# Unlocking Rare 3- O -Sulfation Patterns in Heparan Sulfate via ARSG-Directed Regioselective Desulfation and HILIC-MS

Salomé Poyer, Agnès Denys, Nesrine El Omrani, Mai-Britt Ilse, Fabrice

Allain, Markus Damme, Torben Lübke, Régis Daniel

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Salomé Poyer, Agnès Denys, Nesrine El Omrani, Mai-Britt Ilse, Fabrice Allain, et al. Unlocking Rare 3- O -Sulfation Patterns in Heparan Sulfate via ARSG-Directed Regioselective Desulfation and HILIC-MS. Analytical Chemistry, In press, 10.1021/acs.analchem.5c05985 . hal-05518239

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# Unlocking Rare 3-O-Sulfation Patterns in Heparan Sulfate via ARSG-Directed Regioselective Desulfation and HILIC-MS.

Salomé Poyer,¹,†* Agnès Denys,³ Nesrine El Omrani,¹ Mai-Britt Ilse,² Fabrice Allain,³ Markus Damme,⁴ Torben Lübke,² Régis Daniel¹*

1. Université Paris-Saclay, Univ Evry Paris-Saclay, CY Cergy Paris Université, CNRS, LAMBE, 91025 Evry-Courcouronnes, France

2. Department of Chemistry, Biochemistry III, Bielefeld University, 33615 Bielefeld, Germany

3. Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France.

4. Institute of Biochemistry, Christian-Albrechts-University Kiel, 24118 Kiel, Germany

**KEYWORDS:** 3-O-sulfate, heparan sulfate, arylsulfatase G, mass spectrometry, HILIC

**ABSTRACT:**

Deciphering rare 3-O-sulfated motifs in heparan sulfate (HS) is the key to understanding critical biological processes such as viral infection and tumor progression. However, the structural analysis of these elusive structures remains highly challenging due to their low abundance and the limitations of conventional analytical approaches. In this study, we report the application of human lysosomal arylsulfatase G (ARSG), a sulfatase specifically targeting 3-O-sulfated glucosamine residues, as a novel enzymatic tool, which in combination with heparinase and heparanase digestion, facilitates the identification of 3-O-sulfated glucosamine moieties within HS. Coupling ARSG-mediated regioselective desulfation with hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) enables the efficient detection and quantification of 3-O-sulfated oligosaccharides in HS and heparin derivatives. This approach overcomes a major analytical barrier in HS characterization and also provides new insights into the distribution and functional roles of 3-O-sulfation. The integration of ARSG expands the enzymatic toolkit available for HS structural characterization and represents a significant advance toward elucidating the structure-function relationships of HS in health and disease.

Glycosaminoglycans (GAGs) are anionic polysaccharides involved in physiological processes such as embryonic development, cell growth and differentiation, homeostasis, and cerebral plasticity, and in pathological disorders such as inflammation, neurodegeneration, and tumor growth.¹,² Among GAGs, heparan sulfate (HS) is a critical component of proteoglycans found in the extracellular matrix and at cellular membranes, through which communication necessary for biological and physical interactions between cells is carried out. HS is composed of repeating disaccharide units consisting of a hexuronic acid, either iduronic (IdoA) or glucuronic acid (GlcA), and the hexosamine glucosamine (GlcN). These disaccharides can be modified at multiple positions by sulfation and acetylation, creating HS motifs with isomeric heterogeneity making their sequence identification a real analytical challenge.³ Deciphering the mechanisms of interaction of HS with different biological factors is crucial to understanding the origin of these polysaccharides biological action and exploiting them as targets for therapeutic applications.⁴ This objective requires, among others, the establishment of structure-function relationships of HS, which is still a challenge today due to their high structural diversity.

The sequencing of HS and, in particular, the determination of the distribution of sulfate groups is an essential step to elucidate the biological properties of HS and determine their bioactive sequences. Considerable progress in the structural elucidation of GAGs has been made over the last two decades thanks to advances in mass spectrometry (MS)

and its coupling with separative methods.⁵ Although these methods have proven effectiveness in identifying the most abundant motifs, most of them only allow an analysis of the disaccharide composition, thus without easy-to-extract sequence information and blind to rare motifs. These latter are nonetheless crucial for the biological activity of GAGs. They include the 3-O-sulfation of glucosamine in HS, which is only present at a few percent (<4%) and yet is involved in or governs biological processes.⁶ Actually, 3-O-sulfation appears to be crucial in the cellular entry of the type-I Herpes simplex virus (HSV-I),⁷⁻⁹ in the propagation and cellular internalization of the aggregated tau protein in Alzheimer's disease,¹⁰, ¹¹ in neuronal growth,¹² in cell growth via interaction with growth factors and stabilization of complex with their receptors,¹³, ¹⁴ in the cellular clearance of heparin (HP) and HS by interacting with Stabilin-2 and hyaluronic acid receptor for endocytosis (HARE),¹⁵ and in cancer through the generation of pro-oncogenic determinants.¹⁶⁻¹⁸ Of all the biological processes involving 3-O-sulfation, only the one concerning the inhibition of the coagulation cascade could be supported by a precise structure/activity relationship that highlighted a unique pentasaccharide sequence featuring a central 3-O-sulfated GlcN unit required for anti-coagulant activity via interaction with antithrombin and factor Xa.¹⁹⁻²¹

The advent of a wide variety of MS fragmentation techniques has prompted considerable progress in the structural elucidation of GAGs.²²⁻²⁷ However, rare structural motifs such as 3-O-sulfation remain the blind spot in these devel-
opments. In recent years, more sophisticated methodological developments have emerged to target 3-O-sulfation.²⁸, ²⁹ The use of isotopically labeled ¹³C-oligosaccharides as calibrants combined with AMAC-labeled HP/HS digestion products has been recently reported to determine the level of 3-O-sulfated oligosaccharides.³⁰ A synthetic route to access 3-O-sulfated HS disaccharide standards has also been described for the liquid chromatography (LC) compositional analysis at the disaccharide level of clinical HPs and alterations in HS profiles.³¹ The negative electron transfer dissociation capabilities to distinguish 3-O-sulfation have been reported using a hybrid Qh-FTICR mass spectrometer.³² Ultraviolet photodissociation (UVPD) of 3-O-sulfated pentasaccharide carried out on an Orbitrap mass spectrometer equipped with UVPD at 213 nm allowed the identification of a 3-O-sulfated monosaccharide diagnostic ion.³³ More recently, ion mobility-mass spectrometry has been introduced as a complementary MS-based approach to specifically target and resolve 3-O-sulfated HS motifs.³⁴

Another way to progress in the knowledge of GAGs is the exploitation of enzymes that, combined with MS, are valuable tools for structural characterization.³⁵ The microbial depolymerization enzymes heparinases (Hep) are thus widely used to reduce the structural complexity and to produce fractions enriched according to their sequence specificities. The sulfate monoester-cleaving sulfatases allow modulating regioselectively the sulfate content as the sulfatases HSulf, which target the 6-O-sulfation in HP/HS,³⁶ and 4-O-sulfatase, targeting the 4-O-sulfation of chondroitin.³⁷ The arylsulfatase G (ARSG) specifically hydrolyzing the 3-O-sulfation of the monosaccharide GlcN has been reported.³⁸ In eukaryotic cells, lysosomes are equipped with the full set of GAG-degrading enzymes, offering a toolbox for GAG-analysis.³⁹ ARSG is active only on the 3-O-sulfated glucosamine residue carrying an N-substituent, either an N-sulfate or an N-acetyl group, with a clear preference for N-sulfated substrates.³⁸ Experimentally validated

substrates include GlcNS3S, GlcNS3S6S, GlcNAc3S6S and GlcNAc3S, whereas ARSG shows no detectable activity toward the 3-O-sulfate of N-unsubstituted glucosamine. This strict regioselectivity has been firmly established in previously reported studies.³⁸, ⁴⁰ The regioselectivity of ARSG is very interesting in exploring the molecular space of rare 3-O-sulfation sparsely dispatched in HP/HS. Here, we extend the study of the lysosomal enzyme ARSG to its action on sulfated oligosaccharides and its combination with MS to determine the level of 3-O-sulfation in HP/HS. We show that its exploitation combined with MS-coupled hydrophilic interaction liquid chromatographic (HILIC) separation proves to be a potent tool to identify and quantify rare 3-O-sulfate groups.⁴¹

## RESULTS AND DISCUSSION

1. ARSG enzymatic activity on 3-O-sulfated oligosaccharides *HILIC-MS analysis of 3-O-sulfated oligosaccharides.* The regioselective activity of ARSG has been previously reported on 3-O-sulfated monosaccharide.³⁸, ⁴⁰ To study the action of ARSG on substrates longer than monosaccharides, we developed a HILIC-MS method allowing the separation of HP sulfated oligosaccharides. This method is compatible with high salt concentrations commonly used in enzymatic reactions. It uses a positively charged stationary phase, separating HP oligosaccharides according to their size and sulfate location. The quench solvent was adapted to permit the direct analysis of the reaction mixture containing enzymes and oligosaccharide products below about ten micromolar, without prior treatment (filtration, desalination). We obtained efficient separation and online MS analysis of HP oligosaccharides for different sizes (up to dp12) and degrees of sulfation (see Sup. Info). We investigated the ARSG activity on various 3-O-sulfated oligosaccharides containing a 3-O-sulfate group within their sequence (Table 1).

<table><caption>**Table 1.** Reaction products observed by HILIC-MS upon heparanase and/or heparinase I digestion of commercial HS oligosaccharides</caption><thead><tr><th>Oligosaccharide substrates</th><th>Reaction products by heparinase I</th><th>Reaction products by heparanase</th></tr></thead><tbody><tr><td>GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-O-Me</td><td>GlcNS6S-GlcA-GlcNS3S6S +<br>ΔUA2S-GlcNS6S-O-Me</td><td>GlcNS6S-GlcA +<br>GlcNS3S6S-IdoA2S-GlcNS6S-O-Me</td></tr><tr><td>GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td><td>GlcNS6S-GlcA-GlcNS3S6S +<br>ΔUA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td><td>GlcNS6S-GlcA +<br>GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td></tr><tr><td>GlcNAc6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td><td>GlcNAc6S-GlcA-GlcNS3S6S +<br>ΔUA2S-GlcNS6S +<br>ΔUA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td><td>GlcNAc6S-GlcA +<br>GlcNS3S6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td></tr><tr><td>GlcNAc6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td><td>GlcNAc6S-GlcA-GlcNS6S +<br>ΔUA2S-GlcNS6S +<br>ΔUA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td><td>GlcNAc6S-GlcA +<br>GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl</td></tr><tr><td colspan="3">Reaction products heparinase I and heparanase</td></tr><tr><td>GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-O-Me</td><td>GlcNS6S-GlcA + GlcNS3S6S + ΔUA2S-GlcNS6S-O-Me</td><td></td></tr></tbody></table>

<footnote>* oligosaccharides containing a 3-O-SO₃ are underlined</footnote>
To increase the substrate diversity offered to ARSG, these 3-*O*-sulfated oligosaccharides were further submitted to the action of bacterial heparinases or heparanase. Indeed, thanks to the distinct regioselectivity of heparinases and human heparanase (HPSE),⁴²⁻⁴⁶ we formed additional 3-*O*-sulfated oligosaccharides either with a terminal 3-*O*-sulfate group at the reducing end (with heparinase I and/or II) or at the non-reducing end (with heparanase) (Table 1).

Four different synthetic oligosaccharides (Table 1) were submitted to reaction by heparinase I and heparanase to form additional 3-*O*-sulfated oligosaccharides. Their identification was determined by HILIC-MS experiments (Figure 1 and SI), and the sulfate assignation was established by tandem mass spectrometry performed on sodiated ions (SI).⁴⁷

<figure><img src="image_2.png" /><figcaption>a) Fondaparinux pentasaccharide digested by Hep-I</figcaption></figure>

**Figure 1.** HILIC-MS analysis of GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-O-Me digested by a) heparinase I, b) heparanase or c) heparanase and ARSG. Chromatograms correspond to the extracted ion chromatogram (EIC) overlay of the [disaccharide-H]⁻ at a) *m/z* 589 or b) and c) *m/z* 514 and [trisaccharide-2H]²⁻ at a) *m/z* 456, b) *m/z* 503 or c) *m/z* 463. Gradient LC of 35 to 55% of A in 15min, injection 5µL.

We were then able to identify and produce six additional 3-*O*-sulfated saccharides from mono- (GlcNS3S6S) to hexasaccharide (GlcNS3S6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl) with two of them bearing a 3-*O*-sulfate at the reducing end (RE) and the four others at the non-reducing end (NRE), the latter being the physiological substrate of ARSG. Trisaccharides with 3-*O*-sulfated RE were produced by heparinase I digestion, and tri-, tetra-, and hexasaccharides with 3-*O*-sulfated NRE were produced

by heparanase digestion as expected. The monosaccharide GlcNS3S6S was obtained from the tandem digestion of the synthetic Factor Xa inhibitor Fondaparinux pentasaccharide by heparanase and heparinase I.

ARSG is active on 3-*O*-sulfated non-reducing end oligosaccharides. The activity of ARSG on the monosaccharide GlcNS3S6S as determined by HILIC-MS, showed partial desulfation of this monosaccharide by ARSG after 24h of reaction in agreement with previously reported results,⁴⁸,⁴⁹ confirming that the HILIC-MS method is suitable to monitor the ARSG enzymatic activity. We observed a partial 3-*O*-desulfation of the trisaccharide GlcNS3S6S-IdoA2S-GlcNS6S-O-Me issued from the digestion by heparanase of the Fondaparinux pentasaccharide, which was confirmed by MS/MS analysis (Figure S1). Interestingly, the 3-*O*-desulfation of this trisaccharide was total in the same reaction time when ARSG was added together with heparanase to the Fondaparinux pentasaccharide (Figure 1c, Figure S2).

These different desulfation rates could be explained by the formation of a conformational intermediate generated by heparanase, which would favor the subsequent sulfate hydrolysis by ARSG. These results demonstrate that ARSG can accept longer substrates than the previously characterized monosaccharides.³⁸ It also suggests that heparanase and ARSG may be connected somehow, potentially through a functional relationship and/or as part of a multi-enzyme complex, a hypothesis that warrants further investigation. To investigate the action of ARSG on longer oligosaccharides, we measured its activity on the tetrасасcharide (GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl) and the hexasaccharide (GlcNS3S6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl) produced by heparanase (Figure S3 and S4). In these experiments, ARSG was added with heparanase to maximize sulfatase activity. Both tetra- and hexasaccharides lost one sulfate group after incubation with ARSG. The regioselective 3-*O*-sulfate hydrolysis was confirmed by the lack of ARSG activity on the non-3-*O*-sulfated analog hexasaccharide GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-O-p-nitrophenyl. We did not observe any enzymatic desulfation on oligosaccharides carrying a 3-*O*-sulfate group located within their sequences, as for the Fondaparinux pentasaccharide GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-O-Me without prior heparanase digestion. Moreover, when the 3-*O*-sulfate group was located at the reducing end, as for the trisaccharide GlcNS6S-GlcA-GlcNS3S6S released from Fondaparinux after heparinase digestion, no ARSG activity was observed (Figure S5). These results show that ARSG catalyzes the regioselective hydrolysis of the 3-*O*-sulfate group of oligosaccharides as long as a hexasaccharide. Moreover, this activity is carried out only at the non-reducing end, confirming its activity as an exclusive exosulfatase.

This study shows the combined use of heparanase and heparinase I as a practical approach to generate ARSG substrate saccharides. It should be noted that no heparinase can produce such ARSG substrate sequences alone, since they form oligosaccharides with a 3-*O*-sulfated reducing end. Nevertheless, the characterization of low molecular weight HP (LMWH) most often uses heparinases, which, thus, does not allow the exploitation of ARSG to de-
termine the 3-O-sulfation of these HP. However, only the combined action of heparanase and heparinase I enables the generation of the 3-O-sulfated monosaccharide substrate of ARSG, namely GlcNS3S6S. Therefore, as we demonstrated that heparinases and heparanase can be easily used together without time-consuming setups, in the following experiments, we used the combination of these enzymes plus ARSG to highlight 3-O-sulfated motifs in LMWH.

## 2. Determination of the 3-O-SO₃ motif in heparin.

3-O-sulfation is one of the final modifications in HS biosynthesis, explaining why 3-O-sulfated motifs are predominantly found in the highly sulfated regions of HS, known as S-domains. HP, primarily composed of sulfated disaccharides, is therefore commonly used as a structural surrogate for the S-domains of HS. We first performed heparinase II digestion of LMWH (Enoxaparin and Dalteparin) and of full-length HP (Celsius) to form a wide variety of disaccharide products, given the broad specificity of heparinase II and also to take advantage of the reported resistance of 3-O-sulfated sequences to this enzyme.⁵⁰ In particular, we looked for the formation of heparinase II-resistant tetrasaccharides potentially containing the 3-O-SO₃ motif. The HILIC analysis of the digestion mixture showed the presence of tetrasaccharides, including those with the unnatural LMWH-specific reducing end ΔUA2S-RE (Scheme 1).⁵¹,⁵² All the disaccharides separated by HILIC were quantified by UV and annotated based on their retention times and comparison with disaccharide standards (Table 2 and Figure S6). The observation of the LMWH-specific reducing end disaccharide ΔUA2S-RE indicates that the action of heparinase II is not affected by this modification and, consequently, that this unnatural reducing end is not the cause of the heparinase II-resistant tetrasaccharides.

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Scheme 1.** ΔUA2S-RE of a) Enoxaparin, and b) Dalteparin.</figcaption></figure>

<table><caption>**Table 2.** Disaccharide composition of heparin samples quantified from HILIC-UV experiments based on retention times (Figure S6) and *m/z* values. Heparinase II digestion performed in triplicate (Enoxaparin and Dalteparin) or in duplicate (Heparin Celsius).</caption><thead><tr><th></th><th>Enoxaparin</th><th>Dalteparin</th><th>Heparin Celsius</th></tr></thead><tbody><tr><td>Δ UA2S-GlcNAc6S (I-A)</td><td>0.8 ± 0.1</td><td>1.0 ± 0.1</td><td>0.8 ± 0.1</td></tr><tr><td>Δ UA-GlcNAc6S (II-A)</td><td>2.9 ± 0.2</td><td>2.9 ± 0.2</td><td>2.4 ± 0.2</td></tr><tr><td>Δ UA2S-GlcNAc (III-A)</td><td>1.3 ± 0.3</td><td>0.8 ± 0.4</td><td>1.7 ± 0.1</td></tr><tr><td>Δ UA-GlcNAc (IV-A)</td><td>0.9 ± 0.1</td><td>0.1 ± 0.1</td><td>1.0 ± 0.3</td></tr><tr><td>Δ UA2S-GlcNS6S (I-S)</td><td>66.1 ± 0.8</td><td>61.3 ± 0.6</td><td>69.8 ± 0.1</td></tr><tr><td>Δ UA-GlcNS6S (II-S)</td><td>11.0 ± 0.7</td><td>10.3 ± 0.3</td><td>9.8 ± 0.1</td></tr><tr><td>Δ UA2S-GlcNS (III-S)</td><td>7.3 ± 0.4</td><td>4.5 ± 0.3</td><td>8.3 ± 0.1</td></tr><tr><td>Δ UA-GlcNS (IV-S)</td><td>3.1 ± 0.1</td><td>1.4 ± 0.1</td><td>3.7 ± 0.1</td></tr></tbody></table>

<table><thead><tr><th></th><th>Δ UA2S-RE</th><th>2.1 ± 0.5</th><th>6.5 ± 2.9</th><th>ND</th></tr></thead><tbody><tr><td>Digestion-resistant tetrasaccharides</td><td>4.5 ± 1.4</td><td>11.2 ± 3.4</td><td>2.5 ± 0.4</td></tr></tbody></table>

Heparinase II catalyzes the hydrolysis of the glycosidic bond between the glucosamine and the iduronic and glucuronic acid,⁵⁰ yielding ΔUA-GlcN disaccharides with various sulfation degrees measurable by UV. Based on the retention times of standard disaccharides and utilizing UV detection, we determined the proportion of the different disaccharides in these various HP samples (Figure S6). The disaccharide composition (Table 2) of these LMWHs is in agreement with the literature.⁴⁵,⁵²

a) Enoxaparin digested by Hep-II

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

**Figure 2.** EIC overlays from HILIC-MS experiments of enoxaparin digested by a) heparinase II, b) heparinase II and heparanase and c) heparinase II, heparanase, and ARSG. Grey peaks correspond to disaccharides summarized in Table 2; the black peak corresponds to ΔUA2S-RE; the red peak corresponds to ΔUA-GlcNAc-GlcA(1S); and the blue peaks corresponds to [GlcNS3S6S-SO₃]⁻. The use of heparanase and ARSG after heparinase II digestion enables the release and identification of 3-O-SO₃ monosaccharides in Enoxaparin by HILIC-MS analysis. More details are provided in Figure S7.

In addition to the main disaccharides, we were also able to detect two minor disaccharides (2% for enoxaparin and 6.5% for Dalteparin) in each LMWH sample, one corre-
sponding to ΔUA2S-RE of Dalteparin and enoxaparin and was observed at *m/z* 480.9957 and 477.9959, respectively (Scheme 1). Finally, we observed tetrasaccharides that we attributed to heparinase II-resistant species bearing a 3-*O*-sulfate group. As expected, 3-*O*-SO₃-resistant tetrasaccharides were observed more in LMWH samples than in full-length HP. Because 3-*O*-sulfation prevents complete enzymatic depolymerization into disaccharides, 3-*O*-sulfated dp2 species are not directly detected and reported in Table 2. Instead, the extent of 3-*O*-sulfation is inferred from the relative abundance of digestion-resistant tetrasaccharides generated upon enzymatic treatment.

We determined the 3-*O*-sulfated motive in the heparinase II-resistant tetrasaccharides by combining their hydrolysis by heparanase and subsequent regioselective 3-*O*-desulfation by ARSG and HILIC-MS analysis. The heparanase digestion led to the formation of a saturated trisulfated monosaccharide which loses one sulfate upon ARSG reaction, showing that this monosaccharide is 3-*O*-sulfated and corresponds to GlcNS3S6S (Figure 2). It should be pointed out that heparinase II-resistant tetrasaccharides presenting a sulfated IdoA unit were not substrate of heparanase (especially tetrasaccharides bearing the initial RE of LMWH).⁵³ This specificity allowed us to highlight a high proportion of the disaccharide IdoA2S-GlcNS3S6S in the analyzed HP samples, corresponding to resistant 3-*O*-SO₃-tetrasaccharides not digested by heparinase (last entry Table 2). This result clearly shows that heparinase II-resistant 3-*O*-sulfated tetrasaccharides contain this disaccharide motif; meanwhile, other 3-*O*-sulfated motifs are also certainly present, as indicated by the detection of resistant tetrasaccharides with lower degrees of sulfation (Figure S6). In fact, for the three HS analyzed, even if the 3-*O*-SO₃ motive was clearly observed from the saturated monosaccharide formed, no significant signal variation was observed from the heparinase II-tetrasaccharide even after multiple heparanase additions.

The 3-*O*-sulfated content in LMWH could thus be identified and quantified by HILIC-MS analysis of the tandem digestion of resistant tetrasaccharides by heparanase and ARSG.

Although most heparinase-resistant tetrasaccharides contained an IdoA2S adjacent to GlcNS6S3S, heparanase digestion also revealed rarer motives such as IdoA2S-GlcNAc-GlcA-GlcNS3S6S and GlcA-GlcNAc6S-GlcA-GlcNS3S6S. These motives were revealed in enoxaparin, dalteparin, and HP Celsus by the detection of trisaccharides ΔUA-GlcNAc-GlcA(1S) after digestion with heparanase (Figure 2, Figures S7 and S8). The presence of a unique sulfate group on the trisaccharides avoids its digestion by heparanase,⁵⁴,⁵⁵ and MS/MS performed on this species allows the sulfate location either on the ΔUA or on the GlcNAc (Figure S8) of these low-sulfated trisaccharides observed concomitantly with GlcNS3S6S after heparanase digestion.

### 3. Determination of the 3-*O*-sulfation in cellular HS

With seven isoenzymes identified in humans, HS 3-*O*-sulfotransferases (HS3STs) represent the largest family among HS-modifying enzymes. Under normal physiological conditions, the expression levels of these enzymes are low or non-detectable in adult tissues.⁶ As a result, 3-*O*-sulfated units are present in only a limited number of HS chains or may be entirely absent. Moreover, two distinct 3-*O*-sulfated

motifs can be synthesized by these isoenzymes: HS3ST1 and HS3ST5 participate in the generation of anticoagulant-active HS/HP binding sequences for antithrombin-III (AT-type), while HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, and HS3ST6 were involved in the production of HS-binding motifs for the viral envelope glycoprotein gD of herpes simplex virus-1 (HSV-1) (gD-type). Since then, only a few ligands and biological systems have been identified to be selectively influenced by 3-*O*-sulfated motifs, apart from their roles in the anticoagulant properties of antithrombin and HSV-1 infection.⁶,¹⁸

To illustrate ARSG as a tool of choice for determining 3-*O*-sulfation in a biological context, we combined this enzyme with HILIC-MS analysis to identify 3-*O*-sulfated motifs in HS expressed at the cell surface. For this purpose, HS was purified from a clonal cell line derived from the MDA-MB-231 cell line, which was modified to stably overproduce the HS 3-*O*-sulfotransferase HS3ST3B. To show that the method can track variations in the 3-*O*-sulfation, we also purified HS from parental cells.⁵⁶ As described in our previous work, overexpression of HS3ST3B in these cells resulted in the production of HS containing 3-*O*-sulfated sequences. Analysis by confocal microscopy confirmed that a recombinant form of the HSV-1 gD protein, reported to specifically interact with 3-*O*-sulfated HS motifs, bound to the surface of HS3ST3B-expressing cells, whereas no binding was observed on parental MDA-MB-231 cells.¹⁷,⁵⁶ Following HS depolymerization, analysis of fluorescently-labelled disaccharides by C18 RP-HPLC revealed the presence of new sulfated products in HS3ST3B-overexpressing cells, which were attributed to 3-*O*-sulfated species. Cellular HS displayed a disaccharide profile distinct from LMWH, as revealed by HILIC-MS analysis following heparinase II digestion (Figure 3).
<figure><img src="image_8.png" /><figcaption>**Figure 3.** EIC overlays from HILIC-MS experiments of a) and b) MDA-MB-231 parental cells, or c) and d) HS3ST3B-overexpressing cells digested by heparanase (HPSE) (a and c) and ARSG (b and d) after heparinase II (Hep II) treatment. Grey peaks correspond to disaccharides listed in **Table 2**, and blue peaks to [GlcNS3S-SO₃]⁻. See **Figure S9** for further details.</figcaption></figure>

This difference was consistent with the heterogeneous
sulfation pattern characteristic of cellular HS. Quantification
was not performed due to the low sensitivity of UV detec-
tion. Compared to their MDA-MB-231 parental cells, the
HS3ST3B-overexpressing cells exhibited a significantly
higher proportion of 3-O-sulfated monosaccharides after
digestion with heparanase and ARSG (**Figure 3**). This ob-
servation indicates that the overexpressed sulfotransferase
effectively altered the sulfation pattern of cell membrane
HS. These monosaccharides are, however, less sulfated than
those observed with LMWH digestions. The presence of a
diagnostic fragment ion observed at *m/z* 137.99 from
HILIC-MS experiments using 20 eV of collisional energy in
the collision cell indicates a NS sulfation (**Figure S10**).
Indeed, this ion was observed at retention times of species
annotated as GlcNS3S and GlcNS on **Figure 3**, but also for all
HS disaccharides of the S series. In addition, the disappear-
ance of the peak GlcNS3S to the benefit of the peak of GlcNS
after digestion by ARSG confirms the 3-O-SO₃ motive. The
3-O-SO₃ enrichment of this motif was also supported by the
relative decrease of IV-S disaccharide unit (ΔUA-GlcNS) in
HS3ST3B-overexpressing cells compared to parental ones
(**Figure 3**).

HS3ST3B introduces a sulfate group at the 3-OH position of
a GlcNS residue linked to an adjacent upstream 2-O-sulfated
IdoA. According to the known specificities of heparanase,
this hydrolase catalyzes the hydrolysis of glycosidic bonds
exclusively between GlcNS residues and non-sulfated IdoA
units. The observed increase in 3-O-sulfation following
digestion with heparanase suggests that the specificity of
HS3ST3B may be broader than previously anticipated.

CONCLUSIONS

This study highlights the analytical power of the lysosomal
enzyme arylsulfatase G (ARSG), used in combination with
heparinase and heparanase, to reveal and quantify rare 3-
O-sulfation motifs in HP and HS sequences. Through an
optimized HILIC-MS approach, we demonstrated the
exosulfatase specificity of ARSG for non-reducing ends bear-
ing a 3-O-sulfate group and confirmed its activity on oligo-
saccharides up to six units in length. The analysis of
tetrasaccharides resistant to heparinase II digestion ena-
bled the identification of 3-O-sulfated motifs in low-
molecular-weight HP, and this methodology was further
extended to detect these motifs in cell-derived HS.

These results underscore the value of ARSG as an enzymatic
tool for fine mapping of 3-O-sulfation in HS, paving the way
for a better understanding of their functional roles in patho-
physiological processes.⁵⁷ The present workflow, combined
with targeted MS/MS analyses, opens the way of discovering
previously uncharacterized 3-O-sulfated patterns in HS and
identification of rare or novel local environments surrounding
3-O-sulfated GlcN, thereby extending its potential application
to HS samples of unknown structure. This approach adds to
the still-limited set of tools available for the detailed struc-
tural analysis of GAGs. It provides a valuable complement to
recently developed methods aimed at deciphering the mo-
lecular determinants of GAG-protein interactions,⁵⁸ which
remain largely unexplored despite their central role in many
biological processes. These advances lay the groundwork
for improved insight into GAG structure-function relation-
ships and for the identification of new bioactive determi-
nants that could be exploited more specifically for diagnos-
tic or therapeutic purposes. Beyond quantifying rare 3-O-
sulfated glucosamine units, this enzymatic-MS workflow
also provides a foundation for future sequence-level charac-
terization of the regions flanking these motifs. In particular,
targeted MS/MS analysis of the oligosaccharides generated
by the combined action of heparinases and heparanase
could enable the identification of distinct surrounding se-
quences, a critical yet still unexplored aspect of 3-O-
sulfotransferase specificity.

EXPERIMENTAL METHODS

Production of 3-O-sulfated HS and purification of cellular
HS.

Human breast cancer MDA-MB-231 cells (ATCC® HTB-26™) were routinely cultured at 37°C in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (Lonza, Verviers, Belgium), in an atmosphere containing 5% CO₂. The clonal cell lines with stable overexpression of HS3ST3B were constructed as described.¹⁷, ⁵⁶ Following transfection with the plasmid encoding human
HS3ST3B, MDA-MB-231 cells were cultured in the presence of the antibiotic G418 and isolated by limiting dilution. Then, resistant colonies were maintained in a culture medium supplemented with G418 to obtain cellular clones with stable overexpression of HS3ST3B. In parallel, MDA-MB-231 cells were stably transfected with the empty vector to obtain control parental cells.

The extraction of HS was performed essentially as described.<sup>17,56</sup> Briefly, 75 × 10<sup>6</sup> cells were collected and resuspended in 2 mL of lysis buffer (Tris-HCl 50 mM, pH 7.9, 100 mM NaCl, Triton X100 1%) at 4°C for 6 h. After centrifugation, the supernatant was treated with Pronase E (Merck Millipore, Darmstadt, Germany) (1 mg/mL) at 50°C for 16 h. Following the inactivation of the enzyme at 100°C, sample was clarified by centrifugation and treated with benzonase (250 mU/mL) (Sigma-Aldrich, Schnelldorf, Germany) at 37°C for overnight. The final concentration of NaCl was adjusted to 1 M, after which the mixture was complemented with chloroform (vol/vol) and methanol (1/10 vol) and stirred vigorously. After centrifugation, the aqueous phase was dialysed overnight at 4°C (Slide-A-Lyser 2000 Da, Thermo Fischer Scientific) in H<sub>2</sub>O, lyophilized and the sample was then treated with chondroitinase ABC (300 mU/mL) at 37°C for 2 h in buffer containing 50 mM of NaHCO<sub>3</sub>, 2 mM CaCl<sub>2</sub>, Tris 50 mM, pH 8. After inactivation of the enzyme at 100°C and centrifugation, the supernatant was dialyzed in H<sub>2</sub>O and lyophilized. The amount of purified HS was quantified by using a carbazole assay.

## Purification of recombinant murine ARSG

C-terminally His-tagged mouse ARSG was purified as described previously and purified fractions were checked by a sulfatase assay using para-nitrocatechol sulfate (pNCS) as artificial substrate.<sup>38</sup>

## Enzymatic digestions

All oligosaccharides presented in Table 1 were purchased from Iduron except for Fondaparinux pentasaccharide which was purchased from Sigma Aldrich and were used without purification. Enoxaparin was used form LOVENOX 4 000 UI (40 mg) / 4.0 ml from Biogaran Laboratory, Dalteparin from Sigma Aldrich and HP from Celsius Laboratories Inc.

For the Heparanase enzymatic reaction, 2.5 µL of Heparanase 0.11 g/L were mixed to 45 µL NaOAc 40 mM pH 5.0, containing 100 µM of oligosaccharide, and incubated overnight at 37 °C in a dry bath. For the Heparinase I and II reactions, 2.5 µL of Heparinase I 5 mU/µL were mixed to 45 µL of a buffer TrisHCl 20 mM / CaCl<sub>2</sub> 3 mM at pH 7.5, containing 100 µM of oligosaccharide, and incubated overnight at 37°C. For the ARSG enzymatic reaction, 5 µL of ARSG 332 mU was mixed with 45 µL NaOAc 100 mM pH 5.0, containing 100 µM of oligosaccharide, and incubated overnight at 37 °C. When tandem reactions were involved, the previous reaction was diluted by three using the buffer corresponding to the second enzyme without other action or after boiling of the first reaction 5 min at 95 °C if noted. For each enzymatic reaction, 5µL of the overnight resulting mixture was quenched by diluting them in a mixture containing 20 µL of H<sub>2</sub>O and 50 µL of MeCN and were

analyzed by HILIC-MS without further purification nor dilution.

## HILIC-MS/MS experiments

HILIC-MS experiments were carried out using a quadrupole-time of flight (Q-TOF) mass spectrometer (Xevo G2-S QTOF, Waters) operating in the negative ionization mode. MS and MS/MS experiments were performed using the resolution mode in the *m/z* 50 to 1200 mass range. Electrospray (ESI) source parameters were set as follows: capillary voltage 2.5 kV, sampling cone voltage: 50 V, source temperature 80°C, desolvation gas flow: 800 L/Hr at 250 °C. The TOF analyzer was calibrated daily using a sodium formate solution and leucine encephalin was used as an internal standard for all MS experiments.

HILIC separation of HS analogues was achieved using a ZIC-cHILIC column (SeQuant® ZIC®-cHILIC, 3µm, 100 Å 150 × 2.1 mm, VWR, France) fitted on an Acquity H-class system. Solvent A consisted of ammonium formate at 7.5 mM adjusted at pH 4.0 with formic acid, and solvent B was 100 % acetonitrile. The autosampler was set at 8 °C, and 1 to 3 µL µL of HS mixtures were injected depending on the experiment. The flow rate was 300 µL/min, and the column was thermostated at 30°C. The gradient used to analyze HS oligosaccharides was 35 % to 55 % solvent A ramping in 15 min, and was adjusted to 23 % to 60 % solvent A ramping in 20 min for disaccharide and enzymatic digestion of LMWH. Both HILIC gradients were followed by 100% solvent A to remove salts and then 10 % A activation before getting back to initial conditions for a total runtime of 32 and 37 min, respectively. MassLynx V4.1 software was used to process data. All quantitative data in **Table 2** were obtained from the area of identified UV peaks and normalized to 100 %

# ASSOCIATED CONTENT

## Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

MS/MS mass spectra of oligosaccharides and HILIC-MS spectra of enzyme reactions (PDF)

# AUTHOR INFORMATION

## Corresponding Author

Salomé Poyer†, Université Paris-Saclay, Univ Evry, CY Cergy Paris Université, CNRS, LAMBE, 91025, Evry-Courcouronnes, France. https://orcid.org/0000-0003-0628-7383. E-mail: salome.poyer@cnrs.fr

Régis Daniel, Université Paris-Saclay, Univ Evry, CY Cergy Paris Université, CNRS, LAMBE, 91025, Evry-Courcouronnes, France. https://orcid.org/0000-0003-0725-5439 E-mail: regis.daniel@univ-evry.fr

## Present Addresses

†Université Paris-Saclay, CNRS, ICSN, UPR 2301, 91198, Gif-sur-Yvette, France
## Author Contributions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

## ACKNOWLEDGMENT

This work was supported by the Université Evry Paris-Sacaly, the Université de Lille and the CNRS

## ABBREVIATIONS

Glycosaminoglycans (GAGs); heparan sulfate (HS), heparin (HP) glucuronic (GlcA), iduronic acid (IdoA), glucosamine (GlcN), polymerization degree (dp), type-I Herpes simplex virus (HSV-I), mass spectrometry (MS), liquid chromatography (LC), 2-aminoacridone (AMAC), ultraviolet photodissociation (UVPD), arylsulfatase G (ARSG), hydrophilic interaction liquid chromatography (HILIC), reducing end (RE), non-reducing end (NRE), extracted ion chromatogram (EIC), heparinase-I (Hep-I), heparinase-II (Hep-II), heparanase (HPSE) low molecular weight heparin (LMWH), Dulbecco's Modified Eagle Medium (DMEM), quadrupole-time of flight (Q-TOF), Electrospray (ESI), tandem mass spectrometry (MS/MS).

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