Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection

One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end-labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.     

Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase

An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.     

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.     

The solution structure of heparan sulfate differs from that of heparin: implications for function

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments degree of polymerization 6-18 (dp6-dp18) and dp24, corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s(20,)(w) of HS dp6-dp24 showed a small rotor speed dependence, where similar s(20,)(w) values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6-dp24 gave radius of gyration (R(G)) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (R(XS)) values from 0.31 to 0.65 nm, and maximum lengths (L) from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5000-8000 conformationally randomized HS structures gave best fit dp6-dp16 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dp18 or dp24, indicating their higher flexibility. We conclude that HS displays an extended bent conformation that is significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.     

Crystal Structure of a Bacterial Unsaturated Glucuronyl Hydrolase with Specificity for Heparin

Extracellular matrix molecules such as glycosaminoglycans (GAGs) are typical targets for some pathogenic bacteria, which allow adherence to host cells. Bacterial polysaccharide lyases depolymerize GAGs in β-elimination reactions, and the resulting unsaturated disaccharides are subsequently degraded to constituent monosaccharides by unsaturated glucuronyl hydrolases (UGLs). UGL substrates are classified as 1,3- and 1,4-types based on the glycoside bonds. Unsaturated chondroitin and heparin disaccharides are typical members of 1,3- and 1,4-types, respectively. Here we show the reaction modes of bacterial UGLs with unsaturated heparin disaccharides by x-ray crystallography, docking simulation, and site-directed mutagenesis. Although streptococcal and Bacillus UGLs were active on unsaturated heparin disaccharides, those preferred 1,3- rather than 1,4-type substrates. The genome of GAG-degrading Pedobacter heparinus encodes 13 UGLs. Of these, Phep_2830 is known to be specific for unsaturated heparin disaccharides. The crystal structure of Phep_2830 was determined at 1.35-Å resolution. In comparison with structures of streptococcal and Bacillus UGLs, a pocket-like structure and lid loop at subsite +1 are characteristic of Phep_2830. Docking simulations of Phep_2830 with unsaturated heparin disaccharides demonstrated that the direction of substrate pyranose rings differs from that in unsaturated chondroitin disaccharides. Acetyl groups of unsaturated heparin disaccharides are well accommodated in the pocket at subsite +1, and aromatic residues of the lid loop are required for stacking interactions with substrates. Thus, site-directed mutations of the pocket and lid loop led to significantly reduced enzyme activity, suggesting that the pocket-like structure and lid loop are involved in the recognition of 1,4-type substrates by UGLs.     

Heterogeneity of depolymerized heparin SEC fractions: to pool or not to pool?

In the structural analysis of heparin and heparan sulfate, it is customary to combine or pool like-sized fractions obtained by size-exclusion chromatography (SEC) of enzymatically derived heparin oligosaccharides. In this study, we examine the heterogeneity of preparative-scale SEC fractions obtained from enzymatic digests of porcine intestinal mucosa heparin. Each fraction was profiled by capillary electrophoresis with UV detection (CE−UV) using a 60 mM formic acid running buffer at pH 3.43. Differences in the composition and relative concentration of components of the SEC fractions were observed for disaccharides and larger oligosaccharides. The heterogeneity of the fractions becomes more pronounced when heparin is digested using a heparin lyase cocktail. The heterogeneity of preparative SEC fractions was further investigated by reversed-phase ion-pairing ultraperformance liquid chromatography coupled with mass spectrometry (RPIP−UPLC−MS) using the ion-pairing reagent, tributylamine (Bu₃N). Our results suggest that preliminary profiling of preparative SEC fractions prior to pooling may simplify efforts to identify and/or isolate rare structures.     

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Acidolysis-based component mapping of glycosaminoglycans by reversed-phase high-performance liquid chromatography with off-line electrospray ionization–tandem mass spectrometry: Evidence and tags to distinguish different glycosaminoglycans

Diverse monosaccharide analysis methods have been established for a long time, but few methods are available for a complete monosaccharide analysis of glycosaminoglycans (GAGs) and certain acidolysis-resistant components derived from GAGs. In this report, a reversed-phase high-performance liquid chromatography (RP–HPLC) method with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization was established for a complete monosaccharide analysis of GAGs. Good separation of glucosamine/mannosamine (GlcN/ManN) and glucuronic acid/iduronic acid (GlcA/IdoA) was achieved. This method can also be applied to analyze the acidolysis-resistant disaccharides derived from GAGs, and the sequences of these disaccharides were confirmed by electrospray ionization–collision-induced dissociation–tandem mass spectrometry (ESI–CID–MS/MS). These unique disaccharides could be used as markers to distinguish heparin/heparan sulfate (HP/HS), chondroitin sulfate/dermatan sulfate (CS/DS), and hyaluronic acid (HA).     

Semi-synthetic heparin derivatives: chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/N-acetyl groups

The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide-protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS-protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.     

Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans

A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.     

A systems biology approach for the investigation of the heparin/heparan sulfate interactome

A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.     

The Solution Structure of Heparan Sulfate Differs from That of Heparin: IMPLICATIONS FOR FUNCTION*

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6–dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s_20,w of HS dp6–dp24 showed a small rotor speed dependence, where similar s_20,w values of 0.82–1.26 S (absorbance optics) and 1.05–1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6–dp24 gave radii of gyration R_G values from 1.03 to 2.82 nm, cross-sectional radii of gyration R_XS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6–dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.     

Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy

Biological and pharmacological interactions of heparin and structurally related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues. Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by high-performance liquid chromatographic analysis of fragments obtained by enzymatic or chemical degradation. Nuclear magnetic resonance (NMR) spectroscopy is also currently used for structural characterization of GAGs. However, the use of monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not allow reliable quantitative measurements. Using magnetically equivalent signals, the higher resolution achieved by two-dimensional NMR methods could be also exploited for quantitative applications. In this work, heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and HS mimics obtained by chemical modification of the Escherichia coli K5 polysaccharide (K5-PS) structurally related to the common biosynthetic precursor of heparin and HS. Heparin was used as a model for assessing the influence of 1H-13C spin-spin couplings on "volumes" of the corresponding signals. For major signals, the HSQC approach permitted quantification of additional structural features both in heparins and in a typical HS. The method was applied to profile the substitution patterns of K5-PS derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.     

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.     

Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C(5)-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments.     

Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography–mass spectrometry

Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography–mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3 wt% of heparin and from 6.2 to 8.3 wt% of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group-containing tetrasaccharides in a heparin correlated positively (r = 0.8294) to heparin’s anticoagulant activity.     

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

Quantitation of heparosan with heparin lyase III and spectrophotometry

Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected.     

Oligosaccharide Substrate Preferences of Human Extracellular Sulfatase Sulf2 Using Liquid Chromatography-Mass Spectrometry Based Glycomics Approaches

Sulfs are extracellular endosulfatases that selectively remove the 6-O-sulfate groups from cell surface heparan sulfate (HS) chain. By altering the sulfation at these particular sites, Sulfs function to remodel HS chains. As a result of the remodeling activity, HSulf2 regulates a multitude of cell-signaling events that depend on interactions between proteins and HS. Previous efforts to characterize the substrate specificity of human Sulfs (HSulfs) focused on the analysis of HS disaccharides and synthetic repeating units. In this study, we characterized the substrate preferences of human HSulf2 using HS oligosaccharides with various lengths and sulfation degrees from several naturally occurring HS sources by applying liquid chromatography mass spectrometry based glycomics methods. The results showed that HSulf2 preferentially digests highly sulfated HS oligosaccharides with zero acetyl groups and this preference is length dependent. In terms of length of oligosaccharides, HSulf2 digestion induced more sulfation decrease on DP6 (DP: degree of polymerization) compared to DP2, DP4 and DP8. In addition, the HSulf2 preferentially digests the oligosaccharide domain located at the non-reducing end (NRE) of the HS and heparin chain. In addition, the HSulf2 digestion products were altered only for specific isomers. HSulf2 treated NRE oligosaccharides also showed greater decrease in cell proliferation than those from internal domains of the HS chain. After further chromatographic separation, we identified the three most preferred unsaturated hexasaccharide for HSulf2.