Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion.

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria.

Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Southern hybridization analysis of the alpha-galactosidase gene in affected hemizygous males from 130 unrelated families with Fabry disease revealed six with different gene rearrangements and one with an exonic point mutation resulting in the obliteration of an Msp I restriction site. Five partial gene deletions were detected ranging in size from 0.4 to greater than 5.5 kb. Four of these deletions had breakpoints in intron 2, a region in the gene containing multiple Alu repeat sequences. A sixth genomic rearrangement was identified in which a region of about 8 kb, containing exons 2 through 6, was duplicated by a homologous, but unequal crossover event. The Msp I site obliteration, which mapped to exon 7, was detected in an affected hemizygote who had residual enzyme activity. Genomic amplification by the polymerase chain reaction and sequencing revealed that the obliteration resulted from a C to T transition at nucleotide 1066 in the coding sequence. This point mutation, the first identified in Fabry disease, resulted in an arginine356 to tryptophan356 substitution which altered the enzyme's kinetic and stability properties. The detection of these abnormalities provided for the precise identification of Fabry heterozygotes, thereby permitting molecular pedigree analysis in these families which revealed paternity exclusions and the first documented new mutations in this disease.

Late-onset X-linked sideroblastic anemia. Missense mutations in the erythroid delta-aminolevulinate synthase (ALAS2) gene in two pyridoxine-responsive patients initially diagnosed with acquired refractory anemia and ringed sideroblasts.

X-linked sideroblastic anemia (XLSA) is caused by mutations of the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) resulting in deficient heme synthesis. The characteristic hypochromic, microcytic anemia typically becomes manifest in the first three decades of life. Hematologic response to pyridoxine is variable and rarely complete. We report two unrelated cases of highly pyridoxine-responsive XLSA in geriatric patients previously diagnosed with refractory anemia and ringed sideroblasts. A previously unaffected 77-yr-old male and an 81-yr-old female were each found to have developed severe hypochromic, microcytic anemia with ringed sideroblasts in the bone marrow, which responded dramatically to pyridoxine with normalization of hemoglobin values. Sequence analysis identified an A to C transversion in exon 7 (K299Q) of the ALAS2 gene in the male proband and his daughter. In the female proband a G to A transition was identified in exon 5 (A172T). This mutation resulted in decreased in vitro stability of bone marrow delta-aminolevulinate synthase activity. Each patient's recombinant mutant ALAS2 enzyme had marked thermolability. Addition of pyridoxal 5'-phosphate in vitro stabilized the mutant enzymes, consistent with the observed dramatic response to pyridoxine in vivo. This late-onset form of XLSA can be distinguished from refractory anemia and ringed sideroblasts by microcytosis, pyridoxine-responsiveness, and ALAS2 mutations. These findings emphasize the need to consider all elderly patients with microcytic sideroblastic anemia as candidates for XLSA, especially if pyridoxine responsiveness is demonstrated.

Plasma alpha-galactosidase A:properties and comparisons with tissue alpha-galactosidases.

The human plasma form of alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was highly purified and exhibited apparent Km values of 1.9 mM with 4-methylumbelliferyl-alpha-D-galactopyranoside and 0.23 mM with globotriglycosylceramide. Its inhibition with myo-inositol (Ki = 0.29 M) was similar to that observed with alpha-galactosidase A from various tissues. The plasma form of this lysosomal enzyme has a lower molecular weight of 96 600, a lower pI of 3.7 and faster electrophoretic mobility in polyacrylamide gels than the enzyme obtained from human liver. These data and the increased pI obtained after neuraminidase treatment suggest that the plasma form is an isoenzyme with a more highly sialylated carbohydrate moiety than the tissue isoenzymes.

Pilot scale purification of alpha-galactosidase A from Cohn fraction IV-1 of human plasma.

Human plasma alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified 7000-fold over plasma levels from Cohn Fraction IV-1. The yield per kg starting material averaged 11 000 units (nmol galactose liberated per h) and the specific activity was about 600 units per mg protein with 4-methylumbelliferyl-alpha-D-galactoside. The ratio of 4-methylumbelliferyl-alpha-galactosidase to ceramide trihexosidase activities was 6.2. Both activities were heat labile and exhibited the same relative mobilities on polyacrylamide gel electrophoresis. Enzymatic activity was stable for at least 4 months at 4 and -20 degrees C. The endotoxin concentration of this preparation averaged 0.26 mg per mg protein.

Molecular cloning of a cDNA for human delta-aminolevulinate dehydratase.

A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library. Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen. Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences. Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert. This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension. The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues.

delta-Aminolevulinic acid dehydratase isozymes and lead toxicity.

ALAD is a zinc metalloenzyme whose inhibition by lead is the first and most sensitive indicator of lead exposure and whose decreased activity has been implicated in the pathogenesis of lead poisoning. This heme biosynthetic enzyme is encoded by a gene located at chromosome 9q34, which has two codominant alleles, ALAD1 and ALAD2. The occurrence of two frequent alleles for ALAD stimulated an investigation into the possible pharmacogenetic role of the enzyme polymorphism in lead poisoning. In a New York City population at high risk for lead exposure, individuals heterozygous or homozygous for the less common allele, ALAD2, had blood lead levels greater than or equal to 30 micrograms/dl more frequently than expected. These findings suggest a potential genetic susceptibility to lead poisoning in individuals with the ALAD 1-2 and 2-2 phenotypes.

[Partial deletion of alpha-galactosidase A gene in a Japanese mutant of Fabry disease].

We identified a structural defect of alpha-galactosidase A (alpha-Gal A) gene in a Japanese patient with Fabry disease. A partial deletion approximately 0.4 kilobase-pairs in size was delineated by restriction endonuclease mapping; whole exon 3 sequence was removed. alpha-Gal A mRNA was deficient in the mRNA preparation from the lymphoblastoid cells derived from the patient, and a faulty transcription resulting in an unstable alpha-Gal A message was suggested in this case. Molecular pedigree analysis was successfully performed in identifying heterozygotes and the ancestry of the mutant allele in this family.

The molecular biology and pyridoxine responsiveness of X-linked sideroblastic anaemia.

Pyridoxine-responsive, X-linked sideroblastic anaemia (XLSA) has been shown to be caused by missense mutations in the erythroid-specific ALA synthase gene, ALAS2. These are scattered widely across the part of the gene encoding the catalytic domain and in half the cases affect residues conserved throughout evolution. Only a loose correlation has been found between the in vitro kinetics and stability of the catalytic activity of the recombinant variant enzymes and the in vivo severity and pyridoxine-responsiveness of the anaemia. Enhanced instability in the absence of pyridoxal phosphate (PLP) or decreased PLP and substrate binding have been noted. A detailed explanation of the anaemia and its response to pyridoxine, however, requires greater insight into the structure-function relationships of this protein than we have at present. Knowledge of its tertiary structure and further knowledge of intracellular factors which impinge on the ability of normal and variant ALAS2 to contribute to haemoglobin production are also required. Mutations in the same gene which affect mitochondrial processing, terminate translation prematurely, or are thought to abolish function altogether cause an XLSA that is refractory to treatment with pyridoxine. A major complication of this disorder is its accompanying increased iron absorption and iron overload which occurs in patients and female heterozygotes. Mutation detection enables the early diagnosis of those affected, targeted education of families, early treatment with pyridoxine and prevention of iron overload. It also allows for a distinction to be made between late-onset variants of this condition and the more insidious refractory anaemia with ring sideroblasts. The next few years of investigation should be illuminating as tools now exist to study all aspects of this protein from the gene to the mitochondrial matrix.

Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms.

The substrate analog alpha-D-galactosylamine was synthesized, linked to 6-aminohexanoic acid, and coupled to carboxyhexyl-Sepharose. This affinity support permitted the purification of human alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from spleen, placenta, and plasma. When used in conjunction with conventional procedures, affinity chromatography enabled the rapid and specific purification of alpha-galactosidase A from each source. Significantly, pyrogenic endotoxins were eliminated from enzyme preparations by the use of the affinity column. Splenic alpha-galactosidase A was purified in high yield (38%) with a specific activity of 1.9 X 10(6) units/mg. The purified enzyme was a homodimer with a native molecular weight of 101,000 and a subunit weight of 49,800. The UV absorption coefficient was E280 1% = 18 and the lambda max was 282 nm. The plasma form was purified with a markedly improved yield to a specific activity (229,000 units/mg) which was 3 times greater than that achieved previously. The enzymes from plasma, spleen, and placenta were immunologically identical. The physical and kinetic properties of the purified enzymes were consistent with and confirmed previous findings.

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene.

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.

Coupled-enzyme and direct assays for uroporphyrinogen III synthase activity in human erythrocytes and cultured lymphoblasts. Enzymatic diagnosis of heterozygotes and homozygotes with congenital erythropoietic porphyria.

Rapid and reproducible assays for uroporphyrinogen III synthase (URO-S; EC 4.2.1.75) have been developed and used to determine the enzymatic activity in human erythrocytes and cultured lymphoid cells. In the coupled-enzyme assay, porphobilinogen was first converted to hydroxymethylbilane, the natural substrate for URO-S, by hydroxymethylbilane synthase which was conveniently obtained from heat-treated erythrocyte lysates. In the direct assay, synthetic hydroxymethylbilane was used as substrate. In both assays, the uroporphyrinogen reaction products were oxidized to their respective uroporphyrin isomers, which were then resolved and quantitated by reversed-phase high-pressure liquid chromatography. Both assays were optimized for pH, substrate concentration, and linearity with time and protein concentration. The mean URO-S activities in normal human erythrocyte lysates determined by the coupled-enzyme and direct assays were 7.41 +/- 1.35 and 7.64 +/- 1.73 units/mg protein, respectively. In normal human cultured lymphoid cells, the mean activities were 13.7 +/- 1.39 and 17.6 +/- 1.15 units/mg protein for the coupled-enzyme and direct assays, respectively. In four families with congenital erythropoietic porphyria, both assays reliably identified the markedly decreased URO-S activities in erythrocytes and cultured lymphoid cells from affected homozygotes and the half-normal activities in these sources from obligate heterozygotes. The coupled-enzyme assay was easier to perform and was suited for clinical diagnostic assays and for monitoring enzyme purification procedures, while the direct assay, which required substrate preparation and technical dexterity, was best for kinetic studies of URO-S.

Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region.

Human alpha-galactosidase A (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) is a lysosomal hydrolase encoded by a gene localized to the chromosomal region Xq22. The deficient activity of this enzyme results in Fabry disease, an X chromosome-linked recessive disorder that leads to premature death in affected males. For studies of the structure and function of alpha-galactosidase A and for characterization of the genetic lesions in families with Fabry disease, the full-length cDNA was isolated, sequenced, and used to screen human genomic libraries. The 1393-base-pair full-length cDNA had a 60-nucleotide 5' untranslated region and encoded a precursor peptide of 429 amino acids including a signal peptide of 31 residues. Three overlapping lambda clones spanning 32 kilobases were identified that contained the entire approximately equal to 12-kilobase chromosomal gene as well as approximately equal to 9 and approximately equal to 11 kilobases of 5' and 3' flanking sequence, respectively. The gene had seven exons. The genomic exonic and full-length cDNA sequences were identical. All intron-exon splice junctions conformed to the GT/AT consensus sequence. The 5' flanking region of this lysosomal housekeeping gene contained Sp1 and CCAAT box promoter elements as well as sequences corresponding to the activator protein 1 (AP1), octanucleotide ("OCTA"), and "core" enhancer elements. There was an upstream "HTF" island (Hpa II tiny fragments) followed by four direct repeats of the "chorion box" enhancer. The unique lack of a 3' untranslated sequence in the alpha-galactosidase A cDNA was confirmed by sequencing additional cDNA clones and the genomic 3' region.

Human uroporphyrinogen III synthase: molecular cloning, nucleotide sequence, and expression of a full-length cDNA.

Uroporphyrinogen III synthase [URO-synthase; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75], the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10(6) recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The authenticity of this clone was established by colinearity of the predicted amino acid sequence with 81 microsequenced residues from the purified enzyme. In addition, high levels of enzymatic activity and immunoreactive protein were expressed when a blunt-ended 971-base-pair Ava II cDNA fragment containing the entire coding region was inserted into vectors for expression in Escherichia coli. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

Treatment of trichloroethylene (TCE) in a membrane biofilter.

This article reports on the biodegradation of trichloroethylene (TCE) in a hollow-fiber membrane biofilter. Air contaminated with TCE was passed through microporous hollow fibers while an oxygen-free nutrient solution was recirculated through the shell side of the membrane module. The biomass was attached to the outside surface of the microporous hollow fibers by initially supplying toluene in the gas phase that flows through the fibers. While studies on TCE biodegradation were conducted, there was no toluene present in the gas phase. At 20-ppmv inlet concentration of TCE and 36-s gas-phase residence time, based on total internal volume of the hollow fibers, 30% removal efficiency of TCE was attained. At higher air flow rates or lower gas-phase residence times, lower removal efficiencies were observed. During TCE degradation, the pH of the liquid phase on the shell side of the membrane module decreased due to release of chloride ions. A mathematical model was developed to describe the synchronous aerobic/anaerobic biodegradation of TCE.

Human alpha-galactosidase A: high plasma activity expressed by the -30G-->A allele.

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the lysosomal exoglycosidase responsible for the hydrolysis of terminal alpha-galactosyl residues from glycoconjugates and is the defective enzyme causing Fabry disease (McKusick 301500). An unusally elevated level of plasma alpha-Gal A activity (> 2.5 times the normal mean) was detected in two unrelated normal males and the elevated activities were inherited as X-linked traits in their families. Sequencing of the alpha-Gal A coding region, intron/exon boundaries and 5'-flanking region from the proband identified a single mutation, a G-->A transition 30 nt upstream from the initiation of translation codon in exon 1. The -30G-->A mutation occurred in a putative NF kappa B/Ets consensus binding site that was recently shown to inhibit protein binding to the 5'-untranslated region of the gene, providing a possible explanation for its high activity. To further characterize the mutation, the mRNA and protein expressed by this variant allele were studied. Purified plasma and lymphoblast alpha-Gal A activity from individuals with the -30G-->A mutation had normal physical and kinetic properties. In vitro translation of mRNAs from the cloned normal and high plasma activity alleles resulted in similar levels of alpha-Gal A protein, indicating that this mutation did not enhance translation. These findings suggest that the -30G-->A mutation in the 5'-untranslated region of the alpha-Gal A gene enhances transcription, presumably by interfering with the binding of negatively-acting transcription factors which normally decrease alpha-Gal A expression in various cells. Preliminary studies of the frequency of the -30G-->A mutation in 395 unrelated normal males of mixed ancestry revealed two additional unrelated individuals who had high plasma enzymatic activity and the mutation, confirming the effect of this mutation on enzyme expression and suggesting that about 0.5% of normal individuals have high plasma alpha-Gal A activity due to this variant allele.

X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley.

In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family in which the inheritance of the disease was documented through six generations. Almost 40 years later the enzymatic defect in XLSA was identified as the deficient activity of the erythroid-specific form of delta-aminolevulinate synthase (ALAS2), the first enzyme in the heme biosynthetic pathway. To determine the nature of the mutation in the ALAS2 gene causing XLSA in Cooley's original family, genomic DNAs were isolated from two affected hemizygotes, and each ALAS2 exon was PCR amplified and sequenced. A single transversion (A to C) was identified in exon 5. The mutation predicted the substitution of leucine for phenylalanine at residue 165 (F165L) in the first highly conserved domain of the ALAS2 catalytic core shared by all species. No other nucleotide changes were found by sequencing each of the 11 exons, including intron/exon boundaries, 1 kb of 5'-flanking and 350 nucleotides of 3'-flanking sequence. The mutation introduced an Mse I site and restriction analysis of PCR-amplified genomic DNA confirmed the presence of the lesion in the two affected brothers and in three obligate heterozygotes from three generations of this family. Carrier diagnosis of additional family members identified the mutation in one of the proband's sisters. After prokaryotic expression and affinity purification of both mutant and normal ALAS2 fusion proteins, the specific activity of the F165L mutant enzyme was about 26% of normal. The cofactor, pyridoxal 5'-phosphate, activated and/or stabilized the purified mutant recombinant enzyme in vitro, consistent with the pyridoxine-responsive anemia in affected hemizygotes from this family.

Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc, E.C. 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties from glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of alpha-GalNAc is the enzymatic defect in Schindler disease, an inherited neuroaxonal dystrophy. To isolate a full-length cDNA, the enzyme from human lung was purified to homogeneity, 129 non-overlapping amino acids were determined by microsequencing the N terminus and seven tryptic peptides, and four synthetic oligonucleotide mixtures were used to screen a human fibroblast cDNA library. A full-length cDNA, pAGB-3, isolated from a placental lambda gt11 cDNA library, had a 2158-base pair (bp) insert with an open reading frame which predicted an amino acid sequence that was colinear with all 129 microsequenced residues of the purified enzyme. The pAGB-3 insert had a 344-bp 5'-untranslated region, a 1236-bp open reading frame encoding 411 amino acids, a 514-bp 3'-untranslated region, and a 64-bp poly(A) tract. A signal peptide sequence of 17 amino acids as well as six N-glycosylation sites were predicted. The pAGB-3 cDNA was subcloned into the p91023(B) mammalian expression vector and human alpha-GalNAc activity was transiently expressed in COS-1 cells, demonstrating the functional integrity of the full-length cDNA. Northern hybridization analysis of mRNA revealed two transcripts of about 3.6 and 2.2 kilobases (kb), and primer extension studies indicated a cap site at nucleotide -347 for the 2.2-kb transcript. The 3.6-kb cDNA (pAGB-35) was isolated; the 3598-bp pAGB-35 insert was identical to that of the 2.2-kb insert but had additional 5'- and 3'-untranslated sequences including a second downstream polyadenylation signal at nucleotide 3100-3105. Isolation of a genomic clone, gAGB-1, and sequencing the 2048-bp region including pAGB-3 revealed a 1754-bp intron between codons 319 and 320, which also was the site of a 70-bp insertion and a45-bp deletion in other cDNA clones. Notably, the alpha-GalNAc cDNA had remarkable amino acid homology with the human alpha-galactosidase A (alpha-Gal A) cDNA suggesting the evolutionary relatedness of these genes. The alpha-GalNAc cDNA had 46.9-64.7% amino acid identity in sequences (codons 1-319) corresponding to alpha-Gal A exons 1 through 6, while the comparable exon 7 sequence (pAGB-3 codons 320-411) had only 15.8% homology with numerous gaps.(ABSTRACT TRUNCATED AT 400 WORDS)