Trypsin inhibitory activity of bovine fetuin de-O-glycosylated by endo-alpha-N-acetylgalactosaminidase.
(57/897)
The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined. De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-alpha-N-acetylgalactosaminidase, respectively. De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl beta1,3 N-acetylgalactosamine (Gal beta1-->3GalNAc). Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio. In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively. (+info)
Arterial lysosomes and connective tissue in primate atherosclerosis and hypertension.
(58/897)
The cellular events that occur in the vessel wall consequent to changes in endothelial permeability result in the progression of vascular disease, particularly atherosclerosis. Female rhesus monkeys were fed an atherogenic diet or were made hypertensive for 6-8 months; and their vessels were then compared with vessels from control monkeys. Length-defined segments of coronary vessels, the thoracic aorta, and the abdominal aorta showed significant increases in total connective tissue in the atherosclerotic and hypertensive groups; pulmonary vessels did not. The diseased aortic segments had increased levels of two lysosomal enzymes, acid phosphatase and beta-N-acetylglucosaminidase; pulmonary vessels were not diseased and did not show these changes. Coronary vessels from the atherosclerotic and hypertensive groups did not show an increase in enzyme levels on biochemical measurements, but focal accumulations of lysosomes were identified by cytochemical techniques. In atherosclerotic lesions, a doubling of cholesterol and more than a tenfold increase in cholesterol ester were found. These connective tissue and lysosomal changes are early features of primate vascular disease and may result from the accumulation of excessive substrate (cholesterol ester) in the lysosomes of vascular smooth muscle cells. (+info)
Human chorionic gonadotropin beta-subunit affects the folding and glycosylation of alpha-cys mutants.
(59/897)
Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta-subunits is rigid, whereas that of alpha-subunit is flexible and can be molded by a beta-subunit. Previously, we reported that secreted forms of a mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta-subunit. This finding indicated that hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha-subunits was less than 5% of total alpha-subunits. In contrast to free alpha-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants. (+info)
Release of soluble peptidoglycan from growing gonococci: hexaminidase and amidase activities.
(60/897)
Peptidoglycan (PG) turnover in exponentially growing Neisseria gonorrhoeae RD5 type 4 was accompanied by release of soluble PG fragments into the medium. Turnover of the D-[14C]glucosamine-labeled glycan moiety and of the meso-[3H]diaminopimelic acid (DAP)-labeled peptide region occurred at similar rates (ca. 35% per generation). Turnover of D-[14C]alanine-labeled sites within the peptide side chain of PG occurred at roughly twice this rate; no turnover of L-[3H]proline-labeled protein was detected. Gel filtration of supernatants of cultures grown in the presence of labeled DAP, glucosamine, and D-alanine as described above and paper chromatography of hydrolyzed peak fractions revealed four major types of soluble PG. Two of these contained both peptide and glycan moieties and appeared to represent forms of disaccharide peptide monomers and dimers. The other two were (i) a 3H-labeled product lacking 14C and (ii) a 14C-containing product lacking 3H, which were similar in size to that expected for free tetrapeptides and free disaccharides, respectively. Together the appearance of these PG fragments and the concurrent turnover of glycan and peptide regions indicate that both glycan splitting and amidase PG hydrolase activities are involved in the turnover of PG in growing gonococci. If released during gonococcal infections, similar soluble PG fragments might influence the consequences of host-gonococcus interactions. (+info)
Interaction of immunoglobulin glycopeptides with concanavalin A.
(61/897)
A number of intact and partially degraded immunoglobulin glycopeptides have been tested for their ability to interact with concanavalin A. The degraded glycopeptides were prepared by using purified glycosidases to remove sugar residues from the nonreducing ends of the oligosaccharide chains of intact glycopeptides. A quantitative and sensitive assay was devised to measure the potency of the glycopeptides as haptene inhibitors of 125I-concanavalin A binding to guinea pig erythrocytes. The most potent haptene, derived from an immunoglobulin G glycopeptide, had a branched chain oligosaccharide with two GlcNAc (see article) Man (see article) nonreducing termini linked to a mannose residue in the core. The other very potent glycopeptide was an immunoglobulin E high mannose glycopeptide which contained 3 terminal alpha-mannose residues and 1 internal 2-O-mannose residue. Removal of terminal beta-N-acetylglucosamine residues or alpha-mannose residues reduced the activity of these and other glycopeptides as inhibitors of 125I-concanavalin A binding. It was concluded that the ability of these glycopeptides to interact with concanavalin A is dependent on their content of terminal beta-N-acetylglucosamine residues, terminal alpha-mannose residues, and also internal mannose residues substituted on the C-2 hydroxyl group, and that the saccharide combining site of concanavalin A must be able to bind several sugar residues. (+info)
The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis.
(62/897)
The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins. (+info)
Stoichiometry and kinetics of transport vesicle fusion with Golgi membranes.
(63/897)
The in vitro complementation assay established by Rothman and co-workers continues to be an important tool to study intra-Golgi transport. In this study, kinetic modeling is used to identify four main parameters that, together, explain the basic features of an assay that is a modification of the original assay. First, the assay signal depends on the ratio of Golgi membranes to transport intermediates in the assay. Secondly, an inactivation rate describes how the activity of transport intermediates decreases over time. Thirdly, the rate at which transport intermediates irreversibly bind to Golgi membranes is measured independently of membrane fusion, thus allowing a quantitative distinction between these two steps. Fourthly, a single rate constant describes the remaining reactions, which result in membrane fusion. This approach of kinetic modeling of experiments is generally applicable to other in vitro assays of cell biological phenomena, permitting quantitative interpretations and an increased resolution of the experiments. (+info)
Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?
(64/897)
Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological symptoms. Both sibs are homozygous for the E325K mutation, the same genotype that was found in the most severe form of alpha-NAGA deficiency presenting as infantile neuroaxonal dystrophy. Thus, at the age of 7 years the same genotype of alpha-NAGA may present as a 'non-disease' (present healthy case) and can be associated with the vegetative state (the first two patients described with alpha-NAGA deficiency). The clinical heterogeneity among the 11 known individuals with alpha-NAGA deficiency is extreme, with a 'non-disease' (two cases) and infantile neuroaxonal dystrophy (two cases) at the opposite sides of the clinical spectrum. The broad spectrum is completed by a very heterogeneous group of patients with various degrees of epilepsy/behavioural difficulties/psychomotor retardation (four patients) and a mild phenotype in adults without overt neurological manifestations who have angiokeratoma and clear vacuolisation in various cell types (three cases). These observations are difficult to reconcile with a straightforward genotype-phenotype correlation and suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the 11 patients with alpha-NAGA deficiency. (+info)