Purification and characterization of recombinant murine sulfamidase.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the lysosomal enzyme sulfamidase, which is required for the degradation of heparan sulfate. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. A naturally occurring mouse model to MPS IIIA exhibits a similar disease progression to that observed in patients. Disease in the mice results from a base substitution at codon 31 in the sulfamidase gene, altering an aspartic acid to an asparagine (D31N). This aspartic 31 is involved in binding of the divalent metal ion needed for catalytic function, and as such reduces the specific activity of the enzyme to about 3% of that of wild-type. The mutant protein has decreased stability and shows increased degradation over a 24 h chase period when compared to wild-type mouse sulfamidase. Mouse sulfamidase that was purified using a two-step ion exchange procedure was shown to have similar kinetic properties to that of purified human sulfamidase. Recombinant murine sulfamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.

Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.

Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic, clinical, and biological implications.

Mucopolysaccharidosis (MPS) types IIIA, B, C, and D are a group of autosomal recessive lysosomal storage diseases caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. The progressive lysosomal storage of heparan sulfate eventually results in the clinical onset of disease, which is predominantly characterized by severe central nervous system degeneration. MPS-IIIA and MPS-IIIB involve deficiencies of heparan sulfate sulfamidase (SGSH) and alpha-N-acetylglucosaminidase (NAGLU), respectively. Both the SGSH and NAGLU genes have been cloned and characterized, thereby permitting mutation analysis of MPS-IIIA and MPS-IIIB patients. A total of 62 mutations have now been defined for MPS-IIIA consisting of 46 missense/nonsense mutations, 15 small insertions/deletions, and one splice site mutation. A total of 86 mutations have been identified in the NAGLU gene of MPS-IIIB patients; 58 missense/nonsense mutations, 27 insertions/deletions, and one splice site mutation. Most of the identified mutations in the SGSH and NAGLU genes are associated with severe clinical phenotypes. Many of the missense, nonsense, and insertion/deletion mutations have been expressed in mammalian cell lines to permit the characterization of their effects on SGSH and NAGLU activity and intracellular processing and trafficking. For MPS-IIIA and MPS-IIIB many of the reported mutations are unique making screening the general population difficult. However, molecular characterization of MPS-IIIA patients has revealed a high incidence of particular mutations of different geographical origins, which will be beneficial for the molecular diagnosis of MPS-IIIA.

Glycosidase active site mutations in human alpha-L-iduronidase.

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.

Expression and characterization of human recombinant and alpha-N-acetylglucosaminidase.

Mucopolysaccharidosis type IIIB (MPS-IIIB, Sanfilippo type B Syndrome) is a heterosomal, recessive lysosomal storage disorder resulting from a deficiency of [alpha]-N-acetylglucosaminidase (NAGLU). To characterize this enzyme further and evaluate its potential for enzyme replacement studies we expressed the NAGLU-encoding cDNA in Chinese hamster ovary cells (CHO-K1 cells) and purified the recombinant enzyme from the medium of stably transfected cells by a two-step affinity chromatography. Two isoforms of recombinant NAGLU with apparent molecular weights of 89 and 79 kDa were purified and shown to differ in their glycosylation pattern. The catalytic parameters of both forms of the recombinant enzyme were indistinguishable from each other and similar to those of NAGLU purified from various tissues. However, compared to other recombinant lysosomal enzymes expressed from CHO-K1 cells, the mannose-6-phosphate receptor mediated uptake of the secreted form of recombinant NAGLU into cultured skin fibroblasts was considerably reduced. A small amount of phosphorylated NAGLU present in purified enzyme preparations was shown to be endocytosed by MPS-IIIB fibroblasts via the mannose-6-phosphate receptor-mediated pathway and transported to the lysosomes, where they corrected the storage phenotype. Direct metabolic labeling experiments with Na(2) (32)PO(4) confirmed that the specific phosphorylation of recombinant NAGLU secreted from transfected CHO cells is significantly lower when compared with a control lysosomal enzyme. These results suggest that the use of secreted NAGLU in future enzyme and gene replacement therapy protocols will be severely limited due to its small degree of mannose-6-phosphorylation.

Mucopolysaccharidosis type IIIB: characterisation and expression of wild-type and mutant recombinant alpha-N-acetylglucosaminidase and relationship with sanfilippo phenotype in an attenuated patient.

Mucopolysaccharidosis type IIIB (MPS-IIB) is a lysosomal storage disorder characterised by the defective degradation of heparan sulfate due to a deficiency of alpha-N-acetylglucosaminidase (NAG). The clinical severity of MPS-IIIB ranges from an attenuated to severely affected Sanfilippo phenotype. This paper describes the expression and characterisation of wild-type recombinant NAG and the molecular characterisation of a previously identified R297X/F48L compound heterozygous MPS-IIIB patient with attenuated Sanfilippo syndrome. We have previously shown R297X to be the most common mutation in a cohort of Dutch and Australian patients, occurring at a frequency of approximately 12.5%. To date F48L has only been described in the proband. To determine the contribution of each mutation to the overall clinical phenotype of the patient, both mutant alleles were engineered into the wild-type NAG cDNA and expressed in Chinese hamster ovary cells. The wild-type NAG and F48L mutant alleles were also retrovirally expressed in MPS-IIIB skin fibroblasts. Residual NAG activity and the stability and maturation of immunoprecipitated NAG were determined for wild-type NAG and mutant NAG. The combined biochemical phenotypes of the two NAG mutant alleles demonstrated a good correspondence with the observed attenuated Sanfilippo phenotype of the patient.

Expression and characterization of wild type and mutant recombinant human sulfamidase. Implications for Sanfilippo (Mucopolysaccharidosis IIIA) syndrome.

Mucopolysaccharidosis IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and storage of heparan sulfate. This paper reports the production and characterization of monoclonal and polyclonal antibodies against recombinant human sulfamidase (rhNS) to quantitate and characterize normal and mutant sulfamidase produced from the wild type NS expression vector. Glycosylation and phosphorylation studies of immunoprecipitated rhNS show that all five potential glycosylation sites are utilized, with three high mannose/hybrid oligosaccharides and two simpler chains, with at least one functional mannose 6-phosphate group. An NS quantification system was developed to determine the effect of the three most common and severe patient mutations: S66W (Italy), R74C (Poland), and R245H (The Netherlands). The quantity and specific activity of expressed mutant rhNS was significantly lower than expressed normal rhNS, with 0.3, 0.2, and 0.05% of normal rhNS produced and 15, 17, and 83% of normal specific activity for S66W, R74C, and R245H observed, respectively. The recent structural elucidation of N-acetylgalactosamine-4-sulfatase was utilized to postulate the effect on the structure-function relationship of NS. The characterization of normal and mutated rhNS has relevance for efficient diagnosis and therapeutic developments for MPS-IIIA patients.

Regulation of N-acetylgalactosamine 4-sulfatase expression in retrovirus-transduced feline mucopolysaccharidosis type VI muscle cells.

As a preliminary step toward muscle-mediated gene therapy in the mucopolysaccharidosis (MPS) type VI cat, we have analyzed the transcriptional regulation of feline N-acetylgalactosamine 4-sulfatase (f4S) gene expression from various retroviral constructs in primary cultures of muscle cells. Two retroviral constructs were made containing the f4S cDNA under the transcriptional control of the human polypeptide chain-elongation factor 1alpha (EF1alpha) gene promoter or the cytomegalovirus (CMV) immediate-early promoter. Two further retroviral constructs were made with the murine muscle creatine kinase (mck) enhancer sequence upstream of the internal promoter. Virus made from each construct was used to transduce feline MPS VI myoblasts. The mck enhancer significantly upregulated f4S gene expression from both the EF1alpha promoter and the CMV promoter in transduced myoblasts and in differentiated myofibers. The highest level of 4S activity was observed in myoblasts and myofibers transduced with the retroviral construct Lmckcmv4S, in which the f4S gene is under the transcriptional regulation of the mck enhancer and CMV immediate-early promoter. Lmckcmv4S-transduced myofibers demonstrated correction of glycosaminoglycan storage and contained a 58-fold elevated level of 4S activity compared with normal myofibers. Recombinant f4S secreted from Lmckcmv4S-transduced myofibers was endocytosed by feline MPS VI myofibers, leading to correction of the biochemical storage phenotype.

Evaluation of fibroblast-mediated gene therapy in a feline model of mucopolysaccharidosis type VI.

Fibroblast-mediated ex vivo gene therapy was evaluated in the N-acetylgalactosamine 4-sulfatase (4S) deficient mucopolysaccharidosis type VI (MPS VI) cat. Skin biopsies were obtained at birth from severely affected MPS VI kittens and used to initiate fibroblast outgrowths for retroviral transduction with the 4S cDNA. 4S gene expression in transduced cells was under the transcriptional control of the MoMLV long terminal repeat promoter or the cytomegalovirus (CMV) immediate-early promoter. Characterisation of gene-transduced fibroblasts demonstrated the cells to be over-expressing 4S activity. Twenty-four to forty million autologous, gene-corrected fibroblasts were implanted under the renal capsule of three MPS VI kittens at 8-16 weeks of age. Transient, low levels of 4S activity were detected in peripheral blood leukocytes shortly after implantation but were not detectable within 3-8 weeks' post-implantation. Long-term biochemical and clinical evaluation of these cats demonstrated identical disease progression to that previously described in untreated, clinically severe MPS VI cats.

Mild feline mucopolysaccharidosis type VI. Identification of an N-acetylgalactosamine-4-sulfatase mutation causing instability and increased specific activity.

The missense mutation, L476P, in the N-acetylgalactosamine 4-sulfatase (4S) gene, has previously been shown to be associated with a severe feline mucopolysaccharidosis type VI (MPS VI) phenotype. The present study describes a second mutation, D520N, in the same MPS VI cat colony, which is inherited independently of L476P and is associated with a clinically mild MPS VI phenotype in D520N/L476P compound heterozygous cats. Biochemical and clinical assessment of L476P homozygous, D520N/L476P compound heterozygous, and D520N homozygous cats demonstrated that the entire range of clinical phenotypes, from severe MPS VI, to mild MPS VI, to normal are clustered within a narrow range of residual 4S activity from 0. 5% to 4.6% of normal levels. When overexpressed in CHO-KI cells, the secreted form of D520N 4S was inactivated in neutral pH conditions. In addition, intracellular D520N 4S protein was rapidly degraded and corresponded to 37%, 14.5%, and 0.67% of normal 4S protein levels in the microsomal, endosomal, and lysosomal compartments, respectively. However, the specific activity of lysosomal D520N 4S was elevated 22. 5-fold when compared with wild-type 4S. These results suggest that the D520N mutation causes a rapid degradation of 4S protein. The effect of this is partially ameliorated as a result of a significant elevation in the specific activity of mutant D520N 4S reaching the lysosomal compartment.

Two mutations within a feline mucopolysaccharidosis type VI colony cause three different clinical phenotypes.

Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase (4S). A feline MPS VI model used to demonstrate efficacy of enzyme replacement therapy is due to the homozygous presence of an L476P mutation in 4-sulfatase. An additional mutation, D520N, inherited independently from L476P and recently identified in the same family of cats, has resulted in three clinical phenotypes. L476P homozygotes exhibit dwarfism and facial dysmorphia due to epiphyseal dysplasia, abnormally low leukocyte 4S/betahexosaminidase ratios, dermatan sulfaturia, lysosomal inclusions in most tissues including chondrocytes, corneal clouding, degenerative joint disease, and abnormal leukocyte inclusions. Similarly, D520N/D520N and L476P/D520N cats have abnormally low leukocyte 4S/betahexosaminidase ratios, mild dermatan sulfaturia, lysosomal inclusions in some chondrocytes, and abnormal leukocyte inclusions. However, both have normal growth and appearance. In addition, L476P/D520N cats have a high incidence of degenerative joint disease. We conclude that L476P/D520N cats have a very mild MPS VI phenotype not previously described in MPS VI humans. The study of L476P/D520N and D520N/ D520N genotypes will improve understanding of genotype to phenotype correlations and the pathogenesis of skeletal dysplasia and joint disease in MPS VI, and will assist in development of therapies to prevent lysosomal storage in chondrocytes.

Feline mucopolysaccharidosis type VI: correction of glycosaminoglycan storage in myoblasts by retrovirus-mediated transfer of the feline N-acetylgalactosamine 4-sulfatase gene.

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal storage disorder characterised by the deficiency of N-acetylgalactosamine 4-sulfatase (4S). MPS VI has also been described in the cat. As an initial step toward muscle-mediated gene therapy in the MPS VI cat, we have made two retroviral constructs (pLf4S and pLf4SSN) that transduce the feline 4S gene. Both constructs were designed to express the feline 4S sequence from the viral long terminal repeat promoter. In addition pLf4SSN expressed the neomycin resistance gene from the SV40 early promoter. Amphotrophic virus was produced for each construct and used to transduce feline MPS VI myoblasts. Lf4S- and Lf4SSN-transduced MPS VI feline myoblasts demonstrated correction of glycosaminoglycan storage and contained 55-fold and 3.5-fold elevated levels of 4S activity when compared with normal feline myoblasts respectively. Recombinant feline 4S (rf4S) secreted by Lf4S-transduced MPS VI myoblasts was shown to be endocytosed by MPS VI feline cells via the mannose-6-phosphate receptor system, leading to metabolic correction. The results from this study demonstrate that muscle-mediated gene replacement therapy may be a viable method for achieving circulating levels of recombinant f4S (rf4S) in the MPS VI cat.

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.

Mutational analysis of the carboxy-terminal region of UDP-glucuronosyltransferase 2B1.

UDP-glucuronosyltransferases (UGTs) are membrane-bound glycoproteins that are resident in the endoplasmic reticulum with a type I topology. The roles of the membrane-spanning and membrane-proximal cytoplasmic domains in UGT activity were investigated. Site-directed and deletional mutagenesis techniques were used to generate truncated forms of the enzyme, forms with altered residues, or forms with heterologous tails appended to the carboxyl terminus. The presence of the transmembrane domain was a critical requirement for UGT activity whereas the cytoplasmic domain seemed to be a modulator of activity but was not essential. Truncation of the protein did not appear to lead to scavenging and degradation, although appending long heterologous tails to the cytoplasmic domain did seem to trigger proteolysis. Analysis of enzyme kinetic parameters and enzyme latency allowed us to discount substrate binding or substrate transport defects as the cause of ameliorated UGT activity in the mutants.

Expression and distribution of surfactant proteins and lysozyme after prolonged hyperpnea.

We have induced prolonged hyperpnea in rats and examined the distribution of surfactant-associated proteins (SP-A and SP-B) and lysozyme in lamellar bodies (lb) and two alveolar fractions, one tubular myelin rich (alv-1) and the other tubular myelin poor (alv-2). We have also examined the expression of SP-A, SP-B, SP-C, and lysozyme mRNA in lung tissue and alveolar type II cells. Hyperpnea resulted in significant increases in lb SP-A, lysozyme, and phospholipid (PL) but no change in the protein-to-PH ratios, suggesting that lb stoichiometry is constant. The SP-A and SP-B-to-PL ratios were 33 and 18 times greater, respectively, in control alv-1 than in lb, suggesting that alv-1 is enriched with these proteins. In contrast, the lysozyme-to-PL ratio was similar in control alv-1 and lb. Hyperpnea did not alter the alv-1 SP-A or SP-B-to-PL ratios, suggesting some constant stoichiometry to their lipid association; however, the lysozyme-to-PL ratio was reduced. Whereas hyperpnea significantly elevated the PL, SP-A, and lysozyme levels in alv-2, the SP-B level was unchanged. We suggest that surfactant-associated lysozyme is secreted with lb, the majority of SP-A is linked to lipid secretion but not necessarily with lb, and the majority of SP-B secretion is independent of PL secretion. Hyperpnea did not alter the mRNA expression of SP-A, SP-B, SP-C, or lysozyme in alveolar type II cells, but expression of SP-A and SP-B mRNA was significantly increased in lung tissue.