1. Purification, characterisation and cDNA cloning of an antimicrobial peptide from Macadamia integrifolia
J P Marcus, K C Goulter, J L Green, S J Harrison, J M Manners Eur J Biochem. 1997 Mar 15;244(3):743-9. doi: 10.1111/j.1432-1033.1997.00743.x.
An antimicrobial peptide with no significant amino acid sequence similarity to previously described peptides has been isolated from the nut kernels of Macadcamia integrifolia. The peptide, termed MiAMP1, is highly basic with an estimated pI of 10.1, a mass of 8.1 kDa and contains 76 amino acids including 6 cysteine residues. A cDNA clone containing the entire coding region corresponding to the peptide was obtained. The deduced amino acid sequence of the cDNA indicated a 26-amino-acid signal peptide at the N-terminus of the preprotein. Purified MiAMP1 inhibited the growth of a variety of fungal, oomycete and gram-positive bacterial phytopathogens in vitro. Some pathogens exhibited close to 100% inhibition in less than 1 microM peptide (5 microg/ml). Antimicrobial activity was diminished against most, but not all, microbes in the presence of calcium and potassium chloride salts (1 mM and 50 mM, respectively). MiAMP1 was active against bakers yeast, was inactive against Escherichia coli and was non-toxic to plant and mammalian cells. Analysis of genomic DNA indicated that MiAMP1 was encoded on a single copy gene containing no introns. The MiAMP1 gene may prove useful in genetic manipulations to increase disease resistance in transgenic plants.
2. Len c 1, a major allergen and vicilin from lentil seeds: protein isolation and cDNA cloning
Gema López-Torrejón, Gabriel Salcedo, Manuel Martín-Esteban, Araceli Díaz-Perales, Cristina Y Pascual, Rosa Sánchez-Monge J Allergy Clin Immunol. 2003 Dec;112(6):1208-15. doi: 10.1016/j.jaci.2003.08.035.
Background: Lentils are among the main plant foods causing allergic reactions in pediatric patients in the Mediterranean area and in many Asian communities. However, very few reports have been devoted to identifying lentil allergens. Seed storage proteins of the vicilin family have been characterized as major allergens in several seed legumes and tree nuts. Objective: We sought to evaluate the role of lentil vicilins as food allergens. Methods: A serum pool and individual sera from 22 patients with lentil allergy were used in different IgE-binding assays. Mature lentil vicilin was isolated by means of cation-exchange chromatography, followed by reverse-phase HPLC, and characterized by means of N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) analysis, complex asparagine-linked glycan detection, specific IgE immunodetection with individual sera, and ELISA inhibition assays. Complete cDNAs encoding lentil vicilin variants were isolated by means of PCR with primers based on the amino acid sequence of the allergen. Results: A major IgE-binding component of approximately 50 kd was detected in lentil extracts. This component was isolated and characterized, showing a single N-terminal amino acid sequence homologous to those of legume vicilins and a broad peak (maximum at 48613 d) in MALDI analysis. The purified allergen was recognized by 77% (17/22) of the individual sera from patients with lentil allergy and reached up to 65% inhibition of the IgE binding to the crude lentil extract. The allergen showed 3 isoforms varying in their degree of N-glycosylation. Two cDNA clones encoding different allergen variants were isolated. The amino acid sequences deduced from both clones (415 and 418 residues; 47.4 and 47.8 kd) showed greater than 50% identity with major peanut (Ara h 1) and soybean (conglutinin subunits) allergens belonging to the vicilin family. Furthermore, these sequences included those of the previously characterized lentil allergen Len c 1.02 (108 amino acid residues of the C-terminal domain) and those of a novel lentil IgE-binding protein of 26 kd. Conclusion: The mature 48-kd lentil vicilin, designated Len c 1.01, is a major allergen. Two of its processing fragments, corresponding to subunits of 12 to 16 kd (previously named Len c 1) and 26 kd, are also relevant lentil IgE-binding proteins. The sequence homology of Len c 1.01 to those of major allergens from peanut, soybean, walnut, and cashew can help to investigate potential cross-reactions among these plant foods.
3. Vicilin-like seed storage proteins in the gymnosperm interior spruce (Picea glauca/engelmanii)
C H Newton, B S Flinn, B C Sutton Plant Mol Biol. 1992 Oct;20(2):315-22. doi: 10.1007/BF00014500.
A seed storage protein cDNA was characterized from a library of interior spruce (Picea glauca/engelmanii complex) cotyledonary stage somatic embryos. The deduced amino acid sequence predicts a 448 amino acid (50 kDa) polypeptide with 28-38% identity with angiosperm vicilin-like 7S globulins. XXC/G codon usage is low (47%) relative to monocot angiosperms while pairwise comparisons show that spruce, monocot, and dicot vicilins are approximately equal in amino acid divergence. Although small by comparison, the spruce vicilin contains an N terminal hydrophilic region characteristic of angiosperm 'large' vicilins. Genomic Southern blotting predicts that the cDNA is encoded by a gene family.
