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Identification and characterization of acid and alkaline phosphatases and protein phosphatases in L. catesbeianus tail during metamorphosis

Abstract

Amphibian metamorphosis is a tightly regulated transformation involving the participation of hormones and other biomolecules in cell death and tail absorption. Among the regulators, phosphate is essential for various processes, such as cell death and survival and enzyme activity regulation. Therefore, identification and characterization of phosphatases in L. catesbeianus tail may contribute to understand the events involved in cell death and nutrient release during metamorphosis. Differential centrifugation was used to separate soluble proteins from membrane proteins and analyzed by phosphomonohydrolases, serine/threonine protein phosphatase 2A (PP2A), and protein tyrosine phosphatase (PTP) assays. Mitochondrial fractioning was used to evaluate PP2A and alkaline phosphatase activities. Tandem mass spectrometry (MS/MS) analysis was performed using the crude extract. Phosphomonohydrolase activity was assayed by p-nitrophenylphosphate (pNPP) hydrolysis, whereas PP2A and PTP were assayed by peptides phosphorylated in threonine and tyrosine, respectively; inhibitor-2 was used to identify the serine/threonine protein phosphatase type 1 (PP1). The enzymatic activities and kinetic parameters of pNPP hydrolysis revealed three distinct phosphomonohydrolases. MS/MS analysis of the crude extract revealed three protein phosphatases, viz., PP2A, PP1, and a PTP, which was confirmed by in vitro assays. The results may relate PP2A activity to membrane-bound alkaline phosphatase, PTP activity to soluble acid phosphatase, and PP1 activity to membrane acid phosphatase, although more detailed studies are needed to confirm this hypothesis. We propose a model providing information on the role of PP1 and PP2A in the signaling events leading to cell death and the role of these enzymes in anuran tail absorption during metamorphosis.

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Data availability

Not applicable.

Abbreviations

BAD:

BCL-2 antagonist of cell death

BAX:

BCL-2 Associated X

BCL-2:

B cell lymphoma 2 – family of regulator proteins

BCL-XL:

B cell lymphoma-extra large

BSA:

Bovine serum albumin

ESI:

Electrospray ionization

FWHM:

Full Width at Half Maximum

GPI:

Glycosylphosphatidylinositol

HCD:

Higher-energy C-trap dissociation

IL-2:

Interleukin-2

LMW-PTP:

Low molecular weight phosphotyrosine protein phosphatase

MIT:

Mitochondrial fraction

MOMP:

Mitochondrial outer membrane permeabilization

PDGF:

Platelet derived growth factor

PIPLC:

Phospholipase C

pNPP:

p-nitrophenylphosphate

PPP:

Phosphoprotein phosphatases

PP1:

Protein phosphatase 1

PP2A:

Protein phosphatase 2A

PP2B:

Protein phosphatase 2B

PP4:

Protein phosphatase 4

PP5:

Protein phosphatase 5

PP6:

Protein phosphatase 6

PP7:

Protein phosphatase 7

PPM:

Magnesium dependent protein phosphatases

PSMs:

Peptide-spectrum matches

PTP:

Protein tyrosine phosphatase

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SMP:

Fraction containing soluble and cytoplasmic membrane proteins

TH:

Thyroid hormone

TNF:

Tumor necrosis factor

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Acknowledgments

We are grateful to all the members of LEIA (Laboratório de Enzimologia e Imunoquímica Aplicadas).

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Funding

This work was supported by CAPES and FAPESP.

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Contributions

AMG, CCS, RRC and LFJS and classified the animals, executed the experiments and participated on results discussion. AMG performed the statistical analyses. TSB performed mass spectrometry analyses and result interpretation. AMG and JMPJ conceived of the study and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Adriano Marques Gonçalves.

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The authors state that all procedures were performed with the approval of the Ethics Committee on Animal Use of the University (Protocol No. 010537/11).

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Supplementary Information

Supplementary Fig. 1
figure6

Scheme for obtaining soluble and membrane proteins (PNG 65 kb)

Supplementary Fig. 2
figure7

Scheme of mitochondrial isolation from the tail of L. catesbeianus tadpole (PNG 30 kb)

Supplementary Fig. 3
figure8

Effect of pNPP concentration on p-nitrophenylphosphatase activity of the phosphatases present in the tail of stage 44 L. catesbeianus tadpoles. A- Alkaline phosphatase solubilized with PIPLC. B- Membrane-bound acid phosphatase. C- Soluble acid phosphatase. (PNG 41 kb)

Supplementary Fig. 4
figure9

Apparent optimum pH of alkaline phosphatase activity present in the tail membrane fraction of stage 44 L. catesbeianus tadpoles. A- pNPP 1 mM. B- pNPP 0.1 mM (PNG 311 kb)

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Gonçalves, A.M., Santana, C.C., Santos, L.F.J.D. et al. Identification and characterization of acid and alkaline phosphatases and protein phosphatases in L. catesbeianus tail during metamorphosis. Biologia (2021). https://doi.org/10.1007/s11756-021-00877-9

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Keywords

  • Anura
  • Cell death
  • Metamorphosis
  • Mitochondria
  • Phosphatases
  • PP2A
  • PP1