Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

Lee, K. J.; Kim, J.-B.; Ha, Bo‐Keun; Kim, S. H.; Kang, Si-Yong; Lee, B.-M.; Kim, D. S.

doi:10.1007/s00726-011-1092-y

Cited by 6 publications

(7 citation statements)

References 28 publications

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“…Plant Kunitz inhibitors are characterized by molecular mass of about 20,000 Da (for the whole protein or a domain), low content of cysteine residues, and the presence of one or two reactive sites that are responsible for their inhibitory activity. They are frequently present in several polymorphic variants, exemplified by the STI isoforms, Tia and Tib, which differ by nine amino acids [12].…”

Section: Introductionmentioning

confidence: 99%

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

et al. 2013

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A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1′ pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.

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Section: Introductionmentioning

confidence: 99%

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

et al. 2013

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“…A total of 42% of these data correspond to storage proteins, as shown in Figure 1C. From TSP, 78% of the identified species correspond to glycinin and beta-conglycinin chains: GLYG1_SOYBN-Glycinin and Q549Z4_SOYBN-Proglycinin A2B1 correspond to 18% and 12% of TSP, respectively [33]; 13% correspond to inhibitors, including Q9ATY1_SOYBN kunitz type (2.35% of TSP) and Q9SBA9_SOYBN Bowman Birk proteinase inhibitor (0.06% of TSP) [34]; 16% correspond to energy-related proteins, such as C0J370_SOYBN Ribolose (0.33% TSP), Q42795_SOYBN beta-amylases (0.57%) and B3TDK4_SOYBN lipoxygenase (1.14% TSP); and 17% are associated with abiotic stress, including Q7XAW0_SOYBN dehydrin (1% TSP) and LEA proteins (<0.01 % TSP) [35]. An additional 13% correspond to putative uncharacterized proteins.…”

Section: Resultsmentioning

confidence: 99%

NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database

Murad

Rech

2012

BMC Biotechnol

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BackgroundRecombinant DNA technology has been extensively employed to generate a variety of products from genetically modified organisms (GMOs) over the last decade, and the development of technologies capable of analyzing these products is crucial to understanding gene expression patterns. Liquid chromatography coupled with mass spectrometry is a powerful tool for analyzing protein contents and possible expression modifications in GMOs. Specifically, the NanoUPLC-MSE technique provides rapid protein analyses of complex mixtures with supported steps for high sample throughput, identification and quantization using low sample quantities with outstanding repeatability. Here, we present an assessment of the peptide and protein identification and quantification of soybean seed EMBRAPA BR16 cultivar contents using NanoUPLC-MSE and provide a comparison to the theoretical tryptic digestion of soybean sequences from Uniprot database.ResultsThe NanoUPLC-MSE peptide analysis resulted in 3,400 identified peptides, 58% of which were identified to have no miscleavages. The experiment revealed that 13% of the peptides underwent in-source fragmentation, and 82% of the peptides were identified with a mass measurement accuracy of less than 5 ppm. More than 75% of the identified proteins have at least 10 matched peptides, 88% of the identified proteins have greater than 30% of coverage, and 87% of the identified proteins occur in all four replicates. 78% of the identified proteins correspond to all glycinin and beta-conglycinin chains.The theoretical Uniprot peptide database has 723,749 entries, and 548,336 peptides have molecular weights of greater than 500 Da. Seed proteins represent 0.86% of the protein database entries. At the peptide level, trypsin-digested seed proteins represent only 0.3% of the theoretical Uniprot peptide database. A total of 22% of all database peptides have a pI value of less than 5, and 25% of them have a pI value between 5 and 8. Based on the detection range of typical NanoUPLC-MSE experiments, i.e., 500 to 5000 Da, 64 proteins will not be identified.ConclusionsNanoUPLC-MSE experiments provide good protein coverage within a peptide error of 5 ppm and a wide MW detection range from 500 to 5000 Da. A second digestion enzyme should be used depending on the tissue or proteins to be analyzed. In the case of seed tissue, trypsin protein digestion results offer good databank coverage. The Uniprot database has many duplicate entries that may result in false protein homolog associations when using NanoUPLC-MSE analysis. The proteomic profile of the EMBRAPA BR-16 seed lacks certain described proteins relative to the profiles of transgenic soybeans reported in other works.

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“…However, information regarding on isoforms of proteolytic enzymes, isoelectric point of peptidases, and even a higher resolution of complex proteolytic profiles cannot be obtained by 1D zymographic systems. In superior eukaryotes, a broader analysis of functional peptidases has been achieved by combining zymographic techniques with proteomic technologies, specifically two-dimensional electrophoresis (2D) and mass spectrometry that enable a better resolution of peptidase arranges and the direct identification of peptidase species (Ong and Chang 1997;Park et al 2002;Zhao and Russell 2003;Wilkesman and Schröder 2007;Lee et al 2011). Nevertheless, this combined approach has been little used in the study of protozoan parasites ).…”

Section: Two-dimensional Zymography Coupled To Peptidase Identificatimentioning

confidence: 99%

“…This technique, coupled with mass spectrometry for protein identification make possible the broader mapping of active proteolytic enzymes present in a protein extract (Zhao et al 2004;De Jesus et al 2009;Saitoh et al 2007;Paes-Leme et al 2009;Larocca et al 2010;Lee et al 2011). Two main strategies are used for 2DZ analysis: the first one consist on the separation of protein sample by 2DZ or 2D reverse zymography in parallel with separation by denaturing or non-denaturing 2D followed by staining with MS compatible stain.…”

Section: Two-dimensional Zymography Coupled To Peptidase Identificatimentioning

confidence: 99%

“…Two main strategies are used for 2DZ analysis: the first one consist on the separation of protein sample by 2DZ or 2D reverse zymography in parallel with separation by denaturing or non-denaturing 2D followed by staining with MS compatible stain. After migration, the comparison and overlapping of both gel images, using appropriated gel image analysis software, allow the assigning of proteolytic spots to protein spots which are carefully excised from the gel and further identified by MS (Métayer et al 2002;Park et al 2002;Choi et al 2004;Taiyoji et al 2009;Lee et al 2011). Using this strategy our group identified active cysteine peptidases in whole extracts of the two Trichomonas vaginalis isolates exhibiting high and low virulence phenotypes (De Jesus et al 2007) (Figure 6).…”

Section: Two-dimensional Zymography Coupled To Peptidase Identificatimentioning

confidence: 99%

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Applications of Zymography (Substrate-SDS-PAGE) for Peptidase Screening in a Post-Genomic Era

d’Avila-Levy¹,

Santos²,

Cuervo³

et al. 2012

Gel Electrophoresis - Advanced Techniques

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Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

Cited by 6 publications

References 28 publications

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database

Applications of Zymography (Substrate-SDS-PAGE) for Peptidase Screening in a Post-Genomic Era

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