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Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors

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Abstract

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural–functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI’s optimization and functional differentiation for future biotechnological applications.

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References

  1. Rane AS, Venkatesh V, Joshi RS, Giri AP (2020) International journal of biological macromolecules molecular investigation of coleopteran specific α-amylase inhibitors from amaranthaceae members. Int J Biol Macromol 163:1444–1450. https://doi.org/10.1016/j.ijbiomac.2020.07.219

    Article  CAS  PubMed  Google Scholar 

  2. Lu S, Deng P, Liu X et al (1999) Solution structure of the major α–amylase inhibitor of the crop plant amaranth *. J Biol Chem 274:20473–20478

    Article  CAS  PubMed  Google Scholar 

  3. Pereira PJB, Lozanov V, Patthy A et al (1999) Specific inhibition of insect α-amylases: yellow meal worm α-amylase in complex with the Amaranth α-amylase inhibitor at 2.0 Å resolution. Structure 7:1079–1088. https://doi.org/10.1016/S0969-2126(99)80175-0

    Article  CAS  PubMed  Google Scholar 

  4. Molesini B, Treggiari D, Dalbeni A et al (2017) Plant cystine-knot peptides: pharmacological perspectives. Br J Clin Pharmacol 83:63–70. https://doi.org/10.1111/bcp.12932

    Article  CAS  PubMed  Google Scholar 

  5. Rane AS, Joshi RS, Giri AP (2020) Molecular determinant for specificity: differential interaction of α-amylases with their Proteinaceous inhibitors. Biochim Biophys Acta (BBA)- General Subj. 1864:129703

    Article  CAS  Google Scholar 

  6. Bhide AJ, Channale SM, Yadav Y et al (2017) Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species. Plant Mol Biol 94:319–332. https://doi.org/10.1007/s11103-017-0609-5

    Article  CAS  PubMed  Google Scholar 

  7. Svensson B, Fukuda K, Nielsen PK, Bønsager BC (2004) Proteinaceous α-amylase inhibitors. Biochim Biophys Acta—Proteins Proteomics 1696:145–156. https://doi.org/10.1016/j.bbapap.2003.07.004

    Article  CAS  Google Scholar 

  8. Nguyen PQT, Ooi JSG, Nguyen NTK et al (2015) Antiviral cystine knot α-amylase inhibitors from Alstonia scholaris. J Biol Chem 290:31138–31150. https://doi.org/10.1074/jbc.M115.654855

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chagolla-Lopez A, Blanco-Labra A, Patthy A et al (1994) A novel α-amylase inhibitor from amaranth (Amaranthus hypocondriacus) seeds. J Biol Chem 269:23675–23680

    Article  CAS  PubMed  Google Scholar 

  10. Price-Carter M, Gray WR, Goldenberg DP (1996) Folding of ω-conotoxins. 1. efficient disulfide-coupled folding of mature sequences in vitro. Biochemistry 35:15537–15546. https://doi.org/10.1021/bi961574c

    Article  CAS  PubMed  Google Scholar 

  11. Price-Carter M, Gray WR, Goldenberg DP (1996) Folding of ω-conotoxins. 2. influence of precursor sequences and protein disulfide isomerase. Biochemistry 35:15547–15557. https://doi.org/10.1021/bi9615755

    Article  CAS  PubMed  Google Scholar 

  12. Buczek O, Olivera BM, Bulaj G (2004) Propeptide does not act as an intramolecular chaperone but facilitates protein disulfide isomerase-assisted folding of a conotoxin precursor †. Biochemistry 43:1093–1101

    Article  CAS  PubMed  Google Scholar 

  13. Berkut AA, Peigneur S, Myshkin MY et al (2015) Structure of membrane-active toxin from crab spider Heriaeus melloteei suggests parallel evolution of sodium channel gating modifiers in Araneomorphae and Mygalomorphae. J Biol Chem 290:492–504. https://doi.org/10.1074/jbc.M114.595678

    Article  CAS  PubMed  Google Scholar 

  14. Xu W, Meng Y, Surana P et al (2015) The knottin-like Blufensin family regulates genes involved in nuclear import and the secretory pathway in barley-powdery mildew interactions. Front Plant Sci 6:1–18. https://doi.org/10.3389/fpls.2015.00409

    Article  CAS  Google Scholar 

  15. Bende NS, Dziemborowicz S, Herzig V et al (2015) The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop. FEBS J282:904–920. https://doi.org/10.1111/febs.13189

    Article  CAS  Google Scholar 

  16. Schwarz E (2017) Cystine knot growth factors and their functionally versatile proregions. Biol Chem 398:1295–1308

    Article  CAS  PubMed  Google Scholar 

  17. Clouse JW, Adhikary D, Page JT et al (2016) The amaranth genome: genome, transcriptome, and physical map assembly. Plant Genome 9:1. https://doi.org/10.3835/plantgenome2015.07.0062

    Article  CAS  Google Scholar 

  18. Madeira F, Pearce M, Tivey ARN et al (2022) Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50:W276–W279. https://doi.org/10.1093/nar/gkac240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kumar S, Stecher G, Li M et al (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320–W324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lobstein J, Emrich CA, Jeans C et al (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11:1. https://doi.org/10.1186/1475-2859-11-56

    Article  CAS  Google Scholar 

  22. Channale SM, Bhide AJ, Yadav Y et al (2016) Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose. Insect Biochem Mol Biol 74:1–11. https://doi.org/10.1016/j.ibmb.2016.04.009

    Article  CAS  PubMed  Google Scholar 

  23. Bhide AJ, Channale SM, Patil SS et al (2015) Biochimica et biophysica acta biochemical, structural and functional diversity between two digestive α -amylases from Helicoverpa armigera. BBA—Gen Subj 1850:1719–1728. https://doi.org/10.1016/j.bbagen.2015.04.008

    Article  CAS  Google Scholar 

  24. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738. https://doi.org/10.1038/nprot.2010.5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30:162–173. https://doi.org/10.1002/elps.200900140

    Article  Google Scholar 

  26. Pierce BG, Wiehe K, Hwang H et al (2014) ZDOCK server: Interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics 30:1771–1773. https://doi.org/10.1093/bioinformatics/btu097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pettersen EF, Goddard TD, Huang CC et al (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. https://doi.org/10.1002/jcc.20084

    Article  CAS  PubMed  Google Scholar 

  28. Pires DEV, Ascher DB, Blundell TL (2014) DUET: A server for predicting effects of mutations on protein stability using an integrated computational approach. Nucleic Acids Res 42:314–319. https://doi.org/10.1093/nar/gku411

    Article  CAS  Google Scholar 

  29. Jo S, Kim T, Iyer VG, Im W (2008) CHARMMGUI A web-based graphical user interface for CHARMM. J Comput Chem 29:1859–1865

    Article  CAS  PubMed  Google Scholar 

  30. Joshi RS, Wagh TP, Sharma N et al (2014) Way toward “dietary pesticides”: molecular investigation of insecticidal action of caffeic acid against Helicoverpa armigera. J Agric Food Chem 62:10847–10854

    Article  CAS  PubMed  Google Scholar 

  31. Joshi RS, Mishra M, Tamhane VA et al (2014) The remarkable efficiency of a Pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop. J Biomol Struct Dyn 32:13–26. https://doi.org/10.1080/07391102.2012.745378

    Article  CAS  PubMed  Google Scholar 

  32. Lansdowne LR, Beamer S, Jaczynski J, Matak KE (2009) Survival of Escherichia coli after isoelectric solubilization and precipitation of fish protein. J Food Prot 72:1398–1403. https://doi.org/10.4315/0362-028X-72.7.1398

    Article  CAS  PubMed  Google Scholar 

  33. Mi LZ, Brown CT, Gao Y et al (2015) Structure of bone morphogenetic protein 9 procomplex. Proc Natl Acad Sci USA 112:3710–3715. https://doi.org/10.1073/pnas.1501303112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

ASR thanks the Department of Science and Technology under Women Scientist Scheme A (Project code:GAP320726). This project work is supported by the Rajiv Gandhi Science and Technology Commission(RGSTC/File-2018/DPP-184/CR-23), Mumbai, Government of Maharashtra, India. APG and RSJ acknowledge the Council of Scientific and Industrial Research (CSIR), India, and CSIR-National Chemical Laboratory (CSIR-NCL), Pune, India, for financial support under Project code MLP101526 and MLP036626.VSN and RSJ acknowledge the Science and Engineering Research Board, Department of Science and Technology, Government of India for financial support under Project code GAP336726. The authors acknowledge Nivedita Rai and Ananya Chouhan for their technical help.

Funding

Department of Science and Technology, India, GAP320726, Ashwini Rane, Council for Scientific and Industrial Research, India, MLP101526, Ashok P Giri, Council of Scientific and Industrial Research, India, MLP036626, Rakesh S. Joshi, Rajiv Gandhi Science and Technology Commission, RGSTC/File-2018/DPP-184/CR-23, Ashok P Giri, Science and Engineering Research Board, GAP336726, Rakesh S. Joshi

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Authors and Affiliations

  1. Biochemical Sciences Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, Maharashtra, 411008, India

    Ashwini S. Rane, Vineetkumar S. Nair, Rakesh S. Joshi & Ashok P. Giri

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, 201002, India

    Ashwini S. Rane, Rakesh S. Joshi & Ashok P. Giri

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  1. Ashwini S. Rane
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  2. Vineetkumar S. Nair
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  3. Rakesh S. Joshi
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Correspondence to Rakesh S. Joshi or Ashok P. Giri.

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Rane, A.S., Nair, V.S., Joshi, R.S. et al. Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors. Protein J 42, 519–532 (2023). https://doi.org/10.1007/s10930-023-10148-y

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