Structural requirements of Holothuria floridana fucosylated chondroitin sulfate oligosaccharides in anti-SARS-CoV-2 and anticoagulant activities

Abstract

Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from Pentacta pygmaea (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species Holothuria floridana (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional 1H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin, and heparin cofactor II) in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration–response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.

Nuclear magnetic resonance‐based structural elucidation of novel marine glycans and derived oligosaccharides

“…Five major fractions (Fr1-Fr5), with MW distribution of Fr1 > Fr2 > Fr3 > Fr4 > Fr5, were obtained. 38 The 1D 1 H NMR spectra of all HfFucCS-derived oligosaccharides (HdHfFucCS and Fr1-Fr5) showed the characteristic 1 H signals of all three sulfated fucosyl branches (α-Fuc-2,4S, α-Fuc-3,4S, and α-Fuc-4S) (Figure 4b and Table 6), indicating the same structure of the native HfFucCS molecule within all low MW samples. The methyl 1 H signals from GalNAc and Fuc units were also observed at the same δ H values like native HfFuCS (Figure 4b and Table 6).…”

“…The 1 H- 13 C HSQC spectrum allows assessment of the proton-carbons pairs directly bound to each other therefore ultimately allowing assessment of chemical shifts of both nuclei. In the following sections, we explain how we can explore the 1 H and 13 C chemical shifts in assessment of the structural details of four recently characterized polysaccharides of our laboratory, the α-glucan extracted from the bivalve Marcia hiantina (MhαG), 35 two SGs extracted from the red alga Botryocladia occidentalis (BoSG), 36 and the FucCS isolated from the sea cucumber Pentacta pygmaea (PpFucCS), 20 as well as the structures of three sets of oligosaccharides derived from two marine glycans of two holothurian species, PpFucCS, 37 and the FucCS from Holothuria floridana (HfFucCS), 38 and the SF from this later species (HfSF). 39…”

“…39,[41][42][43][44] Free-radical depolymerization technique, on the other hand, requires other reagents for controlled digestion, such as copper (Fenton-based reaction) to provide selectivity of cleavage solely on the FucCS backbones and not on the chemical bonds of sulfate esters and Fuc branches. 37,38 One-dimensional 1 H NMR comprises a simple, quick, and reliable method to monitor the progress of such digestive reactions and to assess the structural integrity or modifications of the produced oligosaccharides during depolymerization. Below, we describe how we used 1D 1 H NMR spectroscopy to monitor oligosaccharide production of PpFucCS, 37 HfFucCS, 38 and HfSF.…”

“…37,38 One-dimensional 1 H NMR comprises a simple, quick, and reliable method to monitor the progress of such digestive reactions and to assess the structural integrity or modifications of the produced oligosaccharides during depolymerization. Below, we describe how we used 1D 1 H NMR spectroscopy to monitor oligosaccharide production of PpFucCS, 37 HfFucCS, 38 and HfSF. 39 As stated above, PpFucCS is structurally…”

Heparan-6-O-endosulfatase 2 promotes invasiveness of head and neck squamous carcinoma cell lines in co-cultures with cancer associated fibroblasts

“…The fucosylated chondroitin sulfates exhibit biological actions including inhibition of blood clotting and inhibition of severe acute respiratory syndrome coronavirus infection. These biological effects depend on molecular weight (MW), number of fucosyl branches, and sulfation of both the branching as well as the backbone residues [33,34]. What specific structural features define the inhibition of the Sulf-2 enzyme by HfFucCS is as yet unknown and requires further study.…”