Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars

Sagu, Sorel Tchewonpi; Landgräber, Eva; Rackiewicz, Michal; Huschek, Gerd; Rawel, Harshadrai M.

doi:10.3390/molecules25245982

Cited by 9 publications

(10 citation statements)

References 51 publications

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“…The extraction protocol using ammonium bicarbonate buffer containing urea is the one optimized and applied in our previous work [ 28 , 49 , 50 ]. Briefly, 100 mg of each defatted nut sample were mixed with 1.5 mL of extraction buffer (100 mM ammonium bicarbonate containing 4 M urea) and the whole was shaken at 95 rpm at room temperature for 60 min, followed by the centrifugation (10,000× g , 4 °C for 5 min).…”

Section: Methodsmentioning

confidence: 99%

“…Candidates with less than 95% protein sequence similarity to other species were in-silico digested with the Skyline software using trypsin as enzyme. The additional settings are described in our previous works [ 28 , 49 , 50 ]. The species selectivity of the identified individual nut peptides was analyzed once more by the BLAST algorithm (UniProt Universal Protein Resource, ; accessed on 13 June 2021).…”

Section: Methodsmentioning

confidence: 99%

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Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

et al. 2021

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The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

et al. 2021

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“…The CM-proteins are small proteins of 12-16 kDa containing four to five well-conserved disulfide bonds and can be either monomeric or composed of two or four subunits (Carbonero and García-Olmedo, 1999). They are found in seeds of a wide range of cereal crops; wheat, barley, oats, rye, finger millet, barnyard millet, corn, rice, and sorghum (Garcia-Olmedo et al, 1987;Feng et al, 1991;Chen et al, 1992;Maskos et al, 1996;Carbonero and García-Olmedo, 1999;Altenbach et al, 2011;Wang et al, 2014;Gadge et al, 2015;Gazza et al, 2016;Panwar et al, 2018;Sagu et al, 2020). Many isoforms have been identified for example in barley and wheat (Østergaard et al, 2004;Guo et al, 2016;Bose et al, 2020;di Francesco et al, 2020;Geisslitz et al, 2020).…”

Section: General Characteristics Occurrence and Phylogeny Of Cm-proteinsmentioning

confidence: 99%

Structure, Function and Protein Engineering of Cereal-Type Inhibitors Acting on Amylolytic Enzymes

Møller

Svensson

2022

Front. Mol. Biosci.

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Numerous plants, including cereals, contain seed proteins able to inhibit amylolytic enzymes. Some of these inhibitors, the CM-proteins (soluble in chloroform:methanol mixtures)—also referred to as cereal-type inhibitors (CTIs)—are the topic of this review. CM-proteins were first reported 75 years ago. They are small sulfur-rich proteins of the prolamine superfamily embracing bifunctional α-amylase/trypsin inhibitors (ATIs), α-amylase inhibitors (AIs), limit dextrinase inhibitors (LDIs), and serine protease inhibitors. Phylogenetically CM-proteins are predicted across poaceae genomes and many isoforms are identified in seed proteomes. Their allergenicity and hence adverse effect on humans were recognized early on, as were their roles in plant defense. Generally, CTIs target exogenous digestive enzymes from insects and mammals. Notably, by contrast LDI regulates activity of the endogenous starch debranching enzyme, limit dextrinase, during cereal seed germination. CM-proteins are four-helix bundle proteins and form enzyme complexes adopting extraordinarily versatile binding modes involving the N-terminal and different loop regions. A number of these inhibitors have been characterized in detail and here focus will be on target enzyme specificity, molecular recognition, forces and mechanisms of binding as well as on three-dimensional structures of CM-protein–enzyme complexes. Lastly, prospects for CM-protein exploitation, rational engineering and biotechnological applications will be discussed.

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“…Damougari and S35 sorghum cultivars (Sorghum bicolor L. Moench) were obtained from the Institute of Research and Agronomic Development, IRAD, Maroua, Cameroon. Both sorghum and wheat samples were selected on the basis of their ATI content, determined by mass spectrometry according to the methods reported in our previous works [30,39]. Wheat Siyazan and sorghum Damougari contained low amounts of ATIs, whereas wheat Esperya and sorghum S35 samples had high ATI contents.…”

Section: Biological Materials and Chemicalsmentioning

confidence: 99%

“…Table 1 presents the main characteristics of the selected samples. * total protein content of samples was evaluated according to the Kjeldahl method (N × 6.25); ** ATI amounts are given in terms of relative content, as described in our previous works [30,39]. Tables S1-S3 (Supplementary Data) present the optimized conditions of the HPLC-MS/MS method used for the quantification of wheat, sorghum and rice ATIs.…”

Section: Biological Materials and Chemicalsmentioning

confidence: 99%

Effect of Cereal α-Amylase/Trypsin Inhibitors on Developmental Characteristics and Abundance of Digestive Enzymes of Mealworm Larvae (Tenebrio molitor L.)

Sagu

Landgräber

Henkel³

et al. 2021

Insects

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The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.

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Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars

Cited by 9 publications

References 51 publications

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

Structure, Function and Protein Engineering of Cereal-Type Inhibitors Acting on Amylolytic Enzymes

Effect of Cereal α-Amylase/Trypsin Inhibitors on Developmental Characteristics and Abundance of Digestive Enzymes of Mealworm Larvae (Tenebrio molitor L.)

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