Recombinant dermatan sulfate is a potent activator of heparin cofactor II-dependent inhibition of thrombin

Abstract The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.

These glycosaminoglycan-derived anticoagulants are far from ideal drugs due to their (i) animal origin, (ii) narrow therapeutic window and (iii) inherent structural dishomogeneity. The latter problems were exposed in the 2008 heparin crisis where oversulfated chondroitin sulfate illegally added to heparin caused fatal anaphylactic reactions (Guerrini et al. 2008). To address some of these problems, we initiated a study to produce structurally well-defined recombinant DS (recDS) with anticoagulant activity from the microbial K4 polysaccharide, using the recombinant enzymes DS-epi1, D4ST1, UST and GalNAc4S-6ST.

Results and discussion
Structurally well-defined recombinant dermatan sulfate can be produced from a bacterial polysaccharide substrate As previously shown, incubation of chondroitin with DS-epi1 alone results in short stretches of IdoA-containing sequences . In order to produce long IdoA-blocks (Tykesson et al. 2018), DS-epi1 and D4ST1 were co-incubated with chondroitin and PAPS for 24 h ( Figure 1A and B) to give a product (recDS-4) containing 96% UA-GalNAc-4S, determined by disaccharide analysis after chondroitinase ABC ( Figure 1C). The presence of IdoA and GalNAc-4S was shown by disaccharide analysis after chondroitinase B (data not shown) and confirmed by NMR spectroscopy ( Figure 1D). Further characterization by SEC-MALS showed a weight average molecular weight (M w ) of 35.5 kDa with a dispersity of 1.14 ( Figure 1E). Based on the sulfation degree and the size of the polymers it can be concluded that the 4% of non-sulfated disaccharide structures in recDS-4 are most likely positioned on the reducing and non-reducing ends of the polymer, in agreement with the substrate specificity of both DS-epi1 and D4ST1 (Evers et al. 2001;Mikami et al. 2003;Tykesson et al. 2016). Depending on the potential future applications of the recDS, we could also show that it is also possible to tune the degree of 4-O-sulfated residues by varying the enzymatic incubation time ( Figure 1B). In contrast to the heterogeneity of commercial 4-O-sulfated DS preparations isolated from animal sources, the unique recDS-4 preparation is homogenously composed of IdoA-GalNAc-4S along the polymer and can be used to evaluate the biological properties and functions of 4-O-sulfated DS.

A recombinant 2,4-O-sulfated DS is a potent activator of HCII-dependent inhibition of thrombin
To investigate the functionality of our recDS preparations, an assay for the HCII-dependent inactivation of thrombin was set up. The 4-O-sulfated recDS showed no activity. However, the 4,6-Osulfated recDS with 86% IdoA-GalNAc-4,6 sulfate exhibited a HCII-dependent inactivation of thrombin with similar potency as heparin, i.e., IC 50 values of 479 ± 95 ng/mL and 348 ± 25 ng/mL, respectively ( Figure 2B). A DS preparation with 9% 4,6-O-sulfated structures has previously been shown to weakly inhibit thrombin via HCII, suggesting that a significantly higher proportion of disulfated structures is necessary for efficient inhibition (Halldórsdóttir et al. 2006). Most importantly, we found that a preparation with 71% of IdoA-2S-GalNAc-4S had an IC 50 value of 19 ± 4 ng/mL, i.e., 15-20 times better inhibition compared to heparin (Maimone and Tollefsen 1990). To our knowledge, this is the most potent native polysaccharide activator of HCII to date. Trisulfated recDS had an IC 50 value of 19 ± 6 ng/mL, suggesting that additional sulfation does not improve activation.
In order to verify these results in in vivo-like settings, we analyzed the ability of the 2,4-O-sulfated recDS to prolong the activated partial thromboplastin time (aPTT) ( Figure 2C). The concentration required to double the aPTT was 13.0 ± 0.8 μg/mL for 2,4-O-sulfated recDS, compared to 3.5 ± 0.2 μg/mL for heparin. Even though the 2,4-O-sulfated recDS only acts via HCII, and not ATIII (Figure 2A), the activity is four times lower than that of heparin when comparing mass concentrations and only one point five times lower when comparing molar concentrations (~16 kDa versus 40 kDa for heparin and recDS-2,4, respectively). While chemoenzymatic synthesis is highly developed for heparin/ heparan sulfate, the production of recombinant dermatan sulfate has so far gained less attraction (Xu et al. 2011;Zhang et al. 2017). In this article we report that functional DS, of non-eukaryotic origin, with well-defined chemical structures can be produced, enabling production of DS with different amounts of IdoA, 2-O-, 4-O-, and 6-O-sulfate.
The control of blood coagulation is of outermost importance, and we show that recombinant DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. Based on previously reported functions of mammalian DS, our method can potentially be extended to preparation of structures that are of importance for binding and activation of cytokines, control of collagen matrix structure, inflammation and metastasis involving P-selectin mediated extravasation and cancer cell migration (Maccarana et al. 2009;Kozlowski et al. 2011;Thelin et al. 2012;Mizumoto et al. 2013;Westergren-Thorsson et al. 2016).

Materials
PAPS and chondroitin was prepared as described previously (Hannesson et al. 1996;Zhou et al. 2011). Unfractionated heparin sodium salt with a weight average molecular weight (M w ) of 15.5 kDa and a dispersity of 1.28 (determined by multi-detection SEC, as below) was from porcine intestinal mucosa (Sigma-Aldrich H3393, Grade I-A, 179 USP units/mg).
Cloning and expression of DS-epi1, D4ST1, UST and GalNAc4S-6ST DS-epi1 and D4ST1 were cloned and expressed as previously described (Tykesson et al. , 2018. The part of the open reading frame of the human uronyl 2-Osulfotransferase (UST) gene UST (sequence harmonized, Genewiz, USA) corresponding to the lumenal amino acids 71 to 406 was subcloned together with a C-terminal 8xHIS tag into the NheI and NotI sites of a pCEP-Pu/BM40 (Kohfeldt et al. 1997 Size determination of recDS-4   Transfection, expression and purification was performed as previously described ).

Disaccharide analysis
Disaccharide analysis was essentially performed as described previously (Stachtea et al. 2015). In short, samples were buffer exchanged into an ammonium acetate buffer (50 mM, pH 7.5) and to each sample, in approximately 30 μL, chondroitinase ABC (10 mIU, Sigma-Aldrich) was added to depolymerize the polysaccharide products to Δ 4,5 -unsaturated uronic acid containing disaccharides. Depolymerization was achieved by incubation at 37°C for 4 h, after which the samples were boiled, centrifuged at 20,000 × g for 10 min and the supernatant was dried in a centrifugal concentrator and saved for future analysis. Pre-column, 2-aminoacridone-labeled DS disaccharides were analyzed on a Thermo Scientific UltiMate 3000 Quaternary Analytical system equipped with an FLD-3400RS fluorescence detector. For recDS-4, the polysaccharides were also degraded using chondroitinase B (2 mIU, R&D Systems) in 30 μL ammonium acetate buffer (50 mM, pH 7.5) overnight at 37°C. Disaccharide standards were from Iduron (Manchester, UK).

Multi-detection SEC
SEC was performed using a Malvern Panalytical OMNISEC system (Malvern, UK) consisting of Refractive Index (RI), Right Angle and Low Angle light scattering (RALS/LALS) and differential viscometer. All data was collected and processed using OMNISEC v10. For chromatographic separation, a Malvern Panalytical PLS3030 column (300 Å, 3 μM, 7.8 × 300 mm) was used with PBS buffer. For analysis of the polysaccharides a dn/dc of 0.12 ml/g was used as it was assumed that all samples had a composition like that of heparin.

NMR analysis
Experiments were performed at 298 K on a Bruker Avance III HD 800 MHz equipped with a TXO cryo probe and referenced to HDO at 4.70 ppm, with the following settings:
aPTT analysis aPTT was measured in triplicates by a SWEDAC accredited (ISO 15189) medical laboratory at the Skåne University Hospital on a CS-5100 automated analyzer (Siemens, Marburg, Germany) using the Actin FSL reagent (Siemens). The reference interval was 26-33 s with a coefficient of variation of <4%.

Statistical analysis
Data are expressed as mean values ± one standard deviation of experiments performed in triplicates, calculated using GraphPad Prism version 8.0.0.