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.     

Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells.

Previously isolated lysosomal alpha-glucosidase cDNA clones were ligated to full-length constructs for expression in vitro and in mammalian cells. One of these constructs (pSHAG1) did not code for functional enzyme, due to an arginine residue instead of a tryptophan residue at amino acid position 402. The mutation does not affect the rate of enzyme synthesis, but interferes with post-translational modification and intracellular transport of the acid alpha-glucosidase precursor. Using immunocytochemistry it is demonstrated that the mutant precursor traverses the endoplasmic reticulum and the Golgi complex, but does not reach the lysosomes. Pulse-chase experiments suggest premature degradation. The Trp-402-containing enzyme (encoded by construct pSHAG2) is processed properly, and has catalytic activity. A fraction of the enzyme is localized at the plasma membrane. It is hypothesized that membrane association of the acid alpha-glucosidase precursor, as demonstrated by Triton X-114 phase separation, is responsible for transport to this location. Transiently expressed acid alpha-glucosidase also enters the secretory pathway, since a catalytically active precursor is found in the culture medium. This precursor has the appropriate characteristics for use in enzyme replacement therapy. Efficient uptake via the mannose 6-phosphate receptor results in degradation of lysosomal glycogen in cultured fibroblasts and muscle cells from patients with 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.     

Glycogenosis type II: protein and DNA analysis in five South African families from various ethnic origins.

The molecular nature of lysosomal alpha-glucosidase deficiency was studied in five South African families with glycogenosis type II. Distinct ethnic origins were represented. Two new mutant acid alpha-glucosidase alleles were discovered. In two infantile patients from a consanguineous Indian family we found for the first time an acid alpha-glucosidase precursor of reduced size. The mutant precursor appeared normally glycosylated and phosphorylated but was not processed to mature enzyme. Abnormalities of the mRNA were not obvious, but digestion of genomic DNA with HindIII, BglII, and StuI revealed for each enzyme a fragment of increased length. Heterozygosity was demonstrated in the parents. Complete lack of acid alpha-glucosidase mRNA, as well as deficiency of precursor synthesis, was observed in two black baby girls from unrelated families. In these cases the length of all restriction-enzyme fragments was normal. Reduced enzyme synthesis but normal processing was registered in juvenile and young adult Cape colored patients. The extensive heterogeneity of glycogenosis type II is emphasized in these studies on various ethnic groups. The newly discovered mutants are valuable for the understanding of clinical diversity as a result of allelic variation.     

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.     

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.     

Human alpha-L-fucosidase: complete coding sequence from cDNA clones

The human lysosomal storage disorder fucosidosis results from the deficiency of alpha-L-fucosidase, a lysosomal enzyme essential for the catabolism of oligosaccharides containing alpha-L-fucosides. cDNA clones coding for human alpha-L-fucosidase have been isolated from lambda gt10 and lambda gt11 cDNA libraries derived from human liver, placenta and colon. Compilation of cDNA sequences results in a nucleotide sequence of 2053 base pairs encoding alpha-L-fucosidase. The sequence contains an open reading frame of 461 amino acids beginning with the first in-frame methionine and includes 439 amino acids which comprise the mature protein in addition to a hydrophobic signal peptide sequence of 22 amino acids.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

Biochemical, immunological, and cell genetic studies in glycogenosis type II.

Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cultured fibroblasts from patients were determined. Residual enzyme activities varying from 7%-22% were detected in fibroblasts from patients with the adult form but not from patients with the infantile form of glycogenosis II. An inverse correlation was found between the severity of the clinical manifestation and the degree of residual enzyme activity in the fibroblasts. The kinetic and electrophoretic properties of acid alpha-glucosidase in fibroblasts from the adult patients and from control individuals were similar. Immunological studies suggested that the decrease of acid alpha-glucosidase activity is caused by a mutation that affects the production or degradation of the enzyme rather than its catalytic activity. Complementation studies were carried out by fusing fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis II, but neither conventional assays on multikaryons nor enzyme assays on single binuclear heterokaryons gave any evidence for genetic heterogeneity among these forms.     

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

Structural requirements for efficient processing and activation of recombinant human UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an alpha2beta2gamma2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the alpha- and beta-subunits are encoded by a single gene as a precursor, whereas the gamma-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the alpha- and beta-subunits but not the gamma-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the alpha- and beta-subunits. Because this modification prevented precursor processing to mature alpha- and beta-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified alpha/beta-subunits (alpha'/beta'-subunits) precursor and wild type gamma-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an alpha'2beta'2gamma2-subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAc-phosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid alpha-glucosidase in vitro.     

Isolation and characterization of three alpha-glucosidases from the Japanese quail

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

Lysosomal glycogen storage mimicking the cytological picture of Pompe’s disease as induced in rats by injection of an α-glucosidase inhibitor

The present paper describes an animal model of lysosomal glycogenosis as induced by a competitive inhibitor of alpha-glucosidase. Rats received intraperitoneal injections of the inhibitor, a pseudotetrasaccharide (Acarbose, Bay g 5421); liver tissue was examined by light and electron microscopy. Substrate-histochemical and enzyme-cytochemical methods were used to demonstrate intralysosomal glycogen storage within hepatocytes and Kupffer cells. The cytological picture closely resembled that occurring in glycogenosis type II (Pompe's disease) of humans. After cessation of drug treatment, the glycogen storage was slowly reversible. The present results point to the physiological role of the lysosomal apparatus for intracellular glycogen turnover. On the cellular level, this experimentally induced glycogenosis may be useful as a model of Pompe's disease.     

Novel alpha-glucosidase from Aspergillus nidulans with strong transglycosylation activity

Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.     

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.