucosidase of synanthropic mites are suitable targets for inhibitor-based strategies of mite control.
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Digestive α-amylases of the flour moth Ephestia kuehniella-adaptation to alkaline environment and plant inhibitors
Article
The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment was investigated in the flour moth Ephestia kuehniella, an important stored-product pest. Three digestive alpha-amylases [Ephestia kuehniella alpha-amylase isoenzymes 1-3 (EkAmy1-3)] with an alkaline pH optimum were purified from larvae and biochemically characterized. These isoenzymes differ significantly in their sensitivity to alpha-amylase inhibitors of plant origin that are directed against herbivores as antifeedants. Such functional variability renders the amylolytic system less vulnerable to suppression by plant defensive molecules. Moreover, we found that expression of alpha-amylases is upregulated in larvae feeding on a diet enriched with an alpha-amylase inhibitor. The alpha-amylases are secreted into the larval midgut by an exocytotic mechanism, as revealed by immunogold microscopy. The cDNA sequence of EkAmy3 was determined, and a homology model of EkAmy3 was built in order to analyze the structural features responsible for adaptation to alkaline pH. First, the overall fold was found to be stabilized by remodeling of ion pairs. Second, molecular simulations supported by activity measurements showed that EkAmy3 does not bind a Cl(-), owing to an Arg-to-Gln mutation in a conserved binding site. The Cl(-)-binding residues are in contact with the catalytic residues, and this change might help to fine-tune the catalytic pK(a) values to an alkaline pH optimum. We conclude that lepidopteran alpha-amylases are evolutionarily adapted in terms of structure and expression dynamics for effective functioning in the digestive system.
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Mite Amylase from Blomia tropicalis (Blo t 4): Differential Allergenicity Linked to Geographical Regions
Article
  • Dec 2008
  • INT ARCH ALLERGY IMM
Blomia tropicalis is an important domestic dust mite in the tropics and subtropics. This study describes cDNA cloning of the group 4 allergen of B. tropicalis, and the evaluation of the sensitization of this allergen in atopic populations from 2 geographic regions. cDNA cloning was carried out using the Smart RACE cDNA amplification kit. The full-length Blo t 4 cDNA was isolated by cDNA library screening, 5' and 3' rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of the Clustal W, CGC and Blast program packages. The cDNA was expressed in Pichia pastoris yeast. The skin prick test was used to evaluate the sensitization profile of recombinant Blo t 4, crude dust mite allergen extracts and major B. tropicalis recombinant allergen Blo t 5. The cloned Blo t 4 had a molecular weight of 56 kDa and had 68% amino acid homology with group 4 allergens of Dermatophagoides pteronyssinus and 65% with those of Euroglyphus maynei. A sensitization profile to the expressed recombinant Blo t 4 allergen (28%) showed an unusually higher frequency than to the major allergen Blo t 5 (22%) in allergic subjects from Chengdu, PR China. In comparison, the subjects from Singapore showed very low sensitization to Blo t 4 (4%) compared with Blo t 5 (84%). Group 4 allergens of B. tropicalis may be an important dust mite allergen in certain distinct populations.
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