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. Author manuscript; available in PMC: 2023 May 30.
Published in final edited form as: Food Chem. 2021 Dec 29;377:131959. doi: 10.1016/j.foodchem.2021.131959

Amaranthus hypochondriacus seeds as a rich source of cysteine rich bioactive peptides

Tessa B Moyer a, Wyatt J Schug a, Leslie M Hicks a,*
PMCID: PMC8821138  NIHMSID: NIHMS1768928  PMID: 34995961

Abstract

Amaranthus hypochondriacus is a nutritious alternative grain native to Central and South America. Increased interest in the impact of A. hypochondriacus on the human body has driven characterization of bioactive secondary metabolites. The seeds are known to contain bioactive small molecules but little is known regarding endogenous peptides. Cysteine-rich peptides (CRPs) in foodstuffs are particularly relevant because they are stabilized by disulfide bonds enhancing resistance to digestion. Here, in silico predictions, proteomics, and simulated gastrointestinal digestions are leveraged to identify digestion resistant CRPs within A. hypochondriacus seeds. Thirteen in silico predicted CRPs were detected in a seed extract providing evidence for the translation of five CRP families. Mature forms of six CRPs were characterized via top-down proteomics revealing multiple post-translational modifications. All six peptides demonstrated resistance to simulated gastrointestinal digestion, suggesting that A. hypochondriacus CRPs may exhibit bioactivity after consumption and should be prioritized for further characterization.

Keywords: Amaranthus hypochondriacus, proteomics, mass spectrometry, peptidomics, cysteine rich peptides

Graphical Abstract

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1. INTRODUCTION

Plants produce a wide range of secondary metabolites which can exert bioactivity when consumed as food (e.g. antioxidant, anti-inflammatory, allergens) (Kumar et al., 2021; Tam et al., 2015). Therefore, compositional knowledge of bioactive compounds in foodstuffs is critical to understand their potential impact on the human body. In general, the effects of small molecules are better characterized than endogenous peptides which have functional roles within their species of origin (e.g. signaling and defense) (Matsubayashi, 2011; Tam et al., 2015) and can exert bioactivity upon consumption. While endogenous peptides can be susceptible to inactivation and degradation during gastrointestinal digestion, cysteine-rich peptides (CRPs) are stabilized by multiple disulfide bonds conferring stability against proteases, temperature, and pH (Tam et al., 2015). Despite this potential capacity for longer lasting bioactivity, CRPs are underexplored within functional foods (Huang et al., 2021). Furthermore, CRP activity could range from desirable (e.g. anti-inflammatory or advantageous antimicrobial activity in gut) to deleterious (e.g. unwanted antimicrobial activity in gut or allergenic) (Tam et al., 2015). Genome sequencing and improvements in in silico prediction algorithms are enabling higher throughput mass spectrometry-based characterization of CRPs in non-model plant species, though post-translational processing complicates accurate prediction of mature peptidoforms (Figure 1) (Moyer et al., 2021).

Figure 1.

Figure 1.

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Cysteine-rich peptides undergo a variety of processing steps in order to generate the mature bioactive peptide. First, they must be excised from their precursor proteins which can have a variety of structures. (A) One of the most common scenarios is that the precursor protein contains an N-terminal signal peptide (grey) which must be cleaved from the CRP domain (green). (B) Alternatively, the precursor can also contain can also contain a C-terminal pro-domain (blue) which adds an addition proteolytic processing requirement. (C) In other cases, multiple CRP domains are encoded within the same precursor and are joined by linker regions (purple). (D) After excision, CRPs are often modified to contain PTMs (stars) and disulfide bonds (black lines). Variable proteolytic processing can generate multiple CRPs from the same domain which contain different termini (dark green). CRPs derived from the same precursor domain that vary in PTMs or proteolytic processing are peptidoforms.

Amaranthus hypochondriacus (amaranth), once a staple crop of the Aztec empire, is an increasingly popular pseudo-cereal. It is known for its strong nutritional value, including high insoluble fiber, digestible protein, essential amino acids, and mineral content (Ballabio et al., 2011; Nardo et al., 2020). Further, desirable agricultural traits including drought/herbicide resistance make it an attractive crop for food-scarce regions (Deb et al., 2020; Lightfoot et al., 2017). Renewed interest in amaranth grain has encouraged broader characterization of its secondary metabolites, including bioactive peptides. The majority of bioactive peptides identified from A. hypochondriacus are digest products, or proteolytic peptides produced from larger, functional proteins (Ayala-Niño et al., 2019; Montoya-Rodríguez et al., 2014; Sandoval-Sicairos et al., 2021). Comparatively fewer studies have examined endogenous bioactive peptides. One targeted ELISA assay identified a 5 kDa anticancer lunasin-like peptide in A. hypochondriacus seeds (Silva-Sánchez et al., 2008). The sole CRP detected to date in A. hypochondriacus is AC-AMP2 (also known as Ay-AMP) (Rivillas-Acevedo & Soriano-García, 2007), a 3 kDa anti-fungal hevein-like peptide originally identified in the seeds of A. caudatus (Broekaert et al., 1992).

Herein, in silico prediction and proteomics were used to identify and characterize CRPs from A. hypochondriacus seeds. In silico prediction from the amaranth proteome revealed 89 putative CRPs, 13 of which were confirmed via bottom-up proteomics within a seed peptide extract. Mature peptidoforms of two known hevein-like peptides (AC-AMP1 and AC-AMP2) and four novel CRPs, including one hevein (Ay-AMP2), one defensin (Ay-DEF1), and two α-hairpinins (Ay-AMP3 and Ay-AMP4), were characterized via top-down peptidomics. Simulated gastrointestinal digestion revealed the CRPs were resistant to proteolytic cleavage. This study emphasizes that A. hypochondriacus seeds are a rich source of CRPs which may remain intact after digestion, exhibit bioactivity, and contribute to the utility of amaranth grain a functional food.

2. MATERIALS AND METHODS

2.1. CRP predictions.

A database containing the genomic proteome of A. hypochondriacus was accessed via Phytozome (v2.1; www.phytozome.net; 08/15/2018) (Clouse et al., 2016) and submitted to SignalP-5.0 (Almagro Armenteros et al., 2019) to identify proteins with signal peptides and predict cleavage sites. A FASTA file with these proteins after signal peptide cleavage was exported and submitted to Cysmotif Searcher (version 3.31) (A. A. Shelenkov et al., 2018) to predict CRPs in the A. hypochondriacus proteome. Translation of input sequences and SPADA analysis (Zhou et al., 2013) were excluded from the Cysmotif Searcher computational pipeline.

2.2. Peptide extraction and fractionation.

A. hypochondriacus seeds were purchased from Strictly Medicinal Seeds (Williams, USA). Peptide extracts were created as previously described (Moyer et al., 2019) with modifications noted herein. Briefly, seeds were extracted in an acetic acid solution (5 g seeds/150 mL) with size exclusion steps to remove large proteins (>30 kDa) and small molecules (<1 kDa). The crude extract was concentrated to 1.5 mL and fractionated via strong cation exchange chromatography (SCX) to remove neutral and negatively charged molecules and reduce sample complexity. Fractionation was performed by injecting 0.5 mL aliquots on a PolySulfethyl A column (100 mm × 4.6 mm, 3 μm particles, PolyLC, Columbia, USA). A 30 min gradient (0.5 mL/min) was used starting from 100% mobile phase A [5 mM ammonium formate (Honeywell Fluka, Charlotte, USA) 20% acetonitrile (VWR, Radnor, USA), pH 2.7] with the following steps: 0–5 min: 0% B (500 mM ammonium formate, 20% acetonitrile, pH 3.0); 5–17 min: 0–100% B; 17–30 min: 100% B. The first two 5-min fractions were discarded after which four 5-min fractions were collected from 10 – 30 min. SCX fractions were desalted using offline Sep-Pak C18 cartridges (Waters, Milford, USA, 500 mg) sequentially eluting in 80/20/0.1 water (VWR, Radnor, USA) /acetonitrile/formic acid (Sigma-Aldrich, Burlington, USA), 60/40/0.1 water/acetonitrile/formic acid, and 40/60/0.1 water/acetonitrile/formic acid. Sep-Pak eluate was collected, producing a total of 12 fractions (Supplementary Figure S1) and concentrated to dryness in a vacuum centrifuge. Fractions were resuspended in 30 μL LC-MS grade water for further analysis.

2.3. Reduction, alkylation, and trypsin digestion.

Reduced (dithiothreitol, Sigma-Aldrich, Burlington, USA) and alkylated (iodoacetamide, Sigma-Aldrich, Burlington, USA) or reduced, alkylated, and trypsin (Promega, Madison, USA) digested peptide fractions were prepared for LC-MS/MS as previously described (Moyer et al., 2021).

2.4. MS data acquisition and processing.

LC-MS/MS data for bottom-up proteomic and top-down peptidomic characterization of A. hypochondriacus seed fractions were acquired using an Acquity M-class UPLC system (Waters, Milford, USA) coupled to a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, USA) as previously described (Al-Mohanna et al., 2021). Collected raw MS data (*.raw) were converted to Mascot Generic Files (*.mgf) using ProteoWizard (Chambers et al., 2012).

In vitro gastrointestinal digestion samples were analyzed using a nanoAcquity UPLC (Waters, Milford, USA) coupled to a TripleToF 5600 (Sciex, Framingham, USA), as previously described (Moyer et al., 2021).

2.5. Database searching and antimicrobial peptide identification.

Database searching was performed against a database of A. hypochondriacus proteins (23,879 entries, v2.1; www.phytozome.net; 08/15/2018) appended with the sequences for common laboratory contaminants (www.thegpm.org/cRAP; 116 entries). Digests were searched (Matrix Science, version 2.5.1) against the aforementioned database and a decoy database using peptide/fragment mass tolerances 15 ppm/0.02 Da, trypsin specificity, three possible missed cleavages, a fixed modification of cysteine carbamidomethylation, and two variable modifications (N-terminal acetylation and methionine oxidation). Peptide false discovery rates (FDR) were adjusted to ≤1% using the Mascot Percolator algorithm (Brosch et al., 2009). Peptides with a Mascot score > 13, matching to A. hypochondriacus entries, and at least one unique tryptic peptide were considered for further analysis. Identified proteins were manually parsed for predicted CRPs. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaíno et al., 2016) with the data set identifier PXD025965 (Reviewer account details: Username: reviewer_pxd025965@ebi.ac.uk, Password: rtu7XPbq).

2.6. Spectral annotations.

Fragmentation spectra were annotated using peak lists exported from the FreeStyle (Thermo Scientific) spectral viewer and imported into the Interactive Peptide Spectral Annotator (Brademan et al., 2019). Annotated spectra were generated using appropriate peptide sequences, charges, and 10 ppm error. Fragment assignments were manually validated.

2.7. Identification of similar antimicrobial peptides and sequence alignments.

The sequences of Cysmotif Searcher predicted peptides that were identified in A. hypochondriacus seed fractions as classified as “Cys-rich” were compared to known the Uniprot database of reviewed plant proteins using the Basic Local Alignment Search Tool (BLAST) to identify any similarities with CRP (Bateman et al., 2021).

Sequences of predicted CRPs AH022535, Ay-AMP2, Ay-DEF1, Ay-AMP3, and Ay-AMP4 were submitted to the Antimicrobial Peptide Database APD3: Calculator and Predictor Tool (Wang et al., 2016) for alignment with AMPs with validated activity. Sequence alignment figures were prepared using the ClustalW Multiple Sequence Alignment tool to identify highly conserved residues (Madeira et al., 2019).

2.8. In vitro gastrointestinal digestion

In vitro gastrointestinal digestions of fractions containing AC-AMP1, AC-AMP2, Ay-AMP2, Ay-DEF1, Ay-AMP3, and Ay-AMP4 were conducted as previously described with the following modifications (Sandoval-Sicairos et al., 2021). 1 μg of human insulin was added to 1 μL of fraction and diluted to a final volume of 12.5 μL with water (pH 2). Porcine pepsin was added (Sigma-Aldrich, Burlington, USA, 1 μg) and incubated for 2 h (37 °C, 850 rpm, Thermal Sake Touch, VWR, Radnor, USA). After pepsin digestion the sample was concentrated to dryness, resuspended in 10 μL of water (pH 7.5), and incubated with pancreatin (Sigma-Aldrich, Burlington, USA, 1 μg) for 2 h (37 °C, 850 rpm). Samples were immediately acidified to pH < 3. In tandem, negative controls composed of 1 μg of insulin and 1 μL of fraction were subjected to all sample handling steps excluding the addition of proteases. All samples were desalted using Sep-Pak C18 column (Waters, Milford, USA, 50 mg) prior to LC-MS analysis.

3. RESULTS AND DISCUSSION

3.1. CRP prediction and proteomics profiling

SignalP and Cysmotif Searcher algorithms were used in concert to predict 89 CRPs within the A. hypochondriacus proteome (Almagro Armenteros et al., 2019; Clouse et al., 2016; A. A. Shelenkov et al., 2018) representing five peptide families (Supplementary Table S1). Prediction of 89 CRPs is congruent with a recent analysis of 1267 plant transcriptomes revealing approximately 50–150 CRPs per plant species (A. Shelenkov et al., 2020).

Tryptic digests of A. hypochondriacus seed fractions analyzed via LC-MS/MS produced 168 identifications (Supplementary Table S2) including 13 of the 89 predicted CRPs (Table 1). Comparison of each “Cys-rich” protein identified in the proteomics data with the Uniprot database of reviewed plant proteins revealed sequence similarity with known CRP families (Supplementary Table S3). Of note, the predicted protein reassigned as a hevein-like peptide (PA: AH003978) had sequence similarity with a hevein-like peptide precursor protein from Stellaria media (chickweed) containing two hevein domains connected by a linker sequence. Additionally, all predicted peptides re-categorized as α-hairpinins had similarities with vicilin-like proteins which can contain multiple α-hairpinin domains within a single precursor protein (Zhang et al., 2019).

Table 1.

Putative CRPs identified in seed fractions.

Accession of precursor protein Family Protein Score Predicted sequence
AH012328 α-hairpinin* 6574 FVPNQDEVQRELQQCIQRCQRERGQMGQMKCIHECAEEIAEESRGERRQYNPIGELQECIQRRCESQGGQQQMVKCVAECVREQTTGGRWESEEWIEEVIRRDPQQQYEECRSECERQGRRGQSQKWQCLKSCQEKYQEGERYGYGQQEEGSESWNQGHIHGDPRERYFECQQRCQRQGRGYECQRRCKQEMQGQREQVGRFDPQEECERRCQRQGRGYECIRSCQHEMRGQRGLVGRFDPQEECERKCQRQGRGYECQKRCQQGMRDEIERFDPQEECRQRCQRQGRGYECQKSCQREMQGQRGQVGRFDPLEECQQRCQRQGRGYECQRSCQQEIQGQRGQRGQVGRFDPQEQCKQRCQRQGRGFECQMRCQKEFQGQRDQPGRYTDEVENPQQEPLRRFQECQQRCQMKQGQRMGCERRCKEQLQRGNQS
AH019498 α-hairpinin* 2342 IRFPNQDSPIRELEQCVQRCQSQSGKQQLECVLECAEDIPSEQRRSNEAGPQRELQQCIHSCQREQGGEQQIECVHQCVEKVGKPGSNQYSELEQCVQRRCGSRQSRWERAECVQHCIEEIEGGGRKWVISDDDLLEAMINPYRPEQEQYEECRRDCERRGSRGQSQQWQCQKRCQEEFERESHGRRHFDNQAHFEPFQICQQRCQGKSTRSQRTQCQKQCMDQFQGERGLDIYVDDSENPDIYGICMDQCRRLPLSERDTCTWACSPLQFMVENPTRDPQQQKYRQCQQRCQRQGRGFECQMKCQQEFQGQRGQGSRFGFPEESENEYQDPQKRFQECQQRCQMQPRQQKMQCQKMCRQQLQTETGISPRQTILDSFLDLTAGWF
AH002580 α-hairpinin* 887 RFEEEQKLQECQHQCKQQRQFDSKDRHECERACERYIKEKERREKEHDQERERRSRDEIVGRMREREEEEEEEVGGESGAPYVFDEQHFETKFREQEGNVRVLKRFSKRSRLLEGIENYRVLIFEANPQTFVVPNHWDADVVLFVAQGEGTVSLVYTDRRESFNIERGHVMVIPAGVTAYLVNRGNNEKLVIVKLLNPVSNPSGKFETFFGAGGQNPQSFLNAFSTEILEAAYKTSGDRLKRIFSQQSEGAIIRASEEQISALTHEKSSHWPFGGKSSRDSGPIQLFRKDPKQSNAFGTLFETDFDDRRLGQQLQNLDIAVAFANITQGSMHTPYYNSRATKIAVVINGRGHFEMACPHVSKSGHSRHQQQHQHHQYRDGKTWRGDESTTPVHYERITAQLREGTVFVVPPGHPFVTLASEDQNLEVVCFEINAQNNHKFPLAGQRNIFKNFKREAQELAFASSAEEIERVFETQEEEFFFPGPRQQGGGRGRGRRGYYSII
AH012327 α-hairpinin* 383 IRLPNEDINYIEEKGGLKLLGFENLKNQGDDDRAYRAYLRCLEICEEVAPRPVCARKCVGSRRRNAHLELQEQHPECQKRCRKTETGYECEMWCYEQIPAQKK
AH011853 α-hairpinin* 132 KEREWDPHQKKDIQRRCEQECERQQPYFEEHLCRRKCEKSEYYDHDDHDDHINVRYDDDDYYSKEEYERCQQRCPGTGKKQWECQQMCKDEYEHAEPHHGHDHPHGGGGDKRYPQRHDYTHCLRECEDREEGFPRQRQCKLRCEEEFGERRERHDHQHRDDKRGHRKGVLGNINKSKDKKKNDNPYYFDSESFESIYSTQEGKMSVLQRFSEKSKLLLGIDRFRIGNAVITLLMQENRKTFNLERGDVLMIPAGTTAYLVNSHDDEKLEIAELLRPVNNLGGAGPKSIFNSFSPELLQAALNIPKEQLKKMLSQQREGAIIKASQEQVRSLAMSGRQFGESRDGPFKLLYEPLYSNEHGDFYEVTPNDYQPLQDLDVSIGYCNIKQGSMMAPHYNSRTTYVVLVEEGSGYIEMISPHAGSESNKKHSTSSKKYEKIRSRLSKGDVFVIPAGYPIILVASEDEFRTIGFGINAQNNQRNFLAGKENIINQLDEEAMELTFNMAAREVEEMFQQQGQSYFMAAPQESKKEDLVSILDFAGF
AH015980 Snakin* 35 VEVTPPAPQASAPSDPFPPAPAVAPYPPKSPFVAPYPPKSPVVAPYPPKSPVIAPYPPKSPVVAPYPPKAPSIAPYPPKAPVVAPYPPKAPVVAPYPPKAPVVAPYPPKAPIVAPTTPYYGHLPPVKKEDCVPLCEKRCSLHSRKRLCVRACSTCCLRCKCVPPGTYGNKEVCGRCYTDMTTHGGRLKCP
AH002202 Snakin 88 MESETTSNHVGSNTGNVSESKIDCGAACAVRCSATKRPNLCKRACGSCCSRCNCVPPGTSGNYEACPCYAGLTTHGTRKKCP
AH013438 Snakin 29 VFFSSPQTPAQQPAAAAAPDGFCDGKCSVRCKLKGRDSRCFKYCIMCCGKCRCVPSGTSGNLNECPCYRDWKSPNGRPKCP
AH012467 Lipid transfer protein 37 AMNCGLVTKNLAQCLSYLTSPGGAPAAACCNGIKTLNNMASTPADRKTACTCLKSAANSMKKMDYAKAAGLPSKCGVRIPYAISPKTDCSRFVLAAY
AH003978 Hevein* 495 GYQCGWQSGGKRCSGGLCCSRYGFCGTTPEYCGRGQCQSQCLLNITDKQVQAQAQQEAAQSNDVPKTRKIPAMKSVALIALLLILLMGKSRAGFRCGKLAHNSTCPVGFCCSMSGYCGTTPEFCGKDRCHSQCTSSNNIGAIKSRKIPTDKLTTVKQFAP
AH022535 Hevein 2694 VGECVRGRCPSGMCCSQFGYCGKGPKYCGRASTTVDHQADVAATKTAQNPTDAKLAGAGSP
AH008931 Defensin 610 ATCAKPSKYFKGVCGGNGACRNACSREGWPSGRCIGPKVLIFQKCQCERPC
AH003396 Defensin 14 SRIDKVEVVPEMGQKQVFNLCSPMPYCNLPECRIRCGLDAKNSHCSGNHVCCCQS
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Tryptic peptide identified in Mascot search are red. Asterisks indict proteins which were originally classified as “Cys-rich” but were manually reassigned

Proteomic analysis of A. hypochondriacus seeds thus provided direct evidence for the translation of a wide variety of CRPs. However, it is still necessary to characterize intact mature peptidoforms, especially in cases where peptide excision from the precursor protein requires multiple proteolytic cleavage events or PTMs (Figure 1).

3.2. Characterization via top-down peptidomics

A general strategy was implemented to identify possible intact, mature sequences of putative CRPs identified via bottom-up proteomics. First, varying N- and C-terminal cleavages were predicted in silico for each peptide. Then, the theoretical monoisotopic mass of each possible peptidoform was calculated assuming the formation of the proper number of peptide family specific conserved disulfide bonds. A. hypochondriacus seed fractions were analyzed intact via a top-down peptidomics strategy to detect predicted peptide masses. This approach led to the identification of three hevein-like peptides (AC-AMP1, AC-AMP2, Ay-AMP2), one defensin (Ay-DEF1) and two α-hairpinins (Ay-AMP3 and Ay-AMP4).

3.2.1. Hevein-like peptide characterization

Hevein-like peptides are ~3–4.5 kDa CRPs expressed in precursor proteins composed of variable domains which are processed to form mature peptides with six-, eight-, or ten-Cys (three to five disulfide bonds) (Slavokhotova et al., 2017). Hevein-like peptides exhibit a variety of activities but are most often associated with antifungal activity as they contain a conserved chitin binding motif which targets the fungal cell wall (Kini et al., 2017). Additionally, hevein domain containing proteins can contribute to food allergies (Barre et al., 2020).

Two precursor proteins with hevein Cys-motifs were identified in A. hypochondriacus seed fractions via bottom-up proteomics (Table 1). The peptide predicted from A. hypochondriacus protein AH022535 contained a six-Cys hevein motif and was expected to form three conserved disulfide bonds (CysI-CysIV, CysII-CysV, CysIII-CysVI) (Slavokhotova et al., 2017). Sequence comparison with hevein-like peptides deposited in the Antimicrobial Peptide Database (APD) (Wang et al., 2016) revealed that AH022535 contained the sequences of AC-AMP1/AC-AMP2 originally identified in A. caudatus (Broekaert et al., 1992) (Supplementary Figure 2A). AC-AMP1/AC-AMP2 are peptidoforms produced from the same precursor, varying only in the C-terminal cleavage site (Broekaert et al., 1992). In our analysis, both mature peptides were detected intact and their identity was confirmed by HCD fragmentation of the reduced and alkylated peptides (Supplementary Figure 2B–G).

The other hevein identifier, AH003978, contained two eight-Cys hevein motifs (Figure 2A). No tryptic peptides from the second hevein motif (Cys96 – Cys133) were identified by bottom-up proteomics, suggesting that only the first motif (Cys4 – Cys41) was processed to produce a mature hevein-like peptide, deemed Ay-AMP2. Ay-AMP2 was detected with four disulfide bonds and leader peptide cleavage between residues Cys41 | Leu42 of the predicted sequence [theoretical monoisotopic mass (MW−0) 4352.667 Da, Figure 2B]. Subsequent reduction and alkylation produced a mass shift of 464.234 Da confirming the presence of four disulfide bonds (MW−0 4820.348 Da, Figure 2C). MS/MS sequencing of reduced and alkylated Ay-AMP2 verified the primary sequence (Figure 2D). Ay-AMP2 is the first eight-Cys hevein identified in an Amaranthus spp. Sequence comparison of Ay-AMP2 with hevein-like peptides deposited in the APD confirmed that Ay-AMP2 shares a chitin binding and Cys-motif with other eight-Cys hevein-like peptides (Figure 2E).

Figure 2.

Figure 2.

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(A) The predicted CRP derived from AH003978 contains two 8-Cys hevein domains (green) connect by a linker regions (B) Ay-AMP2, produced when the first hevein domain is cleaved from the precursor protein between Cys41 and Leu42 and the formation four disulfide bonds, was detected during top-down peptidomic analysis of fraction 5. (C) Ay-AMP2 was reduced with DTT and alkylated using IAM producing a 464.2344 Da mass shift and confirming the presence of four disulfide bonds. (D) HCD fragmentation of reduced and alkylated Ay-AMP2 produced 53 percent sequence coverage (21/40 bonds). (D) Sequence alignment of Ay-AMP2 with similar hevein-like peptides from the APD (including percent sequence similarity with Ay-AMP2) confirmed a conserved Cys-motif and chitin binding motif (grey, SXΦXΦCGX4Φ, where X represents any amino acid and Φ represents Trp, Tyr or Phe). Cysteines forming disulfide bonds are green, basic residues are red, and acidic residues are blue. Asterisks note fully conserved residues, two dots note positions with highly similar residues, and single dots note positions with weakly similar residues.

3.2.2. Defensin characterization

Defensins are an extensively studied AMP family with more than 1200 known members from plant species (Shafee et al., 2016). They are approximately 50 residues in length and typically contain four conserved disulfide bonds (CysI–CysVIII, CysII–CysV, CysIII–CysVI, and CysIV–CysVII) (Shafee et al., 2016). The mature peptides form a characteristic antiparallel β-sheet bound to an α-helix by two disulfide bonds and contain a highly conserved γ-core motif (Vriens et al., 2014). Although plant defensin antifungal activity is the most well studied, they can also exhibit other functions such as antimicrobial activity and α-amylase inhibition (Sathoff & Samac, 2019).

Two defensins were identified in seed fractions via bottom-up proteomics (Table 1), but only one was detected intact. Top-down analysis of seed fractions identified a peptide which corresponded to the mass of the predicted defensin in protein AH008931 with four disulfide bonds and no additional proteolytic processing (MW−0 5455.510 Da, Figure 3A), deemed Ay-DEF1. Reduction and alkylation produced a mass shift of 464.234 Da (MW−0 5919.729 Da, Figure 3B) thus supporting the presence of four disulfide bonds. Fragmentation of the reduced and alkylated peptide confirmed its identity (Figure 3C). Ay-DEF1 was aligned with the five most similar plant defensins in the APD and Atr-DEF1 (Figure 3D). Atr-DEF1, which is not yet included in the APD and was identified in an A. tricolor aerial tissue (Moyer et al., 2021), is the only other defensin from Amaranthus spp. whose mature peptidoform has been characterized. As anticipated, these peptides contain the conserved defensin cysteine motif which are expected to form four highly conserved disulfide bonds and the defensin γ-core motif. Ay-DEF1 is the first defensin characterized in the seeds of Amaranthus spp.

Figure 3.

Figure 3.

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(A) Mature Ay-DEF1 includes four disulfide and was detected during top-down peptidomic analysis of fraction 8. (B) Ay-DEF1 was reduced with DTT and alkylated with IAM resulting in a 464.220 Da mass shift and confirming the presence of four disulfide bonds. (C) MS/MS fragmentation of reduced and alkylated Ay-DEF1 resulted in 26 percent sequence coverage of Ay-DEF1 (13/50 bonds). (D) Sequence alignment of Ay-DEF1 with similar defensins from the APD (including percent sequence similarity with Ay-DEF1) and A. tricolor defensin Atr-DEF1 supported the presence of a conserved Cys-motif and γ-core motif (GXCX3–9C, where Xn is the number of residues between cysteines, shaded grey). Cysteines forming disulfide bonds are green, basic residues are red, and acidic residues are blue. The γ-core motif is shaded grey. Asterisks note fully conserved residues, two dots note positions with highly similar residues, and single dots note positions with weakly similar residues.

3.2.3. α-Hairpinin characterization

The α-hairpinins (AKA: vicilin-buried peptides) are a family of CRPs with more than 20 known members that have been identified in edible species such as Zea mays, Macadamia integrifolia, and Triticum kiharae (Slavokhotova & Rogozhin, 2020; Zhang et al., 2019). Antibacterial, antifungal, and trypsin inhibition are the most common activities reported for α-hairpinins (Slavokhotova & Rogozhin, 2020). Precursor proteins often encode multiple α-hairpinin motifs which are proteolytically released to form two antiparallel α-helices stabilized by a pair of disulfide bonds (CysI-CysIV and CysII-CysIII) (Slavokhotova & Rogozhin, 2020). α-Hairpinins can be further modified to contain pyroglutamic acid or hydroxyproline residues (Rogozhin et al., 2018; Slavokhotova & Rogozhin, 2020).

In total, two intact α-hairpinins derived from two precursors (AH012328 and AH019498) were identified in A. hypochondriacus seeds (Figure 4A and B). Ay-AMP3 was derived from the second α-hairpinin motif of AH012328 by N-terminal cleavage at Ile270 | Glu271, C-terminal processing at Glu299 | Met300, and conversion of Glu271 to pyroglutamic acid (MW−0 3609.557 Da). The second α-hairpinin motif of A. hypochondriacus protein AH019498 produced Ay-AMP4 as a result of C-terminal cleavage at Gly83 | Lys84, N-terminal cleavage at Arg53 | Glu54, and conversion of Glu54 to pyroglutamic acid (MW−0 3687.640 Da). Each of these α-hairpinins were identified in A. hypochondriacus seed fractions and reduction and alkylation produced 232.117 Da mass shifts confirming the presence of two disulfide bonds (Figure 4C–F). Their identities were confirmed by HCD fragmentation of the reduced and alkylated peptides (Figure 4G and H). The sequences of A. hypochondriacus α-hairpinins were compared with those deposited in the APD (Figure 4I) (Wang et al., 2016). Overall, sequence alignment between A. hypochondriacus α-hairpinins was poor suggesting high sequence diversity.

Figure 4.

Figure 4.

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(A) AH012328 contained ten α-hairpinin motifs. The seventh motif (orange) is processed to produced Ay-AMP3. (B) AH019498 contained eight α-hairpinin motifs the second (pink) of which was cleavage to yield Ay-AMP4. Yellow stars indicate pyroglutamic acid. Top-down analysis of seed fractions revealed (C) Ay-AMP3 (fraction 8), and (D) Ay-AMP4 (fraction 6). Reduced and alkylated (RA) (E) Ay-AMP3 and (F) Ay-AMP4. MS/MS fragmentation of reduced and alkylated (G) Ay-AMP3 and (H) Ay-AMP4. (I) Sequence alignment of A. hypochondriacus α-hairpinins with similar α-hairpinins from the APD. Cysteines are green, basic residues are red, and acidic residues are blue. Asterisks note fully conserved residues and single dots note positions with weakly similar residues. Bolded residues are modified to pyroglutamic acid.

3.3. Stability assessment through simulated gastrointestinal digestion

General resistance to proteolysis is widely reported for CRPs but rarely examined within the context of foodstuffs and gastrointestinal digestion (Huang et al., 2021; Tam et al., 2015). NaD1 (defensin), SBI6 (defensin) and hevein (hevein-like) have demonstrated resistance in in vitro gastrointestinal digestions providing direct evidence that peptides from these families may impact the bioactivity of foods. (Parisi et al., 2020; Yagami et al., 2000). Peptide fractions containing AC-AMP1, AC-AMP2, Ay-AMP2, Ay-DEF1, Ay-AMP3, and Ay-AMP4 were subjected to in vitro gastrointestinal digestion with pepsin and pancreatin. Exogenous human insulin was added to each fraction as a positive control to confirm protease activity. All six A. hypochondriacus peptides were detected intact post-proteolysis (Figure 5 A–E) while human insulin was eliminated (Figure 5F) This preliminary screening provides evidence for resistance to degradation after consumption and the potential for prolonged activity.

Figure 5.

Figure 5.

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Extracted ion chromatograms (XIC) of (A) AC-AMP1, (B) AC-AMP2, (C) Ay-AMY2, (D) Ay-DEF1, (E) Ay-AMP3, and (F) Ay-AMP4 before and after simulated gastrointestinal digestion. Human insulin was added to each fraction to confirm protease activity. (G) A representative insulin XIC confirms protease activity.

4. CONCLUSIONS

Bioactive CRPs whose disulfide bonds enhance stability in gastrointestinal digestive conditions are underexplored in foodstuffs. This study details the first untargeted analysis of CRPs from A. hypochondriacus seeds. Cysteine motif-based algorithms were used to predict 89 putative CRPs within the A. hypochondriacus proteome. Bottom-up proteomics provided evidence for the translation of 13 of the predicted AMPs. Further analysis of intact samples facilitated the mature sequence characterization of four novel peptides (Ay-AMP2, Ay-DEF1, Ay-AMP3 and Ay-AMP4) including enumerating disulfide bonds, accurate proteolytic maturation, and modified termini. Gastrointestinal digestion simulation suggested that AC-AMP1, AC-AMP2, Ay-AMP2, Ay-DEF2, Ay-AMP3, Ay-AMP4 have enhanced stability. These peptides have the potential to evade digestion and exert a range of bioactivities. For example, hevein-like peptides such as Ay-AMP2 can have a wide range of activities including antifungal and allergenicity. Understanding the activities of this and other CRPs detailed herein could provide key insights into the impact of A. hypochondriacus flour on the gut microbiome or potential allergic reactions to such products. Further studies on isolated CRPs will yield insight into the impact of A. hypochondriacus food products after consumption as well as the potential value of each peptide as a purified product.

Supplementary Material

1

Supplementaryfile_1.doc: Contains Supplementary Figures S1 and S2 describing the generation of seed fractions and characterization of known hevein-like peptides.

2

Supplementaryfile_2.xls: Contains Supplementary Tables S1–S3 listing predicted CRPs, Mascot identifications, and sequence comparisons of CRPs with known proteins.

HIGHLIGHTS.

  • Mass spectrometric analysis of Amaranthus hypochondriacus seed extracts reveals cysteine-rich peptide (CRPs) from five families.

  • CRPs from three families (hevein-like peptides, defensins, and α-hairpinins) were characterized intact revealing multiple post-translational modifications which may improve stability.

  • A simulated gasterointestinal digestion demonstrated that CRPs were resistance to proteolytic digestion increasing the likelihood that they can exert bioactivity following the consumption of A. hypochondriacus foodstuffs.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (R01 GM125814), NSF Graduate Research Fellowship program (DGE-1650016), an NSF Major Research Instrumentation award (CHE-1726291) for the Q Exactive HF-X mass spectrometer, and the American Chemical Society Division of Analytical Chemistry Graduate Fellowship program.

Footnotes

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DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT AUTHOR STATEMENT

Tessa B. Moyer: Conceptualization, Investigation, Writing – Original Draft. Wyatt J. Schug: Investigation, Visualization, Writing – Review & Editing. Leslie M. Hicks: Conceptualization, Supervision, Writing – Review & Editing

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplementaryfile_1.doc: Contains Supplementary Figures S1 and S2 describing the generation of seed fractions and characterization of known hevein-like peptides.

NIHMS1768928-supplement-1.docx (758KB, docx)
2

Supplementaryfile_2.xls: Contains Supplementary Tables S1–S3 listing predicted CRPs, Mascot identifications, and sequence comparisons of CRPs with known proteins.

NIHMS1768928-supplement-2.xlsx (125.9KB, xlsx)

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