ABSTRACT
Two alpha-N-acetylgalactosaminidases, alpha-N-acetylgalactosaminidase (alpha-GalNAcase) I and II, were purified from the digestive organ of starfish. Purified alpha-GalNAcase I and II gave nearly single protein bands on SDS-polyacrylamide gel electrophoresis, individually. Even the final preparation of alpha-GalNAcase I contained alpha-galactosidase activity, while alpha-GalNAcase II was almost free from that activity with p-nitrophenyl and 4-methylumbelliferyl alpha-N-acetylgalactosaminides as substrates. alpha-GalNAcase I and II both hydrolyzed terminal alpha-N-acetylgalactosaminyl linkages of the natural compounds investigated: Forssman hapten glycolipid, blood group A active oligosaccharide and GalNAc-alpha1-O-serine. On the other hand, oligosaccharides, and glycolipid containing alpha-galactosyl terminals were hydrolyzed by alpha-GalNAcase I but not by alpha-GalNAcase II. The substrate specificities and other enzymatic properties of alpha-GalNAcase I were similar to those of human placental alpha-GalNAcase, but distinct from alpha-GalNAcase II.
Subject(s)
Oligosaccharides/isolation & purification , Starfish/enzymology , alpha-N-Acetylgalactosaminidase/isolation & purification , Animals , Electrophoresis, Polyacrylamide Gel , Substrate SpecificityABSTRACT
A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.
Subject(s)
Enzyme Replacement Therapy/methods , Fabry Disease/drug therapy , alpha-N-Acetylgalactosaminidase/chemistry , alpha-N-Acetylgalactosaminidase/therapeutic use , Amino Acid Substitution , Animals , Binding Sites , CHO Cells , Catalysis , Cells, Cultured , Cricetinae , Cricetulus , Culture Media, Conditioned/chemistry , DNA, Complementary/metabolism , Disease Models, Animal , Drug Stability , Fabry Disease/enzymology , Fabry Disease/metabolism , Fibroblasts/drug effects , Fluorescent Dyes/metabolism , Galactosides/metabolism , Genetic Vectors , Humans , Hydrogen-Ion Concentration , Hymecromone/analogs & derivatives , Hymecromone/metabolism , Immunohistochemistry , Kidney/drug effects , Kidney/pathology , Kidney/ultrastructure , Liver/drug effects , Liver/pathology , Liver/ultrastructure , Mice , Mice, Knockout , Models, Molecular , Molecular Weight , Myocardium/pathology , Myocardium/ultrastructure , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , Retroviridae/genetics , Transfection , Trihexosylceramides/metabolism , alpha-N-Acetylgalactosaminidase/genetics , alpha-N-Acetylgalactosaminidase/isolation & purificationABSTRACT
Based on the inherent alpha-galactosidase activity, squid liver contains two different alpha-N-acetylgalactosaminidases (alpha-GalNAcases): alpha-N-acetylgalactosaminidase I (alpha-GalNAcase I), which typically exhibits the alpha-galactosidase activity and alpha-N-acetylgalactosaminidase II (alpha-GalNAcase II), which is devoid of such activity. The molecular properties of the alpha-GalNAcases that may account for their enzymological differences are as yet unknown. In this study, we have characterized and compared the chemical and immunological properties of alpha-GalNAcase I and alpha-GalNAcase II. Analysis of the N-terminal sequence of the first twenty amino acids revealed the striking homology between alpha-GalNAcase I and alpha-GalNAcase II. Digestion of alpha-GalNAcase I and alpha-GalNAcase II generated the peptide maps that display similarities in peptide pattern, indicating their close relationship in structure. Polyclonal antibodies were generated in rabbits against the purified alpha-GalNAcase I and alpha-GalNAcase II for comparison of the immunological properties. Both Western blot and surface plasmon resonance (SPR) studies showed that the anti-alpha-GalNAcase II antibody reacted with both alpha-GalNAcase I and alpha-GalNAcase II, whereas the anti-alpha-GalNAcase I antibody reacted only with alpha-GalNAcase I, indicating the presence of common as well as unique antigenic determinants on alpha-GalNAcase I and alpha-GalNAcase II. Taken together, these results suggest that alpha-GalNAcase I and alpha-GalNAcase II are closely related with regard to structure and that their nonhomologous domains are possibly responsible for the differences in enzymatic properties.
Subject(s)
Decapodiformes/enzymology , Epitopes/immunology , Liver/enzymology , alpha-N-Acetylgalactosaminidase/chemistry , alpha-N-Acetylgalactosaminidase/immunology , Amino Acid Sequence , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Epitope Mapping , Isoenzymes , Molecular Sequence Data , Peptide Mapping , Sequence Homology, Amino Acid , Surface Plasmon Resonance , alpha-N-Acetylgalactosaminidase/isolation & purificationABSTRACT
We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research.
Subject(s)
Bacterial Proteins/chemistry , Disaccharides/chemistry , Enterococcus faecalis/enzymology , Trisaccharides/chemistry , alpha-N-Acetylgalactosaminidase/chemistry , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Catalysis , Cloning, Molecular , Glycosylation , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Substrate Specificity , alpha-N-Acetylgalactosaminidase/biosynthesis , alpha-N-Acetylgalactosaminidase/isolation & purificationABSTRACT
The complete coding sequences of 3 porcine genes - ASPA, NAGA, and HEXA - were amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referenced pig ESTs. These 3 novel porcine genes were then deposited in the NCBI database and assigned GeneIDs: 100142661, 100142664 and 100142667. The phylogenetic tree analysis revealed that the porcine ASPA, NAGA, and HEXA all have closer genetic relationships with the ASPA, NAGA, and HEXA of cattle. Tissue expression profile analysis was also carried out and results revealed that swine ASPA, NAGA, and HEXA genes were differentially expressed in various organs, including skeletal muscle, the heart, liver, fat, kidney, lung, and small and large intestines. Our experiment is the first one to establish the foundation for further research on these 3 swine genes.
Subject(s)
Amidohydrolases/genetics , Hexosaminidase A/genetics , Sus scrofa/genetics , alpha-N-Acetylgalactosaminidase/genetics , Amidohydrolases/isolation & purification , Amidohydrolases/metabolism , Animals , DNA Primers/chemistry , DNA, Complementary/genetics , Hexosaminidase A/isolation & purification , Hexosaminidase A/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tissue Distribution , alpha-N-Acetylgalactosaminidase/isolation & purification , alpha-N-Acetylgalactosaminidase/metabolismABSTRACT
A set of filamentous fungi (42 strains) was screened for alpha-N-acetylgalactosaminidase activity, and a series of inducers and different cultivation conditions were tested. Enzyme production by the best producer Aspergillus niger CCIM K2 was optimized and scaled up. alpha-N-Acetylgalactosaminidase was purified to apparent homogeneity by cation exchange chromatography, gel filtration, and chromatofocusing, and basic biochemical data of the enzyme were determined: The native molecular weight was estimated by gel filtration to be approximately 440 kDa, the molecular weight of the subunit was determined to be 76 kDa and the pI = 4.8. The K (M) was 0.73 mmol/l for o-nitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (o-NP-alpha-GalNAc), and optimum enzyme activity was achieved at pH 1.8 and 55 degrees C. This alpha-N-acetylgalactosaminidase is a retaining-type glycosidase, and it was N-deglycosylated without any loss of activity.
Subject(s)
Aspergillus niger/enzymology , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , alpha-N-Acetylgalactosaminidase/chemistry , alpha-N-Acetylgalactosaminidase/isolation & purification , Aspergillus niger/metabolism , Enzyme Activation , Enzyme Stability , Fungal Proteins/metabolism , Kinetics , Molecular Weight , alpha-N-Acetylgalactosaminidase/metabolismABSTRACT
alpha-N-acetylgalactosaminidase has been isolated from liquid culture of micromycete Aspergillus niger and purified 600 times by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels with specific activity 10.5 U/mg of protein. The preparation was homogenic: its molecular mass by the data of gel-filtration on Sepharose 6B was 430 kDa, on PAAGE in the system of DDSNa--70 kDa. That gives every reason to suppose oligomeric structure of the enzyme molecule. The carbohydrate component, including mannose, galactose, glucosamine and two nonidentified hexosamines was observed in alpha-N-acetylgalactosaminidase. Thermo- and pH- optima were 60 degrees C and pH 3.5, respectively. The enzyme was thermo- and pH-stable, resistant in storage. The enzyme was found to exhibit strict specificity in respect ofglycon. It was shown that enzyme was competitively inhibited by substrate and reaction product. Km and Vmax with respect to nitrophenyl substrate were 1.25 mM and 10.5 mkmole/min/mg of protein. The activity of glycosidase tested was independent of the presence of metal ions. The presence of carboxylic group of C-terminal aminoacid and imidazol group of hystidine in active centre of molecule was established. A number of natural and synthetic substrates were able to activate (50-200%) production of A. niger alpha-N-acetylgalactosaminidase.
