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. 2025 Apr 23;35(6):cwaf025.
doi: 10.1093/glycob/cwaf025.

Heparan-6-O-endosulfatase 2, a cancer-related proteoglycan enzyme, is effectively inhibited by a specific sea cucumber fucosylated glycosaminoglycan

Marwa Farrag  1   2 , Reem Aljuhani  3   4 , Julius Benicky  4   5 , Hoda Al Ahmed  1 , Sandeep K Misra  1 , Sushil K Mishra  1   6 , Joshua S Sharp  1   7   8 , Robert J Doerksen  1   7 , Radoslav Goldman  3   4   5 , Vitor H Pomin  1   7
Affiliations

Heparan-6-O-endosulfatase 2, a cancer-related proteoglycan enzyme, is effectively inhibited by a specific sea cucumber fucosylated glycosaminoglycan

Marwa Farrag et al. Glycobiology. .

Abstract

Heparan-6-O-endosulfatase 2 (Sulf-2) is a proteoglycan enzyme that modifies sulfation of heparan sulfate proteoglycans. Dysregulation of Sulf-2 is associated with various pathological conditions, including cancer, which makes Sulf-2 a potential therapeutic target. Despite the key pathophysiological roles of Sulf-2, inhibitors remain insufficiently developed. In previous work, a fucosylated chondroitin sulfate from the sea cucumber Holothuria floridana (HfFucCS) exhibited potent Sulf-2 inhibition. This study investigates the structural basis of HfFucCS-mediated Sulf-2 inhibition, examines the binding profile of HfFucCS to Sulf-2, and explores the mode of inhibition. Additionally, a structurally diverse library of sulfated poly/oligosaccharides, including common glycosaminoglycans and unique marine sulfated glycans, was screened for Sulf-2 inhibition. Results from a high-throughput arylsulfatase assay and specific 6-O-desulfation assay have proved that HfFucCS is the most potent among the tested sulfated glycans, likely due to the presence of the unique 3,4-disulfated fucose structural motif. HfFucCS demonstrated non-competitive inhibition, and inhibitory analysis of its low-molecular-weight fragments suggests a minimum length of ~7.5 kDa for effective inhibition. Surface plasmon resonance analyses revealed that Sulf-2 binds to surface heparin with high affinity (KD of 0.817 nM). HfFucCS and its derivatives effectively disrupt this interaction. Results from mass spectrometry-hydroxyl radical protein footprinting and repulsive scaling replica exchange molecular dynamics indicate similarities in the binding of heparin and HfFucCS oligosaccharides to both the catalytic and hydrophilic domains of Sulf-2. These findings reveal the unique inhibitory properties of a structurally distinct marine glycosaminoglycan, supporting its further investigation as a selective and effective inhibitor for Sulf-2-associated cancer events.

Keywords: enzyme inhibitor; fucosylated chondroitin sulfate; glycosaminoglycan; heparan-6-O-endosulfatase 2; proteoglycan.

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Conflict of interest statement

J.S.S. discloses a significant financial interest in GenNext Technologies, Inc., a company that commercializes instrumentation and software for HRPF analysis. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The funders had no role in the design, writing, or decision of this publication.

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