Growth factor signaling governs essential cellular processes, and its precision relies on interactions with heparan sulfate on the cell surface. The sulfation pattern of heparan sulfate dictates its capacity to bind specific growth factors and their receptors, thereby controlling the signaling strength and specificity. Extracellular endosulfatases, including sulfatase 1 and sulfatase 2, further modulate these interactions by selectively removing sulfate groups from defined regions of heparan sulfate. Although these enzymes are known to influence developmental- and disease-related signaling, their direct effects on growth factor recognition have remained unclear. Using a panel of bioengineered heparan sulfate conjugates with defined sulfation compositions, this study examines how the structural features of heparan sulfate govern its regulation by the sulfatases. By tracking enzyme binding and catalytic remodeling, we found that both enzymes rely on two coordinated mechanisms: catalytic desulfation of heparan sulfate and competitive binding that transiently prevents growth factor association. The balance between catalytic remodeling and competitive binding depends on the sulfation characteristics of the heparan sulfate substrate and the identity of the growth factor and differs between the two enzyme isoforms. These findings provide a new framework for understanding how extracellular sulfatases shape growth factor signaling in both development and disease.

The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (μCs) cultures. Additionally, rHS surfaces reduced β-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.

Membrane-associated heparan sulfate (HS) proteoglycans (PGs) contribute to the regulation of extracellular cellular signaling cues, such as growth factors (GFs) and chemokines, essential for normal organismal functions and implicated in various pathophysiologies. PGs accomplish this by presenting high affinity binding sites for GFs and their receptors through highly sulfated regions of their HS polysaccharide chains. The composition of HS, and thus GF-binding specificity, are determined during biosynthetic assembly prior to installation at the cell surface. Two extracellular 6-O-endosulfatase enzymes (Sulf-1 and Sulf-2) can uniquely further edit mature HS and alter its interactions with GFs by removing specific sulfation motifs from their recognition sequence on HS. Despite being implicated as signaling regulators during development and in disease, the Sulfs have resisted structural characterization, and their substrate specificity and effects on GF interactions with HS are still poorly defined. Using a panel of PG-mimetics comprising compositionally-defined bioengineered recombinant HS (rHS) substrates in combination with GF binding and enzyme activity assays, we have discovered that Sulfs control GF-HS interactions through a combination of catalytic processing and competitive blocking of high affinity GF-binding sites, providing a new conceptual framework for understanding the functional impact of these enzymes in biological context. Although the contributions from each mechanism are both Sulf- and GF-dependent, the PG-mimetic platform allows for rapid analysis of these complex relationships. Significance Statement Cells rely on extracellular signals such as growth factors (GFs) to mediate critical biological functions. Membrane-associated proteins bearing negatively charged heparan sulfate (HS) sugar chains engage with GFs and present them to their receptors, which regulates their activity. Two extracellular sulfatase (Sulf) enzymes can edit HS and alter GF interactions and activity, although the precise mechanisms remain unclear. By using chemically defined HS-mimetics as probes, we have discovered that Sulfs can modulate HS by means of catalytic alterations and competitive blocking of GF-binding sites. These unique dual activities distinguish Sulfs from other enzymes and provide clues to their roles in development and disease.

Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair.

Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme-substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.

Accumulations of glycosaminoglycans (GAGs) that result from deficiencies in lysosomal hydrolases are characteristic of mucopolysaccharidoses (MPS). Enzyme replacement therapies (ERTs) are now available for several MPS diseases (MPS I, MPS II, MPS IVA, MPS VI, and MPS VII), but assessment of the efficacy of treatment can be challenging because these are rare, progressive, and highly heterogeneous diseases; because some clinical manifestations may be irreversible if treatment initiation is delayed; and because determining the benefits of a treatment to prevent those manifestations may take prolonged periods of time. In addition to accumulation of GAGs in tissues, elevated urinary GAG (uGAG) levels are evident and are reduced rapidly after initiation of ERT. Studies in MPS animal models and clinical studies in subjects with MPS diseases have revealed correlations between reductions of uGAG levels and clinical effects of ERTs. In this article, we review the growing body of evidence to support the potential for the use of uGAG levels as predictive biomarkers of treatment efficacy.

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities.

Lysosomal storage diseases result in various developmental and physiological complications, including cachexia. To study the causes for the negative energy balance associated with cachexia, we assessed the impact of sulfamidase deficiency and heparan sulfate storage on energy homeostasis and metabolism in a mouse model of type IIIa mucopolysaccharidosis (MPS IIIa, Sanfilippo A syndrome). At 12-weeks of age, MPS IIIa mice exhibited fasting and postprandial hypertriglyceridemia compared with wildtype mice, with a reduction of white and brown adipose tissues. Partitioning of dietary [3H]triolein showed a marked increase in intestinal uptake and secretion, whereas hepatic production and clearance of triglyceride-rich lipoproteins did not differ from wildtype controls. Uptake of dietary triolein was also elevated in brown adipose tissue (BAT), and notable increases in beige adipose tissue occurred, resulting in hyperthermia, hyperphagia, hyperdipsia, and increased energy expenditure. Furthermore, fasted MPS IIIa mice remained hyperthermic when subjected to low temperature but became cachexic and profoundly hypothermic when treated with a lipolytic inhibitor. We demonstrated that the reliance on increased lipid fueling of BAT was driven by a reduced ability to generate energy from stored lipids within the depot. These alterations arose from impaired autophagosome-lysosome fusion, resulting in increased mitochondria content in beige and BAT. Finally, we show that increased mitochondria content in BAT and postprandial dyslipidemia was partially reversed upon 5-week treatment with recombinant sulfamidase. We hypothesize that increased BAT activity and persistent increases in energy demand in MPS IIIa mice contribute to the negative energy balance observed in patients with MPS IIIa.

Heparan sulfate (HS) is a glycosaminoglycan produced by all mammalian cells that plays important roles in physiology and various pathologies. Heparin is a highly sulfated form of HS that is used clinically as an anticoagulant. Heparin and HSs may also have therapeutic benefits for a wide variety of other indications. Cultured mammalian cells produce HS and, through genetic modification, have been used to elucidate the biosynthetic pathway. Recently, metabolic engineering has been used to produce HS from cultured mammalian cells for clinical purposes. This review describes the HS biosynthetic pathway and its manipulation through metabolic engineering to produce bioengineered HSs. We also discuss current challenges and opportunities to advance the field of HS metabolic engineering.

Heparan sulfate is a polysulfated, unbranched polysaccharide that plays a role in numerous developmental, physiological and pathophysiological processes via its interaction with hundreds of heparan sulfate‐binding proteins. Heparin is a highly sulfated variant of heparan sulfate, used routinely in the clinic as an anticoagulant. Both molecules share a common biosynthetic pathway that includes numerous glycosyltransferases, sulfotransferases and an epimerase. Researchers in the field typically use heparan sulfate purified from porcine intestines or bovine kidney for experimentation. This material suffers from contamination, batch‐to‐batch variation and fails to reproduce the diversity of heparan sulfate structures found in nature. Heparin used in the clinic is also purified from pig intestines with variable compositions, risks of contamination and supply deficits. TEGA Therapeutics is developing a library of heparan sulfate products derived from cell culture. Genetic engineering gives rise to a diversity of heparan sulfate compositions. Each type of heparan sulfate is characterized with respect to disaccharide composition, chain length and protein binding affinity. This work paves the way for development of recombinant heparin bioengineered in cultured cells with a secure supply chain and the potential for enhanced properties and reduced side effects. Support or Funding Information This work is supported by NSF STTR Phase I grant 1622959 to C.G and S.S. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Iduronidase (IDUA)-deficient mice accumulate glycosamino-glycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III-targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1). ApoC-III ASO treatment lowered plasma TGs in mice lacking lipoprotein lipase (LPL), hepatic heparan sulfate proteoglycan (HSPG) receptors, LDLR, or LRP1 and in animals with combined deletion of the genes encoding HSPG receptors and LDLRs or LRP1. However, the ApoC-III ASO did not lower TG levels in mice lacking both LDLR and LRP1. LDLR and LRP1 were also required for ApoC-III ASO-induced reduction of plasma TGs in mice fed a high-fat diet, in postprandial clearance studies, and when ApoC-III-rich or ApoC-III-depleted lipoproteins were injected into mice. ASO reduction of ApoC-III had no effect on VLDL secretion, heparin-induced TG reduction, or uptake of lipids into heart and skeletal muscle. Our data indicate that ApoC-III inhibits turnover of TG-rich lipoproteins primarily through a hepatic clearance mechanism mediated by the LDLR/LRP1 axis.

Binding of proteins to heparan sulfate is driven predominantly by electrostatic interactions between positively charged amino acid residues in the protein and negatively charged sulfate groups located at various positions along the polysaccharide chain. Although many heparin/heparan-sulfate-binding proteins have been described, few exhibit preferential binding for heparan sulfates containing relatively rare 3-O-sulfated glucosamine residues. To expand the "3-O-sulfate proteome," affinity matrices were created from Chinese hamster ovary (CHO) cell heparan sulfate engineered in vitro with and without 3-O-sulfate groups. Fractionation of different animal sera yielded several proteins that bound specifically to columns containing 3-O-sulfated heparan sulfate modified by two members of the heparan sulfate 3-O-sulfotransferase superfamily, Hs3st1 and Hs3st2. Neuropilin-1 was analyzed in detail because it has been implicated in angiogenesis and axon guidance. We show that 3-O-sulfation enhanced the binding of neuropilin-1 to heparan sulfate immobilized on plastic plates and to heparan sulfate present on cultured cells. Chemoenzymatically synthesized 3-O-sulfated heparan sulfate dodecamers protected neuropilin-1 from thermal denaturation and inhibited neuropilin-1-dependent, semaphorin-3a-induced growth cone collapse of neurons derived from murine dorsal root ganglia. The effect of 3-O-sulfation was cell autonomous and specific to Hs3st2 based on collapse assays of neurons derived from Hs3st1- and Hs3st2-deficient mice. Finally, 3-O-sulfated heparan sulfate enhanced the inhibition of endothelial cell sprouting by exogenous heparan sulfate. These findings demonstrate a reliable method to identify members of the 3-O-sulfate proteome and that 3-O-sulfation of heparan sulfate can modulate axonal growth cone collapse and endothelial cell sprouting.

Heparan sulfate structural heterogeneity is driven, in part, by the placement of sulfate groups at various positions in the polysaccharide. While many ligands bind to heparan sulfate without strict requirements for the positions of sulfate groups, binding of a small number of known ligands is influenced by the presence of a sulfate at the C3 position of a glucosamine residue. In mammals, seven enzymes can catalyze the addition of 3‐O‐sulfate groups, suggesting the possibility of other, previously unidentified, ligands whose binding is influenced by 3‐O‐sulfated sequences. To identify these ligands, affinity matrices were created using CHO‐S heparan sulfate, with and without modification by recombinant 3‐O‐sulfotransferases. Serum was fractionated on the matrices and several proteins binding specifically to the 3‐O‐sulfated resins were identified by mass spectrometry. Neuropilin‐1, a modulator of angiogenesis and axonal guidance, bound specifically to affinity matrices with 3‐O‐sulfation. In addition, high affinity binding of neuropilin‐1 to the cell surface required 3‐O‐sulfation. In cell culture, 3‐O‐sulfation modulated two neuropilin‐1 dependent processes. (1) In VEGF‐induced endothelial cell sprouting, 3‐O‐sulfation endowed CHO‐S heparan sulfate with greater inhibitory potential than native heparan sulfate. (2) In semaphorin‐3a‐induced neuronal growth cone collapse, a 3‐O‐sulfated heparan sulfate dodecamer was a potent inhibitor of collapse. An identical dodecamer lacking 3‐O‐sulfation inhibited with much less efficiency. Furthermore, genetic removal of a 3‐O‐sulfotransferase (Hs3st‐2) attenuated the response of the growth cone to semaphorin‐3a. In conclusion, these results demonstrate that 3‐O‐sulfation endows heparan sulfate with enhanced affinity to neuropilin‐1 and modulates two neuropilin‐1 dependent processes, angiogenesis and axonal guidance.Grant Funding Source: Supported by GM93131 and T32CA067754

Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by type I herpes simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.

The effects of botulinum neurotoxin A on the passive mechanical properties of skeletal muscle have not been investigated, but may have significant impact in the treatment of neuromuscular disorders including spasticity. Single fiber and fiber bundle passive mechanical testing was performed on rat muscles treated with botulinum neurotoxin A. Myosin heavy chain and titin composition of single fibers was determined by gel electrophoresis. Muscle collagen content was determined using a hydroxyproline assay. Neurotoxin-treated single fiber passive elastic modulus was reduced compared to control fibers (53.00 kPa vs. 63.43?kPa). Fiber stiffness and slack sarcomere length were also reduced compared to control fibers and myosin heavy chain composition shifted from faster to slower isoforms. Average titin molecular weight increased 1.77% after treatment. Fiber bundle passive elastic modulus increased following treatment (168.83 kPa vs. 75.14 kPa). Bundle stiffness also increased while collagen content per mass of muscle tissue increased 38%. Injection of botulinum neurotoxin A produces an effect on the passive mechanical properties of normal muscle that is opposite to the changes observed in spastic muscles. (C) 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:497502, 2012

Joubert syndrome is a developmental disease affecting multiple parts of the body, including the cerebellum in the brain. Now, Joseph Gleeson and his colleagues show that mouse models of Joubert syndrome show deficient Wnt-dependent cerebellar proliferation and midline fusion and that these phenotypes can be rescued by lithium, a Wnt agonist. The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear1,2,3. To investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome–associated gene4,5. These mice show cerebellar hypoplasia with a vermis-midline fusion defect early in development. This defect is concomitant with expansion of the roof plate and is also evident in a mouse mutant for another Joubert syndrome–associated gene, Cep2906,7. Furthermore, fetal magnetic resonance imaging (MRI) of human subjects with Joubert syndrome reveals a similar midline cleft, suggesting parallel pathogenic mechanisms. Previous evidence has suggested a role for Jouberin (Jbn), the protein encoded by Ahi1, in canonical Wnt signaling8. Consistent with this, we found decreased Wnt reporter activity at the site of hemisphere fusion in the developing cerebellum of Ahi1-mutant mice. This decrease was accompanied by reduced proliferation at the site of fusion. Finally, treatment with lithium, a Wnt pathway agonist9, partially rescued this phenotype. Our findings implicate a defect in Wnt signaling in the cerebellar midline phenotype seen in Joubert syndrome that can be overcome with Wnt stimulation.