Identification of Echinacea Purpurea (L.) Moench Root LysM Lectin with Nephrotoxic Properties
Abstract
:1. Introduction
2. Results
2.1. Protein Extraction, Lectin Purification, and Evaluation of Hemagglutinating Activity
2.2. Biochemical Characterisation of Purified Lectin
2.2.1. Hemagglutinating Activity Inhibition by Carbohydrates Test Results
2.2.2. Purified Hemagglutinating Active Fraction Analysis by SDS-PAGE and Western Blot
2.2.3. Lectin Identification
2.3. Purified LysM Lectin Impact to Kidney Morphology
3. Discussion
4. Materials and Methods
4.1. Herbal Material Preparation
4.2. Preparation of Crude Extract and Protein Precipitation
4.3. Lectin Separation by Affinity Chromatography
4.4. Biochemical Characterisation of Purified Lectin
4.4.1. Hemagglutination Assay
4.4.2. Hemagglutination Inhibition Assay
4.4.3. SDS-PAGE and Western Blotting
4.4.4. Protein In-Gel Digestion, Sample Preparation, and Mass Spectrometry Analysis
4.5. Evaluation of LysM Lectin Biological Activity In Vivo
4.5.1. Animals
4.5.2. Experimental Model
4.5.3. Histological Analysis of Kidney Samples
4.6. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
NCBI Blast Annotation | ||||||||
---|---|---|---|---|---|---|---|---|
Database Accession | MW, kDa | pI | Mascot Score | Number of Peptides | SC, % | RMS90, ppm | NCBI RefSeq Accession | Protein Name |
Echinacea purpurea transcriptome | ||||||||
epa_locus_7790_iso_1_len_1277_ver_2 | 40.1 | 4.5 | 184.6 | 2 | 9.5 | 6.21 | XP_023762850.1 | lysM domain-containing GPI-anchored protein 1-like |
epa_locus_17753_iso_4_len_2582_ver_2 | 82.3 | 5 | 431.0 | 6 | 8.8 | 5.51 | XP_022020643.1 | glycerophosphodiester phosphodiesterase GDPDL3-like |
Helianthus annus genome | ||||||||
Ha412v1r1_08g009630 | 43.3 | 4.7 | 182.0 | 2 | 8.8 | 6.21 | XP_021976452.1 | lysM domain-containing GPI-anchored protein 1-like |
Ha412v1r1_11g016880 | 37.6 | 6.3 | 259.2 | 2 | 5.4 | 4.09 | XP_021992913.1 | early nodulin-like protein 2 isoform X4 |
Appendix B
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Total Protein (mg) | Specific Activity a (HU, mg Protein−1) | Amount of Lectin b (μg mL−1) | Purification Fold c | |
---|---|---|---|---|
Crude extract | 90.58 ± 1.74 | 5.66 | 113.22 | 1 |
F60-80 | 11.38 ± 0.71 | 0.35 | 35.56 | 3.18 |
Purified LysM lectin | 0.04 ± 0.008 | 0.00204 | 1.02 | 111.00 |
Minimum Inhibitory Concentration (mM) | |
---|---|
Lactose | 3 |
D-mannose | 21 |
D-galactose | 21 |
Fructose | 42 |
Sucrose | 42 |
Maltose | 42 |
N-Acetylglucosamine | 42 |
Ribose | NI |
D-glucose | NI |
D-Xylose | NI |
Acetyl-galactosamine | NI |
Description | Max Score | Total Score | Query Coverage | E value | Percent Identity | Accession Number in NCBI Protein Database | |
---|---|---|---|---|---|---|---|
epa_locus_7790_iso_1_len_1277 | LysM domain-containing GPI-anchored protein [Helianthus annuus] | 676 | 676 | 96% | 0.0 | 90 | XP_021976452.1 |
LysM domain-containing GPI-anchored protein [Lactuca sativa] | 629 | 629 | 97% | 0.0 | 85 | XP_023762850.1 | |
LysM domain-containing GPI-anchored protein [Cynara cardunculus var. scolymus] | 646 | 646 | 99% | 0.0 | 83 | XP_024991685.1 |
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Balciunaite, G.; Haimi, P.-J.; Mikniene, Z.; Savickas, G.; Ragazinskiene, O.; Juodziukyniene, N.; Baniulis, D.; Pangonyte, D. Identification of Echinacea Purpurea (L.) Moench Root LysM Lectin with Nephrotoxic Properties. Toxins 2020, 12, 88. https://doi.org/10.3390/toxins12020088
Balciunaite G, Haimi P-J, Mikniene Z, Savickas G, Ragazinskiene O, Juodziukyniene N, Baniulis D, Pangonyte D. Identification of Echinacea Purpurea (L.) Moench Root LysM Lectin with Nephrotoxic Properties. Toxins. 2020; 12(2):88. https://doi.org/10.3390/toxins12020088
Chicago/Turabian StyleBalciunaite, Gabriele, Perttu-Juhani Haimi, Zoja Mikniene, Girius Savickas, Ona Ragazinskiene, Nomeda Juodziukyniene, Danas Baniulis, and Dalia Pangonyte. 2020. "Identification of Echinacea Purpurea (L.) Moench Root LysM Lectin with Nephrotoxic Properties" Toxins 12, no. 2: 88. https://doi.org/10.3390/toxins12020088
APA StyleBalciunaite, G., Haimi, P. -J., Mikniene, Z., Savickas, G., Ragazinskiene, O., Juodziukyniene, N., Baniulis, D., & Pangonyte, D. (2020). Identification of Echinacea Purpurea (L.) Moench Root LysM Lectin with Nephrotoxic Properties. Toxins, 12(2), 88. https://doi.org/10.3390/toxins12020088