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

Cellular and Molecular Life Sciences : CMLS
Amal SeffouhRomain R Vivès

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 ...Continue Reading

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Citations

May 20, 2020·Frontiers in Immunology·Ditmer T TalsmaUNKNOWN COMBAT Consortium
Apr 23, 2020·Frontiers in Immunology·Rana El MasriRomain R Vivès
Aug 29, 2019·American Journal of Physiology. Lung Cellular and Molecular Physiology·Kaori OshimaEric P Schmidt
Sep 18, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Thibault AnnavalHugues Lortat-Jacob
Jan 10, 2021·Scientific Reports·Cathy DanesinCathy Soula
Jun 20, 2021·Colloids and Surfaces. B, Biointerfaces·Raul Diaz-SalmeronKawthar Bouchemal

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Datasets Mentioned

BETA
CR749319.1

Methods Mentioned

BETA
PCR
size-exclusion chromatographies
NMR
electrophoresis
electron microscopy
ELISA
size exclusion chromatography
nickel column chromatography
protein core folding

Software Mentioned

Clustal
Modeller
ClustalW
Robiomol
PROCHECK
omega

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