Primary structure of Vicia angustifolia proteinase inhibitor

Abstract

The complete amino acid sequence (72 amino acid residues) of a double-headed proteinase inhibitor from seeds of Vicia angustifolia L. var. segetalis Koch has been determined and compared with those of other double-headed inhibitors of known structure. Sequencing was performed by conventional methods with the aid of the fragments produced by reduction and S-carboxymethylation of the enzymatically modified inhibitors, and also using tryptic and chymotryptic peptides.The positions of the 14 half-cystine residues agreed among all the reported primary structures of the legume double-headed inhibitors. However, V. angustifolia inhibitor possessed extensive amino acid differences compared to the others. The phylogenetic relationship among these inhibitors was established using the unweighted pair-group method and revealed that the V . angustifolia inhibitor and the peanut inhibitor B-I11 had diverged at a relatively earlier stage compared to the other inhibitors. The inhibitor consists of 72 amino acid residues and inhibits both trypsin and chymotrypsin. As shown for the other proteinase inhibitors [6], limited hydrolysis at either reactive site also occurs in V . angustifolia inhibitor by attack of the corresponding enzyme under acidic conditions [7,8]. However, in constrast to the others, an additional cleavage other than that of the antitryptic site was observed during the incubation of the inhibitor with trypsin at pH 3 [9].Using the fragments produced by reduction and S-carboxymethylation of the enzymatically modified derivatives and also using tryptic and chymotryptic peptides, the complete amino acid sequence of the inhibitor has been determined. The result is compared with those of the other doubleheaded inhibitors whose primary structures have been reported [lo -211 and the phylogenetic relationships between them are discussed. MATERIALS AND METHODS MaterialsVicia angustifolia inhibitor was prepared as reported previously [5]. Bovine a-chymotrypsin (Boehringer) was treated with N-tosyl-L-lysine chloromethyl ketone (TosLysCH,CI) [22] (Nakarai Chemicals, Japan) and purified by passing through a column of lima bean proteinase inhibitor linked to Sepharose 4B which was prepared by the method of Knights and Light [23]. DEAE-Sephadex A-25 and DEAE-Sephacel were obtained from Pharmacia Fine Chemicals. Polyamide thin-layer sheets were from Cheng Chin Trading Co. Preparation of enzymatically modified inhibitor derivatives and their fragmentsAll the inhibitor derivatives produced by the limited hydrolyses of the inhibitor by either trypsin or chymotrypsin, are schematically presented in Fig. 1 as well as their fragments separated after reduction and S-carboxymethylation. Details of the preparations, isolations, and partial sequence determinations of the trypsin-modified derivatives have been described previously [7,9]. The chymotrypsin-modified inhibitor (CmV) was prepared at pH 3 with a catalytic amount of chymotrypsin under similar conditions to those of the trypsinmodified derivative, TmV-I [7]. After the reduction...

WIP1, a wound-inducible gene from maize with homology to Bowman-Birk proteinase inhibitors

“…The sequences for wheat germ [38], Arachis hypogea [36], and Viciafaba [2] are complete sequences starting with respective amino acid 1. The sequence of Vicia angustifolia [50] entends from amino acid 1 through 45 out of 72 in total. found to be around 700.…”

Amino acid sequence of a Bowman-Birk proteinase inhibitor from pea seeds

“…Moreover, over the 20 residues determined in their N-terminal sequence, PSTI I-V were identical to each other and also to those of Pisum abyssinicum TI1 and TI2 inhibitors (Domoney et al, 1993). Moreover, they were extremely similar to those of trypsin inhibitors from Vicia faba (Asao et al, 1991) and Vicia angustifolia (Shimokawa et aL, 1984). According to this sequence similarity as well as their molecular weight, it may be expected that these six pea protease inhibitors may belong, like as both V. faba and V. angustifolia inhibitors, to the Bowman-Birk class of trypsin inhibitors.…”

“…The invariant amino acids are boxed. VAI, Vicia angustifolia inhibitor (Shimokawa et al, 1984); FBI, Vicia faba inhibitor (Asao et al, 1991); BBI, soybean Bowman-Birk inhibitor (Odany and Ikenaka, 1972); SBI-C-II and SBI D-II, soybean inhibitor C-II (Odany and Ikenaka, 1977b) and D-II (Odany and Ikenaka, 1978); GBI-II', garden bean inhibitor II' (Wilson and Laskowsky, 1975); ABI I, adzuky bean inhibitor (Ishikawa et al, 1979); Cp TI flV, cowpea trypsin inhibitor fIV (Hilder et al, 1989); MAI DE3 and DE4, Macrotyloma axillare inhibitors DE3 and DE4 (Joubert et al, 1979); PI B-III and B-II, peanut inhibitor B-Ill. (Norioka and Ikenaka, 1983a) and B-II (Norioka and Ikenaka, 1983b).…”

The amino acid sequence of a cereal Bowman‐Birk type trypsin inhibitor from seeds of Jobs' tears (Coix lachryma‐jobi L.)

“…The composition calculated from the sequence is also very similar to that reported for the Coix 12 kDa trypsin inhibitor by Ohtsubo et al [24] when the latter is recalculated for a protein of 7 kDa. Fig.3 shows the clear sequence homology which exists between the Coix trypsin inhibitor, rice bran inhibitor [23], wheat germ inhibitors [22] and Bowman-Birk proteinase inhibitors from a number of legumes [31][32][33][34][35][36][37]. The legume inhibitors have …”

“…Homology of the amino acid sequences of the Bowman-Birk family of proteinase inhibitors from legumes and cereals. (1) Inhibitor CII from soybean (Giycine max) [31]; (2) inhibitor AII from peanut (Arachis hypogaea) [32]; (3) adzuki bean (Phaseolus angularis) [33]; (4) Macrotyioma axiilare [34]; (5) Vicia angustifolia [35]; (6) mung bean (Phaseolus radiata) [36]; (7) alfalfa leaf (Medicago sativa) [37]; (8) rice bran (Oryza sativa) residues 1-69 [23], residues 70-133 are an internal duplication homologous with residues 3-69, and are shown as 8*; (9) wheat germ (Triticum aestivum) II-4 incomplete sequence [22]; (10) wheat I-2h incomplete sequence [22]; (11) Jobs' tears (Coix lachryma-jobO. On the numbering of the soybean (top line) and the Coix (bottom) sequences are shown.…”