Identification of a point mutation in the human lysosomal alpha-glucosidase gene causing infantile glycogenosis type II

Two patients in a consanguineous Indian family with infantile glycogenosis type II were found to have a G to A transition in exon 11 of the human lysosomal alpha-glucosidase gene. Both patients were homozygous and both parents were heterozygous for the mutant allele. The mutation causes a Glu to Lys substitution at amino acid position 521, just three amino acids downstream from the catalytic site at Asp-518. The mutation was introduced in wild type lysosomal alpha-glucosidase cDNA and the mutant construct was expressed in vitro and in vivo. The Glu to Lys substitution is proven to account for the abnormal physical properties of the patients lysosomal alpha-glucosidase precursor and to prevent the formation of catalytically active enzyme. In homozygous form it leads to the severe infantile phenotype of glycogenosis type II.     

Defects in synthesis, phosphorylation, and maturation of acid alpha-glucosidase in glycogenosis type II

Glycogenosis type II is an inherited lysosomal storage disease with acid alpha-glucosidase deficiency as the primary defect. Using cultured skin fibroblasts, we have studied the biosynthesis of acid alpha-glucosidase in clinically different forms of this disease. Three unrelated patients were identified (one with an infantile, one with a juvenile, and one with an adult form of the disease) producing normal quantities of the 110-kDa precursor form of acid alpha-glucosidase. However, post-translational modification to mature 76-kDa enzyme protein was either completely deficient or extremely inefficient. No abnormalities were observed in glycosylation of the mutant precursors, as measured by the incorporation of [3H]mannose, but phosphorylation was only detectable for the precursor synthesized by fibroblasts from the juvenile patient. In three other patients (one with a juvenile and two with adult forms of glycogenosis type II) apparently reduced synthesis of precursor protein was observed, but the processing to mature enzyme seemed to be undisturbed. Finally, neither precursor nor mature forms of acid alpha-glucosidase were detectable in one particular case of infantile glycogenosis type II. The studies reveal an unexpected degree of genetic heterogeneity in this disease and identify various mutants which could be of importance to further elucidate the biosynthetic events during lysosomal enzyme formation.     

Bovine generalised glycogenosis type II. Uptake of lysosomal alpha-glucosidase by cultured skeletal muscle and reversal of glycogen accumulation

acid alpha-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid alpha-glucosidase activity was internalised with an uptake constant of 300 nM and a Vmax of uptake of 133 nmol/h per mg protein. The glycogen concentration in affected cultures treated with exogenous acid alpha-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals.     

Cell-free translation of messenger RNA coding for a precursor of human calcitonin

Polyadenylated RNA, extracted from a human medullary thyroid carcinoma, was translated in cell-free systems prepared from wheat germ and reticulocyte lysates. The major product of the translations was a protein of 15,000 M_R which was immunoprecipitated specifically with an antiserum to synthetic human calcitonin. Addition to the translation reactions of microsomal membranes, prepared from canine pancreas, resulted in the partial disappearance of the 15,000 M_R polypeptide and the concomitant appearance of a smaller peptide (11,000 M_R), also immunoprecipitated specifically by antisera to calcitonin. These results indicate that human calcitonin is synthesized in the form of a precursor of 15,000 M_R and suggest that the precursor contains a leader sequence that is cleaved from the polypeptide by enzymes associated with microsomal membranes.     

Adult forms of glycogenosis type II. A defect in an early stage of acid alpha-glucosidase realization

The activity of acid alpha-glucosidase in cultured fibroblasts from adult patients with the lysosomal storage disease glycogenosis type II is only 10% of normal. A normal activity per molecule is found for the mature as well as for the precursor form of acid alpha-glucosidase in adult mutant fibroblasts. Excessive lysosomal breakdown of mature enzyme purified from mutant fibroblasts and taken up by acceptor cells does not occur. However, the NH4Cl-stimulated secretion of a precursor form of acid alpha-glucosidase by adult mutant fibroblasts is markedly reduced. The results are indicative of a defect during the production of acid alpha-glucosidase.     

Biosynthesis of acid alpha-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease)

Cultured human skin fibroblasts from control persons and from patients with the generalized and late-onset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid alpha-glucosidase and cathepsin D was analysed by immunoprecipitation, gel electrophoresis and fluorography. When the labelling was carried out for 6-12 h in the presence of NH4Cl, the labelling of secreted alpha-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the late-onset form of Pompe's disease was less than 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for less than 1 h, the relative rate of incorporation of radioactivity into acid alpha-glucosidase was rather similar in the two types of fibroblasts. In fibroblasts from patients with the generalized form of Pompe's disease no incorporation of radioactivity into acid alpha-glucosidase could be detected.     

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.     

Identification of a missense mutation in an adult-onset patient with glycogenosis type II expressing only one allele.

The lysosomal enzyme acid alpha glucosidase (GAA) or acid maltase is deficient in glycogen storage disease type II. We sought to determine the molecular basis for the disease in an adult-onset patient, unusual for very low enzyme activity similar to that seen with the infantile-onset form and with a previously reported defect in phosphorylation. We constructed cDNA and genomic DNA libraries from the patient's cell line (GM 1935) and determined the nucleotide sequence of the coding region. There were three base-pair substitutions in one allele (C1935 to A; G2446 to A and C2780 to T), all predicting amino acid changes (Asp-645 to Glu; Val-816 to Ile and Thr-927 to Ile). To determine which of the three base-pair substitutions resulted in loss of enzyme activity, we next utilized primer-directed mutagenesis and transient gene expression in an SV40-immortalized GAA-deficient fibroblast cell line. Only the construct containing the G2446 to A mutation (Val-816 to Ile) lost GAA enzyme activity, while the other two substitutions (including the Thr-927 to Ile change that predicts a loss of a potential site for N-linked glycosylation and mannose phosphorylation) each resulted in enzyme activity equal to the control. Analysis of RFLPs in genomic DNA, as well as sequence analysis for the three base-pair alterations, indicated that the patient was a genetic compound. We next digested PCR-amplified cDNA (reverse-transcribed from RNA) with Aat II to detect the base-pair 1935 substitution and found that virtually all of the mRNA was derived from the allele with the three base-pair substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody to human lysosomal alpha-glucosidase in immunocytochemistry: unexpected reactivity with cytoskeletal structures

Summary A well-characterized monoclonal antibody against human lysosomal -glucosidase has been used for the immunohistochemical localization of the enzyme in cultured human skin fibroblasts. Under conditions that are routinely used for the preparation of cells for immunocytochemistry, this monoclonal antibody does not react with acid -glucosidase but in contrast with components of the cytoskeleton. Double-labelling experiments with the monoclonal antibody and rabbit anti-vimentin antiserum identified the cytoskeletal components as intermediate filaments. The implications of this observation for the use of monoclonal antibodies in immunocytochemistry in general are discussed.     

Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.     

Bovine generalised glycogenosis type II: Uptake of lysosomal α-glucosidase by cultured skeletal muscle and reversal of glycogen accumulation

Acid α-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid α-glucosidase activity was internalised with an uptake constant of 300 nM and a V_max of uptake of 133 per mg protein. The glycogen concentration in affected cultures treated with exogenous acid α-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals.Glycogenosis type IIa-GlucosidaseEnzyme uptakePompe's diseaseCultured muscle     

Translational control of immunoglobulin synthesis: II. Cell-free interaction of myeloma immunoglobulin with mRNA

Repression of heavy chain synthesis by myeloma protein has been demonstrated (Stevens & Williamson, 1973) and shown to result from an interaction of the myeloma protein molecule with the H-chain² mRNA.Oocytes from Xenopus laevis, when injected with polyribosomes from myeloma cells and increasing amounts of H₂L₂ protein, were shown to produce decreasing amounts of H-chain protein in the presence of increasing amounts of H₂L₂. This repression of H-chain synthesis was shown to result from the interaction of H₂L₂ with the added mRNA and not with mRNA-associated proteins. Under proper conditions of temperature and salt concentration, a population of mRNA molecules specifically and reversibly binds to H₂L₂ protein. This RNA was shown to contain sequences coding for H-chain protein when assayed by injection into oocytes.     

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

EPR evidence for an acceptor functioning in photosystem II when the pheophytin acceptor is reduced

Photosystem II particles have been poised at redox potentials where the pheophytin acceptor is reduced. Illumination of these particles at 200K results in the formation of radical signal in the g?2.00 region. This is attributed to the photoreduction of another acceptor. This acceptor may function between the primary donor, P₆₈₀, and pheophytin in forward electron transfer.     

Direct evidence for expression of type II flavoprotein subunit in human complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in aerobic respiration and the tricarboxylic acid cycle. We recently identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual and demonstrated mRNAs of these two isoforms, Type I Fp and Type II Fp, in skeletal muscle, liver, brain, heart, and kidney. Type I Fp was expressed at higher levels than Type II Fp in all cases. In the present study, the biochemical properties of Type II Fp-containing complex II in Raji cells predominantly expressing Type II Fp were investigated. Complex II having Type II Fp was separated from that having Type I Fp by isoelectric focusing in the presence of sucrose monolaurate. Together with the fact that succinate-ubiquinone reductase activity of mitochondria prepared from Raji cell was almost identical to that from human liver, these results clearly indicate the presence of two distinct isoforms of active complex II in human mitochondria.     

Synthesis and intracellular localization of chick acid alpha-glucosidase in chick erythrocyte-human fibroblast heterokaryons. A model system for the study of lysosomal enzyme synthesis

The synthesis and localization of chick acid alpha-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid alpha-glucosidase were used. It was found that the acid alpha-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.     

Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.