Mutational spectrum of the iduronate-2-sulfatase gene in Mexican patients with Hunter syndrome.

OBJECTIVE: Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which is responsible for degrading heparan and dermatan sulfate. The IDS gene is located on chromosome Xq28; pathological variants in this gene mostly consist of missense mutations and small and larger deletions, which produce different phenotypes. However, there is only one record in our population concerning the molecular mechanism of this disease; a genotype-phenotype description is not available. PATIENTS AND METHODS: There were included 24 unrelated male patients; clinical features were recorded at a database, fluorometric IDS enzyme activity testing was done for each individual, followed by Sanger sequencing to identify mutations. RESULTS: The mutational spectrum was found in 16 out of 24 Mexican patients with MPS II, and its range of phenotypes was described. The most frequent variants were of the missense type. The most affected exons were exon 3 (c.275T>G, c.284_287del, c.325T>C), exon 8 (c.1035G>C, c.550G>A), exon 9 (c.1403G>C, c.1229_1229del), and exon 7 (c.979A>C; this variant has not been previously reported). Exon 5 (c.438C>T, a non-pathogenic variant) was the least frequent. It was also found that the most severely affected patients were those with large deletions (2 out of 24) [rsaIDS: IDSP1 (P164)x0, FMR1, AFF2 (P164)x2] involving genes and pseudogenes. We found 2 patients with a synonymous mutation in exon 4. CONCLUSIONS: Our results confirmed reports in the literature, since the most frequent variants were reported in exons 3 and 8. However, this result varies from one previous report in our population, which mentions large deletions and rearrangements as the most frequent alterations, since complex rearrangements were not found. According to what has been previously found, the most severely affected patients are those in which a whole gene has been deleted.

Population-Based Newborn Screening for Mucopolysaccharidosis Type II in Illinois: The First Year Experience.

OBJECTIVES: To assess the outcome of population-based newborn screening for mucopolysaccharidosis type II (MPS II) during the first year of screening in Illinois. STUDY DESIGN: Tandem mass spectrometry was used to measure iduronate-2-sulfatase (I2S) activity in dried blood spot specimens obtained from 162 000 infant samples sent to the Newborn Screening Laboratory of the Illinois Department of Public Health in Chicago. RESULTS: One case of MPS II and 14 infants with pseudodeficiency for I2S were identified. CONCLUSIONS: Newborn screening for MPS II by measurement of I2S enzyme activity was successfully integrated into the statewide newborn screening program in Illinois.

Low-scale expression and purification of an active putative iduronate 2-sulfate sulfatase-Like enzyme from Escherichia coli K12.

The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.

Depiction of the forces participating in the 2-O-sulfo-alpha-L-iduronic acid conformational preference in heparin sequences in aqueous solutions.

2-O-Sulfo-alpha-l-iduronic acid (IdoA2S) is one of the main components of heparin, an anticoagulant and antithrombotic polysaccharide able to potentiate the inhibitory effect of antithrombin over plasma serine proteases. This monosaccharide unit adopts an equilibrium between chair (1C4) and skew-boat (2SO) forms as a function of heparin sequence size and composition. Although the prevalence of the 1C4 chair conformation in monosaccharides is understood, the reasons for the increase in 2SO contribution in the whole polysaccharide chain are still uncertain. In this context, 0.2 mus molecular dynamics simulations of IdoA2S-containing oligosaccharides indicated that stabilization due to intramolecular hydrogen bonds around IdoA2S is highly correlated (p0.001) with the expected conformational equilibrium for this residue in solution. This behavior explains the known effect of different heparin compositions, at the monosaccharide level, on IdoA2S conformation in biological solutions.

A small fraction of dermatan sulfate with significantly increased anticoagulant activity was selected by interaction with the first complement protein.

Dermatan sulfate (DS) is a member of the family of structurally complex, sulfated, linear heteropolysaccharides called glycosaminoglycans (GAGs). It has a similar structure to heparin and heparan sulfate (HS), but with acetylgalactosamine replacing glucosamine, and the uronic acid moiety, mainly iduronic, joined 1-->3 to the hexosamine. We are studying the relationships between structure and activities of dermatan sulfate, in particular those associated with the thrombin inhibition mediated by heparin cofactor II (HCII). As we have demonstrated with heparin, a small fraction of dermatan sulfate was isolated by precipitation with the first component of the complement system, under very specific conditions of low ionic strength, and the presence of calcium ions. The sulfate content and the anticoagulant activity of the dermatan sulfate fraction isolated in the precipitate were three and four times greater respectively than the starting material. Our in vivo studies showed that this fraction has threefold higher thrombolytic activity than the DS. All these results suggest that this fraction could be used as a therapeutic agent for thrombi dissolution.

Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis.

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.

Highly sulfated dermatan sulfates from Ascidians. Structure versus anticoagulant activity of these glycosaminoglycans.

Dermatan sulfates with the same backbone structure [4-alpha-L-IdceA-1-->3-beta-D-GalNAc-1]n but with different patterns of sulfation substitutions have been isolated from the ascidian body. All the ascidian dermatan sulfates have a high content of 2-O-sulfated alpha-L-iduronic acid residues but differ in the pattern of sulfation of the N-acetyl-beta-D-galactosamine units. Styela plicata and Halocynthia pyriformis have 4-O-sulfated units, but in Ascidian nigra they are 6-O-sulfated. This collection of ascidian dermatan sulfates (together with native and oversulfated mammalian dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, were tested in anticoagulant assays. Dermatan sulfate from A. nigra has no discernible anticoagulant activity, which indicates that 4-O-sulfation of the N-acetyl-beta-D-galactosamine is essential for the anticoagulant activity of this glycosaminoglycan. In contrast dermatan sulfates from S. plicata and H. pyriformis are potent anticoagulants due to potentiation of thrombin inhibition by heparin cofactor II. These ascidian dermatan sulfates have approximately 10-fold and approximately 6-fold higher activity with heparin cofactor II than native and an oversulfated mammalian dermatan sulfate, respectively. They have no effect on thrombin or factor Xa inhibition by antithrombin. These naturally oversulfated ascidian dermatan sulfates are sulfated at selected sites required for interaction with heparin cofactor II and thus have specific and potent anticoagulant activity.

Chondroitin ABC lyase digestion of an ascidian dermatan sulfate. Occurrence of unusual 6-O-sulfo-2-acetamido-2-deoxy-3-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-beta-D-galactose units.

A dermatan sulfate-like glycosaminoglycan was isolated from the body of the ascidian Ascidia nigra (J. Biol. Chem. 270: 31027-31036, 1995). 1H NMR and fast atom bombardment mass spectrometry (FAB-MS) spectra of the tetra and disaccharides formed by chondroitin ABC lyase digestion support the proposed repeating disaccharide structure for this glycosaminoglycan, [-->4)-alpha-L-IdoA(2SO4)-(1-->3)-beta-D-GalNAc(6SO4)-(1-->] , which differ from mammalian dermatan sulfate in its sulfation at both 2-position of the alpha-L-iduronic acid and the 6-position of the N-acetyl-beta-D-galactosamine residue.