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Cloning and characterization of trehalase: a conserved glycosidase from oriental midge, Chironomus ramosus

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Abstract

Insect trehalase is a multiferous enzyme, crucial for normal physiological functions as well as under stress conditions. In this report, we present a fundamental study of the trehalase gene segment (1587 bp) from Chironomus ramosus (CrTre) encoding for 529 amino acids, using appropriate bioinformatics tools. C. ramosus, a tropical midge is an emerging animal model to investigate the consequences of environmental stresses. We observed that CrTre belongs to GH family 37 in the CAZy database and possess 57–92% identity to dipteran trehalases. In silico characterization provided information regarding the structural, functional and evolutionary aspects of midge trehalase. In the phylogenetic tree, CrTre clustered with the soluble dipteran trehalases. Moreover, domain functional characterization of the deduced protein sequence by InterProScan (IPR001661), ProSite (PS00927 and PS00928) and Pfam (PF01204) indicated presence of highly conserved signature motifs which are important for the identification of trehalase superfamily. Furthermore, the instability index of CrTre was predicted to be < 40 suggesting its in vivo stability while, the high aliphatic index indicated towards its thermal stability (index value 71–81). The modelled 3D tertiary structure of CrTre depicts a (α/α)6 barrel toroidal core. The catalytic domain of the enzyme comprised Glu424 and Asp226 as the putative active site residues. Interestingly, the conserved motifs were observed to be formed by the flexible loopy regions in the tertiary structure. This study revealed essential sequence features of the midge trehalase and offers better insights into the structural aspects of this enzyme which can be correlated with its function.

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Fig. 1
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Abbreviations

CrTre:

C. ramosus trehalase

dNTP:

Deoxy nucleotide triphosphate

MEGA:

Molecular evolutionary genetics analysis

min:

Minutes

NCBI:

National Centre for BIotechnology

PCR:

Polymerase chain reaction

PDB:

Protein Data Bank

pI:

Isoelectric point

References

  • Adhav AS, Kokane SR, Joshi RS (2018) Functional characterization of Helicoverpa armigera trehalase and investigation of physiological effects caused due to its inhibition by Validamycin A formulation. Int J Biol Macromol 112:638–647

    Article  CAS  PubMed  Google Scholar 

  • Barraza A, Sánchez F (2013) Trehalases: A neglected carbon metabolism regulator. Plant Signal Behav 8:24778

    Article  CAS  Google Scholar 

  • Bini D, Cardona F, Forcella M, Parmeggiani C, Parenti P, Nicotra F, Cipolla L (2012) Synthesis and biological evaluation of nojirimycin and pyrrolidine-based trehalase inhibitors. Beilstein J Org Chem 8:514–521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brayer GD, Luo Y, Withers SG (1995) The structure of human pancreatic alpha-amylase at 1.8 Å resolution and comparisons with related enzymes. Protein Sci 4:1730–1742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chou PY, Fasman GE (1973) Structural and functional role of leucine residues in proteins. J Mol Biol 74:3:263–281

    Article  Google Scholar 

  • D’Adamio G, Sgambato A, Forcella M, Caccia S, Parmeggiani C, Casartelli M, Parenti P, Bini D, Cipolla L, Fusi P, Cardona F (2015) New synthesis and biological evaluation of uniflorine A derivatives: towards specific insect trehalase inhibitors. Org Biomol Chem 13(3):886–892

    Article  CAS  PubMed  Google Scholar 

  • D’Adamio G, Forcella M, Fusi P, Parenti P, Matassini C, Ferhati X, Vanni C, Cardona F (2018) Probing the influence of linker length and flexibility in the design and synthesis of new trehalase inhibitors. Molecules 23(2):436

    Article  CAS  PubMed Central  Google Scholar 

  • Daviess G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859

    Article  Google Scholar 

  • Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–D285

    Article  CAS  PubMed  Google Scholar 

  • Forcella M. Cardona F, Goti A, Parmeggiani C, Cipolla L, Gregori M, Schirone R, Fusi P, Parenti P (2010) A membrane-bound trehalase from Chironumus riparius larvae: purification and sensitivity to inhibition. Gycobiology 20(9):1186–1195

    Article  CAS  Google Scholar 

  • Forcella M, Mozzi A, Bigi A, Parenti P, Fusi P (2012) Molecular cloning of soluble trehalase from Chironomus riparius larvae and its heterologous expression in Escherichia coli. Arch Insect Biochem Physiol 81(2):77–89

    Article  CAS  PubMed  Google Scholar 

  • Gibson RP, Gloster TM, Roberts S, Warren AJ, Storch de Gracia I, Garc´ıa A, Chiara JL, Davies GJ (2007) Molecular basis for trehalase inhibition revealed by the structure of trehalase in complex with potent inhibitors. Angew Chem Int Ed Engl 46:4115–4119

    Article  CAS  PubMed  Google Scholar 

  • Ikai A (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895–1898

    CAS  PubMed  Google Scholar 

  • Jones P, Binns D, Chang H, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30(9):1236–1240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar S, Bansal M (1998) Geometrical and sequence characteristics of α helices in globular proteins. Biophys J 75:1935–1944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laskowski RA (2001) PDBsum: summaries and analyses of PDB structures. Nucleic Acids Res 29(1):21–222

    Article  Google Scholar 

  • Liu J, Tan H, Rost B (2002) Loopy proteins appear conserved in evolution. J Mol Biol 322:53–64

    Article  CAS  PubMed  Google Scholar 

  • Lombard V, Golaconda RH, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495

    Article  CAS  PubMed  Google Scholar 

  • Ogiso M, Shinohara Y, Hanaoka K, Kageyama T, Takahashi SY (1985) Further purification and characterization of trehalase from the American cockroach Periplaneta americana. J Comp Physiol B Biochem Syst Environ Physiol 155:553–560

    Article  CAS  Google Scholar 

  • Panigrahi P, Sule M, Ghanate A, Ramasamy S, Suresh CG (2015) Engineering proteins for thermostability with iRDP web server. PLoS One 5(10):10

    Google Scholar 

  • Ramasubbu N, Ragunath C, Mishra PJ (2003) Probing the role of mobile loop in substrate binding and enzyme activity of human salivary amylase. J Mol Biol 325:1061–1076

    Article  CAS  PubMed  Google Scholar 

  • Reyes-DelaTorre A, Peña-Rangel MT, Riesgo-Escovar JR (2012) Carbohydrate metabolism in drosophila: reliance on the disaccharide trehalose. In: Chang C (ed) Carbohydrates-comprehensive studies on glycobiology and glycotechnology (online). InTech, Winchester, pp 317–338

    Google Scholar 

  • Rogers S, Wells R, Rechsteiner M (1986) Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 234:364–368

    Article  CAS  PubMed  Google Scholar 

  • Schrödinger (2016) Release 2016-1: LigPrep, version 3.7, Schrödinger. LLC, New York

    Google Scholar 

  • Shukla E, Thorat LJ, Nath BB, Gaikwad SM (2015) Insect trehalase: Physiological significance and potential applications. Glycobiology 25(4):357–367

    Article  CAS  PubMed  Google Scholar 

  • Shukla E, Thorat LJ, Bhavnani V, Bendre AD, Pal JK, Nath BB, Gaikwad SM (2016) Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster. Int J Biol Macromol 92:282–292

    Article  CAS  PubMed  Google Scholar 

  • Silva MCP, Terra WR, Ferreira C (2010) The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda. Insect Biochem Mol Biol 40:733–741

    Article  CAS  PubMed  Google Scholar 

  • Sitbon E, Pietrokovski S (2007) Occurrence of protein structure elements in conserved sequence regions. BMC Struct Biol 7:3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Terrapon N, Lombard V, Drula E, Coutinho PM, Henrissat B (2017) The CAZy database/the carbohydrate-active enzyme (CAZy) database: principles and usage guidelines. In: Aoki-Kinoshita K (ed) A practical guide to using glycomics databases. Springer, Tokyo

    Google Scholar 

  • The CAZypedia Consortium (2018) Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes. Glyco-Forum Glycobiology 28(1):3–8

    Article  Google Scholar 

  • Thorat LJ, Nath BB (2015) Tolerance to desiccation stress in Chironomus ramosus through plasticity in homeostatic control. Eur J Env Sci 5:86–91

    Article  Google Scholar 

  • Thorat LJ, Nath BB (2016) Quantitative assessment of larval desiccation tolerance in oriental Chironomus species. Curr Sci 11:1448–1449

    Google Scholar 

  • Thorat L, Gaikwad S, Nath BB (2012) Trehalose as an indicator of desiccation stress in Drosophila melanogaster larvae: a potential marker of anhydrobiosis. Biochem Biophys Res Comm 419:638–642

    Article  CAS  PubMed  Google Scholar 

  • Thorat LJ, Oulkar D, Banerjee K, Gaikwad SM, Nath BB (2017) High-throughput mass spectrometry analysis revealed a role for glucosamine in potentiating recovery following desiccation stress in Chironomus. Nat Sci Rep 7:3659

    Article  CAS  Google Scholar 

  • Webb B, Sali A (2014) Comparative protein structure modeling using modeller. Curr Protoc Bioinformatics 54:5–6

    Article  Google Scholar 

Download references

Acknowledgements

This work is supported by funding received from DRDP programme, Department of Biotechnology, SPPU and DST-PURSE grants to JKP and from partial funding received from DST-PURSE and BCUD-UoP grants to BBN. The authors thank Dr. Varsha Bhatia, Gennova Biopharmaceuticals Ltd. and Mr. Ejaj Pathan, CSIR-NCL for their valuable suggestions and timely help. ES and ADB acknowledge University Grants Commission, New Delhi, for Senior Research Fellowships. LT is grateful for financial support received from the University Grants Commission-DS Kothari Postdoctoral Fellowship (UGC-DSK-PDF) and from DBT Bio-CARe Women Scientist Scheme, New Delhi, India.

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Author notes

  1. Ekta Shukla

    Present address: National Centre for Cell Science, Ganeshkhind, Pune, 411007, India

  2. Santosh Jadhav

    Present address: BAIF Central Research Station, Urulikanchan, Pune, 412202, India

  3. Jayanta K. Pal

    Present address: Dr. D.Y. Patil Biotechnology and Bioinformatics Institute, Pune, 411033, India

Authors and Affiliations

  1. Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, 411008, India

    Ekta Shukla, Ameya D. Bendre & Sushama M. Gaikwad

  2. Academy of Scientific and Innovative Research (AcSIR), CSIR-NCL Campus, Pune, 411008, India

    Ekta Shukla, Ameya D. Bendre & Sushama M. Gaikwad

  3. Stress Biology Research Laboratory, Department of Zoology, S.P. Pune University, Pune, 411007, India

    Leena Thorat & Bimalendu B. Nath

  4. Cell and Molecular Biology Laboratory, Department of Biotechnology, S.P. Pune University, Pune, 411007, India

    Santosh Jadhav & Jayanta K. Pal

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  1. Ekta Shukla
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Correspondence to Ekta Shukla.

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Shukla, E., Thorat, L., Bendre, A.D. et al. Cloning and characterization of trehalase: a conserved glycosidase from oriental midge, Chironomus ramosus. 3 Biotech 8, 352 (2018). https://doi.org/10.1007/s13205-018-1376-y

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