Immunohistochemical characterization of transgenic mice highly expressing human lysosomal α-galactosidase

AbstractHuman lysosomal α-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc–TgN(GLA) 1951 Rin, highly expressing human α-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of α-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and α-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of α-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54–60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h−1 mg−1 protein) and heart (39,906 nmol h−1 mg−1 protein), followed by in the kidney tissue (9177 nmol h−1 mg−1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed α-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information

Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies

Spatial reorganization of metabolic enzymes to form the “metabolic enzymes transiently assembling (META) body” is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27–42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed “META body-forming sequences (METAfos).” METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation

Production of Adh1p conjugated with foci-forming peptides and domains in <i>S</i>. <i>cerevisiae</i> under hypoxia.

A: Overview of constructed yeast strains. ADH1 was fused with tag sequences (SC2, SC3, scENO, FUSN, or Sup35p) and FusionRed then Cu2+-dependently expressed using the CUP1 promoter. As a control, FusionRed or ADH1 fused with FusionRed were expressed using the CUP1 promoter. Within the genome of the host strain (CDC19-GFP), GFP is fused with CDC19. Green fluorescence indicates the presence of condensates formed by Cdc19p (META body) under hypoxia. B: Localization of tagged Adh1p in CDC19-GFP cells under hypoxia. Green fluorescence indicates subcellular localization of Cdc19p. META bodies are seen as green foci. FusionRed demonstrates subcellular localization of tagged Adh1p. White arrow shows colocalized Adh1p with META bodies, and only foci that overlapped with META body markers were marked with arrows. C: Cu2+-dependent foci formation by tagged proteins. X axis shows CuSO4 concentration (μM). The proportion of foci-forming cells was calculated as follows: foci-forming cells (%) = 100 × number of cells with red foci/number of cells with red fluorescence. n = 3. Error bars show standard deviation.</p

Amino acid sequences of peptides and proteins used in the present study.

Amino acid sequences of peptides and proteins used in the present study.</p

Effect of Adh1p localization in cells on cellular metabolism under hypoxia.

A: Overview of constructed yeast strains. ADH1 was fused with tag sequences (SC3, scENO, FUSN, or Sup35p) and FusionRed then expressed in response to 100 μM Cu2+ using the CUP1 promoter. As a control, ADH1 was fused with FusionRed and expressed using the CUP1 promoter. Within the genome of the host strain (adh1Δ), ADH1 was knocked out by homologous recombination using the kanamycin resistance gene. B: Simplified illustration of glucose metabolism. Most (11 out of 12) enzymes shown to form META bodies under hypoxia are components of the metabolic pathway converting glucose to acetic acid (black arrow). Enzymes catalyzing the production of glycerol (white arrow) do not form condensates. The metabolic reaction catalyzed by Adh1p converts acetaldehyde to ethanol (shaded arrow). Metabolites predicted to be affected the subcellular localization of Adh1p are underlined. C: Proportions of foci-forming cells for each transformant. ADH1, S. cerevisiae BY4741 adh1Δ transformed with p426-CUP1p-Adh1p-FusionRed; X-ADH1, S. cerevisiae BY4741 adh1Δ transformed with p426-CUP1p-X-Adh1p-FusionRed. n = 3. Error bars show standard deviations. *: P <0.05 compared with cells transformed with ADH1. D: Ethanol concentration in culture media. E: Ratio of ethanol to acetic acid concentrations in culture media. D and E: black bars, ADH1; white bars, SC3-ADH1; gray bars, scENO-ADH1; shaded bars, FUSN-ADH1; horizontal striped bars, Sup35p-ADH1. n = 3. Error bars show standard errors. *: P <0.05 compared with cells transformed with ADH1.</p

Summary of the four identified foci-forming peptides; SC1, SC2, SC3 and SC4.

A: Three-dimensional distribution of identified peptides in pyruvate kinase (CDC19, PDB ID:1A3W) [24]. PyMOL ver. 2.5.2 was used to provide a graphical illustration of identified peptides. SC1, pink; SC2, blue; SC3, yellow; SC4, orange; other domains, green. Two dimers are shown. Gray molecule represents the monomer subunit. B: Images of each EGFP-conjugated fragment in cells under normoxia. pUL-ATG-EGFP indicates images of the S. cerevisiae BY4741 wild type strain cells transformed with the plasmid expressing EGFP only (negative control). pULGI2-CDC19 indicates images of cells transformed with the plasmid expressing full-length Cdc19p fused with EGFP. pULGI2-SCX (X = 1–4) indicates images of cells transformed with the pULGI2-SCX plasmid. Bar = 10 μm.</p

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Spatial reorganization of metabolic enzymes to form the “metabolic enzymes transiently assembling (META) body” is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27–42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed “META body-forming sequences (METAfos).” METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.</div

Foci formation by fragmented pyruvate kinase in <i>S</i>. <i>cerevisiae</i>.

A: The ratio of foci formation for each fragment conjugated with EGFP and overexpressed in S. cerevisiae BY4741 wild type strain under normoxic condition. X axis shows the fragments of Cdc19p and the fragments correspond to the location shown in B. n = 3. Error bars show standard deviation. B: Overview of fragmented peptides. Numbers on the left indicate the amino acid residues of Cdc19p. White bars represent Cdc19p fragments fused with FusionRed that did not show foci, gray bars represent Cdc19p fragments that showed foci, and black dotted bars represent Cdc19p fragments with relatively high foci-forming ratios, namely SC1, SC2, SC3, and SC4. Dotted lines connect the bars to the numbers on the left to indicate the location of the fragments within Cdc19p.</p

Strains used in the present study.

Spatial reorganization of metabolic enzymes to form the “metabolic enzymes transiently assembling (META) body” is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27–42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed “META body-forming sequences (METAfos).” METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.</div