Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Toxocara canis infective stage larvae continually produce excretory-secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive posttranslational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.
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PMID:Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae. 145 27

Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo-beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N-glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56-53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor.
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PMID:Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins. 171 81

A glycoprotein reactive with antibodies against corneal keratan sulfate proteoglycan (KSPG) was purified 300-fold from extracts of bovine aorta using DEAE ion-exchange, gel-filtration, hydrophobic interaction, and reverse-phase chromatographic separations. The intact glycoprotein was 70-80 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Deglycosylation with endo-beta-galactosidase and N-glycanase reduced the size to 48 and 37 kDa, respectively, similar to the large isoforms of corneal KSPG. N-terminal amino acid sequence of the arterial KSPG was identical with lumican, the 37B isoform of corneal KSPG, and the arterial KSPG reacted with an antibody to synthetic peptide duplicating this sequence. Arterial KSPG and corneal lumican displayed identical tryptic maps. Arterial lumican contains fucose and mannose in amounts similar to corneal KSPG, but galactose, glucosamine, and sulfate were reduced compared to KSPG from cornea. Treatment of arterial lumican with endo-beta-galactosidase released 8-9 mol of glucosamine and galactose per mol of protein as oligosaccharides. These eluted as neutral, nonsulfated oligosaccharides on high pH anion-exchange chromatography. The size of arterial lumican was not altered by glycosidases having specificity for sulfated keratan sulfate, nor was the charge of the lumican molecule altered by digestion with endo-beta-galactosidase. These data show arterial lumican to be a glycoprotein containing unsulfated lactosaminoglycan chains. Abundance of low sulfate lumican in many tissues indicates that this protein occurs predominantly as a glycoprotein rather than as the more widely studied, highly sulfated proteoglycan present in the cornea.
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PMID:Arterial lumican. Properties of a corneal-type keratan sulfate proteoglycan from bovine aorta. 176 72

Small proteoglycans were dissociatively extracted from normal human thoracic aorta with 4 M guanidine hydrochloride containing protease inhibitors, and purified by Sepharose CL-4B chromatography, dissociative cesium chloride density gradient centrifugation, and diethylaminoethyl cellulose chromatography. The intact proteoglycans migrated in the 270,000-340,000 range on 4-20% sodium dodecyl sulfate polyacrylamide gradient gels. Core proteins prepared following digestion of the intact proteoglycan monomer with chondroitinase ABC consisted of a major Coomassie blue-staining protein band of 50,000 along with a minor band of 44,000. Subsequent studies using endoglycosidases H, F, and N-glycanase demonstrated that mainly complex type N-linked glycans were present on the 50,000 cores while the 44,000 cores appeared to be devoid of N-linked glycans. Western blotting demonstrated that both of these cores were recognized by the monoclonal antibody 2-B-6, indicating the presence of the terminal 4-sulfated unsaturated disaccharide (delta Di-4S) remaining on the linkage region following chondroitinase ABC digestion. In contrast, a diffuse pattern of delta Di-4S epitopes ranging from 50,000 to approximately 60,000 was observed following chondroitinase AC II digestion of the dermatan sulfate proteoglycan, suggesting the presence of iduronate residues in close proximity to the glycosaminoglycan-linkage region. Conversely, the large chondroitin sulfate-proteoglycan core proteins from aorta (Mr 200,000-400,000) did not react with either monoclonal antibody 3-B-3 (recognizing the terminal delta DI-6S) or 2-B-6 following chondroitinase AC II digestion, although both delta DI-4S and delta DI-6S were present on these cores following chondroitinase ABC digestion. Additional studies using antisera against synthetic peptides derived from sequences of the core proteins of human bone small PG I and PG II indicated the presence of both gene products in PG isolated from human thoracic aorta. The Mr approximately 44,000 and 50,000 core proteins represent small PG I type cores while a closely spaced doublet (Mr 49,000 and 51,000) represented small PG II type cores. The results demonstrate that the core proteins of dermatan sulfate proteoglycan from human aorta are heterogeneous in primary structure and in the content of N-linked glycans.
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PMID:Heterogeneity in glycosylation of dermatan sulfate proteoglycan core proteins isolated from human aorta. 212 40

Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family.
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PMID:Isoforms of corneal keratan sulfate proteoglycan. 213 77

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).
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PMID:Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody. 244 76

The low molecular weight proteoglycan fraction extracted from articular discs with 4 M guanidinium chloride was found to consist predominantly of an iduronate-rich dermatan sulphate proteoglycan, together with chondroitin sulphate-containing material. The dermatan sulphate proteoglycan was purified by ion-exchange and gel-filtration chromatography and its core protein isolated after digestion with chondroitinase ABC. The amino acid composition and pattern of cyanogen bromide peptides obtained from this core were closely similar to those of the protein core of bovine skin proteodermatan sulphate. Four monoclonal antibodies raised against bovine skin proteodermatan sulphate also reacted with the disc protein core and its cyanogen bromide peptides. Results of digestion with glycopeptidase F demonstrated the presence of three N-linked oligosaccharides. The combined size of these oligosaccharides appeared to be somewhat less than the size of those on skin proteodermatan sulphate. The glycosaminoglycan chain released by digestion with cathepsin C had a higher molecular weight than that from skin. These differences in glycosylated structures may be responsible for the different effects on collagen fibrillogenesis in vitro; whereas skin proteodermatan sulphate only reduced the rate of fibril growth, disc dermatan sulphate proteoglycan also increased the length of the lag-phase and the final opacity.
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PMID:Proteoglycans of the articular disc of the bovine temporomandibular joint. II. Low molecular weight dermatan sulphate proteoglycan. 279 47

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.
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PMID:The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase. 405 10

A large brain-specific chondroitin sulfate proteoglycan, identified with monoclonal antibody 6B4 (6B4 proteoglycan/phosphacan), was isolated from rat brain. Soluble proteoglycans in the phosphate-buffered saline extract from 20-day-old rat whole brain were fractionated by anion exchange chromatography and CsCl density gradient centrifugation. 6B4 proteoglycan was further purified by gel filtration and additional ion exchange chromatography. The molecular mass of 6B4 proteoglycan shifted from 800 to 300 x 10(3) mol. wt after chondroitinase ABC digestion. The core protein was substituted with chondroitin sulfate chains with an average molecular weight of 21,000, keratan sulfate and HNK-1 carbohydrates. Glycosidase digestion of 6B4 proteoglycan with O-glycanase, N-glycanase, endo-beta-galactosidase, or keratanase did not remove the HNK-1 epitopes. The expression of 6B4 proteoglycan was developmentally regulated in the rat cerebral cortex; appearing first at embryonic day 14, peaking at postnatal day 0, and persisting throughout adulthood at a lower level. Immunohistochemical analysis indicated that 6B4 proteoglycan was distributed along the radial glial fibers and on the migrating neurons in the embryonal rar cerebrum. The radial glial fibers were stained intensely all along their length, but the neurons in the cortical plate were not stained in contrast to the moderate staining of the migrating neurons in the intermediate zone and the subplate. From postnatal day 5 to postnatal day 20, 6B4 proteoglycan was present throughout the cortex. After postnatal day 30, staining of the neuropil was weakened, and the expression of 6B4 proteoglycan was restricted around subsets of neurons. The positive neurons were mostly non-pyramidal cells (> 95%) and were relatively concentrated in layers IV and VI of the primary somatosensory cortex. Immunohistochemical analysis of the dissociated cortical neurons indicated that 6B4 proteoglycan was distributed on the cell bodies and neurites. 6B4 proteoglycan strikingly promoted neurite extension of cortical neurons from embryonic day-16 rat embryos when coated on coverslips as a substrate. 6B4 proteoglycan is a brain-specific chondroitin sulfate proteoglycan which carries keratan sulfate and HNK-1 carbohydrates. The spatiotemporal expression profile and effects on the dissociated cerebral neurons suggest that 6B4 proteoglycan plays important roles in the migration and differentiation of neurons in the immature cortex, and also in the maintenance of subsets of neurons in the mature cortex.
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PMID:Purification, characterization and developmental expression of a brain-specific chondroitin sulfate proteoglycan, 6B4 proteoglycan/phosphacan. 747 3

Immunological and biochemical characteristics of a 100,000 MW biglycan-like haemopoietic factor, purified from thymic myoid cells 871207B, were studied to distinguish them from macrophage colony-stimulating factor (M-CSF), which they resemble in activity and biochemical properties. Rabbit antibody raised against a synthetic peptide fragment (J-1) designed from amino acid sequences specific to the 100,000 MW factor responded to 871207B cells, the conditioned medium of 871207B, and capillary-like structures in the thymus, but not to M-CSF producer L-929 cells or the conditioned medium of L-929 cells. In contrast, M-CSF epitope was detected in L-929 cells and the conditioned medium cells but not in 871207B cells or the conditioned medium, even after enzymatic digestion of glycosaminoglycan chains. Treatment of the 100,000 MW factor with chondroitinase ABC and AC produced a 50,000 MW component. Digestion of this product with N-glycanase resulted in a 40,000 MW protein component. These results suggest that the 100,000 MW factor is a proteoglycan consisting of a core protein with an apparent molecular mass of 40,000 MW, a 50,000 MW chondroitin sulphate chain and 10,000 MW N-linked oligosaccharide chains. A small amount of a 40,000 MW monocytic cell growth activity was also found in the 871207B cell-conditioned medium. An enzymatically obtained 40,000 MW factor, the conditioned medium 40,000 MW factor, and the 100,000 MW factor were specifically eluated from an anti-J-1 IgG-immobilized affinity column with monocytic cell growth activity, suggesting that the biological activity resides in the 40,000 MW core protein. The 100,000 MW factor induced the proliferation and differentiation of monocytic lineage cells from a variety of sources, such as bone marrow cells, peritoneal exudated cells and brain microglia cells.
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PMID:Immunological and biochemical characterization of biglycan-like haemopoietic factor. 754 45


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