ABSTRACT
This study attempts to answer the question of whether mice with biallelic and monoallelic disruption of the St3gal5 (GM3 synthase) gene might benefit from GM1 replacement therapy. The GM3 produced by this sialyltransferase gives rise to downstream GD3 and the ganglio-series of gangliosides. The latter includes the a-series (GM1 + GD1a), which has proved most essential for neuron survival and function (especially GM1, for which GD1a provides a reserve pool). These biallelic mice serve as a model for children with this relatively rare autosomal recessive condition (ST3GAL5-/-) who suffer rapid neurological decline including motor loss, intellectual disability, visual and hearing loss, failure to thrive, and other severe conditions leading to an early death by 2-5 years of age without supportive care. Here, we studied both these mice, which serve as a model for the parents and close relatives of these children who are likely to suffer long-term disabilities due to partial deficiency of GM1, including Parkinson's disease (PD). We find that the movement and memory disorders manifested by both types of mice can be resolved with GM1 application. This suggests the potential therapeutic value of GM1 for disorders stemming from GM1 deficiency, including GM3 synthase deficiency and PD. It was noteworthy that the GM1 employed in these studies was synthetic rather than animal brain-derived, reaffirming the therapeutic efficacy of the former.
Subject(s)
G(M1) Ganglioside , Parkinson Disease , Mice , Animals , Gangliosides , Sialyltransferases/geneticsABSTRACT
Parkinson's disease (PD) is a major neurodegenerative disorder characterized by a variety of non-motor symptoms in addition to the well-recognized motor dysfunctions that have commanded primary interest. We previously described a new PD mouse model based on heterozygous disruption of the B4galnt1 gene leading to partial deficiency of the GM1 family of gangliosides that manifested several nigrostriatal neuropathological features of PD as well as movement impairment. We now show this mouse also suffers three non-motor symptoms characteristic of PD involving the gastrointestinal, sympathetic cardiac, and cerebral cognitive systems. Treatment of these animals with a synthetic form of GM1 ganglioside, produced by transfected E. coli, proved ameliorative of these symptoms as well as the motor defect. These findings further suggest subnormal GM1 to be a systemic defect constituting a major risk factor in sporadic PD and indicate the B4galnt1(+/-) (HT) mouse to be a true neuropathological model that recapitulates both motor and non-motor lesions of this condition.
Subject(s)
Disease Models, Animal , G(M1) Ganglioside/administration & dosage , G(M1) Ganglioside/deficiency , N-Acetylgalactosaminyltransferases/deficiency , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Animals , Female , G(M1) Ganglioside/genetics , Gastrointestinal Diseases/drug therapy , Gastrointestinal Diseases/genetics , Gastrointestinal Diseases/metabolism , Male , Memory Disorders/drug therapy , Memory Disorders/genetics , Memory Disorders/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Motor Skills Disorders/drug therapy , Motor Skills Disorders/genetics , Motor Skills Disorders/metabolism , N-Acetylgalactosaminyltransferases/genetics , Parkinson Disease/geneticsABSTRACT
We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a ß1,3-galactosyl transferase and UDP-Glc(NAc)-4-epimerase, both from Campylobacter jejuni, and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon α-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli.
Subject(s)
Growth Hormone/metabolism , Interferon alpha-2/metabolism , Polysaccharides/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Campylobacter jejuni/enzymology , Escherichia coli/metabolism , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Glycosylation , Growth Hormone/genetics , Humans , Interferon alpha-2/genetics , N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , UDPglucose 4-Epimerase/genetics , UDPglucose 4-Epimerase/metabolism , Polypeptide N-acetylgalactosaminyltransferaseABSTRACT
Juvenile Batten disease (JBD) is an inherited disorder that is characterized by the development of blindness, seizures, and progressive motor, psychiatric, and cognitive impairment. A model of JBD expressing the predominant human mutation (Cln3 ∆ex7/8 ) has been explored. Dissociated brain cultures from Cln3 ∆ex7/8 knock-in mice were compared to wild type (WT) for effects on granules of ceroid lipofuscin (CL) and neuronal structure. Utilizing high content image analysis of CL granules identified with antibodies to mitochondrial ATP synthase subunit c or tripeptidyl peptidase-1, significant increases in the areas for both immunoreactive granules were observed in Cln3 ∆ex7/8 cultures in comparison to WT. CL granules also exhibit autofluorescence at 488 and 560 nm, and the areas of these autofluorescent spots were found to be significantly increased in Cln3 ∆ex7/8 cultures in comparison to WT. Progressive increases in CL granule area in Cln3 ∆ex7/8 cultures were observed during culture development. Because current therapies for JBD provide only symptomatic support, a therapeutic strategy has been explored based on the observations that JBD-related tissues are deficient in ß-galactosyl ceramide. Treatment of cultures for 40 h with a potent analog of ß-galactosyl ceramide (SNB-4050) produced significant decreases in CL granule area in the Cln3 ∆ex7/8 cultures; whereas identical studies on WT cultures produced no detectible changes. Significant decreases in average neurite length and neurite branch point number were also observed in the Cln3 ∆ex7/8 cultures that were attenuated by treatment with 1 nM SNB-4050. These studies indicate Cln3 ∆ex7/8 brain cultures may be useful to screen therapeutic agents for treatment of JBD.
Subject(s)
Brain/cytology , Galactosylceramides/pharmacology , Lipofuscin/metabolism , Membrane Glycoproteins/genetics , Molecular Chaperones/genetics , Neuronal Ceroid-Lipofuscinoses/genetics , Neurons/drug effects , ATP Synthetase Complexes/metabolism , Aminopeptidases/metabolism , Animals , Cells, Cultured , Cytoplasmic Granules/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Mice , Neurons/cytology , Neurons/metabolism , Serine Proteases/metabolism , Tripeptidyl-Peptidase 1ABSTRACT
Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we found that ST6GalNAc-I is weakly expressed in normal gastric mucosa, but over-expressed in intestinal metaplasia, co-localized with sialyl-Tn. In gastric carcinomas ST6GalNAc-I was also associated with sialyl-Tn, but with heterogeneous staining and partial co-localization. Our results showed ST6GalNAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues.
Subject(s)
Gastrointestinal Tract/immunology , Sialyltransferases/metabolism , Animals , Antibodies, Monoclonal/immunology , Antigens, Tumor-Associated, Carbohydrate , Base Sequence , CHO Cells , Cricetinae , Cricetulus , DNA Primers , Mice , Mice, Inbred BALB C , Sialyltransferases/immunologyABSTRACT
Mammalian cell surfaces are modified by complex arrays of glycoproteins, glycolipids and polysaccharides, many of which terminate in sialic acid and have central roles in essential processes including cell recognition, adhesion and immunogenicity. Sialylation of glycoconjugates is performed by a set of sequence-related enzymes known as sialyltransferases (STs). Here we present the crystal structure of a mammalian ST, porcine ST3Gal-I, providing a structural basis for understanding the mechanism and specificity of these enzymes and for the design of selective inhibitors.
Subject(s)
Sialyltransferases/chemistry , Animals , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , N-Acetylneuraminic Acid/metabolism , Protein Structure, Secondary , Sialyltransferases/genetics , Sialyltransferases/metabolism , Substrate Specificity , Swine , beta-Galactoside alpha-2,3-SialyltransferaseABSTRACT
BACKGROUND: The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity. RESULTS AND CONCLUSION: In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.
Subject(s)
Escherichia coli/enzymology , Sialyltransferases/metabolism , Animals , Cattle , Escherichia coli/metabolism , Glycoproteins/metabolism , Glycosylation , Humans , Molecular Chaperones/metabolism , Mucins/metabolism , Recombinant Fusion Proteins/metabolism , Sialyltransferases/geneticsABSTRACT
The gene clusters encoding the lipooligosaccharide biosynthesis glycosyltransferases from Campylobacter jejuni have previously been divided in eight classes based on their genetic organization. Here, three variants of the beta1,3-galactosyltransferase CgtB from two classes were purified as fusions with the maltose-binding protein (MalE) from Escherichia coli and their acceptor preference was determined. The acceptor preference of each CgtB variant was directly related to the presence or absence of sialic acid in the acceptor, which correlated with the core oligosaccharide structure in vivo. The three variants were evaluated for their ability to use a derivitized monosaccharide, a GM2 ganglioside mimic, a GA2 ganglioside mimic as well as a peptide containing alpha-linked GalNAc. This characterization shows the flexibility of these galactosyltransferases for diverse acceptors. The CgtB variants were engineered via carboxy-terminal deletions and inversion of the gene fusion order. The combination of a 20 to 30 aa deletion in CgtB followed by MalE at its carboxy terminus significantly improved the glycosyltransferase activity (up to a 51.8-fold increase of activity compared to the full length enzyme) in all cases regardless of the acceptor tested. The improved enzyme CgtB(OH4384)DeltaC-MalE was used to galactosylate a glyco-peptide acceptor based on the interferon alpha2b protein O-linked glycosylation site as confirmed by the CE-MS analysis of the reaction products. This improved enzyme was also used successfully to galactosylate the human therapeutic protein IFNalpha2b[GalNAcalpha]. This constitutes the first report of the in vitro synthesis of the O-linked T-antigen glycan on a human protein by a bacterial glycosyltransferase and illustrates the potential of bacterial glycosyltransferases as tools for in vitro glycosylation of human proteins of therapeutic value.
Subject(s)
Campylobacter jejuni/enzymology , Galactosyltransferases/biosynthesis , Galactosyltransferases/chemistry , Gene Expression Regulation, Enzymologic , Amino Acid Sequence , Gene Deletion , Genotype , Glycosylation , Glycosyltransferases/chemistry , Kinetics , Mass Spectrometry , Molecular Sequence Data , Multigene Family , Plasmids/metabolism , Polysaccharides/chemistry , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
Glycosphingolipids play crucial roles in virtually every stage of the cell cycle, and their clinical administration has been proposed as a treatment for Alzheimer's, Parkinson's, stroke, and a range of other conditions. However, lack of supply has severely hindered testing of this potential. A novel glycosynthase-based synthetic strategy is demonstrated, involving a mutant of an endoglycoceramidase in which the catalytic nucleophile has been ablated. This mutant efficiently couples a range of glycosyl fluoride donors with a range of sphingosine-based acceptors in yields around 95%. This technology opens the door to large-scale production of glycosphingolipids and, thus, to clinical testing.
Subject(s)
Glycoside Hydrolases/metabolism , Glycosphingolipids/chemical synthesis , Catalysis , Glucose/analogs & derivatives , Glucose/chemistry , Glycoside Hydrolases/genetics , Mutation , Sphingosine/chemistryABSTRACT
Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-alpha2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.