A novel missense mutation in lysosomal sulfamidase is the basis of MPS III A in a spontaneous mouse mutant

Sanfilippo syndrome type III A (Mucopolysaccharidosis (MPS) III A) is a rare, autosomal recessive, lysosomal storage disease, characterized by the accumulation of heparan sulfate and the loss of function of lysosomal heparan N-sulfatase activity. The disease leads to devastating mental and physical consequences and a mouse model that can be used to explore gene therapy and enzyme or cell replacement therapies is needed. We have previously identified a mouse with low sulfamidase activity and symptoms and pathologies typical of MPS III A (Bhaumik, M., Muller, V. J., Rozaklis, T., Johnson, L., Dobrenis, K., Bhattacharyya, R., Wurzelmann, S., Finamore, P., Hopwood, J. J., Walkley, S. U., and Stanley, P. [1999] A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology 9, 1389--1396). We now show that the sulfamidase gene of the MPS III A mouse carries a novel mutation (G91A) that gives an amino acid change (D31N) likely to interfere with the coordination of a divalent metal ion in the active site of this sulfatase. This spontaneous mouse mutant is an excellent model for MPS III A in humans as this disease often arises due to a missense mutation in lysosomal sulfamidase.     

Defects in the medial entorhinal cortex and dentate gyrus in the mouse model of Sanfilippo syndrome type B

Sanfilippo syndrome type B (MPS IIIB) is characterized by profound mental retardation in childhood, dementia and death in late adolescence; it is caused by deficiency of α-N-acetylglucosaminidase and resulting lysosomal storage of heparan sulfate. A mouse model, generated by homologous recombination of the Naglu gene, was used to study pathological changes in the brain. We found earlier that neurons in the medial entorhinal cortex (MEC) and the dentate gyrus showed a number of secondary defects, including the presence of hyperphosphorylated tau (Ptau) detected with antibodies raised against Ptau in Alzheimer disease brain. By further use of immunohistochemistry, we now show staining in neurons of the same area for beta amyloid, extending the resemblance to Alzheimer disease. Ptau inclusions in the dentate gyrus of MPS IIIB mice were reduced in number when the mice were administered LiCl, a specific inhibitor of Gsk3β. Additional proteins found elevated in MEC include proteins involved in autophagy and the heparan sulfate proteoglycans, glypicans 1 and 5, the latter closely related to the primary defect. The level of secondary accumulations was associated with elevation of glypican, as seen by comparing brains of mice at different ages or with different mucopolysaccharide storage diseases. The MEC of an MPS IIIA mouse had the same intense immunostaining for glypican 1 and other markers as MPS IIIB, while MEC of MPS I and MPS II mice had weak staining, and MEC of an MPS VI mouse had no staining at all for the same proteins. A considerable amount of glypican was found in MEC of MPS IIIB mice outside of lysosomes. We propose that it is the extralysosomal glypican that would be harmful to neurons, because its heparan sulfate branches could potentiate the formation of Ptau and beta amyloid aggregates, which would be toxic as well as difficult to degrade.     

Female mucopolysaccharidosis IIIA mice exhibit hyperactivity and a reduced sense of danger in the open field test

Reliable behavioural tests in animal models of neurodegenerative diseases allow us to study the natural history of disease and evaluate the efficacy of novel therapies. Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo A), is a severe, neurodegenerative lysosomal storage disorder caused by a deficiency in the heparan sulphate catabolising enzyme, sulfamidase. Undegraded heparan sulphate accumulates, resulting in lysosomal enlargement and cellular dysfunction. Patients suffer a progressive loss of motor and cognitive function with severe behavioural manifestations and premature death. There is currently no treatment. A spontaneously occurring mouse model of the disease has been described, that has approximately 3% of normal enzyme activity levels. Behavioural phenotyping of the MPS IIIA mouse has been previously reported, but the results are conflicting and variable, even after full backcrossing to the C57BL/6 background. Therefore we have independently backcrossed the MPS IIIA model onto the C57BL/6J background and evaluated the behaviour of male and female MPS IIIA mice at 4, 6 and 8 months of age using the open field test, elevated plus maze, inverted screen and horizontal bar crossing at the same circadian time point. Using a 60 minute open field, we have demonstrated that female MPS IIIA mice are hyperactive, have a longer path length, display rapid exploratory behaviour and spend less time immobile than WT mice. Female MPS IIIA mice also display a reduced sense of danger and spend more time in the centre of the open field. There were no significant differences found between male WT and MPS IIIA mice and no differences in neuromuscular strength were seen with either sex. The altered natural history of behaviour that we observe in the MPS IIIA mouse will allow more accurate evaluation of novel therapeutics for MPS IIIA and potentially other neurodegenerative disorders.     

A Genetic Model of Substrate Reduction Therapy for Mucopolysaccharidosis

Inherited defects in the ability to catabolize glycosaminoglycans result in lysosomal storage disorders known as mucopolysaccharidoses (MPS), causing severe pathology, particularly in the brain. Enzyme replacement therapy has been used to treat mucopolysaccharidoses; however, neuropathology has remained refractory to this approach. To test directly whether substrate reduction might be feasible for treating MPS disease, we developed a genetic model for substrate reduction therapy by crossing MPS IIIa mice with animals partially deficient in heparan sulfate biosynthesis due to heterozygosity in Ext1 and Ext2, genes that encode the copolymerase required for heparan sulfate chain assembly. Reduction of heparan sulfate by 30–50% using this genetic strategy ameliorated the amount of disease-specific biomarker and pathology in multiple tissues, including the brain. In addition, we were able to demonstrate that substrate reduction therapy can improve the efficacy of enzyme replacement therapy in cell culture and in mice. These results provide proof of principle that targeted inhibition of heparan sulfate biosynthetic enzymes together with enzyme replacement might prove beneficial for treating mucopolysaccharidoses.     

Identification of a mutation causing mucopolysaccharidosis type IIIA in New Zealand Huntaway dogs

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disease that occurs due to a deficiency of heparan sulfate sulfamidase (SGSH). The deficiency of SGSH results in the lysosomal accumulation and urinary excretion of the glycosaminoglycan heparan sulfate. The clinical severity of MPS IIIA is predominantly characterized by severe central nervous system degeneration. Naturally occurring MPS IIIA has recently been described in New Zealand Huntaway dogs, with similar disease progression and biochemical characteristics observed in severely affected MPS IIIA patients. Here, we identify the disease-causing mutation in the MPS IIIA Huntaway dog as 708-709insC. The frequency of the 708-709insC mutation in a sample group of 203 New Zealand Huntaway dogs was determined to be 3.8%. The identification of the 708-709insC mutation will permit the identification of heterozygous carriers as an initial step toward establishing a breeding colony of MPS IIIA dogs for the study of various therapeutic strategies targeted to the central nervous system.     

Mouse model of Sanfilippo syndrome type B: relation of phenotypic features to background strain

Sanfilippo syndrome type B or mucopolysaccharidosis type III B (MPS IIIB) is a lysosomal storage disorder that is inherited in autosomal recessive manner. It is characterized by systemic heparan sulfate accumulation in lysosomes due to deficiency of the enzyme alpha-N-acetylglucosaminidase (Naglu). Devastating clinical abnormalities with severe central nervous system involvement and somatic disease lead to premature death. A mouse model of Sanfilippo syndrome type B was created by targeted disruption of the gene encoding Naglu, providing a powerful tool for understanding pathogenesis and developing novel therapeutic strategies. However, the JAX GEMM Strain B6.129S6-Naglutm1Efn mouse, although showing biochemical similarities to humans with Sanfilippo syndrome, exhibits aging and behavioral differences. We observed idiosyncrasies, such as skeletal dysmorphism, hydrocephalus, ocular abnormalities, organomegaly, growth retardation, and anomalies of the integument, in our breeding colony of Naglu mutant mice and determined that several of them were at least partially related to the background strain C57BL/6. These background strain abnormalities, therefore, potentially mimic or overlap signs of the induced syndrome in our mice. Our observations may prove useful in studies of Naglu mutant mice. The necessity for distinguishing background anomalies from signs of the modeled disease is apparent.     

Analysis of the Biogenesis of Heparan Sulfate Acetyl-CoA:α-Glucosaminide N-Acetyltransferase Provides Insights into the Mechanism Underlying Its Complete Deficiency in Mucopolysaccharidosis IIIC

Heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) catalyzes the transmembrane acetylation of heparan sulfate in lysosomes required for its further catabolism. Inherited deficiency of HGSNAT in humans results in lysosomal storage of heparan sulfate and causes the severe neurodegenerative disease, mucopolysaccharidosis IIIC (MPS IIIC). Previously we have cloned the HGSNAT gene, identified molecular defects in MPS IIIC patients, and found that all missense mutations prevented normal folding and trafficking of the enzyme. Therefore characterization of HGSNAT biogenesis and intracellular trafficking became of central importance for understanding the molecular mechanism underlying the disease and developing future therapies.In the current study we show that HGSNAT is synthesized as a catalytically inactive 77-kDa precursor that is transported to the lysosomes via an adaptor protein-mediated pathway that involves conserved tyrosine- and dileucine-based lysosomal targeting signals in its C-terminal cytoplasmic domain with a contribution from a dileucine-based signal in the N-terminal cytoplasmic loop. In the lysosome, the precursor is cleaved into a 29-kDa N-terminal α-chain and a 48-kDa C-terminal β-chain, and assembled into active ∼440-kDa oligomers. The subunits are held together by disulfide bonds between at least two cysteine residues (Cys¹²³ and Cys⁴³⁴) in the lysosomal luminal loops of the enzyme. We speculate that proteolytic cleavage allows the nucleophile residue, His²⁶⁹, in the active site to access the substrate acetyl-CoA in the cytoplasm, for further transfer of the acetyl group to the terminal glucosamine on heparan sulfate. Altogether our results identify intralysosomal oligomerization and proteolytic cleavage as two steps crucial for functional activation of HGSNAT.     

Blood-brain barrier impairment in an animal model of MPS III B

Background

Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of α-N-acetylglucosaminidase enzyme, leading to accumulation of heparan sulfate within lysosomes and eventual progressive cerebral and systemic multiple organ abnormalities. However, little is known about the competence of the blood-brain barrier (BBB) in MPS III B. BBB dysfunction in this devastating disorder could contribute to neuropathological disease manifestations.

Methodology/Principal Findings

In the present study, we investigated structural (electron microscope) and functional (vascular leakage) integrity of the BBB in a mouse model of MPS III B at different stages of disease, focusing on brain structures known to experience neuropathological changes. Major findings of our study were: (1) endothelial cell damage in capillary ultrastructure, compromising the BBB and resulting in vascular leakage, (2) formation of numerous large vacuoles in endothelial cells and perivascular cells (pericytes and perivascular macrophages) in the large majority of vessels, (3) edematous space around microvessels, (4) microaneurysm adjacent to a ruptured endothelium, (6) Evans Blue and albumin microvascular leakage in various brain structures, (7) GM3 ganglioside accumulation in endothelium of the brain microvasculature.

Conclusions/Significance

These new findings of BBB structural and function impairment in MPS III B mice even at early disease stage may have implications for disease pathogenesis and should be considered in current and future development of treatments for MPS III B.     

Mucopolysaccharidosis 3 A (Sanfilippo A disease): deficiency of a heparin sulfamidase in skin fibroblasts and leucocytes

Cultured skin fibroblasts and peripheral leucocytes from patients with Sanfilippo A disease are strikingly deficient in sulfamidase activity (sulfamatase, EC 3.1.6.?), as measured with heparin - N³⁵SO₄. A partial sulfamidase deficiency was found in the cells of the heterozygote carriers. Since Sanfilippo A fibroblasts have normal sulfate ester hydrolase activities towards oligosaccharides prepared from ³⁵SO₄-labelled heparan sulfate by nitrous acid treatment, the basic defect in Sanfilippo A disease is considered to be the inactivity of a heparin (heparan sulfate) sulfamidase.     

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.     

Lysosomal storage of heparan sulfate causes mitochondrial defects,altered autophagy,and neuronal death in the mouse model of mucopolysaccharidosis III type C

The genetic metabolic disease mucopolysaccharidosis III type C (MPS IIIC, Sanfilippo disease type C) causes progressive neurodegeneration in infants and children, leading to dementia and death before adulthood. MPS IIIC stands out among lysosomal diseases because it is the only one caused by a deficiency not of a hydrolase but of HGSNAT (heparan--glucosaminide N-acetyltransferase), which catalyzes acetylation of glycosaminoglycan heparan sulfate (HS) prior to its hydrolysis.     

Genomic basis of mucopolysaccharidosis type IIID (MIM 252940) revealed by sequencing of GNS encoding N-acetylglucosamine-6-sulfatase

Mucopolysaccharidosis type IIID (MPS IIID; Sanfilippo syndrome type D; MIM 252940) is caused by deficiency of the activity of N-acetylglucosamine-6-sulfatase (GNS), which is normally required for degradation of heparan sulfate. The clinical features of MPS IIID include progressive neurodegeneration, with relatively mild somatic symptoms. Biochemical features include accumulation of heparan sulfate and N-acetylglucosamine-6-sulfate in the brain and viscera. To date, diagnosis required a specific lysosomal enzyme assay for GNS activity. From genomic DNA of a subject with MPS IIID, we amplified and sequenced the promoter and 14 exons of GNS. We found a homozygous nonsense mutation in exon 9 (1063C --> T), which predicted premature termination of translation (R355X). We also identified two common synonymous coding single-nucleotide polymorphisms and genotyped these in samples from four ethnic groups. This first report of a mutation in GNS resulting in MPS IIID indicates the potential utility of molecular diagnosis for this rare condition.     

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes

Background aimsMucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA.MethodsIn the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1α (hEF-1α) promoter to compare the stability of transgene expression.ResultsDuring differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1α promoter resulted in more homogeneous eGFP expression, with ～ 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors.ConclusionsOur data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1α is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.     

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN LIPIDS AND LYSOSOMAL ENZYMES IN PATIENTS WITH FOUR TYPES OF MUCOPOLYSACCHARIDOSIS AND IN NORMAL CONTROLS

Abstract— Lipids and certain lysosomal enzymes were measured in the cerebral gray and white matter and in the liver of unaffected controls and six patients with mucopolysaccharidosis (MPS). Three of the patients had MPS Type I (Hurler), one Type II (Hunter), one Type IIIA (Sanfilippo A) and one Type V (Scheie). The glycosaminoglycans (GAG) of those tissues have been fully characterized previously (C onstantopoulos et al. , 1976).
Results of the present study: the normally minor brain monosialogangliosides G_M2 and G_M3 were markedly increased in the gray and to a lesser extent in the white matter of all the patients, except the patient with MPS Type V. On an average G_M2 comprised 8.2 and 6.3, and G_M3 11.8 and 6.0% of the total ganglioside neuraminic acid of the gray and white matter respectively in all patients with MPS I, II, and IIIA (normal subjects had less than 1).
Ceramide dihexoside was also increased in the gray matter of the patients with MPS I, MPS II and MPS IIIA.
The sphingolipid abnormalities were found only in tissues containing excessive amounts of partially degraded dermatan and heparan sulfates or heparan sulfate alone.
Of the six acid hydrolases assayed, the activity of /f-glucosaminidase was increased in both brain and liver, while that of α-galactosidase and β-galactosidase was diminished, particularly in the liver.
These results suggest that the partially degraded heparan sulfate (and perhaps the dermatan sulfate) which accumulate in the tissues of the patients with MPS may inhibit catabolic enzymes of various sphingolipids. In turn, accumulation of sphingolipids could be responsible at least for some of the brain damage and the mental retardation in MPS I, II and IIIA.     

A new biochemical subtype of the Sanfilippo syndrome: characterization of the storage material in cultured fibroblasts of Sanfilippo C patients.

Fibroblasts cultured from the skin of three unrelated patients with the clinical symptoms of the Sanfilippo syndrome (mucopolysaccharidosis III) accumulated intracellularly excessive amounts of heparan sulfate and showed a lengthened turnover time for this mucopolysaccharide. They exhibited, however, neither a deficiency of heparan sulfate sulfamidase or alpha-N-acetylglucosaminidase nor of any other known glycosaminoglycan-degrading hydrolase. This new mucopolysaccharidosis was therefore designated as type C of the Sanfilippo syndrome. The abnormal heparan sulfate metabolism of Sanfilippo C fibroblasts could not be normalized by addition of crude urinary proteins or concentrated secretions from normal fibroblasts to the culture medium or by cocultivation with normal fibroblasts. The accumulated heparan sulfate was characterized by a reduced negative net charge. A small proportion of it could be adsorbed onto a cation exchange resin. It was sensitive to nitrous acid degradation under conditions where glucosamine residues with free amino groups are attacked. It is therefore suggested that the primary defect in this new mucopolysaccharidosis concerns the step which follows the hydrolysis of N-sulfonate groups in heparan sulfate degradation.     

Protein Misfolding as an Underlying Molecular Defect in Mucopolysaccharidosis III Type C

Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT, EC 2.3.1.78), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the complete absence of HGSNAT protein.In order to understand the effects of the missense mutations in HGSNAT on its enzymatic activity and biogenesis, we have expressed 21 mutant proteins in cultured human fibroblasts and COS-7 cells and studied their folding, targeting and activity. We found that 17 of the 21 missense mutations in HGSNAT caused misfolding of the enzyme, which is abnormally glycosylated and not targeted to the lysosome, but retained in the endoplasmic reticulum. The other 4 mutants represented rare polymorphisms which had no effect on the activity, processing and targeting of the enzyme. Treatment of patient cells with a competitive HGSNAT inhibitor, glucosamine, partially rescued several of the expressed mutants.Altogether our data provide an explanation for the severity of MPS IIIC and suggest that search for pharmaceutical chaperones can in the future result in therapeutic options for this disease.     

Effect of high dose,repeated intra‐cerebrospinal fluid injection of sulphamidase on neuropathology in mucopolysaccharidosis type IIIA mice

Mucopolysaccharidosis type IIIA (MPS IIIA) is an inherited neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of sulphamidase (N‐sulphoglucosamine sulphohydrolase; SGSH; EC 3.10.1.1) results in intracellular accumulation of heparan sulphate (HS), with the brain as the primary site of pathology. We have used a naturally occurring MPS IIIA mouse model to determine the effectiveness of SGSH replacement through the cerebrospinal fluid (CSF) to decrease neuropathology. This is a potential therapeutic option for patients with this disorder. Mice received intra‐CSF injections of recombinant human SGSH (30, 50 or 70 μg) fortnightly from 6 to 18 weeks of age, and the cumulative effect on neuropathology was examined and quantified. Anti‐SGSH antibodies detected in plasma at euthanasia did not appear to impact upon the health of the mice or the experimental outcome, with significant but region‐dependent and dose‐dependent reductions in an HS‐derived oligosaccharide observed in the brain and spinal cord using tandem mass spectrometry. SGSH infusion reduced the number of storage inclusions observed in the brain when visualized using electron microscopy, and this correlated with a significant decrease in the immunohistochemical staining of a lysosomal membrane marker. Reduced numbers of activated isolectin B4‐positive microglia and glial fibrillary acidic protein‐positive astrocytes were seen in many, but not all, brain regions. Significant reductions in the number of ubiquitin‐positive intracellular inclusions were also observed. These outcomes show the effectiveness of this method of enzyme delivery in reducing the spectrum of neuropathological changes in murine MPS IIIA brain.     

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.     

Renal failure associated with mucopolysaccharidosis type I in a cat from a MPS I research colony

Renal failure was diagnosed in an 11-mo-old male domestic shorthair cat from a colony with mucopolysaccharidosis type I lysosomal storage disease. Grossly, the kidneys were enlarged and bulged on cut section. Histology revealed tubular necrosis and regeneration with severe interstitial macrophage accumulation. Tubular epithelial cells and interstitial macrophages were distended by abundant, large cytoplasmic vacuoles. Electron microscopy demonstrated severe tubular epithelial vacuolar degeneration with lysosomes distended by granular debris and mineral precipitates. Interstitial macrophages contained similarly distended lysosomes. Although the initial cause of the tubular injury was not identified, the presence of macrophages laden with storage product most likely exacerbated the disease. The macrophage infiltrate may have caused tubular ischemia by compressing peritubular capillaries and separating tubules from their blood supply. Because the kidney is not normally affected in MPS I, this case is an unusual presentation of a well-characterized disease. Furthermore, this report documents the diagnostic workflow used to investigate a single case of feline acute renal failure in the setting of numerous at-risk laboratory animals.     

A defect in exodegradative pathways provides insight into endodegradation of heparan and dermatan sulfates

Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.