P. Di Natale

Effect of ‘attenuated’ mutations in mucopolysaccharidosis IVA on molecular phenotypes of N-acetylgalactosamine-6-sulfate sulfatase

SummaryMucopolysaccharidosis IVA is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed for seven MPS IVA patients with attenuated phenotypes from three unrelated families. Four of 5 missense mutations identified in this study (p.F167V, p.R253W, p.R380S, p.P484S) and two reported (p.F97V, p.N204K), associated with attenuated phenotypes, were characterized using in vitro stable expression experiments, enzyme kinetic study, protein processing and structural analysis. The stably expressed mutant enzymes defining the attenuated phenotype exhibited a considerable residual activity (1.2–36.7% of the wild-type GALNS activity) except for p.R380S. Enzyme kinetic studies showed that p.F97V, p.F167V and p.N204K have lower affinity to the substrate compared with other mutants. The p.F97V enzyme was the most thermolabile at 55°C. Immunoblot analyses indicated a rapid degradation and/or an insufficiency in processing in the mutant proteins. Tertiary structure analysis revealed that although there was a tendency for ‘attenuated’ mutant residues to be located on the surface of GALNS, they have a different effect on the protein including modification of the hydrophobic core and salt-bridge formation and different potential energy. This study demonstrates that ‘attenuated’ mutant enzymes are heterogeneous in molecular phenotypes, including biochemical properties and tertiary structure.

Maroteaux-lamy syndrome: five novel mutations and their structural localization.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

Genetic complementation of steroid sulphatase after somatic cell hybridization of X-linked ichthyosis and multiple sulphatase deficiency

SummaryComplementation studies of steroid sulphatase were carried out in the heterokaryon population of fibroblasts derived from patients with X-linked ichthyosis and multiple sulphatase deficiency. The activity of steroid sulphatase of the fused cell population was constantly higher (approximately 2–5 fold) than that of the unfused cocultivated cells. The occurrence of complementation further supports the hypothesis that at least two different loci control the expression of steroid sulphatase in the human genome.

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a congenital storage disorder resulting from mutations on the iduronate-2-sulfatase (IDS) gene. The disease shows variable clinical phenotypes from severe to mild with progressive neurological dysfunction. The therapeutic options for treatment of MPS II are limited and currently no specific therapies are available; the problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system (CNS). In this work, as a potential treatment for this disease, the transfer of the recombinant IDS enzyme into brain cells has been studied in vitro. Two different approaches to obtain recombinant IDS have been utilized: production of the recombinant enzyme by a transfected human clone (Bosc 23 cells); production of the recombinant enzyme by adenoviral transduction of neuronal (SK-N-BE) or glial (C6) cells. Our data indicate that the transfected as well as the infected cells produce a large amount of the IDS enzyme, which is efficiently endocytosed into neuronal and glial cells through the mannose 6-phosphate (M6P) receptor system. Somatic gene therapy appears therefore to be suitable to correct IDS deficiency in brain cells.

In vitro correction of iduronate-2-sulfatase deficiency by adenovirus-mediated gene transfer.

Hunter syndrome is a lethal lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase and characterized by severe skeletal and neurological symptoms. Only symptomatic treatments are available and, although bone marrow transplantation has been suggested, no encouraging results have been obtained so far. Therefore, gene therapy might be a route to be pursued for treatment of the disease. In this respect, one major goal to achieve is the generation of an overexpressing vector able to correct, in particular, central nervous system (CNS) cells. Adenoviruses have been shown to infect CNS cells efficiently with minor or even absent immunological response. We describe the generation of a replication-defective adenoviral vector, AdRSVIDS, which is able to express in vitro high levels of iduronate-2-sulfatase. After infection, accumulation of mucopolysaccharides in treated Hunter cells was normalized. Furthermore, endocytosis of the transduced IDS did occur via the mannose-6-phosphate (M6P) receptor. Since no animal model for the disease is available, we developed a system based on the generation of derma-equivalents which enabled us to verify the expression of high levels of sulfatase up to 30 days after infection.

Bone marrow transplantation in Hunter syndrome

Hunter syndrome, or mucopolysaccharidosis (MPS-osis) type II (McKusick 309900), is an X-linked recessive lysosomal storage disease, caused by a deficiency in the enzyme iduronate sulphatase. Several therapeutic approaches have been tried unsuccessfully on patients with MPS-oses. Recently bone marrow transplantation (BMT) has been proposed. To date BMT has been utilized only in few cases of Hunter syndrome (Krivit et al 1992). We present the preliminary results of an allogenic BMT for Hunter syndrome followed for a period of 2 years. The patient, B.S., was first referred to us aged 19 months for coarse facial features and chronic mucopurulent coryza. The urinary GAG/creatinine ratio was elevated: 579mg GAG/g creatinine (normal <94), qualitatively characterized by electrophoresis as dermatan-sulphate and heparan-sulphate. Iduronate sulphatase activity was not detectable in either serum or lymphoblasts. The biochemical investigation conducted on a brother soon after birth showed normal levels of iduronate sulphatase and an HLA pattern compatible with that of the patient. BMT was performed on 9 June 1992, with the patient aged 2.75 years. Physical examination showed hirsutism, hepatosplenomegaly, joint limitation, global mental age corresponding to 25 months, multiple dysostosis, left ventricular dilation and a slight thickening of the mitral valve sheets observed at heart ultrasonography, and significant sensorineural hearing loss. Cerebral MRI showed whitematter alterations of the posterior horns of lateral ventricles. The ultrastructural aspect of the liver biopsy was typical of lysosomal storage disease, showing all hepatocytes to have multiple vacuoles. Evaluation at regular intervals was carried out for 2 years following transplantation. Over this period the patient has grown 16 cm in height (25th-50th centile), and has shown normalization of liver and spleen size, a progressive reduction of cutaneous thickness and hirsutism, and an improvement of joint mobility. At 20 months post-transplantation there was a partial recovery of motor and social performances (developmental age corresponding to 30 months). Over the same period, the bone survey was unchanged, as were cerebral MRI and audiometry. Echocardiography showed normalization of ventricular size. A needle biopsy performed 14 months after transplantation showed normal hepatocytes with only occasional remnants of isolated vacuoles. Total urinary GAG excretion progressively decreased during the first 6 months, with stabilization in the following months at nearly normal levels. Meanwhile, a normal pattern of excretion of GAGs was observed. Leukocyte and lymphoblast iduronate sulphatase activity were

Animal models for lysosomal storage diseases: a new case of feline mucopolysaccharidosis VI.

SummaryTwo long-haired Siamese cats are reported with clinical manifestations of human mucopolysaccharidosis VI (Maroteaux-Lamy disease): facial dysmorphia, dysostosis multiplex, paralysis. Urine of the two affected animals contained a high concentration of glycosaminoglycans, as detected by the dimethylmethylene blue test. Qualitative analysis, performed by thin-layer chromatography of the cetylpyridinium chloride-precipitable material, showed dermatan sulphate. Excessive incorporation of [35S]sulphate in the intracellular mucopolysaccharide of cultured fibroblasts and deficiency of arylsulphatase B in such cells indicate that these cats are affected by Maroteaux-Lamy disease. They should thus be considered the first European case of feline mucopolysaccharidosis VI.

Analysis of Sanfilippo A gene mutations in a large pedigree.

Mucopolysaccharidosis type IIIA, also known as Sanfilippo A disease, results from mutations in the sulfamidase gene. To date, a total of 62 mutations have been described underlying this lysosomal disorder. Expression studies on missense mutations have shown that each alteration was disease‐causing and helped to clarify the genotype–phenotype correlation. In this report we describe a large pedigree where the mutations have been identified in two second cousins: one with severe disease (E369K/R433Q) and the other with a mild form of the illness (E369K/P128L). This study places R433Q as a severe mutation underlying Sanfilippo A disease.

The pyrophosphate exchange reaction of histidyl-tRNA synthetase from Salmonella typhimurium: Reaction parameters and inhibition by transfer ribonucleic acid

The reactions generally require magnesium as a cofactor [2,3]. Reaction 1 is the amino acid activation reaction and is usually studied by the ATP-PP, isotope exchange reaction. Reaction parameters may be very different for each activating enzyme and also for enzymes from different sources which activate the same amino acid [3] . It has been shown that the first reaction may be affected by the presence of the specific tRNA. However, the effect of tRNA on the reaction is quite different depending on the particular synthetase consider-

Echistatin inhibits pp72syk and pp125FAK phosphorylation in fibrinogen-adherent platelets.

The adhesion of ADP-stimulated platelets to immobilized fibrinogen induces the tyrosine phosphorylation of multiple proteins which include pp72syk and pp125FAK. The phosphorylation of these two proteins increases as function of time of platelet adhesion to fibrinogen; however, pp72syk results strongly phosphorylated already after 15 min, whereas pp125FAK reaches high levels of phosphorylation after 1 h of platelet adhesion. Phosphorylation of both proteins is only slightly detectable when platelets are held in suspension or when platelets are allowed to adhere to bovine serum albumin, a non-specific substrate. Echistatin, an Arg-Gly-Asp (RGD)-containing snake-venom protein, affects protein tyrosine phosphorylation promoted by platelet adhesion to fibrinogen, by causing an approximately 44% and 39% decrease of pp72syk and pp125FAK phosphorylation, respectively. The interaction of echistatin with fibrinogen receptor glycoprotein IIb-IIIa on platelet surface might be responsible for the block of integrin-mediated signaling cascade, including pp72syk and pp125FAK inactivation.