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

ABSTRACT: 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.

SUBMITTER: Hoefsloot LH

PROVIDER: S-EPMC1149726 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Reporters transiently transfected into mammalian cells are highly sensitive to translational repression induced by dsRNA expression.

Project description:In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells--expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

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Characterization of the human lysosomal alpha-glucosidase gene.

Project description:The gene coding for human lysosomal alpha-glucosidase was cloned and its structure was determined. The gene is approx. 20 kb long, and contains 20 exons. The first exon is non-coding. The coding sequence of the putative catalytic site domain is interrupted in the middle by an intron of 101 bp. This intron is not conserved in the highly similar region of the human and rabbit isomaltase genes. The promoter region was defined by a CAT assay and the start of the mRNA was determined by primer extension. The promoter has features characteristic of a 'housekeeping' gene. The GC content is high (80%) and distinct TATA and CCAAT motifs are lacking. Two potential binding sites for the AP-2 transcription factor are present. Four potential Sp-1 binding sites are located downstream of the 5' end of the mRNA.

| S-EPMC1149727 | biostudies-other

Carnitine is a pharmacological allosteric chaperone of the human lysosomal <i>α</i>-glucosidase.

Project description:Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

| S-EPMC8477953 | biostudies-literature

Human lysosomal alpha-glucosidase: functional characterization of the glycosylation sites.

Project description:N-linked glycosylation is one of the important events in the post-translational modification of human lysosomal alpha-glucosidase. Phosphorylation of mannose residues ensures efficient transport of the enzyme to the lysosomes via the mannose 6-phosphate receptor. The primary structure of lysosomal alpha-glucosidase, as deduced from the cDNA sequence, indicates that there are seven potential glycosylation sites. We have eliminated these sites individually by site-directed mutagenesis and thereby demonstrated that all seven sites are glycosylated. The sites at Asn-882 and Asn-925 were found to be located in a C-terminal propeptide which is cleaved off during maturation. Evidence is presented that at least two of the oligosaccharide side chains of human lysosomal alpha-glucosidase are phosphorylated. Elimination of six of the seven sites does not disturb enzyme synthesis or function. However, removal of the second glycosylation site at Asn-233 interferes dramatically with the formation of mature enzyme. The mutant precursor is synthesized normally and assembles in the endoplasmic reticulum, but immunoelectron microscopy reveals a deficiency of alpha-glucosidase in the Golgi complex and in the more distal compartments of the lysosomal transport pathway.

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Structure of human lysosomal acid α-glucosidase-a guide for the treatment of Pompe disease.

Project description:Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level.

| S-EPMC5653652 | biostudies-literature

Tracking the Evolution of Transiently Transfected Individual Cells in a Microfluidic Platform.

Project description:Transient gene expression (TGE) technology enables the rapid production of large amount of recombinant proteins, without the need of fastidious screening of the producing cells required for stable transfection (ST). However, several barriers must be overcome before reaching the production yields using ST. For optimizing the production yields from suspended cells using TGE, a better understanding of the transfection conditions at the single cell level are required. In this study, a universal droplet microfluidic platform was used to assess the heterogeneities of CHO-S population transiently transfected with cationic liposomes (CL) (lipoplexes) complexed with GFP-coding plasmid DNA (pDNA). A single cell analysis of GFP production kinetics revealed the presence of a subpopulation producing higher levels of GFP compared with the main population. The size of high producing (HP) cells, their relative abundance, and their specific productivity were dependent on the charge and the pDNA content of the different lipoplexes: HPs showed increased cell size in comparison to the average population, lipoplexes with positive charge produced more HPs, and lipoplexes carrying a larger amount of pDNA yielded a higher specific productivity of HPs. This study demonstrates the potential for time-resolved single-cell measurements to explain population dynamics from a microscopic point of view.

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WITHDRAWN: Clearance of lysosomal glycogen accumulation by Transcription factor EB (TFEB) in muscle cells from lysosomal alpha-glucosidase deficient mice.

Project description:This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

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Transiently transfected purine biosynthetic enzymes form stress bodies.

Project description:It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.

| S-EPMC3566086 | biostudies-literature

Sequences necessary for trans-splicing in transiently transfected Brugia malayi.

Project description:Many genes in parasitic nematodes are both cis- and trans-spliced. Previous studies have demonstrated that a 7nt element encoded in the first intron of the Brugia malayi 70kDa heat shock protein (BmHSP70) gene was necessary to permit trans-splicing of transgenic mRNAs in embryos transfected with constructs encoding portions of the BmHSP70 gene. Here we demonstrate that this element (the B. malayi HSP70 trans-splicing motif, or BmHSP70 TSM) is necessary and sufficient to direct trans-splicing of transgenic mRNAs derived from two genes naturally containing this motif. Mutations introduced into any position of the BmHSP70 TSM abrogated its ability to direct trans-splicing. Transgenic mRNAs derived from embryos transfected with constructs containing promoters and associated downstream domains from two normally trans-spliced genes that lack a BmHSP70 TSM homologue (the B. malayi 12kDa small subunit ribosomal protein (BmRPS12) gene and the B. malayi RNA-binding protein (BmRBP1) gene), were not trans-spliced. Transfer of the BmHSP70 TSM into the first intron of the BmRPS12 gene rendered it competent for trans-splicing. Insertion of the BmHSP70 TSM into the single intron of the BmRBP1 gene did not render it trans-splicing competent. However, tagged constructs of the full-length BmRBP1 gene were trans-splicing competent. An analysis of the first exons and introns of over 200 trans-spliced B. malayi genes found homologues for the BmHSP70 TSM in roughly 25%. Thus, while the BmHSP70 TSM is necessary and sufficient to direct trans-splicing in some genomic contexts, independent trans-splicing signals are employed by other genes.

| S-EPMC2039923 | biostudies-literature

Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

Project description:Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.

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