Elsevier

Journal of Proteomics

Volume 224, 30 July 2020, 103818
Journal of Proteomics

The role of the quaternary structure in the activation of human L-asparaginase

https://doi.org/10.1016/j.jprot.2020.103818Get rights and content

Abstract

Human L-asparaginase-like protein 1 (ASRGL1) has hydrolytic activity against L-asparagine and isoaspartyl dipeptides. As an N-terminal nucleophile hydrolase family member, its activation depends on an intramolecular autoprocessing step between G167 and T168. In vitro, autoprocessing reaches only 50% completion, which restrains the activity and hampers the full understanding of the activation process. The ASRGL1 dimer interface plays a critical role in intramolecular processing, and the interactions within oligomers can offer relevant information about autoprocessing. In this work, a fully processed trimeric conformation of ASRGL1 was observed for the first time, and we combined biophysical and structural proteomics assays to characterize trimeric ASRGL1. Our analyses show that oligomerization is critical for autoprocessing, hydrolytic activity and thermal stability. The newest trimeric ASRGL1 conformation enhances protein activity and presents a melting temperature deviation of 4.33 °C in comparison to the monomeric conformation. The interaction of the third monomer in the trimeric conformation is driven by an α-helix comprising residues KVNLARLTLF (227–236).

Section snippets

Significance

ASRGL1 has therapeutic potential in reducing collateral effects associated with acute lymphoid leukemia treatment. However, the human enzyme presents low in vitro activity due to its autoprocessing activation step, which is still elusive. The complete knowledge about this step is crucial for generating active variants of ASRGL1. This work contributes to the understanding of autoprocessing and highlights the role of oligomerization in ASRGL1.

Molecular cloning and protein expression and purification

The ASRGL1 gene (RefSeq: NM_001083926) was cloned into a pET28a-TEV vector between the NdeI and XhoI restriction sites. ASRGL1 was expressed in E. coli BL21 Star (DE3) cells. ASRGL1 expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, and cultures were incubated for an additional 4 h at 37 °C.

Affinity chromatography

For the protein purification, cells were harvested by centrifugation for 15 min at 6000 xg and resuspended in buffer A (Tris-HCl 50 mM at

ASRGL1 autoprocessing might involve dimerization through disulfide bond formation

Western blotting with the anti-His primary antibody showed autoprocessing of the partially purified recombinant ASRGL1 (Fig. 1A). Undoubtedly, the ~30 kDa band indicated in Fig. 1 corresponds to the unprocessed protein, and the ~21 kDa band corresponds to the α-chain (Fig. 1A). Notwithstanding, the identification of the β-strand was not possible by western blotting since the His-tag was at the N-terminal portion of the protein.

In addition to the 30 and 21 kDa bands, the observation of the

Discussion

Autoprocessing is crucial in the activation of ASRGL1, and its hydrolytic activity over L-asparagine [11]. The autoprocessing event is also essential for the activation in other Ntn-hydrolase family members [15]. However, the mechanism and residues involved in autoprocessing are different for each Ntn-hydrolase.

ASRGL1 autoprocessing is a slow process in vitro, in which only 50% of the autoprocessing rate is observed without an external activator [11,45]. Oligomerization is one of the factors

Conclusions

ASRGL1 oligomerization is critical for autoprocessing, enzymatic activity and thermal stability. The newest fully processed trimeric ASRGL1 conformation enhances the protein activity and increases the thermal stability in comparison to the monomeric conformation. MD simulations along with SAXS and HDX experiments revealed that residues KVNLARLTLF (227 to 236) within the ASRGL1 trimer may lead to an arrangement that is favorable for autoprocessing.

Authors’ Contributions

SBM conducted most of experiments. NFF and ML performed molecular dynamics simulation. RP and FCG contributes to HDX experiment and analysis. TACBS conceived the idea for the project and contributed to the experimental design, provide technical assistance and wrote the paper together with SBM.

Acknowledgments

We are grateful to the Brazilian Synchrotron Light Laboratory (LNLS) for the provision of time on the SAXS2 beamlines.

Funding

This work was supported by FIOCRUZ, Brasil and Fundação Araucária, Brazil.

Declaration of Competing Interests

The authors have no conflicts of interest.

References (47)

  • J.G. Kidd

    Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. I. Course of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum, or rabbit serum

    J. Exp. Med.

    (1953)
  • J.D. Broome

    Evidence that the L-Asparaginase activity of Guinea pig serum is responsible for its Antilymphoma effects

    Nature.

    (1961)
  • J.D. Broome

    Evidence that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects. I. Properties of the L-asparaginase of guinea pig serum in relation to those of the antilymphoma substance

    J. Exp. Med.

    (1963)
  • J.M. Hill et al.

    L-asparaginase therapy for leukemia and other malignant neoplasms

    Remission in human leukemia, JAMA

    (1967)
  • W.L. Salzer et al.

    Development of asparaginase Erwinia chrysanthemi for the treatment of acute lymphoblastic leukemia

    Ann. N. Y. Acad. Sci.

    (2014)
  • V.I. Avramis

    Asparaginases: biochemical pharmacology and modes of drug resistance

    Anticancer Res.

    (2012)
  • M.E. Rytting

    Role of L-asparaginase in acute lymphoblastic leukemia: focus on adult patients

    BLCTT.

    (2012)
  • R. Pieters et al.

    L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase

    Cancer.

    (2011)
  • J.R. Cantor et al.

    The human Asparaginase-like protein 1 hASRGL1 is an Ntn-hydrolase with β-aspartyl peptidase activity

    Biochemistry.

    (2009)
  • J. Nomme et al.

    Structures of apo and product-bound human L-asparaginase: insights into the mechanism of autoproteolysis and substrate hydrolysis

    Biochemistry.

    (2012)
  • W. Li et al.

    Intramolecular cleavage of the hASRGL1 homodimer occurs in two stages

    Biochemistry.

    (2016)
  • J.A. Brannigan et al.

    A protein catalytic framework with an N-terminal nucleophile is capable of self-activation

    Nature.

    (1995)
  • K. Michalska et al.

    Structural aspects of L-asparaginases, their friends and relations

    Acta Biochim. Pol.

    (2006)
    • View full text
    © 2020 Published by Elsevier B.V.