Isolation of Cysteine-Rich Peptides from Citrullus colocynthis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material and Extraction
2.2. Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) Purification
2.3. Reduction, Alkylation and Enzymatic Digestion of Peptides
2.4. MALDI-TOF/TOF Analysis and De Novo Peptide Sequencing
2.5. Amino Acid Composition Analysis
2.6. Trypsin, α-Chymotrypsin and α-Amylase Inhibitory Assays
2.7. Sequence Homology Analysis
3. Results and Discussion
3.1. Preparation of Peptide-Enriched Citcol Extracts
3.2. Reversed-Phase HPLC Fractionation and Purification
3.3. De Novo Sequencing of Citcol Peptides by MALDI-TOF/TOF
3.4. Genome Mining and Sequence Homology Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name | [M + H]+1 | Sequence 2 | Length |
---|---|---|---|
loop 1 2 3 4 5 | |||
citcol-1 | 3658.1 | ----VCLFVGKPCWSDADCPSGCSCKPLPLIDAGYCGFL | 35 aa |
citcol-2 | 3734.1 | ----VCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | 35 aa |
citcol-3 | 3814.4 | --VGVCLFVGKPCWSDADCPSGCSCKPLPLIDAGYCGFL | 37 aa |
citcol-4 | 3890.2 | --VGVCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | 37 aa |
citcol-5 | 4046.9 | -RVGVCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | 38 aa |
citcol-6 | 4084.1 | NRVGVCLFVGKPCWSDADCPSGCSCKPLPLIDAGYCGFL | 39 aa |
citcol-7 | 4101.2 | NRVGVCLFVGKPCKSDANCPSGCYCKPLPLIDAGYCGFL | 39 aa |
citcol-8 | 4160.3 | NRVGVCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | 39 aa |
********* ***:***** *************** |
Species | Sequence 2 | E-Value 3 | Accession No |
---|---|---|---|
C. colocynthis (citcol-8) | NRVGVCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | n.a. | this work |
Citrullus lanatus (chr10 4) | NRVGVCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | 3 × 10−23 | VOOL01000010.1 |
Cucumis melo (chr7 4) | -RLGVCLLVGKPCMSDADCPSGCYCKPVPLLDIGYCGFL | 5 × 10−16 | LN713261.1 |
*:****:***** *************:**:* ****** |
Species | Sequence | Uniprot ID | Function | Cys Loops Pattern | Similarity (%) 1 | Reference |
---|---|---|---|---|---|---|
C. colocynthis (citcol-8) | NRVGVCLFVGKPCWSDADCPSGCYCKPLPLIDAGYCGFL | n.a. | n.a. | CX6CX5CX3CX1CX10C 2 | n.a. | this work |
Momordica cochinchinensis | QRACPRILKKCRRDSDCPGECICKENGYCG | P82410 | trypsin inhibitor | CX6CX5CX3CX1CX5C | 43.6 | [51] |
Solanum tuberosum | HADPICNKPCKTHDDCSGAWFCQACWNSARTCGPY | P01075 | metallocarboxy peptidase inhibitor | CX3CX5CX5CX2CX6C | 38.5 | [52] |
Phytolacca americana | AGCIKNGGRCNASAGPPYCCSSYCFQIAGQSYGVCKNR | P81418 | antimicrobial peptide | CX6CX8CCX3CX10C | 31.7 | [53] |
Spinacia oleracea | EDKCSPSGAICSGFGPPEQCCSGACVPHPILRIFVCQ | P84781 | trypsin inhibitor | CX6CX8CCX3CX10C | 29.5 | [54] |
Amaranthus hypochondriacus | CIPKWNRCGPKMDGVPCCEPYTCTSDYYGNCS | P80403 | α-amylase inhibitor | CX6CX8CCX4CX7C | 27.3 | [55] |
Amaranthus retroflexus | AGECVQGRCPSGMCCSQFGYCGRGPKYCGR | Q5I2B2 | antimicrobial peptide | CX4CX4CCX5CX6C | 26.7 | [17] |
Astragalus sinicus | TYSCGGHIDCKDFCKSEGYRGFKCTPKKTCTCFH | Q07A30 | nodule specific protein | CX5CX3CX9CX5CX1C | 20.0 | [56] |
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Shahin-Kaleybar, B.; Niazi, A.; Afsharifar, A.; Nematzadeh, G.; Yousefi, R.; Retzl, B.; Hellinger, R.; Muratspahić, E.; Gruber, C.W. Isolation of Cysteine-Rich Peptides from Citrullus colocynthis. Biomolecules 2020, 10, 1326. https://doi.org/10.3390/biom10091326
Shahin-Kaleybar B, Niazi A, Afsharifar A, Nematzadeh G, Yousefi R, Retzl B, Hellinger R, Muratspahić E, Gruber CW. Isolation of Cysteine-Rich Peptides from Citrullus colocynthis. Biomolecules. 2020; 10(9):1326. https://doi.org/10.3390/biom10091326
Chicago/Turabian StyleShahin-Kaleybar, Behzad, Ali Niazi, Alireza Afsharifar, Ghorbanali Nematzadeh, Reza Yousefi, Bernhard Retzl, Roland Hellinger, Edin Muratspahić, and Christian W. Gruber. 2020. "Isolation of Cysteine-Rich Peptides from Citrullus colocynthis" Biomolecules 10, no. 9: 1326. https://doi.org/10.3390/biom10091326
APA StyleShahin-Kaleybar, B., Niazi, A., Afsharifar, A., Nematzadeh, G., Yousefi, R., Retzl, B., Hellinger, R., Muratspahić, E., & Gruber, C. W. (2020). Isolation of Cysteine-Rich Peptides from Citrullus colocynthis. Biomolecules, 10(9), 1326. https://doi.org/10.3390/biom10091326