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

Pytelková J, Lepšík M, Sanda M, Talacko P, Marešová L, Mareš M - BMC Biochem. (2012)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Prague, Czech Republic.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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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.
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Figure 2: 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.

Mentions: α-Amylase was purified to homogeneity from the whole body extract of A. siro using an optimized procedure for affinity precipitation with glycogen. The typical yield was approximately 175 μg from 1 g of fresh weight of mites. The purified enzymatically active α-amylase migrated as a single band of 56 kDa on SDS-PAGE (Figure 2). We performed a two-pronged proteomic characterization of this protein: (i) the N-terminal amino acid sequence, XSPYSNPHFTGSR (X is an unidentified residue), was determined by Edman sequencing and (ii) the protein was subjected to enzymatic digestion followed by LC-MS/MS analysis. The data were searched against the UniProt protein database, which revealed identity with the cDNA-derived protein sequence of an A. siro α-amylase homolog denoted Aca s 4 (GenBank: ABL09312). The MS/MS peptide coverage of this sequence was ~31% (Figure 3). A theoretical mass calculated for the mature Aca s 4 (sequence starting at the native N-terminus) is 55956 Da, which is in good agreement with the experimental value obtained for the purified Aca s 4 (Figure 2).


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

Pytelková J, Lepšík M, Sanda M, Talacko P, Marešová L, Mareš M - BMC Biochem. (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306266&req=5

Figure 2: 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.
Mentions: α-Amylase was purified to homogeneity from the whole body extract of A. siro using an optimized procedure for affinity precipitation with glycogen. The typical yield was approximately 175 μg from 1 g of fresh weight of mites. The purified enzymatically active α-amylase migrated as a single band of 56 kDa on SDS-PAGE (Figure 2). We performed a two-pronged proteomic characterization of this protein: (i) the N-terminal amino acid sequence, XSPYSNPHFTGSR (X is an unidentified residue), was determined by Edman sequencing and (ii) the protein was subjected to enzymatic digestion followed by LC-MS/MS analysis. The data were searched against the UniProt protein database, which revealed identity with the cDNA-derived protein sequence of an A. siro α-amylase homolog denoted Aca s 4 (GenBank: ABL09312). The MS/MS peptide coverage of this sequence was ~31% (Figure 3). A theoretical mass calculated for the mature Aca s 4 (sequence starting at the native N-terminus) is 55956 Da, which is in good agreement with the experimental value obtained for the purified Aca s 4 (Figure 2).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Prague, Czech Republic.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus