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Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate

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Abstract

Through their ability to edit 6-O-sulfation pattern of Heparan sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, we unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled us to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, we confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary for the enzyme 6-O-endosulfatase activity. However, we demonstrated for the first time that high-affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, our findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.

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Acknowledgements

The authors would like to thank Elisa Tournebize for technical assistance, Marjolaine Noirclerc-Savoye for her precious advice on molecular biology, Philippe Desprès for providing the SNAP-containing shuttle vector and Kenji Uchimura for the Anti-HSulf-2 antibody. This work used the SPR, Robiomol and amino-acid sequencing platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). This work was also supported by the CNRS and the GDR GAG (GDR 3739), the “Investissements d’avenir” program Glyco@Alps (ANR-15-IDEX-02), and by grants from the Agence Nationale de la Recherche (ANR-12-BSV8-0023 and ANR-17-CE11-0040) and Université Grenoble-Alpes (UGA AGIR program).

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Author notes

  1. Amal Seffouh and Rana El Masri contributed equally to this work.

Authors and Affiliations

  1. Univ. Grenoble Alpes, CNRS, CEA, IBS, 38000 Grenoble, France

    Amal Seffouh, Rana El Masri, Evelyne Gout, Zahra el Oula Hassoun, Jean-pierre Andrieu, Hugues Lortat-Jacob & Romain R. Vivès

  2. Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, Kazan, Russian Federation

    Olga Makshakova

  3. IBS, 71 Avenue des Martyrs CS 10090, 38044, Grenoble Cedex 9, France

    Romain R. Vivès

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  1. Amal Seffouh
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Supplementary Fig.

 1: Expression and purification of recombinant HSulf-2 and HSulf-2ΔHD. A: Cation-exchange separation profile of HSulf-2. Collected fractions are shown by marks inside the X axis. Pooled fractions are indicated by grey/white dashed area and PAGE stained by Coomassie blue of these fractions are shown in the inset. The arrows indicate the band corresponding to HSulf-2 N-terminal chain. B: Purification of HSulf-2ΔHD by affinity chromatography (Nickel column). Fractions analyzed by PAGE followed by Coomassie blue staining correspond to the wash through (WT), Rinse (R) and 350 mM imidazole elution fraction 1 (E1) and 2 (E2). The arrow indicates the band corresponding to HSulf-2ΔHD C: Coomassie blue PAGE analysis of purified HSulf-2ΔHD (5 µg). (TIFF 492 kb)

Supplementary Fig.

 2: Amino acid sequence of HSulf-2. Within the sequence, the HD domain (R416-Q715) is shown in italic and grey, catalytic cysteine modified FGly (C88) is shown in red and R538S furin cleavage site (determined by Edman degradation N-terminal sequencing) is marked by double underlining. Finally, HS-binding epitopes identified using the cross-linking mapping approach (V179KEK and L401KKK) are underlined in orange and yellow, respectively. (TIFF 505 kb)

Supplementary Fig.

 3: Digestion of 4-MUS by WT and mutant HSulf-2. Time course digestion of 4-MUS by (A) HSulf-2ΔHD (open circles) or (B) HSulf-2/VAEA/LAAA (open diamonds) compared to that of HSulf-2 (black circles). The dashed line represents the linear regression line of HSulf-2 data. Error bars represent SEM of triplicate analysis. (TIFF 116 kb)

Supplementary Fig.

 4: Binding of HSulf-2 VAEA/LAAA and HSulf-2ΔHD/VAEA/LAAA to heparin. Immunoassay of HSulf-2 VAEA/LAAA (black squares) and HSulf-2ΔHD/VAEA/LAAA (open circles) interaction with heparin. Curves shown are representative of three and at least three independent experiments. (TIFF 106 kb)

Supplementary material 5 (DOCX 11 kb)

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Seffouh, A., El Masri, R., Makshakova, O. et al. Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate. Cell. Mol. Life Sci. 76, 1807–1819 (2019). https://doi.org/10.1007/s00018-019-03027-2

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