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1.
Cell Metab ; 30(4): 689-705.e6, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31353261

ABSTRACT

Lafora disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a-/- mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a-/- mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy.


Subject(s)
Brain/drug effects , Drug Discovery , Inclusion Bodies/drug effects , Lafora Disease/therapy , Pancreatic alpha-Amylases/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , Brain/pathology , Disease Models, Animal , HEK293 Cells , Humans , Immunoglobulin G/therapeutic use , Mice , Mice, Inbred C57BL , Pancreatic alpha-Amylases/therapeutic use , Rats , Recombinant Fusion Proteins/therapeutic use
2.
Infect Immun ; 83(12): 4884-95, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26438798

ABSTRACT

Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.


Subject(s)
Bacterial Proteins/genetics , Biofilms/drug effects , Campylobacter Infections/veterinary , Campylobacter jejuni/drug effects , Pancreatic alpha-Amylases/pharmacology , Peptide Hydrolases/genetics , Poultry Diseases/microbiology , Animals , Bacterial Proteins/metabolism , Biofilms/growth & development , Caco-2 Cells , Campylobacter Infections/enzymology , Campylobacter Infections/microbiology , Campylobacter Infections/pathology , Campylobacter jejuni/pathogenicity , Campylobacter jejuni/physiology , Cell Line, Tumor , Chickens , Dextrans/biosynthesis , Dextrans/metabolism , Epithelial Cells , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Intestines/microbiology , Intestines/pathology , Moths/microbiology , Pancreatic alpha-Amylases/isolation & purification , Peptide Hydrolases/metabolism , Poultry Diseases/enzymology , Poultry Diseases/pathology , Signal Transduction , Swine
3.
J Periodontal Res ; 49(1): 62-8, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23550921

ABSTRACT

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract. MATERIAL AND METHODS: The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure. RESULTS: The inhibitor was identified as AmyI-1, an α-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, α-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1. CONCLUSIONS: This is the first study to report that α-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of α-amylases in treating periodontal diseases.


Subject(s)
Anti-Bacterial Agents/pharmacology , Porphyromonas gingivalis/drug effects , alpha-Amylases/pharmacology , Animals , Cell Membrane/drug effects , Humans , Microbial Sensitivity Tests , Microscopy, Electron, Scanning , Oryza/enzymology , Pancreatic alpha-Amylases/pharmacology , Periodontal Diseases/microbiology , Plant Extracts/pharmacology , Porphyromonas gingivalis/growth & development , Porphyromonas gingivalis/ultrastructure , Saliva/enzymology , Salivary Proteins and Peptides/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Swine , alpha-Amylases/isolation & purification
4.
J Biol Chem ; 287(27): 23104-18, 2012 Jun 29.
Article in English | MEDLINE | ID: mdl-22584580

ABSTRACT

Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680-4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na(+)/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other.


Subject(s)
Duodenum/metabolism , Glycoproteins/metabolism , Microvilli/metabolism , Pancreatic alpha-Amylases/metabolism , Polysaccharides/metabolism , Animals , Blood Glucose/metabolism , Duodenum/cytology , Enterocytes/enzymology , Galactans/metabolism , Glycomics/methods , Glycoproteins/isolation & purification , Glycoside Hydrolases/metabolism , Glycosylation , Homeostasis/physiology , Humans , Lectins/metabolism , Ligands , Mannans/metabolism , Oligo-1,6-Glucosidase/metabolism , Pancreatic alpha-Amylases/pharmacology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Sodium-Glucose Transporter 1/metabolism , Starch/metabolism , Sucrase/metabolism , Swine
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