Heparan Sulfate Oligosaccharides Research Articles

Heparan sulfate selectively inhibits the collagenase activity of cathepsin K

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in resorption of bone matrix. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS regulates the biological functions of CtsK, remains largely unknown. In this report, we discovered that HS is a multifaceted regulator of the structure and function of CtsK. Structurally, HS forms a highly stable complex with CtsK and induces its dimerization. Co-crystal structures of CtsK with bound HS oligosaccharides reveal the location of the HS binding site and suggest how HS may support dimerization. Functionally, HS plays a dual role in regulating the enzymatic activity of CtsK. While it preserves the peptidase activity of CtsK by stabilizing its active conformation, it inhibits the collagenase activity of CtsK in a sulfation level-dependent manner. These opposing effects can be explained by our finding that the HS binding site is remote from the active site, which allows HS to specifically inhibit the collagenase activity without affecting the peptidase activity. At last, we show that structurally defined HS oligosaccharides effectively block osteoclast resorption of bone in vitro without inhibiting osteoclast differentiation, which suggests that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor for many diseases involving exaggerated bone resorption.

Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

Synthesis of a heparan sulfate tetrasaccharide using automated glycan assembly.

Herein we utilise automated glycan assembly to complete solid-phase synthesis of defined heparan sulfate oligosaccharides, employing challenging D-glucuronate disaccharide donors. Using an orthogonally protected D-GlcN-α-D-GlcA donor, milligram-scale synthesis of a heparan sulfate tetrasaccharide is completed in 18% yield over five steps. Furthermore, orthogonal protecting groups enabled regiospecific on-resin 6-O-sulfation. This methodology provides an important benchmark for the rapid assembly of biologically relevant heparan sulfate sequences.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 μM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

Synergestic interplay of uronic acid and sulfation composition of heparan sulfate on molecular recognition to activity

Heparan sulfate (HS) is ubiquitous polysaccharide on the surface of all mammalian cells and extracellular matrices. The incredible structural complexity of HS arises from its sulfation patterns and disaccharide compositions, which orchestrate a wide range of biological activities. Researchers have developed elegant synthetic methods to obtain well-defined HS oligosaccharides to understand the structure-activity relationship. These studies revealed that specific sulfation codes and uronic acid variants could synergistically modulate HS-protein interactions (HSPI). Additionally, the conformational flexibility of l-Iduronic acid, a uronic acid unit has emerged as a critical factor in fine-tuning the microenvironment to modulate HSPI. This review delineates how uronic acid composition in HS modulates protein binding affinity, selectivity, and biological activity. Finally, the significance of sulfated homo-oligo uronic acid as heparin mimics in drug development is also discussed.

D-Glucuronate and d-Glucuronate Glycal Acceptors for the Scalable Synthesis of d-GlcN-α-1,4-d-GlcA Disaccharides and Modular Assembly of Heparan Sulfate.

Reported herein is a scalable chemical synthesis of disaccharide building blocks for heparan sulfate (HS) oligosaccharide assembly. The use of d-glucuronate-based acceptors for dehydrative glycosylation with d-glucosamine partners is explored, enabling diastereoselective synthesis of appropriately protected HS disaccharide building blocks (d-GlcN-α-1,4-d-GlcA) on a multigram scale. Isolation and characterization of key donor (1,2 glycal)- and acceptor (ortho-ester, anhydro)-derived side products ensure methodology improvements to reduce their formation; protecting the d-glucuronate acceptor at the anomeric position with a para-methoxyphenyl unit proves optimal. We also introduce glycal uronate acceptors, showing them to be comparative in reactivity to their pyranuronate counterparts. Taken together, this gram-scale access offers the capability to explore the iterative assembly of defined HS sequences containing the d-GlcN-α-1,4-d-GlcA repeat, highlighted by completing this for two tetrasaccharide syntheses.

Fibrolamellar carcinomas–growth arrested by paracrine signals complexed with synthesized 3-O sulfated heparan sulfate oligosaccharides

Fibrolamellar carcinomas (FLCs), lethal tumors occurring in children to young adults, have genetic signatures implicating derivation from biliary tree stem cell (BTSC) subpopulations, co-hepato/pancreatic stem cells, involved in hepatic and pancreatic regeneration. FLCs and BTSCs express pluripotency genes, endodermal transcription factors, and stem cell surface, cytoplasmic and proliferation biomarkers.The FLC-PDX model, FLC-TD-2010, is driven ex vivo to express pancreatic acinar traits, hypothesized responsible for this model's propensity for enzymatic degradation of cultures. A stable ex vivo model of FLC-TD-2010 was achieved using organoids in serum-free Kubota's Medium (KM) supplemented with 0.1% hyaluronans (KM/HA). Heparins (10 ng/ml) caused slow expansion of organoids with doubling times of ∼7–9 days. Spheroids, organoids depleted of mesenchymal cells, survived indefinitely in KM/HA in a state of growth arrest for more than 2 months. Expansion was restored with FLCs co-cultured with mesenchymal cell precursors in a ratio of 3:7, implicating paracrine signaling. Signals identified included FGFs, VEGFs, EGFs, Wnts, and others, produced by associated stellate and endothelial cell precursors.Fifty-three, unique heparan sulfate (HS) oligosaccharides were synthesized, assessed for formation of high affinity complexes with paracrine signals, and each complex screened for biological activity(ies) on organoids. Ten distinct HS-oligosaccharides, all 10–12 mers or larger, and in specific paracrine signal complexes elicited particular biological responses. Of note, complexes of paracrine signals and 3-O sulfated HS-oligosaccharides elicited slowed growth, and with Wnt3a, elicited growth arrest of organoids for months. If future efforts are used to prepare HS-oligosaccharides resistant to breakdown in vivo, then [paracrine signal—HS-oligosaccharide] complexes are potential therapeutic agents for clinical treatments of FLCs, an exciting prospect for a deadly disease.

Rational Design and Expedient Synthesis of Heparan Sulfate Mimetics from Natural Aminoglycosides for Structure and Activity Relationship Studies.

Heparan sulfate (HS) contains variably repeating disaccharide units organized into high- and low-sulfated domains. This rich structural diversity enables HS to interact with many proteins and regulate key signaling pathways. Efforts to understand structure-function relationships and harness the therapeutic potential of HS are hindered by the inability to synthesize an extensive library of well-defined HS structures. We herein report a rational and expedient approach to access a library of 27 oligosaccharides from natural aminoglycosides as HS mimetics in 7-12 steps. This strategy significantly reduces the number of steps as compared to the traditional synthesis of HS oligosaccharides from monosaccharide building blocks. Combined with computational insight, we identify a new class of four trisaccharide compounds derived from the aminoglycoside tobramycin that mimic natural HS and have a strong binding to heparanase but a low affinity for off-target platelet factor-4 protein.

Efficient platform for synthesizing comprehensive heparan sulfate oligosaccharide libraries for decoding glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) are abundant, ubiquitous carbohydrates in biology, yet their structural complexity has limited an understanding of their biological roles and structure-function relationships. Synthetic access to large collections of well defined, structurally diverse GAG oligosaccharides would provide critical insights into this important class of biomolecules and represent a major advance in glycoscience. Here we report a new platform for synthesizing large heparan sulfate (HS) oligosaccharide libraries displaying comprehensive arrays of sulfation patterns. Library synthesis is made possible by improving the overall synthetic efficiency through universal building blocks derived from natural heparin and a traceless fluorous tagging method for rapid purification with minimal manual manipulation. Using this approach, we generated a complete library of 64 HS oligosaccharides displaying all possible 2-O-, 6-O- and N-sulfation sequences in the tetrasaccharide GlcN-IdoA-GlcN-IdoA. These diverse structures provide an unprecedented view into the sulfation code of GAGs and identify sequences for modulating the activities of important growth factors and chemokines.

Glomerular endothelial glycocalyx-derived heparan sulfate inhibits glomerular leukocyte influx and attenuates experimental glomerulonephritis.

Proliferative forms of glomerulonephritis are characterized by the influx of leukocytes, albuminuria, and loss of kidney function. The glomerular endothelial glycocalyx is a thick carbohydrate layer that covers the endothelium and is comprised of heparan sulfate (HS), which plays a pivotal role in glomerular inflammation by facilitating endothelial-leukocyte trafficking. We hypothesize that the exogenous glomerular glycocalyx may reduce the glomerular influx of inflammatory cells during glomerulonephritis. Indeed, administration of mouse glomerular endothelial cell (mGEnC)-derived glycocalyx constituents, or the low-molecular-weight heparin enoxaparin, reduced proteinuria in mice with experimental glomerulonephritis. Glomerular influx of granulocytes and macrophages, as well as glomerular fibrin deposition, was reduced by the administration of mGEnC-derived glycocalyx constituents, thereby explaining the improved clinical outcome. HSglx also inhibited granulocyte adhesion to human glomerular endothelial cells in vitro. Notably, a specific HSglx fraction inhibited both CD11b and L-selectin binding to activated mGEnCs. Mass spectrometry analysis of this specific fraction revealed six HS oligosaccharides, ranging from tetra- to hexasaccharides with 2-7 sulfates. In summary, we demonstrate that exogenous HSglx reduces albuminuria during glomerulonephritis, which is possibly mediated via multiple mechanisms. Our results justify the further development of structurally defined HS-based therapeutics for patients with (acute) inflammatory glomerular diseases, which may be applicable to non-renal inflammatory diseases as well.

Computational studies on glycosaminoglycan recognition of sialyl transferases.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1.

A 6-O-endosulfatase activity assay based on synthetic heparan sulfate oligomers.

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

Well-Defined Heparin Mimetics Can Inhibit Binding of the Trimeric Spike of SARS-CoV-2 in a Length-Dependent Manner.

The emergence of new SARS-CoV-2 variants and the dangers of long-covid necessitate the development of broad-acting therapeutics that can reduce viral burden. SARS-CoV-2 employs heparan sulfate (HS) as an initial cellular attachment factor, and therefore, there is interest in developing heparin as a therapeutic for SARS-CoV-2. Its use is, however, complicated by structural heterogeneity and the risk of causing bleeding and thrombocytopenia. Here, we describe the preparation of well-defined heparin mimetics by a controlled head-to-tail assembly of HS oligosaccharides having an alkyne or azide moiety by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Alkyne- and azide-containing sulfated oligosaccharides were prepared from a common precursor by modifying an anomeric linker with 4-pentynoic acid and by enzymatic extension with an N-acetyl-glucosamine having an azide moiety at C-6 (GlcNAc6N3), respectively, followed by CuAAC. The process of enzymatic extension with GlcNAc6N3 followed by CuAAC with the desired alkyne-containing oligosaccharides could be repeated to give compounds composed of 20 and 27 monosaccharides, respectively. The heparin mimetics could inhibit the binding of the SARS-CoV-2 spike or RBD to immobilized heparin or to Vero E6 cells. The inhibitory potency increased with increasing chain length, and a compound composed of four sulfated hexasaccharides linked by triazoles had a similar potency as unfractionated heparin. Sequence analysis and HS microarray binding studies with a wide range of RBDs of variants of concern indicate that they have maintained HS-binding capabilities and selectivities. The heparin mimetics exhibit no or reduced binding to antithrombin-III and platelet factor 4, respectively, which are associated with side effects.

Synthesis of a Systematic 64-Membered Heparan Sulfate Tetrasaccharide Library.

Heparan sulfate (HS) has multifaceted biological activities. To date, no libraries of HS oligosaccharides bearing systematically varied sulfation structures are available owing to the challenges in synthesizing a large number of HS oligosaccharides. To overcome the obstacles and expedite the synthesis, a divergent approach was designed, where 64 HS tetrasaccharides covering all possible structures of 2-O-, 6-O- and N-sulfation with the glucosamine-glucuronic acid-glucosamine-iduronic acid backbone were successfully produced from a single strategically protected tetrasaccharide intermediate. This extensive library helped identify the structural requirements for HS sequences to have strong fibroblast growth factor-2 binding but a weak affinity for platelet factor-4. Such a strategy to separate out these two interactions could lead to new HS-based potential therapeutics without the dangerous adverse effect of heparin-induced thrombocytopenia.

The influenza-injured lung microenvironment promotes MRSA virulence, contributing to severe secondary bacterial pneumonia.

Influenza infection is substantially worsened by the onset of secondary pneumonia caused by bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). The bidirectional interaction between the influenza-injured lung microenvironment and MRSA is poorly understood. By conditioning MRSA exvivo in bronchoalveolar lavage fluid collected from mice at various time points of influenza infection, we found that the influenza-injured lung microenvironment dynamically induces MRSA to increase cytotoxin expression while decreasing metabolic pathways. LukAB, a SaeRS two-component system-dependent cytotoxin, is particularly important to the severity of post-influenza MRSA pneumonia. LukAB's activity is likely shaped by the post-influenza lung microenvironment, as LukAB binds to (and is activated by) heparan sulfate (HS) oligosaccharide sequences shed from the epithelial glycocalyx after influenza. Our findings indicate that post-influenza MRSA pneumonia is shaped by bidirectional host-pathogen interactions: host injury triggers changes in bacterial expression of toxins, the activity of which may be shaped by host-derived HS fragments.

Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides having a Domain Structure.

Heparan sulfate (HS) has a domain structure in which regions that are modified by epimerization and sulfonation (NS domains) are interspersed by unmodified fragments (NA domains). There is data to support that domain organization of HS can regulate binding of proteins, however, such model has been difficult to probe. Here, we report a chemoenzymatic methodology that can provide HS oligosaccharides composed of two or more NS domains separated by NA domains of different length. It is based on the chemical synthesis of a HS oligosaccharide that enzymatically was extended by various GlcA-GlcNAc units and terminated in GlcNAc having an azido moiety at C-6 position. HS oligosaccharides having an azide and alkyne moiety could be assembled by copper catalyzed alkyne-azide cycloaddition to give compounds having various NS domains separated by unsulfonated regions. Competition binding studies showed that the length of an NA domain modulates the binding of the chemokines CCL5 and CXCL8.

Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides having a Domain Structure**

AbstractHeparan sulfate (HS) has a domain structure in which regions that are modified by epimerization and sulfonation (NS domains) are interspersed by unmodified fragments (NA domains). There is data to support that domain organization of HS can regulate binding of proteins, however, such model has been difficult to probe. Here, we report a chemoenzymatic methodology that can provide HS oligosaccharides composed of two or more NS domains separated by NA domains of different length. It is based on the chemical synthesis of a HS oligosaccharide that enzymatically was extended by various GlcA‐GlcNAc units and terminated in GlcNAc having an azido moiety at C‐6 position. HS oligosaccharides having an azide and alkyne moiety could be assembled by copper catalyzed alkyne‐azide cycloaddition to give compounds having various NS domains separated by unsulfonated regions. Competition binding studies showed that the length of an NA domain modulates the binding of the chemokines CCL5 and CXCL8.

Investigation of the pharmacokinetic properties of synthetic heparan sulfate oligosaccharides.

Heparan sulfate (HS) is a sulfated polysaccharide with a wide range of biological activities. There is an increasing interest in the development of structurally homogeneous HS oligosaccharides as therapeutics. However, the factors influencing the pharmacokinetic properties of HS-based therapeutics remain unknown. Here, we report the pharmacokinetic properties of a panel of dodecasaccharides (12-mers) with varying sulfation patterns in healthy mice and uncover the pharmacokinetic properties of an octadecasaccharide (18-mer) in acutely injured mice. In the 12-mer panel, 1 12-mer, known as dekaparin, is anticoagulant, and 3 12-mers are nonanticoagulant. The concentrations of 12-mers in plasma and urine were determined by the disaccharide analysis using liquid chromatography coupled with tandem mass spectrometry. We observed a striking difference between anticoagulant and nonanticoagulant oligosaccharides in the 12-mer panel, showing that anticoagulant dekaparin had a 4.6-fold to 8.6-fold slower clearance and 4.4-fold to 8-fold higher plasma exposure compared to nonanticoagulant 12-mers. We also observed that the clearance of HS oligosaccharides is impacted by disease. Using an antiinflammatory 18-mer, we discovered that the clearance of 18-mer is reduced 2.8-fold in a liver failure mouse model compared to healthy mice. Our results suggest that oligosaccharides are rapidly cleared renally if they have low interaction with circulating proteins. We observed that the clearance rate of oligosaccharides is inversely associated with the degree of binding to target proteins, which can vary in response to pathophysiological conditions. Our findings uncover a contributing factor for the plasma and renal clearance of oligosaccharides which will aid the development of HS-based therapeutics.

HSMotifDiscover: identification of motifs in sequences composed of non-single-letter elements.

The functional sub-string(s) of a biopolymer sequence defines the specificity of its interaction with other biomolecules and is often referred to as motifs. Computational algorithms and software have been broadly developed for finding such motifs in sequences in which the individual elements are single characters, such as those in DNA and protein sequences. However, there are more complex scenarios where the motifs exist in non-single-letter contexts, e.g. preferred patterns of chemical modifications on proteins, DNAs, RNAs or polysaccharides. To search for those motifs, we describe a new method that converts the modified sequence elements to representative single-letter codes and then uses a modified Gibbs-sampling algorithm to define the position specific scoring matrix representing the motif(s). As a proof of principle, we describe the implementation and application of an R package for discovering heparan sulfate (HS) motifs in glycan sequences, which are important in regulating protein-protein interactions. This software can be valuable for analyzing high-throughput glycoprotein binding data using microarrays with HS oligosaccharides or other biological polymers. HSMotifDiscover is freely available as an open source R package released under an MIT license at https://github.com/bioinfoDZ/HSMotifDiscover and also available in the form of an app at https://hsmotifdiscover.shinyapps.io/HSMotifDiscover_ShinyApp/. Supplementary data are available at Bioinformatics online.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid β Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid β (Aβ) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non‐protein components of Aβ deposits that not only modulates Aβ aggregation, but also acts as a receptor for Aβ fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aβ could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6‐O‐phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP‐mediated Aβ fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.