Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 May 25;16(5):e0251805.
doi: 10.1371/journal.pone.0251805. eCollection 2021.

Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis

Mariusz Olczak  1 , Bożena Szulc  1
Affiliations

Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis

Mariusz Olczak et al. PLoS One. .

Abstract

N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Purified recombinant SEAP examined by SDS-PAGE analysis.
SEAP was produced in HEK 293 cells and purified from medium containing 10% serum. Lanes 1–3, SEAP-6×His purified on Ni-NTA magnetic agarose beads; lane 4, molecular weight standards; lanes 5–7, SEAP-HA purified on magnetic beads with immobilized anti-HA antibodies. The method of elution is shown at the top of each lane.
Fig 2
Fig 2. Relative secretion level of SEAP from HEK293T cells transfected with modified psiTEST vector.
The amount of phosphatase was determined using QUANTI-Blue reagent, as described in Materials and Methods. All experiments were performed in triplicate. All values were expressed as means ± SD. The calculated secretion rate for SEAP-6×His with additional N-glycosylation sites introduced at positions 150 and 278 was approximately 12 μg per 1 ml of the medium. The inset at the top presents SDS-PAGE of SEAP-6×His purified on magnetic Ni-NTA beads from serum-free media collected from HEK293T cells, transfected with vector coding wild-type enzyme (lane 1), enzyme with additional glycosylation site at position 278 (lane 2), and double mutant SEAP with glycosylation sites introduced at positions 150 and 278 (lane 3), respectively. 1 μg of the protein was applied into each lane; lane 4 –molecular weight standards. Statistical significance was assigned to p-value < 0.05. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 3
Fig 3. Comparison of three variants of recombined and enzymatically active SEAP.
The enzyme was bound on magnetic beads and released with imidazole. Specific activity was calculated from end-point reactions of p-nitrophenyl phosphate as substrate (see Materials and Methods for details). Statistical significance was assigned to p-value < 0.05. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 4
Fig 4. Analysis of abundance of high-mannose structures.
N-glycan profiles of HEK293T total lysate and SEAP-6×His with new glycosylation sites at 150 and 278 positions, purified on Ni-NTA beads from standard medium, are shown. Top panels–before mannosidase treatment; bottom panels–after mannosidase digestion. Peak on the left shows the product of digestion of all high-mannose type structures (blue, solid square–N-acetylglucosamine, green, solid circle–mannose).
Fig 5
Fig 5. N-glycosylation profiles of modified SEAP, purified from media of HEK293T, HepG2 and CHO cell cultures.
Fig 6
Fig 6. Schematic description of proposed conditions for N-glycan isolation from recombined SEAP.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Schjoldager K. T., Narimatsu Y., Joshi H. J., Clausen H.. Global view of human protein glycoslation pathways and functions. Nature Review Mol Cell Biol, 21 (2020), 729–749. - PubMed
    1. Etzler M.E. (Ed.), Essential of Glycobiology, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2009. - PubMed
    1. An H. J., Gip P., Kim J., Wu S., Park K.W., McVaugh C. T., et al.., Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells, Mol. Cell. Proteomics 11 (2012), M111.010660. 10.1074/mcp.M111.010660 - DOI - PMC - PubMed
    1. Homouda H., Kaup M., Ullach M., Berger M., Sandig V., Tauber R., et al.., Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry, J. Proteom. Res. 13 (2014), 6144–6151. - PubMed
    1. Hamouda H., Ullah M., Berger M., Sittinger M., Tauber R., Ringe J., et al., N-Glycosylation profile of undifferentiated and adipogenically differentiated human bone marrow mesenchymal stem cells: towards a next generation of stem cell markers, Stem. Cells. Dev. 22 (2013), 3100−13. 10.1089/scd.2013.0108 - DOI - PMC - PubMed

Publication types

Grants and funding

This research was supported by funding from National Science Center (NCN), Kraków, Poland, Website: www.ncn.gov.pl, Grant No. 2016/21/B/NZ5/00144 (MO) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.