Characterization of low‐molecular‐mass trypsin isoinhibitors from oil‐rape (Brassica napus var. oleifera) seed

Abstract

A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P 1 -P 1 H reactive site bond (where residues forming the reactive site have been identified as P n ¼P 1 and P 1 H ¼P n H , where P 1 -P 1 H is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1 H-NMR data indicates the presence of aaNOEs and 3JaNH coupling constants, typical of the b-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine b-trypsin and bovine a-chymotrypsin are 3.3 £ 10 9 m 21 and 2.4 £ 10 6 m 21 , respectively, at pH 8.0 and 21.0 8C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.Keywords: trypsin inhibitors; oil-rape seed; Brassica napus var. oleifera; amino acid sequence determination; disulphide bridge location; 1 H-NMR investigation; inhibitory properties.Serine proteinase inhibitors are widespread in the plant kingdom being found particularly in seeds of Cruciferae, Graminaceae and Leguminosae, as well as in tubers of Solanaceae. Their physiological roles include the regulation of endogenous proteinases during seed dormancy, the reserve protein mobilization, and the protection against the proteolytic enzymes of parasites and insects. Moreover, they may also act as storage or reserve proteins. Plant serine proteinase inhibitors are grouped into Soybean (Kunitz), Bowman-Birk, potato I and II, and squash families. Several other inhibitor families, such as barley, ragi 1 and 2, and thaumatin, were also suggested [1±10]. Different types of serine proteinase inhibitors have been identified in the same plant, suggesting that these proteins have evolved separately to perform distinct physiological roles. In this respect, oil-rape (Brassi...

A Chimeric Mini-Trypsin Inhibitor Derived from the Oil Rape Proteinase Inhibitor Type III

“…The identification of protein domains retaining biological activity, and the demonstration that their functional properties are maintained in chimeric macromolecules are essential steps in the engineering of novel biological activities through fusion of small, stable domains to protein partners (9,16,17). Accordingly, we previously reported that the T-knot domain of RTI-III represents the inhibitor reactive site scaffold (12). Here, we demonstrate that RTI-III inhibitor properties are retained even when the reactive site T-knot scaffold (Ser3-Lys35 residues) is fused to a protein partner (i.e., DHFR).…”

“…Anima) of oil rape (Brassica napus var. oleifera) seed as previously reported (12). Bovine trypsin was obtained from Worthington Biochemical Co. (Freehold, NJ).…”

“…1), the nucleotide sequence coding for the 6ϫ His-DHFR domain, present in the pQE13 vector, was ligated in frame to a double-stranded synthetic oligonucleotide coding for the Ser3-Lys35 RTI-III amino acid sequence (12), based on the E. coli preferred codon usage. Moreover, a short nucleotide sequence coding for the Gly-Ser-Arg linker was inserted between DHFR and mini-RTI-III.…”

“…oleifera) seed; mini-RTI-III, recombinant mini-trypsin-inhibitor derived from RTI-III; DHFR, murine dihydrofolate reductase; DHFR-mini-RTI-III, chimeric protein containing 6ϫ His-DHFR, as the N-terminal domain, the Gly-SerArg linker and the mini-RTI-III, as the C-terminal region; MTI-2, RTI-III high homologous low-molecular-mass trypsin inhibitor from white mustard (Sinapis alba L.) seed; trypsin, bovine trypsin. The amino acid sequence numbering of mini-RTI-III refers to that of native RTI-III (12).macromolecules still retaining their specific biological functions.…”

Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction

“…Indeed Ceciliani et al (1994), and Ascenzi et al (1999) have characterized in rapeseed, serine PIs with features common to MTI-2 (Menegatti et al 1992), and it has recently been demonstrated that the oilseed rape nuclear genome encodes a gene family of PIs (Ceci et al 1999).…”