Heparan Sulfate Sulfation Research Articles

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Myeloid deficiency of heparan sulfate 6-O-endosulfatases impairs bone marrow hematopoiesis

The heparan sulfate (HS) 6-O-endosulfatases or the Sulfs (Sulf1 and Sulf2) are the only known enzymes that can modify HS sulfation status extracellularly and have been shown to regulate diverse biological processes. The role of the Sulfs in bone marrow (BM) hematopoiesis is not known. In this study, we generated a novel mouse line with myeloid-specific deletion of the Sulfs by crossing Sulf1/2 double floxed mice with the LysM-cre line. The LysM-Sulf knockout (KO) male mice exhibited age-dependent expansion of hematopoietic stem cells and the granulocyte-monocyte lineages in the BM, whereas common lymphoid progenitors and B lymphocyte populations were significantly reduced. Although megakaryocytic and erythroid progenitors were not reduced in the BM, the LysM-Sulf KO males suffered age-dependent reduction of red blood cells (RBCs) and platelets in the peripheral blood, suggesting that the production of RBCs and platelets was arrested at later stages. In addition, LysM-Sulf KO males displayed progressive splenomegaly with extramedullary hematopoiesis. Compared to males, LysM-Sulf KO females exhibited a much-reduced phenotype, and ovariectomy had little effect. Mechanistically, reduced TGF-β/Smad2 but enhanced p53/p21 signaling were observed in male but not female LysM-Sulf KO mice. Finally, HS disaccharide analysis via LC-MS/MS revealed increased HS 6-O-sulfation in the BM from both male and female LysM-Sulf KO mice, however, the distribution of 6-O-sulfated motifs were different between the sexes with compensatory increase in Sulf1 expression observed only in LysM-Sulf KO females. In conclusion, our study reveals that myeloid deficiency of the Sulfs leads to multilineage abnormalities in BM hematopoiesis in an age- and sex-dependent manner.

CIRCULATING HEPARAN SULFATE PROFILES IN PEDIATRIC ACUTE RESPIRATORY DISTRESS SYNDROME.

Introduction: Sepsis-induced degradation of endothelial glycocalyx heparan sulfate (HS) contributes to the pulmonary microvascular endothelial injury characteristic of acute respiratory distress syndrome (ARDS) pathogenesis. Our objectives were to (1) examine relationships between plasma indices of HS degradation and protein biomarkers of endothelial injury and (2) identify patient subgroups characterized by distinct profiles of HS degradation in children with ARDS. Methods: We analyzed prospectively collected plasma (2018-2020) from a cohort of invasively mechanically ventilated children (aged >1 month to <18 years) with ARDS. Mass spectrometry characterized and quantified patterns of HS disaccharide sulfation. Protein biomarkers reflective of endothelial injury (e.g., angiopoietin-2, vascular cell adhesion molecule-1, soluble thrombomodulin) were measured with a multiplex immunoassay. Pearson correlation coefficients were used to construct a biomarker correlation network. Centrality metrics detected influential biomarkers (i.e., network hubs). K-means clustering identified unique patient subgroups based on HS disaccharide profiles. Results: We evaluated 36 patients with pediatric ARDS. HS disaccharide sulfation patterns, 6S, NS, and NS2S, positively correlated with all biomarkers of endothelial injury (all P < 0.05) and were classified as network hubs. We identified three patient subgroups, with cluster 3 (n = 5) demonstrating elevated levels of 6S and N-sulfated HS disaccharides. In cluster 3, 60% of children were female and nonpulmonary sepsis accounted for 60% of cases. Relative to cluster 1 (n = 12), cluster 3 was associated with higher oxygen saturation index (P = 0.029) and fewer 28-day ventilator-free days (P = 0.016). Conclusions: Circulating highly sulfated HS fragments may represent emerging mechanistic biomarkers of endothelial injury and disease severity in pediatric ARDS.

Specific 3-O-sulfated heparan sulfate domains regulate salivary gland basement membrane metabolism and epithelial differentiation

Heparan sulfate (HS) regulation of FGFR function, which is essential for salivary gland (SG) development, is determined by the immense structural diversity of sulfated HS domains. 3-O-sulfotransferases generate highly 3-O-sulfated HS domains (3-O-HS), and Hs3st3a1 and Hs3st3b1 are enriched in myoepithelial cells (MECs) that produce basement membrane (BM) and are a growth factor signaling hub. Hs3st3a1;Hs3st3b1 double-knockout (DKO) mice generated to investigate 3-O-HS regulation of MEC function and growth factor signaling show loss of specific highly 3-O-HS and increased FGF/FGFR complex binding to HS. During development, this increases FGFR-, BM- and MEC-related gene expression, while in adult, it reduces MECs, increases BM and disrupts acinar polarity, resulting in salivary hypofunction. Defined 3-O-HS added to FGFR pulldown assays and primary organ cultures modulates FGFR signaling to regulate MEC BM synthesis, which is critical for secretory unit homeostasis and acinar function. Understanding how sulfated HS regulates development will inform the use of HS mimetics in organ regeneration.

Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells.

Introduction: Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of trauma on HS structural modifications that could influence mechanisms of vascular injury and repair has not been evaluated. Moreover, the effect of eGC HS shedding on endothelial cell (EC) homeostasis has not been fully elucidated. The objectives of this work were to characterize the impact of trauma on HS sulfation and determine the effect of eGC HS shedding on the transcriptional landscape of vascular ECs. Methods: Plasma was collected from 25 controls and 49 adults admitted to a level 1 trauma center at arrival and 24h after hospitalization. Total levels of HS and angiopoietin-2, a marker of pathologic EC activation, were measured at each time point. Enzymatic activity of heparanase, the enzyme responsible for HS shedding, was determined in plasma from hospital arrival. Liquid chromatography-tandem mass spectrometry was used to characterize HS di-/tetrasaccharides in plasma. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparinase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparinase III treatment. Results: We found that heparanase activity was increased in trauma plasma relative to controls, and HS levels at arrival were elevated in a manner proportional to injury severity. Di-/tetrasaccharide analysis revealed lower levels of 3-O-sulfated tetramers with a concomitant increase in ΔIIIS and ΔIIS disaccharides following trauma. Admission levels of total HS and specific HS sulfation motifs correlated with 24-h angiopoietin-2 levels, suggesting an association between HS shedding and persistent, pathological EC activation. In vitro pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding. Discussion: Taken together, our findings suggest that HS cleavage associated with eGC injury may disrupt homeostatic EC signaling and influence biosynthetic mechanisms governing eGC repair. These results require validation in larger, multicenter trauma populations coupled with in vivo EC-targeted transcriptomic and proteomic analyses.

Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture

Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.

Glycosaminoglycans exhibit distinct interactions and signaling with BMP2 according to their nature and localization

The role of glycosaminoglycans (GAGs) in modulating bone morphogenetic protein (BMP) signaling represents a recent and underexplored area. Conflicting reports suggest a dual effect: some indicate a positive influence, while others demonstrate a negative impact. This duality suggests that the localization of GAGs (either at the cell surface or within the extracellular matrix) or the specific type of GAG may dictate their signaling role. The precise sulfation patterns of heparan sulfate (HS) responsible for BMP2 binding remain elusive. BMP2 exhibits a preference for binding to HS over other GAGs. Using well-characterized biomaterials mimicking the extracellular matrix, our research reveals that HS promotes BMP2 signaling in the extracellular space, contrary to chondroitin sulfate (CS), which enhances BMP2 bioactivity at the cell surface. Further observations indicate that a central IdoA (2S)-GlcNS (6S) tri-sulfated motif within HS hexasaccharides enhances binding. Nevertheless, BMP2 exhibits a degree of adaptability to various HS sulfation types and sequences. Molecular dynamic simulations attribute this adaptability to the BMP2 N-terminal end flexibility. Our findings illustrate the complex interplay between GAGs and BMP signaling, highlighting the importance of localization and specific sulfation patterns. This understanding has implications for the development of biomaterials with tailored properties for therapeutic applications targeting BMP signaling pathways.

Conformational preferences of heparan sulfate to recognize the CXCL8 dimer in aqueous medium: degree of sulfation and hydrogen bonds.

The sulfation pattern and epimerization of the long-chain sulfated polysaccharide heparan sulfate (HS) cause structural diversity and regulate various physiological and pathological processes when binding with proteins. In this work, we performed a series of molecular dynamics simulations of three variants of the octadecasaccharide HS with varying sulfation positions in aqueous medium in their free forms and in the presence of the chemokine CXCL8 dimer. The free energy of binding depicts the sulfation at the 6-O position of GlcNAc (HS6S), and both 3-O and 6-O positions of GlcNAc (HS3S6S) of HS variants are more likely to bind with the CXCL8 dimer than the triply sulfated HS2S3S6S, which is sulfated at the 2-O position of GlcUA additionally along with 3-O and 6-O positions of GlcNAc. Binding between HS and CXCL8 was driven by electrostatic and van der Waals interactions predominantly regardless of the sulfation pattern; however, unfavorable entropic contribution suppressed the interaction between HS and CXCL8. The contribution of different amino acid residues to the binding energetics suggested that basic amino acids line up the binding site of CXCL8. This study further acknowledges the role of interfacial water that is structured and bound with HS through hydrogen bonds, exhibiting differential hydrogen bond relaxation dynamics compared to when the HS molecules are free. Moreover, this study identifies that with the increase in sulfation, the HS-water hydrogen bond relaxation occurs faster with the complexation, while the reverse trend is followed in their free forms. Significant structural adaptation of the different sulfated HS molecules, as verified from the free energy landscapes generated from various reaction coordinates, root-mean-square-deviations, end-to-end distances, including ring pucker angles, dihedral flexibility, and the high conformational entropy cost arising from the glycosidic bonds, suggests that the different sulfated variants of HS undergo significant structural transformation to bind with CXCL8. The presence of a CXCL8 dimer imposes the bound forms of HS to adopt non-linear structures with skew-boat conformations. The atomistic details of the study would help in understanding the selectivity and conformational diversity, as well as the role of solvents in the recognition of CXCL8 by different sulfated variants of HS molecules.

In vivo activities of heparan sulfate differentially modified by NDSTs during development.

Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for growth factor signaling during development. It is well known that the level and patterns of sulfate groups of heparan sulfate (HS) chains, or HS fine structures, have a major impact on HSPG function. On the other hand, the physiological significance of other structural features of HS, including NS/NA domain organization, remains to be elucidated. A blueprint of the HS domain structures is mainly controlled by HS N-deacetylase/N-sulfotransferases (NDSTs). To analyze in vivo activities of differentially modified HS, we established two knock-in (KI) Drosophila strains with the insertion of mouse Ndst1 (mNdst1) or Ndst2 (mNdst2) in the locus of sulfateless (sfl), the only Drosophila NDST. In these KI lines, mNDSTs are expressed from the sfl locus, in the level and patterns identical to the endogenous sfl gene. Thus, phenotypes of Ndst1 KI and Ndst2KI animals reflect the ability of HS structures made by these enzymes to rescue sfl mutation. Remarkably, we found that mNdst1 completely rescued the loss of sfl. mNdst2 showed a limited rescue ability, despite a higher level of HS sulfation compared to HS in mNdst1 KI. Our study suggests that independent of sulfation levels, additional HS structural features controlled by NDSTs play key roles during tissue patterning.

Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.

Development of peptide-based ratiometric fluorescent probe for sensing heparan sulfate and heparin in aqueous solutions at physiological pH and quantitative detection of heparan sulfate in live cells

Heparan sulfate (HS) plays a critical role in various biological processes as a vital component of the extracellular matrix. In this study, we synthesized three fluorescent probes (1–3) comprising Arg-rich peptides as HS receptors and a fluorophore capable of exhibiting red-shifted emissions upon aggregation. All three probes demonstrated ratiometric responses to HS and heparin in aqueous solutions. Remarkably, probe 3 exhibited a unique ratiometric response to HS in both aqueous solutions at physiological pH and HS proteoglycans on live cells. Probe 3 displayed exceptional sensing properties, including high biocompatibility, water solubility, visible light excitation, a large Stokes shift for ratiometric detection and remarkable selectivity and sensitivity for HS (with a low limit of detection: 720 pM). Binding mode studies unveiled the crucial role of charge interactions between probe 3 and negatively charged HS sugar units. Upon binding, the fluorophore segments of the probes overlapped, inducing green and red emission changes through restricted intramolecular rotation of the fluorophore moiety. Importantly, probe 3 was effectively employed to quantify the reduction of HS proteoglycan levels in live cells by inhibiting HS sulfation using siRNA and an inhibitor. It successfully detected decreased HS levels in cells treated with doxorubicin and irradiation, consistent with results obtained from western blot and immunofluorescence assays. This study presents the first ratiometric fluorescent probe capable of quantitatively detecting HS levels in aqueous solutions and live cells. The unique properties of peptide-based probe 3 make it a valuable tool for studying HS biology and potentially for diagnostic applications in various biological systems.

SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

The heparan sulfate modification enzyme, Hs6st1, governs Xenopus neuroectodermal patterning by regulating distributions of Fgf and Noggin

The nervous system has various types of cells derived from three neuroectodermal regions: neural plate (NP), neural crest (NC), and preplacodal ectoderm (PPE). Differentiation of these regions is regulated by various morphogens. However, regulatory mechanisms of morphogen distribution in neural patterning are still debated. In general, an extracellular component, heparan sulfate (HS), is essential to regulate morphogen gradients by modulating morphogen binding. The present study focused on an HS modification enzyme, heparan sulfate 6-O-sulfotransferase 1 (Hs6st1), which is highly expressed during the neurula stage in Xenopus. Our present in situ hybridization analysis revealed that Hs6st1 is expressed in the lateral sensorial layer of neuroectoderm. Overexpression of Hs6st1 expands Sox3 (NP marker gene) expression, and slightly dampens FoxD3 (NC marker) expression. Hs6st1 knockout using the CRISPR/Cas9 system also expands the neural plate region, followed by retinal malformation. These results imply that 6-O sulfation, mediated by Hs6st1, selectively regulates morphogen distribution required for neuroectodermal patterning. Among morphogens required for patterning, Fgf8a accumulates on Hs6st1-expressing cells, whereas a secreted BMP antagonist, Noggin, diffuses away from those cells. Thus, cell-autonomous 6-O sulfation of HS at the sensorial layer of neuroectoderm also affects neuroectodermal patterning in neighboring regions, including neural plate and neural crest, not only through accumulation, but also through dispersal of specific morphogens.

Role of Heparan Sulfate 6-O-Endosulfatases in Hematopoiesis

Heparan sulfate proteoglycans (HSPGs) are expressed at the cell surface and in the extracellular matrix, including within the bone marrow microenvironment. Heparan sulfate (HS) is the glycosaminoglycan side chain of HSPGs, and these side chains bind to a myriad of growth factors, cytokines, chemokines, and extracellular matrix molecules, mediating a diverse range of biological functions. It is well established that HS-protein interactions critically depend on the amount and position of 6-O-sulfate groups, which form binding sites for proteins. The HS 6-O-endosulfatases, the Sulfs (Sulf1 and Sulf2), remove 6-O-sulfates from specific HS intra-chain sites and are the only known enzymes that can modify HS sulfation status extracellularly. A gain or a loss of HS 6-O-sulfation can alter the outcome of HS-ligand interactions, such as with Wnt ligands, fibroblast growth factors (FGF), and transforming growth factor (TGF)-β1. Monocytes and macrophages express the Sulfs, predominantly Sulf2, and their secretion of the Sulfs can influence HS sulfation status in their surrounding microenvironments. The objective of this study was to examine the impact of myeloid lineage Sulf deletion on hematopoiesis. For this investigation, we generated a murine model of Sulf deletion in Lysozyme 2 (Lyz2) positive myeloid lineage cells by crossing Sulf1 and Sulf2 double floxed mice with the LysMcre mouse line (hereinafter LysM-Sulf-/-). At 8-12 weeks of age, the bone marrow of LysM-Sulf-/- male mice displayed myelodysplastic changes with reduced number of megakaryocytes (MK) compared to the control mice (Lyz2-cre+/-). Bone marrow-derived (BMD) macrophages from LysM-Sulf-/- mice exhibited enhanced proliferation but reduced phagocytosis of Escherichia Coli (E. Coli)in vitro. In addition, LysM-Sulf-/- BMD macrophages expressed reduced levels of key inflammatory mediators including inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and interferon gamma-induced protein 10 (IP-10) in response to lipopolysaccharide stimulation compared to control BMD macrophages. Complete blood cell counts revealed decreases in total white blood cells (4.6 ± 0.6 10E9 Cells/L vs. 8.1 ± 1.010E9Cells/L in control mice, P = 0.0193), red blood cells (RBC, 10.9 ± 0.1 10E12 Cells/L vs. 11.4 ± 0.2 10E12 Cells/L in control mice, P = 0.0385), and hematocrit (43.5 ± 0.3% vs. 46.3 ± 0.7% in control mice, P = 0.0054) in LysM-Sulf-/- males. In addition, LysM-Sulf-/- males (8-12 weeks of age) exhibit extramedullary hematopoiesis with splenomegaly (spleen size increased by 2.6-fold, 162.1 ± 11.5 mg vs. 79.1 ± 1.2 mg in control mice, P < 0.0001). Bone and spleen morphologies are shown in the accompanied Figure. In older LysM-Sulf-/- males (8-12 months of age), the reductions in RBC and hematocrits were further exacerbated with increased reticulocyte counts in the peripheral blood. In addition, thrombocytopenia was observed in 8-12 months old LysM-Sulf-/- males (platelet counts 125.8 ± 41.4 vs. 483.3 ± 7.1 in control mice, P = 0.0007). The above changes were accompanied by amplified splenomegaly (spleen size increased by 9.2-fold, 860.4 ± 157.6 mg vs. 93.2 ± 8.2 mg in control mice, P = 0.0152) and erythropoiesis in the liver. In terms of signaling, enhanced Smad2/3 phosphorylation (primary transducers of TGF-β1 signaling) was observed in both the spleens and the lungs of LysM-Sulf-/- male mice. Finally, in contrast to the male mice, complete blood cell counts were normal in both young and old LysM-Sulf-/- females without obvious extramedullary hematopoiesis. Our investigation revealed the importance of HS 6-O-sulfation in supporting normal hematopoiesis in the bone marrow, and alterations in HS 6-O-sulfation status in the bone marrow microenvironment due to LysM-Sulfdeletion leads to not only impairment in monocyte/macrophage development and function, but also age- and sex-dependent deficiencies in erythropoiesis and thrombopoiesis. Our study identifies TGF-β1 as one of the signaling pathways regulated by the Sulfs. The regulatory roles of the Sulfs in TGF-β1 as well as other signaling pathways in hematopoiesis warrant further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Noncovalent hyaluronan crosslinking by TSG-6: Modulation by heparin, heparan sulfate, and PRG4

The size, conformation, and organization of the glycosaminoglycan hyaluronan (HA) affect its interactions with soluble and cell surface-bound proteins. HA that is induced to form stable networks has unique biological properties relative to unmodified soluble HA. AlphaLISA assay technology offers a facile and general experimental approach to assay protein-mediated networking of HA in solution. Connections formed between two end-biotinylated 50 kDa HA (bHA) chains can be detected by signal arising from streptavidin-coated donor and acceptor beads being brought into close proximity when the bHA chains are bridged by proteins. We observed that incubation of bHA with the protein TSG-6 (tumor necrosis factor alpha stimulated gene/protein 6, TNFAIP/TSG-6) leads to dimerization or higher order multimerization of HA chains in solution. We compared two different heparin (HP) samples and two heparan sulfate (HS) samples for the ability to disrupt HA crosslinking by TSG-6. Both HP samples had approximately three sulfates per disaccharide, and both were effective in inhibiting HA crosslinking by TSG-6. HS with a relatively high degree of sulfation (1.75 per disaccharide) also inhibited TSG-6 mediated HA networking, while HS with a lower degree of sulfation (0.75 per disaccharide) was less effective. We further identified Proteoglycan 4 (PRG4, lubricin) as a TSG-6 ligand, and found it to inhibit TSG-6-mediated HA crosslinking. The effects of HP, HS, and PRG4 on HA crosslinking by TSG-6 were shown to be due to HP/HS/PRG4 inhibition of HA binding to the Link domain of TSG-6. Using the AlphaLISA platform, we also tested other HA-binding proteins for ability to create HA networks. The G1 domain of versican (VG1) effectively networked bHA in solution but required a higher concentration than TSG-6. Cartilage link protein (HAPLN1) and the HA binding protein segment of aggrecan (HABP, G1-IGD-G2) showed only low and variable magnitude HA networking effects. This study unambiguously demonstrates HA crosslinking in solution by TSG-6 and VG1 proteins, and establishes PRG4, HP and highly sulfated HS as modulators of TSG-6 mediated HA crosslinking.

Glycosaminoglycan Analysis of FFPE Tissues from Prostate Cancer and Benign Prostate Hyperplasia Patients Reveals Altered Regulatory Functions and Independent Markers for Survival.

Simple SummaryProstate cancer is one of the most frequent cancer types among men. A better understanding of the involved molecular mechanisms is still necessary for the improvement of therapeutic decision making. Glycosaminoglycan disaccharides were investigated on biopsies taken from patients with prostate cancer and benign prostate hyperplasia. We found that the quantity and sulfation of chondroitin sulfate chains significantly differed between benign prostate hyperplasia and the distinct risk groups of prostate cancer. Doubly and triply sulfated heparan sulfate disaccharides were identified as the independent biomarkers for survival.Prostate cancer is one of the most frequent cancer types among men. Several biomarkers and risk assessment methods are already available; however, enhancing their selectivity and sensitivity is still necessary. For improving therapeutic decisions, both basic and clinical research studies are still ongoing for a better understanding of the underlying molecular mechanisms. The enzymatic digests of heparan sulfate (HS) and chondroitin sulfate (CS) chains were investigated in tissue samples taken from patients with prostate cancer (PCa) and benign prostate hyperplasia (BPH) with the HPLC–MS methodology. None of the HS species analyzed showed correlating alterations with currently used markers such as clinical stage, Gleason score, or prostate-specific antigen (PSA) level. The total quantity and sulfation motifs of CS were both significantly different among BPH and different risk groups of PCa. Furthermore, the cancer-specific survival of patients can be predicted based on the levels of non-sulfated and doubly sulfated CS disaccharides as well as the total HS content and the doubly and triply sulfated HS disaccharide ratios. These disaccharide ratios proved to be independent markers from clinical parameters. Further investigations of glycosaminoglycan motifs were proposed for the validation of the results on independent patient cohorts as well.

Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.

Overexpression of NDST1 Attenuates Fibrotic Response in Murine Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) hold tremendous potential for treating diseases and repairing damaged tissues. Heparan sulfate (HS) plays various roles in cellular signaling mechanisms. The importance of HS in stem cell function has been reported and well documented. However, there has been little progress in using HS for therapeutic purposes. We focused on one of the sulfotransferases, NDST1, which influences overall HS chain extent and sulfation pattern, with the expectation to enhance stem cell function by increasing the N-sulfation level. We herein performed transfections of a green fluorescent protein-vector control and NDST1-vector into mouse ADSCs to evaluate stem cell functions. Overexpression of NDST1 suppressed the osteogenic differentiation of ADSCs. There was no pronounced effect observed on the stemness, inflammatory gene expression, nor any noticeable effect in adipogenic and chondrogenic differentiation. Under the tumor necrosis factor-alpha stimulation, NDST1 overexpression induced several chemokine productions that attract neutrophils and macrophages. Finally, we identified an antifibrotic response in ADSCs overexpressing NDST1. This study provides a foundation for the evaluation of HS-related effects in ADSCs undergoing ex vivo gene manipulation.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Effect of high glucose on glycosaminoglycans in cultured retinal endothelial cells and rat retina.

The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.