Nonsense-mediated decay of human HEXA mRNA.

Nonsense-mediated mRNA decay (NMD), the loss of mRNAs carrying premature stop codons, is a process by which cells recognize and degrade nonsense mRNAs to prevent possibly toxic effects of truncated peptides. Most mammalian nonsense mRNAs are degraded while associated with the nucleus, but a few are degraded in the cytoplasm; at either site, there is a requirement for translation and for an intron downstream of the early stop codon. We have examined the NMD of a mutant HEXA message in lymphoblasts derived from a Tay-Sachs disease patient homozygous for the common frameshift mutation 1278ins4. The mutant mRNA was nearly undetectable in these cells and increased to approximately 40% of normal in the presence of the translation inhibitor cycloheximide. The stabilized transcript was found in the cytoplasm in association with polysomes. Within 5 h of cycloheximide removal, the polysome-associated nonsense message was completely degraded, while the normal message was stable. The increased lability of the polysome-associated mutant HEXA mRNA shows that NMD of this endogenous mRNA occurred in the cytoplasm. Transfection of Chinese hamster ovary cells showed that expression of an intronless HEXA minigene harboring the frameshift mutation or a closely located nonsense codon resulted in half the normal mRNA level. Inclusion of multiple downstream introns decreased the abundance further, to about 20% of normal. Thus, in contrast to other systems, introns are not absolutely required for NMD of HEXA mRNA, although they enhance the low-HEXA-mRNA phenotype.

Enzyme-replacement therapy in mucopolysaccharidosis I.

BACKGROUND: Mucopolysaccharidosis I is a lysosomal storage disease caused by a deficiency of the enzyme alpha-L-iduronidase. We evaluated the effect of enzyme-replacement therapy with recombinant human alpha-L-iduronidase in patients with this disorder. METHODS: We treated 10 patients with mucopolysaccharidosis I (age, 5 to 22 years) with recombinant human alpha-L-iduronidase at a dose of 125,000 U per kilogram of body weight given intravenously once weekly for 52 weeks. The patients were evaluated at base line and at 6, 12, 26, and 52 weeks by detailed clinical examinations, magnetic resonance imaging of the abdomen and brain, echocardiography, range-of-motion measurements, polysomnography, clinical laboratory evaluations, measurements of leukocyte alpha-L-iduronidase activity, and urinary glycosaminoglycan excretion. RESULTS: Hepatosplenomegaly decreased significantly in all patients, and the size of the liver was normal for body weight and age in eight patients by 26 weeks. The rate of growth in height and weight increased by a mean of 85 and 131 percent, respectively, in the six prepubertal patients. The mean maximal range of motion of shoulder flexion and elbow extension increased significantly. The number of episodes of apnea and hypopnea during sleep decreased 61 percent. New York Heart Association functional class improved by one or two classes in all patients. Urinary glycosaminoglycan excretion decreased after 3 to 4 weeks of treatment; the mean reduction was 63 percent of base-line values. Five patients had transient urticaria during infusions. Serum antibodies to alpha-L-iduronidase were detected in four patients. CONCLUSIONS: In patients with mucopolysaccharidosis I, treatment with recombinant human alpha-L-iduronidase reduces lysosomal storage in the liver and ameliorates some clinical manifestations of the disease.

Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells.

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.

alpha-L-iduronidase forms semi-crystalline spherulites with amyloid-like properties.

While seeking conditions for single crystals of human alpha-L-iduronidase, solutions were discovered (pH 3.0-8.5 containing calcium or zinc salts) that transform soluble alpha-L-iduronidase to a solid aggregate. This aggregate is a spherulite of semi-crystalline protein. The X-ray diffraction pattern and ability to bind Congo red characterize the alpha-L-iduronidase spherulite as 'amyloid-like', in that it displays two of the characteristics of amyloidogenic proteins. In addition, alpha-L-iduronidase also interacts with heparin, as do some amyloid-forming proteins.

Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B.

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha-N-acetylglucosaminidase.

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding alpha-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu-/- mice were healthy and fertile while young and could survive for 8-12 mo. They were totally deficient in alpha-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G(M2) and G(M3) in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu-/- mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the alpha-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

NAGLU mutations underlying Sanfilippo syndrome type B.

Sanfilippo syndrome type B (mucopolysaccharidosis III B) is a rare autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase, one of the enzymes required for the lysosomal degradation of heparan sulfate. The gene for this enzyme, NAGLU, recently was isolated, and several mutations were characterized. We have identified, in amplified exons from nine fibroblast cell lines derived from Sanfilippo syndrome type B patients, 10 additional mutations: Y92H, P115S, Y140C, E153K, R203X, 650insC, 901delAA, P358L, A664V, and L682R. Four of these mutations were found in homozygosity, and only two were seen in more than one cell line. Thus, Sanfilippo syndrome type B shows extensive molecular heterogeneity. Stable transfection of Chinese hamster ovary cells, by cDNA mutagenized to correspond to the NAGLU missense mutations, did not yield active enzyme, demonstrating the deleterious nature of the mutations. Nine of the 10 amino acid substitutions identified to date are clustered near the amino or the carboxyl end of alpha-N-acetylglucosaminidase, suggesting a role for these regions in the transport or function of the enzyme.

Carbohydrate structures of recombinant human alpha-L-iduronidase secreted by Chinese hamster ovary cells.

alpha-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human alpha-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant alpha-L-iduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-beta-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-beta-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.

Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysaccharidosis I.

Enzyme replacement is a potential therapy for mucopolysaccharidosis I (MPS I), a lysosomal storage disorder caused by alpha-L-iduronidase deficiency. Previous work showed improvement in the tissues of MPS I dogs treated intravenously for 3 months with recombinant human alpha-L-iduronidase (25,000 units or approximately 0.1 mg/kg/week). We have now treated an MPS I-affected dog for 13 months to assess the clinical effects of enzyme replacement. The treated dog gained more weight, was more active, and had less joint stiffness than the untreated littermate. Biochemical and histologic studies demonstrated uptake of alpha-L-iduronidase and decreased lysosomal storage in the liver, kidney, spleen, lymph nodes, synovium, adrenals, and lungs. The brain had detectable enzyme activity and decreased glycosaminoglycan storage although histologic improvement was not evident. Cartilage and heart valve did not show any detectable improvement. A fivefold higher dose (approximately 0.5 mg/kg) administered five times over 10 days to two other dogs resulted in higher tissue enzyme activity and similarly decreased glycosaminoglycan storage and excretion. Antibodies to human alpha-L-iduronidase were induced in all treated dogs and may be associated with immune complex deposition and proteinuria. Recombinant canine alpha-L-iduronidase also induced antibody formation to a similar degree. The results support the conclusion that enzyme replacement is a promising therapy for MPS I though immunologic complications may occur.

The molecular basis of Sanfilippo syndrome type B.

The Sanfilippo syndrome type B is a lysosomal storage disorder caused by deficiency of alpha-N-acetylglucosaminidase; it is characterized by profound mental deterioration in childhood and death in the second decade. For understanding the molecular genetics of the disease and for future development of DNA-based therapy, we have cloned the cDNA and gene encoding alpha-N-acetylglucosaminidase. Cloning started with purification of the bovine enzyme and use of a conserved oligonucleotide sequence to probe a human cDNA library. The cDNA sequence was found to encode a protein of 743 amino acids, with a 20- to 23-aa signal peptide immediately preceding the amino terminus of the tissue enzyme and with six potential N-glycosylation sites. The 8.5-kb gene (NAGLU), interrupted by 5 introns, was localized to the 5'-flanking sequence of a known gene, EDH17B, on chromosome 17q21. Five mutations were identified in cells of patients with Sanfilippo syndrome type B: 503del10, R297X, R626X, R643H, and R674H. The occurrence of a frameshift and a nonsense mutation in homozygous form confirms the identity of the NAGLU gene.

Four novel mutations underlying mild or intermediate forms of alpha-L-iduronidase deficiency (MPS IS and MPS IH/S).

The alpha-L-iduronidase deficiency diseases (Mucopolysaccharidosis I) cover a spectrum of clinical severity ranging from the very severe (Hurler syndrome, MPS IH) through an intermediate (Hurler/Scheie syndrome, MPS IH/S) to a relatively mild form (Scheie syndrome, MPS IS). Numerous mutations of the gene encoding alpha-L-iduronidase (IDUA) are known in Hurler syndrome, but only three in the other disorders. We report on novel mutations of the IDUA gene in one patient with the Scheie syndrome and in three patients with the Hurler/Scheie syndrome. The novel mutations, all single base changes, encoded the substitutions R492P (Scheie), and X654G, P496L, and L490P (Hurler/Scheie). The L490P mutation was apparently homozygous, whereas each of the others was found in compound heterozygosity with a Hurler mutation. The deleterious nature of the mutations was confirmed by absence of enzyme activity upon transfection of the corresponding mutagenized cDNAs into Cos-1 cells. These results provide additional information for genotype-phenotype correlations.

Enzyme replacement in a canine model of Hurler syndrome.

The Hurler syndrome (alpha-L-iduronidase deficiency disease) is a severe lysosomal storage disorder that is potentially amenable to enzyme-replacement therapy. Availability of a canine model of the disease and a sufficient supply of corrective enzyme have permitted a therapeutic trial lasting 3 mo. Recombinant human alpha-L-iduronidase, purified to apparent homogeneity from secretions of a stably transfected Chinese hamster ovary cell line, was administered i.v. to homozygous affected animals in doses of approximately 1 mg. The enzyme rapidly disappeared from the circulation in a biphasic manner, with t1/2 of 0.9 and 19 min, respectively, and was taken up primarily by the liver. Biopsy of the liver before and after a very short trial (seven doses administered over 12 days) showed remarkable resolution of lysosomal storage in both hepatocytes and Kupffer cells. After weekly administration of enzyme to three affected animals over a period of 3 mo, the level of enzyme was about normal in liver and spleen, lower but significant in kidney and lung, and barely detectable (0-5% of normal) in brain, heart valves, myocardium, cartilage, and cornea. Light and electron microscopic examination of numerous tissues showed normalization of lysosomal storage in liver, spleen, and kidney glomeruli, but there was no improvement in brain, heart valves, or cornea. Even though the treated dogs developed complement-activating antibodies against alpha-L-iduronidase, clinical symptoms could be prevented by slow infusion of enzyme and premedication.

Evidence for degradation of mRNA encoding alpha-L-iduronidase in Hurler fibroblasts with premature termination alleles.

Mutations in the gene encoding alpha-L-iduronidase (IDUA) are the cause of Hurler syndrome. Fibroblasts from patients homozygous for nonsense IDUA alleles have much reduced mRNA detectable by Northern analysis, as has been observed in many other instances of premature translation termination. Yet RT-PCR (reverse transcription followed by PCR amplification) showed a normal level of a segment covering exons 1 and 2 in Hurler cells homozygous for alleles bearing the nonsense mutations, Q70X or W402X. The 3' end of the segment was between exons 2 and 4. The results indicate that the nonsense RNA was degraded to fragment(s), independent of the position of the mutation (exon 2 or exon 9, respectively). Treatment of the cells with cycloheximide resulted in some increase of intact mRNA, suggesting that translation is required for mRNA degradation.

Overexpression of the human lysosomal enzyme alpha-L-iduronidase in Chinese hamster ovary cells.

We developed a Chinese hamster ovary (CHO) cell line that produces and secretes large quantities of recombinant human alpha-L-iduronidase, the lysosomal hydrolase deficient in mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes). The alpha-L-iduronidase cDNA was introduced into a vector containing the cytomegalovirus immediate early gene promoter/enhancer, a murine immunoglobulin C alpha region intron, and the bovine growth hormone polyadenylation signal. Following cotransfection with a plasmid containing the neomycin resistance gene, stably transfected lines were selected with G-418. The highest expressing CHO cell line contained 1400-6000 units of alpha-L-iduronidase per milligram of protein, or 0.6-2.4% of total cell protein. Secreted alpha-L-iduronidase was 3000- to 7000 fold increased, with about 5000 units accumulating in 24 h per 10(7) cells. The activity and distribution of five other lysosomal glycosidases were not significantly affected. Metabolic labeling showed that half of the newly synthesized alpha-L-iduronidase was secreted, but generally less was recovered due to its instability in the medium. It was post-translationally processed as previously shown for alpha-L-iduronidase of human fibroblasts. Recombinant alpha-L-iduronidase was efficiently endocytosed by Hurler fibroblasts utilizing a mannose 6-phosphate-dependent mechanism (half maximal uptake at 0.7 nM) and was "corrective" for abnormal glycosaminoglycan accumulation (half-maximal correction at 0.7 pM). The half-life of the recombinant enzyme was 5 days following uptake into Hurler fibroblasts. Production in a 5-liter microcarrier culture system permitted the collection of 15 mg or more per day.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular analysis of Hurler syndrome in Druze and Muslim Arab patients in Israel: multiple allelic mutations of the IDUA gene in a small geographic area.

The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families, five of them Druze, residing in a very small area of northern Israel. Since such clustering suggests a classic founder effect, the presence of three mutant alleles of the IDUA gene was unexpected.

Architecture of the canine IDUA gene and mutation underlying canine mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease caused by deficiency of alpha-L-iduronidase. In addition to the well-known human forms (Hurler, Hurler/Scheie, and Scheie syndromes), there exists a canine model of the disease. By using previously described canine cDNA encoding alpha-L-iduronidase as a probe, the canine IDUA gene has been cloned and characterized. It contains 14 exons spread over 13 kb. An unusual GC dinucleotide was found at the donor splice site of intron 11. A transcriptional start site was identified by primer extension 177 bp upstream of the initiator AUG codon. The upstream region was found to be similar to the promoter region of many housekeeping genes: it is GC rich and has seven potential Sp1 binding sites but no TATA box or CAAT motif. The mutation in canine MPS I was localized to the area of intron 1 by RT-PCR, identified by sequence analysis of amplified genomic DNA, and confirmed by restriction analysis; it is a G-->A transition in the donor splice site of intron 1. The mutation causes retention of intron 1 in the RNA and creates a premature termination codon at the exon-intron junction.