Human extracellular sulfatases use a dual mechanism for regulation of growth factor interactions with heparan sulfate proteoglycans.

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.

Validation of Recombinant Heparan Sulphate Reagents for CNS Repair.

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.

Impaired mitophagy in Sanfilippo a mice causes hypertriglyceridemia and brown adipose tissue activation.

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.

Biologically Derived Neoproteoglycans for Profiling Protein-Glycosaminoglycan Interactions.

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.

Multiplex genome editing of mammalian cells for producing recombinant heparin.

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.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

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.

Metabolic engineering of mammalian cells to produce heparan sulfates.

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.

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans 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.

ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors.

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.

Expanding the 3-O-Sulfate Proteome--Enhanced Binding of Neuropilin-1 to 3-O-Sulfated Heparan Sulfate Modulates Its Activity.

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 3-O-sulfation: a rare modification in search of a function.

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.

Passive mechanical properties and related proteins change with botulinum neurotoxin A injection of normal skeletal muscle.

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.

Defective Wnt-dependent cerebellar midline fusion in a mouse model of Joubert syndrome.

The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear. To investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome-associated gene. 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, Cep290. 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 signaling. 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 agonist, 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.

Inhibitory peptides of the sulfotransferase domain of the heparan sulfate enzyme, N-deacetylase-N-sulfotransferase-1.

N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.

Regional Myosin heavy chain distribution in selected paraspinal muscles.

STUDY DESIGN: Cross-sectional study with repeated measures design. OBJECTIVE: To compare the myosin heavy-chain isoform distribution within and between paraspinal muscles and to test the theory that fiber-type gradients exist as a function of paraspinal muscle depth. SUMMARY OF BACKGROUND DATA: There is still uncertainty regarding the fiber-type distributions within different paraspinal muscles. It has been previously proposed that deep fibers of the multifidus muscle may contain a higher ratio of type I to type II fibers, because, unlike superficial fibers, they primarily stabilize the spine, and may therefore have relatively higher endurance. Using a minimally invasive surgical approach, using tubular retractors that are placed within anatomic intermuscular planes, it was feasible to obtain biopsies from the multifidus, longissimus, iliocostalis, and psoas muscles at specific predefined depths. METHODS: Under an institutional review board-approved protocol, muscle biopsies were obtained from 15 patients who underwent minimally invasive spinal surgery, using the posterior paramedian (Wiltse) approach or the minimally invasive lateral approach. Myosin heavy chain (MyHC) isoform distribution was analyzed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) electrophoresis. Because multiple biopsies were obtained from each patient, MyHC distribution was compared using both within- and between-muscle repeated measures analyses. RESULTS: The fiber-type distribution was similar among the posterior paraspinal muscles and was composed of relatively high percentage of type I (63%), compared to type IIA (19%) and type IIX (18%) fibers. In contrast, the psoas muscle was found to contain a lower percentage of type I fibers (42%) and a higher percentage of type IIA (33%) and IIX fibers (26%; P<0.05). No significant difference was found for fiber-type distribution among 3 different depths of themultifidus and psoas muscles. CONCLUSION: Fiber-type distribution between the posterior paraspinal muscles is consistent and is composed of relatively high percentage of type I fibers, consistent with a postural function. The psoas muscle, on the other hand, is composed of a higher percentage of type II fibers such as in the appendicular muscles. Our data do not support the idea of a fiber-type gradient as a function of depth for any muscle studied.

Passive mechanical properties of the lumbar multifidus muscle support its role as a stabilizer.

The purpose of this study was to compare the passive mechanical properties and titin isoform sizes of the multifidus, longissimus, and iliocostalis muscles. Given our knowledge of each muscle's architecture and the multifidus' operating range, we hypothesized that multifidus would have higher elastic modulus with corresponding smaller titin isoforms compared to longissimus or iliocostalis muscles. Single-fiber and fiber-bundle material properties were derived from passive stress-strain tests of excised biopsies (n=47). Titin isoform sizes were quantified via sodium dodecyl sulfate-vertical agarose gel electrophoresis (SDS-VAGE) analysis. We found that, at the single-fiber level, all muscles had similar material properties and titin isoform sizes. At the fiber-bundle level, however, we observed significantly increased stiffness (approximately 45%) in multifidus compared to longissimus and iliocostalis muscles. These data demonstrate that each muscle may have a different scaling relationship between single-fiber and fiber-bundle levels, suggesting that the structures responsible for higher order passive mechanical properties may be muscle specific. Our results suggest that divergent passive material properties are observed at size scales larger than the single cell level, highlighting the importance of the extracellular matrix in these muscles. In addition to architectural data previously reported, these data further support the unique stabilizing function of the multifidus muscle. These data will provide key input variables for biomechanical modeling of normal and pathologic lumbar spine function and direct future work in biomechanical testing in these important muscles.