Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family.
A fatty acid-binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.
Timanova
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A
Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Science, Sofia, Bulgaria.
Müller
S
S
Marti
T
T
Bankov
I
I
Walter
R D
RD
D000485:Allergens / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001197:Ascaridia / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D003619:Dansyl Compounds / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D005456:Fluorescent Dyes; D015801:Helminth Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D018996:Myelin P2 Protein / Q000737:chemistry*; D009363:Neoplasm Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014212:Tretinoin / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism
D000485:Allergens; D002352:Carrier Proteins; D003619:Dansyl Compounds; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D015801:Helminth Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D014801:Vitamin A; D014212:Tretinoin; C054400:11-(dansylamino)undecanoic acid; D014364:Tryptophan; D002784:Cholesterol