Characterization of a Kunitz Trypsin Inhibitor with One Disulfide Bridge Purified from Swartzia pickellii

Cavalcanti, María Auxiliadora de Queiroz; Oliva, Maria Luíza Vilela; Fritz, Hans; Jochum, Marianne; Mentele, Reinhard; Sampaio, Misako U.; Coelho, Luana Cassandra Breitenbach Barroso; Batista, Isabel de Fátima Correia; Sampaio, Cláudio Hoffmann

doi:10.1006/bbrc.2002.6436

Cited by 46 publications

(5 citation statements)

References 20 publications

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“…It should be noted that the sequence of these proteins represented 30-40% identical residues and contained highly conserved sequences in long chains. 21,22,[51][52][53] The PI from Acacia karroo 50 -AkCI-also presented 2 subunits, as CTI, and it holds the same percentage of identical and highly conserved sequences, including Cys in the positions highlighted for CTI, STI, SwTI, DrTI, AcTI, and ILTI in Fig. 7.…”

Section: Figurementioning

confidence: 97%

“…These measured masses are lower than the calculated value for trypsin inhibitors from other seed species, such as STI (18,501.4 Da), and those of KTI-type inhibitors from different seeds of Leguminosae, indicating that the CTI-1 inhibitor is composed of 2 subunits. The number of amino acids per peptide chain may account for some of the differences in mass; for example, the soybean inhibitor has 167 amino acids, whereas the DrTI from Delonix regia contains 188 amino acids, 52 the SwTI protein from Swartzia pickellii has 178 amino acids, 22 and the AkCI/1 from A. karroo has 2 subunits with 139 and 44 residues, respectively. 50 Peptide fragments produced by trypsin and endoproteinase Glu-C SV8 (spots A and F) were sequenced by ESI-QTOF.…”

Section: Figurementioning

confidence: 99%

“…31 Plant KTI inhibitors are composed of a single polypeptide chain with ;20 kDa, 2 disulfide bridges (Cys39-Cys86 and Cys136-Cys145), and only 1 reactive site. 51,56 However, the CTI inhibitor protein from C. langsdorffii belongs to the subfamily Caesalpinioideae, which are Bauhinia-type trypsin inhibitors, with 2 polypeptide chains and only 1 disulfide bridge, 31,32 as observed for the SWTI (S. pickellii trypsin inhibitor), 22 gBrEI of Bauhinia rufa 57 from the Caesalpinioideae subfamily, and ILTI 21 and ESTI 58 from I. laurina and Entada scandens, respectively, both from the Mimosoideae subfamily.…”

Section: Figurementioning

confidence: 99%

“…KTIs are proteins with a molecular mass of 18-28 kDa, with 1 or 2 subunits and low Cys, normally with 2 disulfide bridges and only 1 reactive site, containing Arg or Glu residues, which are primarily able to inhibit (mammalian) proteases. 2,21,22 The application of mass spectrometry (MS) in combination with 2-dimensional (2D) PAGE for biomolecular analysis has proven to be of great importance for protein analysis. In fact, the M r of protein and its isoelectric point (pI) obtained by 2D gel/MS analysis are the most commonly used features in protein identification performances.…”

Section: Introductionmentioning

confidence: 99%

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Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

Silva

Pompeu

Smolka³

et al. 2015

J Biomol Tech

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In this study, the aim was to determine the complete sequence of the Copaifera langsdorffii trypsin inhibitor (CTI)-1 using 2-dimensional (2D)-PAGE, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), and quadrupole time-of-flight (QTOF) spectrometry. Spots A (CTI-1) and F (CTI-2) were submitted to enzymatic digestions with trypsin, SV8, and clostripain. The accurate mass of the peptide obtained from each digest was determined by mass spectrometry (MS) using MALDI-TOF. The most abundant peptides were purified and sequenced in a liquid chromatograph connected to an electrospray ionization-QTOF MS. When the purified trypsin inhibitor was submitted to 2D electrophoresis, different spots were observed, suggesting that the protein is composed of 2 subunits with microheterogeneity. Isoelectric points of 8.0, 8.5, and 9.0 were determined for the 11 kDa subunit and of 4.7, 4.6, and 4.3 for the 9 kDa subunit. The primary structure of CTI-1, determined from the mass of the peptide of the enzymatic digestions and the sequence obtained by MS, indicated 180 shared amino acid residues and a high degree of similarity with other Kunitz (KTI)-type inhibitors. The peptide also contained an Arg residue at the reactive site position. Its 3-dimensional structure revealed that this is because the structural discrepancies do not affect the canonical conformation of the reactive loop of the peptide. Results demonstrate that a detailed investigation of the structural particularities of CTI-1 could provide a better understanding of the mechanism of action of these proteins, as well as clarify its biologic function in the seeds. CTI-1 belongs to the KTI family and is composed of 2 polypeptide chains and only 1 disulfide bridge.

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Section: Figurementioning

confidence: 97%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

Silva

Pompeu

Smolka³

et al. 2015

J Biomol Tech

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show abstract

“…Most legume KTi proteins are ∼20 kDa, with one or two disulfide bonds and a single reactive site. The three-dimensional (3D) structure of KTi reveals that the first disulfide bridge, which surrounds the reactive loop, is necessary for trypsin inhibitory activity. , Most legume BBi are composed of approximately ∼100 amino acids and contain 14 cysteine residues . These cysteine residues form conserved disulfide bridges that make BBi stable in high heat and extreme pH.…”

Section: Introductionmentioning

confidence: 99%

Lathyrus sativus Originating from Different Geographical Regions Reveals Striking Differences in Kunitz and Bowman–Birk Inhibitor Activities

Song

et al. 2019

J. Agric. Food Chem.

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Grass pea (Lathyrus sativus L.) is an important legume commonly grown in arid and semi-arid regions. This protein-rich legume performs well even under harsh environmental conditions and is considered to be a strategic famine food in developing countries. Unfortunately, its potential usage is greatly limited as a result of the presence of antinutritional factors, including the neuroexcitatory amino acid β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP) and protease inhibitors. β-ODAP is responsible for a neurodegenerative syndrome that results in the paralysis of lower limbs, while protease inhibitors affect protein digestibility, resulting in reduced growth. Concerted research efforts have led to development of grass pea cultivars with reduced β-ODAP content. In contrast, very little information is available on the protease inhibitors of L. sativus. In this study, we have conducted biochemical characterization of 51 L. sativus accessions originating from different geographical regions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of seed globulins and prolamins revealed striking similarity in their protein profile, although geographic-specific variations in profiles was also evident. Measurement of Bowman–Birk chymotrypsin inhibitor (BBi) and Kunitz trypsin inhibitor (KTi) activities in accessions revealed striking differences among them. Amino acid sequence alignment of grass pea BBi and KTi revealed significant homology to protease inhibitors from several legumes. Real-time polymerase chain reaction analysis demonstrated high-level expression of BBi and KTi in dry seeds and weak expression in other organs. Our study demonstrates substantial variation in BBi and KTi among grass pea accessions that could be exploited in breeding programs for the development of grass pea lines that are devoid of these antinutritional factors.

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A Bowman‐Birk trypsin inhibitor with antiproliferative activity from Hokkaido large black soybeans

2007

Journal of Peptide Science

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A trypsin inhibitor, with an N-terminal sequence highly homologous to those of 8-kDa Bowman-Birk trypsin inhibitors, was isolated from the seeds of Hokkaido large black soybeans. The trypsin inhibitor was unadsorbed on SP-Sepharose but adsorbed on DEAE-cellulose and Mono Q. It inhibited proliferation in breast cancer (MCF-7) cells and hepatoma (Hep G2) cells with an IC50 of 35 and 140 microM, respectively. The trypsin inhibitory activity of the inhibitor was completely preserved after exposure to temperatures up to 100 degrees C for 30 min and to the pH range 2-13 for the same duration. The trypsin inhibitor inhibited HIV-1 reverse transcriptase with an IC50 of 38 microM, but was devoid of antifungal activity toward Fusarium oxysporum and Mycosphaerella arachidicola.

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Characterization of a Kunitz Trypsin Inhibitor with One Disulfide Bridge Purified from Swartzia pickellii

Cited by 46 publications

References 20 publications

Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

Lathyrus sativus Originating from Different Geographical Regions Reveals Striking Differences in Kunitz and Bowman–Birk Inhibitor Activities

A Bowman‐Birk trypsin inhibitor with antiproliferative activity from Hokkaido large black soybeans

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