cDNA Clone of a Putative Peanut (Arachis hypogaea L.) Trypsin Inhibitor Has Homology with Peanut Allergens Ara h 3 and Ara h 4

• Published in: Journal of agricultural and food chemistry Vol. 52; no. 5; pp. 1404 - 1409
• Main Authors: Dodo, Hortense W, Viquez, Olga M, Maleki, Soheila J, Konan, Koffi N
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 10.03.2004
• Subjects: Agronomy. Soil science and plant productions; Allergens - chemistry; Allergens - genetics; Allergens - pharmacology; Amino Acid Sequence; Antigens, Plant; Arachis - chemistry; Arachis hypogaea; Base Sequence; Biological and medical sciences; DNA, Complementary - chemistry; DNA, Plant - chemistry; Economic plant physiology; Fundamental and applied biological sciences. Psychology; Molecular Sequence Data; Morphology. Anatomy. Histology. Cytology; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - pharmacology; Seed Storage Proteins; Sequence Homology; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - genetics; Trypsin Inhibitors - pharmacology; Peanut; Storage protein; Serine endopeptidases; Enzyme; Arachis hypogaea; Enzyme inhibitor; Homology; Arachis hypogaea L; Pathogenesis related protein; cDNA library; Peptidases; Trypsin; seed storage proteins; sequence homology; Leguminosae; Dicotyledones; Angiospermae; Hydrolases; trypsin inhibitor; Spermatophyta; Allergen
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf034765c

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman−Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized 32P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor. Keywords: Allergen; Arachis hypogaea L.; cDNA library; peanut; seed storage proteins; sequence homology; trypsin inhibitor