The predominant role of IP3 type 1 receptors in activation of store-operated Ca²+ entry in liver cells.

Physiologically, hormone induced release of Ca²+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP3) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP3 receptors act as channels, releasing Ca²+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca²+ stores leads to activation of store-operated Ca²+ channels on the plasma membrane which replenishes lost Ca²+ and sustain Ca²+ signalling. There are three isoforms of IP3 receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca²+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca²+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca²+ channels, and Ca²+ release activated Ca²+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP3 receptor but not the others. Here we show that H4IIE liver cells express all three types of IP3 receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca²+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca²+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP3 receptors.

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins.

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (I(CRAC)) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I(CRAC). In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I(CRAC). The results show that cells expressing low Orai1 : STIM1 ratios produce I(CRAC) with strong fast Ca2+-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I(CRAC) with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Mucopolysaccharidosis type VI: Structural and clinical implications of mutations in N-acetylgalactosamine-4-sulfatase.

Mucopolysaccharidosis type VI (MPS-VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-4-sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4-sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS-VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS-VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS-VI alleles will be difficult.

Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase.

Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation factor promoter. The pEFNeo expression vector also contains the human growth hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.17, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell factories. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcFlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by fibroblasts from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.

Feline mucopolysaccharidosis type VI. Characterization of recombinant N-acetylgalactosamine 4-sulfatase and identification of a mutation causing the disease.

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (4S) leading to the lysosomal accumulation and urinary excretion of dermatan sulfate. MPS VI has also been described in the Siamese cat. As an initial step toward enzyme replacement therapy with recombinant feline 4S (rf4S) in MPS VI cats, the feline 4S cDNA was isolated and expressed in CHO-KI cells and rf4S was immunopurified from the culture medium. SDS-polyacrylamide gel electrophoresis analysis showed that the precursor form of immunopurified rf4S was a 66-kDa polypeptide that underwent maturation to a 43-44-kDa polypeptide. Endocytosis of rf4S by cultured feline MPS VI myoblasts was predominantly mediated by a mannose 6-phosphate receptor and resulted in the correction of dermatan sulfate storage. The mutation causing feline MPS VI was identified as a base substitution at codon 476, altering a leucine codon to a proline (L476P). The L476P allele displayed no detectable 4S activity when expressed in CHO-KI cells and was observed only as a "precursor" polypeptide that was not secreted into the medium. Identification of the mutation has allowed the development of a rapid PCR-based screening method to genotype individuals within the cat colony.

Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients.

Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in >1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients.

Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein.

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

Identification of mutations in the alpha-L-iduronidase gene (IDUA) that cause Hurler and Scheie syndromes.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the lysosomal glycosidase alpha-L-iduronidase. Hurler (severe), Scheie (mild), and Hurler/Scheie (intermediate) syndromes are clinical subtypes of MPS-I, but it is difficult to distinguish between these subtypes by biochemical measurements. Mutation analysis was undertaken to provide a molecular explanation for the clinical variation seen in MPS-I. Using chemical cleavage and direct PCR sequencing, we have defined four previously undescribed mutations for MPS-I (delG1702, 1060 + 2t-->c, R89Q, and 678-7g-->a). R89Q and 678-7g-->a were found to be present in 40% of Scheie syndrome alleles. Expression of R89Q demonstrated reduced stability and activity of the mutant protein. The deleterious effect of R89Q may be potentiated by a polymorphism (A361T) to produce an intermediate phenotype. 678-7g-->a was found to be a mild mutation, since it was present in an index Scheie syndrome patient in combination with a severe allele (W402X). This mutation appears to allow a very small amount of normal mRNA to be produced from the allele which is likely to be responsible for the mild clinical phenotype observed. Both the 5' and 3' splice site mutations (1060 + 2t-->c and 678-7g-->a, respectively) result in high proportions of mature mRNAs containing introns, which has not been observed for other splicing mutations. The frameshift mutation (delG1702) and the 5' splice site mutation (1060 + 2t-->c) are both thought to be associated with severe MPS-I. The identification of these MPS-I mutations begins to document the expected genetic heterogeneity in MPS-I and provides the first molecular explanations for the broad range of clinical phenotypes observed.

Long-term clinical progress in bone marrow transplanted mucopolysaccharidosis type I patients with a defined genotype.

Two mucopolysaccharidosis type I (MPS-I) patients, subjected to bone marrow transplantation (BMT) more than 10 years ago, have recently had their alpha-L-iduronidase genotypes defined. Both patients, homozygous for the relatively common W402X mutation, received BMT when they were 14 and 11 months of age, and are now 12 and 14 years old, respectively. Untreated MPS-I patients, homozygous for W402X, have an extremely severe clinical phenotype with rapid clinical deterioration and death before 6 years of age. The 12-year-old patient, with limited mobility, is coping well at school, while the other patient is wheelchair-bound with severe disability in his lower limbs, and attends a school for the physically handicapped. Both patients have less than normal intelligence with slowly continuing losses. A third MPS-I patients, diagnosed at the age of 6 months, was felt, prior to BMT at 14 months, to have a severe phenotype. Twelve years post-BMT, he is ambulatory, albeit with restricted movement, and has normal intelligence. This patient did not have a defined MPS-I genotype and had alpha-L-iduronidase protein and activity consistent with a less severe outcome than the first two patients. We conclude that BMT has significantly slowed down the clinical regression of the W402X phenotype. We propose that if further gains are to be made, BMT should be performed within the first few months of life. Early diagnosis is therefore essential.

PCR detection of two RFLPs in exon I of the alpha-L-iduronidase (IDUA) gene.

Two polymorphisms were detected within exon I of the alpha-L-iduronidase (IDUA) gene both of which create restriction endonuclease sites and one of which changes an amino acid. The polymorphisms may be detected by digesting the same 245-bp polymerase chain reaction product. The polymorphisms can be used diagnostically in families with IDUA deficiency (mucopolysaccharidosis type I) and Huntington disease, which is closely linked to the IDUA locus.

A common mutation for mucopolysaccharidosis type I associated with a severe Hurler syndrome phenotype.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the glycosidase alpha-L-iduronidase which is required for the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Patients with MPS-I store these partially degraded glycosaminoglycans in their lysosomes. MPS-I patients have a wide range of clinical presentations, that makes it difficult to predict patient phenotype which is needed for genetic counselling and also impedes the selection and evaluation of patients undergoing therapy such as bone marrow transplantation. We report the presence of a common mutation accounting for 31% of MPS-I alleles in a study of 64 MPS-I patients. The mutation was originally detected by chemical cleavage and then direct PCR sequencing. The mutation is a single base substitution that introduces a stop codon at position 402 (W402X) of the alpha-L-iduronidase protein and is associated with an extremely severe clinical phenotype in homozygotes. Patients who are compound heterozygotes having one allele carrying the W402X mutation have a wide range of clinical phenotypes. Based on polymorphisms within the alpha-L-iduronidase gene, W402X is associated with three different haplotypes, implying that there is more than one origin for the mutation or that intragenic recombination has occurred. W402X introduces a MaeI restriction endonuclease site into MPS-I alleles enabling its simple detection, which should make possible the assessment of the efficacy of bone marrow transplantation in MPS-I patients homozygous for W402X.

alpha-L-iduronidase mutations (Q70X and P533R) associate with a severe Hurler phenotype.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the glycosidase alpha-L-iduronidase which is required for the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Patients with MPS-I store forms of these partially degraded glycosaminoglycans in their lysosomes. MPS-I patients present with a wide range of clinical phenotypes, which makes prognostic predictions and genetic counselling difficult, therefore impeding the selection and evaluation of patients undergoing experimental therapy, such as bone marrow transplantation. We report the presence of two mutations, one that introduces a stop codon at position 70 (Q70X), and the other that alters the proline at position 533 to an arginine (P533R) in the 653 amino acid alpha-L-iduronidase protein. These mutations were originally detected by chemical cleavage and then by direct PCR sequencing. Allele specific oligonucleotides were used to detect the mutations in a group of 73 MPS-I patients and Q70X was found to account for 15% of all MPS-I alleles and P533R for 3% of MPS-I alleles. Both mutations are associated with an extremely severe clinical phenotype in homozygotes. MPS-I patients heterozygous for either mutation may have a wide range of clinical phenotypes. We have now described three mutations, W402X (Scott et al., 1992c), Q70X, and P533R totalling 53% of MPS-I alleles which together define 28% of MPS-I genotypes.

An N-acetylgalactosamine-4-sulfatase mutation (delta G238) results in a severe Maroteaux-Lamy phenotype.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomally inherited lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (EC 3.1.6.1; 4-sulfatase). In order to determine the gene defect in a clinically severe MPS VI patient, polymerase chain reaction (PCR) products were generated from the patient's fibroblast mRNA and also from a 4-sulfatase cDNA clone and subjected to the chemical cleavage technique to detect mismatched bases, which were then identified by direct DNA sequencing of the PCR products. The patient was homozygous for an early frameshift mutation caused by the deletion of a G at position 238 (delta G238), which produces a truncated 4-sulfatase with an altered amino acid sequence from amino acid 80 to a premature stop codon at codon 113 relative to the normal 4-sulfatase reading frame of 533 amino acids. Since the mutation occurs only 40 amino acids past the signal peptidase cleavage site, it is most likely that this will result in a protein with no 4-sulfatase activity. This is consistent with the severe clinical presentation and the absence of 4-sulfatase enzyme activity or mutant 4-sulfatase protein in the patient. The patient was also found to be homozygous for two polymorphisms, i.e., a G to A transition at nucleotide 1072 resulting in a valine358 to methionine substitution (V358M) and a salient A to G transition in the third base of the proline397 codon at nucleotide 1191.

Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones.

N-Acetylgalactosamine-4-sulphatase (EC 3.1.6.1, G4S) is composed of a 57 kDa species in human liver that dissociates into 43 kDa and 8 kDa subunits under reducing conditions and, when deficient, causes the lysosomal storage disorder, mucopolysaccharidosis type VI. We isolated genomic clones containing the G4S first exon, including the leader peptide and the amino terminus of the 43 kDa polypeptide. Amino-terminal amino acid sequences of the 43 kDa and 8 kDa subunits indicated that the 8 kDa component is linked to the 43 kDa polypeptide by a single disulphide bond, does not contain the mannose-6-phosphate lysosomal targeting signal and is at the carboxyl terminus of G4S.

Chromosomal localization of ARSB, the gene for human N-acetylgalactosamine-4-sulphatase.

A deficiency of N-acetylgalactosamine-4-sulphatase (G4S, gene symbol ARSB), results in the accumulation of undegraded substrate and the lysosomal storage disorder, Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). In situ hybridization using an 3H-labelled human G4S genomic DNA fragment to human metaphase chromosomes localized ARSB to chromosome 5q13-5q14. This location is consistent with, an refines, previous chromosomal assignments based on the expression of human G4S in somatic cell hybrids.

Can combined DES and LHRH depot therapy (ICI 118630) prevent endocrinologic and clinical flare-up in metastatic prostate cancer?

Medical castration obtained with luteinizing hormone releasing hormone (LHRH) analogues in patients with prostate cancer is now well established. To block the initial stimulation of testosterone production and prevent the risk of the so-called flare-up with this medication, we investigated short-term combination therapy with 1 mg of diethylstilbestrol (DES). Fourteen previously untreated patients with histologically proved metastatic prostatic carcinoma were treated with 1 mg DES po daily one week prior to the initiation of therapy with LHRH analogues depot injection of Zoladex (ICI 116630) and continued during four weeks after the first depot injection. LHRH depot form was maintained as long as patients experienced clinical benefit. Endocrinologic results show that in spite of 1 mg of DES a significant increase of testosterone is still observed in the first week after injection of the LHRH depot form. Hence, this combination is not useful to prevent endocrinologic and clinical flare-up in patients with prostate cancer treated with LHRH analogues.