pubmed.ncbi.nlm.nih.gov

Expression of a recombinant bacterial L-asparaginase in human cells - PubMed

  • ️Tue Jan 01 2019

Expression of a recombinant bacterial L-asparaginase in human cells

Raquel Caminha Dantas et al. BMC Res Notes. 2019.

Abstract

Objective: L-Asparaginase (ASNase) is an enzyme used in the treatment of acute lymphoblastic leukemia (ALL). As the therapeutic ASNases has bacterial origin, severe side effects are associated with its use, among them hypersensitivity and inactivation of the enzyme. In this context, the objective of this work was to produce a recombinant ASNase of bacterial origin in human cells in order to determine the presence and consequences of potential post-translational modifications on the enzyme.

Results: Recombinant ASNase was expressed in human cells with a molecular weight of 60 kDa, larger than in Escherichia coli, which is 35 kDa. N-glycosylation analysis demonstrated that the increased molecular weight resulted from the addition of glycans to the protein by mammalian cells. The glycosylated ASNase presented in vitro activity at physiological pH and temperature. Given that glycosylation can act to reduce antigenicity by masking protein epitopes, our data may contribute to the development of an alternative ASNase in the treatment of ALL in patients who demonstrate side effects to currently marketed enzymes.

Keywords: Acute lymphoblastic leukemia; Glycosylation; Hypersensitivity; L-Asparaginase.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1

Bacterial

l

-asparaginase gene expression in mammalian HEK-293 cells. a pASNase vector, containing two in tandem CMV promoters (pCMV), the coding sequence for the E. coli

l

-Asparaginase gene (ASNase) with a milk signal peptide sequence (mSP) and a kanamycin resistance gene (kanr). b Transfection of HEK-293 cells in white light (top line) and under UV light (bottom line) with the pASNase vector in order to produce clones with the stably integrated transgene. (1) Cells transfected with the linear pASNase plasmid, (2) Cells transfected without DNA (negative control), (3) Cells transfected with a commercial GFP plasmid (positive control). c Immunodetection of recombinant

l

-Asparaginase secreted in the medium of HEK-293. (1) Cell culture medium from HEK-293 cells transfected with pASNase plasmid, (2) Cell culture medium from HEK-293 cells transfected with no DNA (negative control), (3) Cellular lysate from HEK-293 cells transfected with pASNase plasmid, (4) Cellular lysate from HEK-293 cells transfected with DNA (negative control), (5) Commercial

l

-Asparaginase from E. coli (positive control)

Fig. 2
Fig. 2

Glycosylation analysis of the bacterial

l

-Asparaginase expressed in the mammalian cell line HEK-293. a Predicted N-glycosylation sites in the

l

-Asparaginase sequence expressed in HEK-293 cells using the NetNGlyc 1.0 software. A position with the potential (vertical lines) crossing the threshold (horizontal line at 0.5) is predicted when glycosylated. b Immunodetection of recombinant

l

-Asparaginase expressed in HEK-293 cells and E. coli after deglycosylation with PNGase-F. (1) HEK-293

l

-Asparaginase treated with PNGase-F, (2) HEK-293

l

-Asparaginase untreated with PNGase-F, (3) E. coli

l

-Asparaginase treated with PNGase-F, (4) E. coli

l

-Asparaginase untreated with PNGase-F

Fig. 3
Fig. 3

Bacterial and HEK-293

l

-Asparaginase relative activity at different pHs and temperatures. a Effect of the pH on the activity of the glycosylated bacterial

l

-Asparaginase expressed in the mammalian cell line HEK-293 (left) and in E. coli (right). b Effect of temperature, at pH 8.0, of the glycosylated bacterial

l

-Asparaginase expressed in the mammalian cell line HEK-293 (left) and in E. coli (right)

Similar articles

Cited by

References

    1. Bhojwani D, Yang JJ, Pui CH. Biology of childhood acute lymphoblastic leukemia. Pediatr Clin North Am. 2015;62:47–60. doi: 10.1016/j.pcl.2014.09.004. - DOI - PMC - PubMed
    1. Shrivastava A, Khan AA, Khurshid M, Kalam MA, Jain SK, Singhal PK. Recent developments in l-asparaginase discovery and its potential as anticancer agent. Crit Rev Oncol Hematol. 2016;100:1–10. doi: 10.1016/j.critrevonc.2015.01.002. - DOI - PubMed
    1. Stams WAG, Den Boer ML, Beverloo HB, Meijerink JPP, Stigter RL, Van Wering ER, et al. Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21) + pediatric ALL. Blood. 2003;101:2743–2747. doi: 10.1182/blood-2002-08-2446. - DOI - PubMed
    1. Rizzari C. Shedding light on the asparaginase galaxy. Blood. 2014;123:1976–1978. doi: 10.1182/blood-2014-02-553040. - DOI - PubMed
    1. Safary A, Moniri R, Hamzeh-Mivehroud M, Dastmalchi S. Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain. Bioimpacts. 2019;9:15–23. doi: 10.15171/bi.2019.03. - DOI - PMC - PubMed

MeSH terms

Substances