Brett E. Crawford

Hereditary multiple exostoses and heparan sulfate polymerization

Hereditary multiple exostoses (HME, OMIM 133700, 133701) results from mutations in EXT1 and EXT2, genes encoding the copolymerase responsible for heparan sulfate (HS) biosynthesis. Members of this multigene family share the ability to transfer N-acetylglucosamine to a variety of oligosaccharide acceptors. EXT1 and EXT2 encode the copolymerase, whereas the roles of the other EXT family members (EXTL1, L2, and L3) are less clearly defined. Here, we provide an overview of HME, the EXT family of proteins, and possible models for the relationship of altered HS biosynthesis to the ectopic bone growth characteristic of the disease.

Biosynthesis of the Linkage Region of Glycosaminoglycans CLONING AND ACTIVITY OF GALACTOSYLTRANSFERASE II, THE SIXTH MEMBER OF THE β1,3-GALACTOSYLTRANSFERASE FAMILY (β3GalT6)

A family of five β1,3-galactosyltransferases has been characterized that catalyze the formation of Galβ1,3GlcNAcβ and Galβ1,3GalNAcβ linkages present in glycoproteins and glycolipids (β3GalT1, -2, -3, -4, and -5). We now report a new member of the family (β3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found inDrosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, β3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the β3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal β-linked galactose residue. This product, Galβ1,3Galβ is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser), indicating that β3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.

Surfen, a small molecule antagonist of heparan sulfate

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.

Essential Alterations of Heparan Sulfate During the Differentiation of Embryonic Stem Cells to Sox1‐Enhanced Green Fluorescent Protein‐Expressing Neural Progenitor Cells

Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe its structural transition from an unusually low‐sulfated variant in ES cells to a more highly sulfated form in fluorescence‐activated cell sorting‐purified neural progenitor cells. The characteristic domain structure of HS was retained during this transformation. However, qualitative variations in surface sulfation patterns between ES and differentiated cells were revealed using HS epitope‐specific antibodies and the HS‐binding growth factor fibroblast growth factor 2 (FGF‐2). Expression profiles of the HS modification enzymes indicated that both “early” (N‐sulfotransferases) and “late” (6O‐ and 3O‐sulfotransferases) sulfotransferases contributed to the alterations in sulfation patterning. An HS‐null ES line was used to demonstrate the necessity for HS in neural differentiation. HS is a coreceptor for many of the protein effectors implicated in pluripotency and differentiation (e.g., members of the FGF family, bone morphogenic proteins, and fibronectin). We suggest that the stage‐specific activities of these proteins are finely regulated by dynamic changes in sulfation motifs in HS chains.

Genistein Improves Neuropathology and Corrects Behaviour in a Mouse Model of Neurodegenerative Metabolic Disease

Background Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein. Methodology/Principal Findings We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed. Conclusions/Significance Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.

Disease-specific non–reducing end carbohydrate biomarkers for mucopolysaccharidoses

A significant need exists for improved biomarkers for differential diagnosis, prognosis and monitoring of therapeutic interventions for mucopolysaccharidoses (MPS), inherited metabolic disorders that involve lysosomal storage of glycosaminoglycans. Here, we report a simple reliable method based on the detection of abundant non-reducing ends of the glycosaminoglycans that accumulate in cells, blood, and urine of MPS patients. In this method, glycosaminoglycans were enzymatically depolymerized releasing unique mono-, di-, or trisaccharides from the non-reducing ends of the chains. The composition of the released mono- and oligosaccharides depends on the nature of the lysosomal enzyme deficiency, and therefore they serve as diagnostic biomarkers. Analysis by liquid chromatography/mass spectrometry allowed qualitative and quantitative assessment of the biomarkers in biological samples. We provide a simple conceptual scheme for diagnosing MPS in uncharacterized samples and a method to monitor efficacy of enzyme replacement therapy or other forms of treatment.

Glycan Antagonists and Inhibitors: A Fount for Drug Discovery

ABSTRACT Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.

Guanidinylated Neomycin Delivers Large, Bioactive Cargo into Cells through a Heparan Sulfate-dependent Pathway

Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.

Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB

Significance Mucopolysaccharidosis type IIIB (MPS IIIB) is a devastating and currently untreatable disease affecting mainly the brain. The cause is lack of the lysosomal enzyme, α–N-acetylglucosaminidase (NAGLU), and storage of heparan sulfate. Using a mouse model of MPS IIIB, we administered a modified NAGLU by injection into the left ventricle of the brain, bypassing the blood–brain barrier. The modification consisted of a fragment of IGFII, which allows receptor-mediated uptake and delivery to lysosomes. The modified enzyme was taken up avidly by cells in both brain and liver, where it reduced pathological accumulation of heparan sulfate and other metabolites to normal or near-normal levels. The results suggest the possibility of treatment for MPS IIIB. Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α–N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood–brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood–brain barrier, the fusion protein (“enzyme”) in artificial cerebrospinal fluid (“vehicle”) was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1–28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Glycan-based biomarkers for mucopolysaccharidoses

The mucopolysaccharidoses (MPS) result from attenuation or loss of enzyme activities required for lysosomal degradation of the glycosaminoglycans, hyaluronan, heparan sulfate, chondroitin/dermatan sulfate, and keratan sulfate. This review provides a summary of glycan biomarkers that have been used to characterize animal models of MPS, for diagnosis of patients, and for monitoring therapy based on hematopoietic stem cell transplantation and enzyme replacement therapy. Recent advances have focused on the non-reducing terminus of the glycosaminoglycans that accumulate as biomarkers, using a combination of enzymatic digestion with bacterial enzymes followed by quantitative liquid chromatography/mass spectrometry. These new methods provide a simple, rapid diagnostic strategy that can be applied to samples of urine, blood, cerebrospinal fluid, cultured cells and dried blood spots from newborn infants. Analysis of the non-reducing end glycans provides a method for monitoring enzyme replacement and substrate reduction therapies and serves as a discovery tool for uncovering novel biomarkers and new forms of mucopolysaccharidoses.