ABSTRACT
Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, ß-hexosaminidase, α-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease.
Subject(s)
Lysosomes/genetics , Mucolipidoses/genetics , Nerve Degeneration/genetics , Animals , Atrophy , Autophagy , Brain/enzymology , Brain/pathology , Disease Models, Animal , Lysosomes/enzymology , Lysosomes/pathology , Mice , Mice, Transgenic , Mucolipidoses/enzymology , Mucolipidoses/pathology , Nerve Degeneration/enzymology , Nerve Degeneration/pathology , Vesicular Transport Proteins/metabolism , alpha-L-Fucosidase/metabolism , alpha-Mannosidase/metabolism , beta-N-Acetylhexosaminidases/metabolismABSTRACT
Rats exposed to early life stress are considered as a valuable model for the study of epigenetic programming leading to mood disorders and anxiety in the adult life. Rats submitted to prenatal restraint stress (PRS) are characterized by an anxious/depressive phenotype associated with neuroadaptive changes in the hippocampus. We used the model of PRS to identify proteins that are specifically affected by early life stress. We therefore performed a proteomic analysis in the hippocampus of adult male PRS rats. We found that PRS induced changes in the expression profile of a number of proteins, involved in the regulation of signal transduction, synaptic vesicles, protein synthesis, cytoskeleton dynamics, and energetic metabolism. Immunoblot analysis showed significant changes in the expression of proteins, such as LASP-1, fascin, and prohibitin, which may lie at the core of the developmental programming triggered by early life stress.
Subject(s)
Hippocampus/metabolism , Prenatal Exposure Delayed Effects/metabolism , Animals , Anxiety/metabolism , Depression/metabolism , Female , Male , Microfilament Proteins/metabolism , Mood Disorders/metabolism , Nerve Tissue Proteins/metabolism , Pregnancy , Prohibitins , Proteomics , Rats , Rats, Sprague-Dawley , Repressor Proteins/metabolism , Restraint, Physical , Stress, Psychological/metabolism , Transferrin/metabolismABSTRACT
Nuclear and cytoplasmic O-GlcNAc transferase (OGT) is a unique and universally expressed enzyme catalyzing O-GlcNAcylation of thousands of proteins. Although OGT interferes with many crucial intracellular processes, including cell cycle, only few studies have focused on elucidating the precise role of the glycosyltransferase during cell cycle entry. We first demonstrated that starved MCF7 cells reincubated with serum quickly induced a significant OGT increase concomitantly to activation of PI3K and MAPK pathways. Co-immunoprecipitation experiments performed upon serum stimulation showed a progressive interaction between OGT and ß-catenin, a major factor in the regulation of cell cycle. OGT expression was also observed in starved HeLa cells reincubated with serum. In these cells, the O-GlcNAcylation status of the ß-catenin-2XFLAG was increased following stimulation. Moreover, ß-catenin-2XFLAG was heavily O-GlcNAcylated in exponentially proliferating HeLa cells when compared to confluent cells. Furthermore, blocking OGT activity using the potent inhibitor Ac-5SGlcNAc prevented serum-stimulated cyclin D1 synthesis and slightly delayed cell proliferation. At last, interfering with OGT expression (siOGT) blocked cyclin D1 expression and decreased PI3K and MAPK activation. Together, our data indicate that expression and catalytic activity of OGT are necessary and essential for G0/G1 transition.
ABSTRACT
The gene DMBT1 (deleted in malignant brain tumour-1) has been proposed to play a role in brain and epithelial cancer, but shows unusual features for a classical tumour-suppressor gene. On the one hand, DMBT1 has been linked to mucosal protection, whereas, on the other, it potentially plays a role in epithelial differentiation. Thus its function in a particular tissue is of mechanistic importance for its role in cancer. Because the former function requires secretion to the lumen and the latter function may depend on its presence in the extracellular matrix, we decided to investigate DMBT1 expression, location and its mode of secretion during malignant transformation in colorectal cancer. Using human colorectal PC/AA cell lines and tissue sections from individual patients, we have examined the expression of DMBT1 and its glycosylation in the adenoma-carcinoma sequence leading to the adenocarcinoma phenotype.
Subject(s)
Adenocarcinoma/genetics , Cell Transformation, Neoplastic , Colorectal Neoplasms/genetics , Receptors, Cell Surface/genetics , Adenoma/genetics , Adenoma/pathology , Calcium-Binding Proteins , Carcinoma/genetics , Carcinoma/pathology , Colorectal Neoplasms/pathology , DNA-Binding Proteins , Gene Expression Regulation, Neoplastic , Glycosylation , Humans , Intestinal Mucosa/pathology , Intestinal Mucosa/physiopathology , Phenotype , Tumor Suppressor ProteinsABSTRACT
The SPASIBA force field has been applied to the determination of the structure and dynamical properties of various disaccharides. It has been shown that the experimental properties (structure, dipole moment, conformational relative energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are confidently reproduced without specific terms for the alpha and beta anomers and the type of glycosidic linkages.
Subject(s)
Algorithms , Carbohydrate Conformation , Disaccharides/chemistry , Glycosides/chemistry , Software , Static Electricity , Stereoisomerism , Structure-Activity Relationship , Sulfhydryl Compounds/chemistry , Thermodynamics , Trehalose/chemistryABSTRACT
Amyloid deposits and neurofibrillary tangles (NFT) are the two hallmarks that characterize Alzheimer's disease (AD). In order to find the molecular partners of these degenerating processes, we have developed antibodies against insoluble AD brain lesions. One clone, named AD46, detects only NFT. Biochemical and histochemistry analyses demonstrate that the labeled protein accumulating in the cytosol of Alzheimer degenerating neurons is the alpha-chain of the ATP synthase. The cytosolic accumulation of the alpha-chain of ATP synthase is observed even at early stages of neurofibrillary degenerating process. It is specifically observed in degenerating neurons, either alone or tightly associated with aggregates of tau proteins, suggesting that it is a new molecular event related to neurodegeneration. Overall, our results strongly suggest the implication of the alpha-chain of ATP synthase in neurofibrillary degeneration of AD that is illustrated by the cytosolic accumulation of this mitochondrial protein, which belongs to the mitochondrial respiratory system. This regulatory subunit of the respiratory complex V of mitochondria is thus a potential target for therapeutic and diagnostic strategies.
Subject(s)
Alzheimer Disease/enzymology , Mitochondrial Proton-Translocating ATPases/metabolism , Neurofibrillary Tangles/enzymology , Alzheimer Disease/pathology , Humans , Mitochondrial Proton-Translocating ATPases/analysis , Mitochondrial Proton-Translocating ATPases/biosynthesis , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/pathology , Neurofibrillary Tangles/chemistry , Neurofibrillary Tangles/pathology , Prospective StudiesABSTRACT
Appearance of a hyposialylated transferrin fraction in the plasma during chronic alcohol exposure is a well-known phenomenon, and it represents the best available marker of chronic alcohol consumption. The mechanisms of its appearance are still not well understood and are extremely complex, involving biosynthesis and catabolism alterations, although the only structural abnormality described corresponds to the loss of an entire glycan chain. We analyzed and compared the oligosaccharides present on the different isoforms of purified transferrin isolated from control and patients with severe alcohol abuse by fluorescent carbohydrate electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Our data indicate that the major modification observed is the loss of an entire oligosaccharide chain; we also demonstrate that there is a modification of terminal sialylation. Carbohydrate-deficient transferrin (CDT) is the result of multiple alterations of glycosylation. These results give a partial explanation to the poor sensitivity of the measurement of CDT and its controversial use as a marker of chronic alcohol consumption.
Subject(s)
Alcoholism/blood , Ethanol/pharmacology , Transferrin/chemistry , Transferrin/metabolism , Alcoholism/metabolism , Animals , Biomarkers/blood , Carbohydrate Sequence , Central Nervous System Depressants/pharmacology , Chromatography, Ion Exchange , Glycosylation/drug effects , Humans , Molecular Sequence Data , Molecular Weight , Oligosaccharides/blood , Oligosaccharides/chemistry , Oligosaccharides/isolation & purification , Protein Isoforms/blood , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Rabbits , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transferrin/isolation & purificationABSTRACT
A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The beta-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44 Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues.
Subject(s)
Glycopeptides/analysis , Mucins/chemistry , Amino Acid Sequence , Catalysis , Dimethylamines , Glycosylation , Humans , Molecular Sequence Data , Spectrometry, Mass, Electrospray Ionization , Tandem Repeat SequencesABSTRACT
Cytosolic and nuclear O-linked N-acetylglucosaminylation has been proposed to be involved in the nuclear transport of cytosolic proteins. We have isolated nuclear and cytosolic N-acetyl-d-glucosamine (GlcNAc)-specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc and identified these lectins, by a proteomic approach, as belonging to the heat-shock protein (HSP)-70 family (one of them being heat-shock cognate 70 stress protein). Two-dimensional electrophoresis indicated that the HSP-70 fraction contained three equally abundant proteins with molecular masses of 70, 65 and 55 kDa. The p70 and p65 proteins are phosphorylated and are themselves O-linked GlcNAc (O-GlcNAc)-modified. The HSP-70 associated into high molecular mass complexes that dissociated in the presence of reductive and chaotropic agents. The lectin(s) present in this complex was (were) able to recognize cytosolic and nuclear ligands, which have been isolated using wheat germ agglutinin affinity chromatography. These ligands are O-GlcNAc glycosylated as demonstrated by [(3)H]galactose incorporation and analysis of the products released by reductive beta-elimination. The isolated lectins specifically recognized ligands present in both the cytosol and the nucleus of human resting lymphocytes. These results demonstrated the existence of endogenous GlcNAc-specific lectins, identified as HSP-70 proteins, which could act as a shuttle for the nucleo-cytoplasmic transport of O-GlcNAc glycoproteins between the cytosol and the nucleus.
Subject(s)
Acetylglucosamine/metabolism , Cell Nucleus/metabolism , Cytosol/metabolism , Heat-Shock Proteins/metabolism , Lectins/chemistry , Liver/metabolism , Animals , Biotinylation , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Glycoproteins/metabolism , Isoelectric Focusing , Ligands , Lymphocytes/metabolism , Phosphorylation , Rats , Rats, Wistar , Sepharose/chemistryABSTRACT
This paper describes structural characterization by NMR, MS and degradative studies of mucin glycans from normal human descending colon obtained freshly at autopsy. The saccharides were mainly based on core 3 (GlcNAcbeta1-3GalNAc). Among the terminal saccharide determinants Sd(a)/Cad-antigen-like structures were prominent, and Lewis x, sialyl Lewis x and sulphated Lewis x were found as minor components, whereas blood group H and A antigenic determinants were absent. The saccharides were markedly different from those of mucins from colon cancers or colon cancer cell lines analysed so far, in which cores 1 and 2 are prominent features, and in which various other terminal determinants have been found, but not Sd(a)/Cad.
Subject(s)
Colon/chemistry , Intestinal Mucosa/chemistry , Mucins/chemistry , Oligosaccharides/chemistry , Autopsy , Carbohydrate Conformation , Carbohydrate Sequence , Histocompatibility Antigens/chemistry , Humans , Lewis X Antigen/chemistry , Mucins/isolation & purification , Nuclear Magnetic Resonance, Biomolecular , Oligosaccharides/isolation & purification , Polysaccharides/chemistry , Sialyl Lewis X Antigen , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
BACKGROUND: The cationic charge of molecules may promote their uptake across epithelia, which are rich in brush border anionic sites. The transport of unsaturated avidin and avidin saturated with a biotinylated compound was investigated across Caco-2 adenocarcinoma cell with fetal enterocyte phenotype. METHODS: The unsaturated avidin and avidin saturated with either biotin or a biotinyl-cobalamin conjugate (biotinyl-Cbl) were iodinated to follow their transport through the cell monolayer. Their apparent permeability coefficient (Papp) and transepithelial pathway were determined and compared to those for control radiolabeled markers [3H]-mannitol, [125I]-beta-lactoglobulin and [57Co]-cobalamin/intrinsic factor (Cbl/IF). RESULTS: The Papp of [125I]-avidin estimated at 2.8 x 10(-7) +/- 0.08 cm/s was close to that for mannitol that uses paracellular pathway. The binding of biotin or biotin conjugate to avidin enhanced its tetrameric conformation. The Papp for [125I]-avidin/biotin and [125I]- avidin/biotinyl-Cbl were respectively increased by 2-fold, compared to that for [125I]-avidin and 4-fold, compared to that for [125I]-beta-lactoglobulin and [54Co]-Cbl/IF. The protein was not accumulated in the cell and was found in intact form in the basolateral side, after its transport across the monolayer. Chloroquine (0.66 micromol/ml) did not significantly decrease the Papp for [125I]-avidin/biotinyl-Cbl. Conversely it decreased by 80% the Papp for Cbl/IF, that uses transepithelial pathway. CONCLUSIONS: Avidin (either saturated or not with biotin and biotinyl-Cbl) was able to cross the monolayer of Caco-2 cell line through a paracellular pathway. This study pointed out the interest for using this protein as a shuttle for increasing the transport rate of biotinylated compounds through fetal epithelial barriers.
Subject(s)
Avidin/metabolism , Intestinal Mucosa/metabolism , Vitamin B 12/metabolism , Biological Transport/drug effects , Biotinylation , Caco-2 Cells , Chloroquine/pharmacology , Edetic Acid/pharmacology , Humans , Intrinsic Factor/metabolism , Lactoglobulins/metabolism , Mannitol/metabolismABSTRACT
A method was developed for the determination of putative lectin activities of cytokines. It involved the immunoblotting measurement of the quantity of these cytokines unbound to a series of different immobilized glycoconjugates and displacement of the bound cytokines with oligosaccharides of known structures. This method allows demonstrating that the following interleukins specifically recognize different oligosaccharide structures in a calcium-independent mechanism: interleukin-1alpha binds to the biantennary disialylated N-glycan completed with two Neu5Acalpha2-3 residues; interleukin-1beta to a GM4 sialylated glycolipid Neu5Acalpha2-3Galbeta1-Cer having very long and unusual long-chain bases; interleukin-4 to the 1,7 intramolecular lactone of N-acetyl-neuraminic acid; interleukin-6 to compounds having N-linked and O-linked HNK-1-like epitopes; and interleukin-7 to the sialyl-Tn antigen. Because the glycan ligands are rare structures in human circulating cells, it is suggested that such activities could be essential for providing specific signaling systems to cells having both the receptors and the oligosaccharide ligands of the interleukin at their cell surface.
Subject(s)
Carbohydrate Metabolism , Interleukin-1/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Interleukin-7/metabolism , Animals , Binding Sites , Bufo bufo , CD57 Antigens/metabolism , Carbohydrate Sequence , Gangliosides/metabolism , Glucuronates/metabolism , Glycoconjugates/metabolism , Humans , Lectins/metabolism , Molecular Sequence Data , Mucins/metabolism , Oligosaccharides/metabolism , Protein Binding , Recombinant Proteins/metabolismABSTRACT
Based on PCR strategies and expression studies, we define the genomic organization of the FUT8b gene. This gene encodes the only known mammalian enzyme transferring fucose in an alpha1-->6 linkage on the asparagine-branched GlcNAc residue of the chitobiose unit of complex N:-glycans. The intron/exon organization of the bovine coding sequence determines five successive functional domains. The first exon encodes a domain homologous to cytoskeleton proteins, the second presents a proline-rich region including a motif XPXPPYXP similar to the peptide ligand of the SH3-domain proteins, the third encodes a gyrase-like domain (an enzyme which can bind nucleotides), and the fourth encodes a peptide sequence homologous to the catalytic domain of proteins transferring sugars. Finally, the last exon encodes a domain homologous to the SH3 conserved motif of the SH2-SH3 protein family. This organization suggests that intramolecular interactions might give a tulip-shaped scaffolding, including the catalytic pocket of the enzyme in the Golgi lumen. Deduced from the published sequence of chromosome 14 (AL109847), the human gene organization of FUT8 seems to be similar to that of bovine FUT8b, although the exon partition is more pronounced (bovine exons 1 and 2 correspond to human exons 1-6). The mosaicism and phylogenetic positions of the alpha6-fucosyltransferase genes are compared with those of other fucosyltransferase genes.
Subject(s)
Evolution, Molecular , Exons/genetics , Fucosyltransferases/genetics , Genes/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , COS Cells , Cattle , DNA/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Fucosyltransferases/chemistry , Gene Expression Regulation, Enzymologic , Humans , Molecular Sequence Data , Phylogeny , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Swine , Tissue DistributionABSTRACT
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ¿M - H(-) ions.
Subject(s)
Fluorescent Dyes , Naphthalenes , Polysaccharides/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Carbohydrate Conformation , Carbohydrate Sequence , Electrophoresis, Polyacrylamide Gel , Molecular Sequence DataABSTRACT
Glycoproteinoses belong to the lysosomal storage disorders group. The common feature of these diseases is the deficiency of a lysosomal protein that is part of glycan catabolism. Most of the lysosomal enzymes involved in the hydrolysis of glycoprotein carbohydrate chains are exo-glycosidases, which stepwise remove terminal monosaccharides. Thus, the deficiency of a single enzyme causes the blockage of the entire pathway and induces a storage of incompletely degraded substances inside the lysosome. Different mutations may be observed in a single disease and in all cases account for the nonexpression of lysosomal glycosidase activity. Different clinical phenotypes generally characterize a specific disorder, which rather must be described as a continuum in severity, suggesting that other biochemical or environmental factors influence the course of the disease. This review provides details on clinical features, genotype-phenotype correlations, enzymology and biochemical storage of four human glycoprotein lysosomal storage disorders, respectively alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency. Moreover, several animal disorders of glycoprotein metabolism have been found and constitute valuable models for the understanding of their human counterparts.
Subject(s)
Congenital Disorders of Glycosylation/enzymology , Fucosidosis/enzymology , Hexosaminidases/deficiency , alpha-Mannosidosis/enzymology , Animals , Carbohydrate Sequence , Disease Models, Animal , Fucosidosis/genetics , Glycoside Hydrolases/deficiency , Humans , Molecular Sequence Data , Phenotype , alpha-Mannosidosis/genetics , alpha-N-AcetylgalactosaminidaseABSTRACT
O-Linked N-Acetylglucosamine (O-GlcNAc) is a major form of post-translational modification found in nuclear and cytoplasmic proteins. Several authors have advanced the hypothesis according to which phosphorylation and O-GlcNAc glycosylation are reciprocally related to one another [1,2]. In order to test this hypothesis we have investigated the effect of a broad spectrum phosphatase inhibitor, okadaic acid (OA), generally used to induce protein hyperphosphorylation, on the GlcNAc content of cellular glycoproteins. We demonstrate that in neuronal cells lines OA decreases the level of O-GlcNAc in both nuclear and cytoplasmic proteins with a greater effect in the nuclear fraction. This phenomenon was demonstrated by the use of three different procedures for the detection of O-GlcNAc in conjunction with a systematic treatment with PNGase F. O-Linked GlcNAc was characterized using respectively lectin staining with WGA, galactosyltransferase labeling and metabolic labeling of cultured cells with [3H]glucosamine. Although the effects on individual proteins varied, a less pronounced effect was observed on HeLa or COS cell total homogenates. When Kelly cells were treated with OA, the major observation was a decrease in O-GlcNAc content of nuclear proteins. The measurement of the UDP-GlcNAc level clearly demonstrates that the decrease on the O-GlcNAc level in the neuroblastoma cell line after treatment with okadaic acid is not a consequence of the modification of the UDP-GlcNAc pool.
Subject(s)
Acetylglucosamine/metabolism , Neuroblastoma/metabolism , Okadaic Acid/pharmacology , Animals , COS Cells , Galactose/metabolism , HeLa Cells , Humans , Neuroblastoma/pathology , Subcellular Fractions/metabolism , Tritium , Tumor Cells, CulturedABSTRACT
Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100% homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab')2gamma specifically bound to psTf. Neutralization by psTf abolished that binding. Although alpha1,3-linked galactose residues (Gal(alpha)1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Gal(alpha)1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.
Subject(s)
Antibodies, Heterophile/immunology , Cytotoxicity, Immunologic , Endothelium, Vascular/immunology , Immunoglobulin G/immunology , Transferrin/immunology , Amino Acid Sequence , Animals , Antigens, Heterophile/genetics , Antigens, Heterophile/immunology , Cell Membrane/immunology , Endothelium, Vascular/pathology , Humans , Molecular Sequence Data , Peptides/genetics , Peptides/immunology , SwineABSTRACT
A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.
Subject(s)
Oligosaccharides/urine , Adult , Carbohydrate Sequence , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes , Gangliosidosis, GM1/urine , Glycogen Storage Disease Type II/urine , Humans , Infant, Newborn , Molecular Sequence Data , Naphthalenes , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , alpha-Mannosidosis/urineABSTRACT
Because of their water-soluble properties, chromogranins (CGs) and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived fragments have been shown to be excreted into the urine in patients with carcinoid tumors and the present study deals with the characterization of the post-translational modifications (phosphorylation and O-glycosylation) located along the largest natural CGA-derived fragment CGA79-439. Using mild proteolysis of peptidic material, high performance liquid chromatography, sequencing, and mass spectrometry analysis, six post-translational modifications were detected along the C-terminal CGA-derived fragment CGA79-439. Three O-linked glycosylation sites were located in the core of the protein on Thr163, Thr165, and Thr233, consisting in di-, tri-, and tetrasaccharides. Three phosphorylation sites were located in the middle and C-terminal domain, on serine residues Ser200, Ser252, and Ser315. These modified sites were compared with sequences of others species and discussed in relation with the post-translational modifications that we have reported previously for bovine CGA.