Amount Of Glycoprotein Research Articles

Verification of the rabies virus glycoprotein lower limit of immunogenicity by serological assay

Rabies lethality is close to 100% and annually 15 million people receive post-exposure prophylaxis. Testing for vaccines against this zoonosis should ensure its quality. A standardized test by the National Institutes of Health (NIH) test, based on mice immunization and challenge, has been used to determine the potency of vaccine lots. It has several disadvantages, the main one being its significant variability. Several in vitro methods like an Enzyme-linked immunosorbent assay (ELISA) have been proposed based on the quality and quantity of glycoprotein (Glptn) of rabies virus, but may also present limitations such as low sensitivity, instability and imprecision. The estimate of immunogenicity based on neutralizing antibody titer (Nab) evaluated by a serological test (ST) such as the Modified Rapid Fluorescent Focus Inhibition Test (mRFFIT), is not yet efectively applied for human vaccine. Nevertheless, a Nab concentration can be used as a predictor of clinical efficacy of this product in vaccinated humans, so, that can be applied in estimating the vaccine potency. The aim of this study was to verify the lower limit of immunogenicity of the viral Glptn content in mice using mRFFIT. The lower Glptn content by ELISA able to induce Nab response was determined. The results were correlated and demonstrated that ST was able to determine the Glptn immunogenicity lower limit. Our findings suggest that a test based on rabies Nabs may represent an additional alternative for the evaluation of rabies vaccines.

Histological Changes of the Mandibular Condyle in the Human Fetus at Early Stages of Gestation

Histochemical studies on the mandibular condyle of the human fetus at gestational ages 12, 14, and 16 weeks were performed. Methods. Histological sections were stained with Schiff’s periodic reaction for glicoproteins, hematoxiline eosine detects mesenchymal tissue and trichhromic stain for collagen. The ANOVA one-way test was used to evaluate the differences during stained zones in the three fetus groups. Results. The percentage of glycoproteins and mesenchymal tissue was denser at 12 weeks. This percentage decreases at 14 weeks and is less at 16 weeks. An increase in the amount of collagen in the studied weeks was observed. The percentages of glycoproteins, mesenchymal tissue, and collagen were significantly different; f = 4373, 9624.8, and 3674, P<0.0001 for the three studied groups. Conclusion. The endochondral bone formation of the mandibular condyle includes modifications of the quantities of glycoproteins, mesenchymal tissue, and collagen.

ＬＣ／ＭＳを用いた糖鎖プロファイリングによるグライコーム解析

Liquid chromatography/mass spectrometry equipped with a graphitized carbon column is useful for the simultaneous analysis of oligosaccharides. By using capillary column and nanoelectrospray ion source, the method can be used for the oligosaccharide profiling of sub microgram quantities of glycoproteins. This oligosaccharide profiling is expected to be a powerful tool for the glycome analysis. We demonstrate a potential application of oligosaccharide profiling in glycomics with two examples, the structural analysis of N-linked oligosaccharides from a gel-separated glycoprotein, and the differential analysis of N-linked oligosaccharides in cells.

An Analytical and Structural Database Provides a Strategy for Sequencing O-Glycans from Microgram Quantities of Glycoproteins

A sensitive, rapid, quantitative strategy has been developed for O-glycan analysis. A structural database has been constructed that currently contains analytical parameters for more than 50 glycans, enabling identification of O-glycans at the subpicomole level. The database contains the structure, molecular weight, and both normal and reversed-phase HPLC elution positions for each glycan. These observed parameters reflect the mass, three-dimensional shape, and hydrophobicity of the glycans and, therefore, provide information relating to linkage and arm specificity as well as monosaccharide composition. Initially the database was constructed by analyzing glycans released by mild hydrazinolysis from bovine serum fetuin, synthetic glycopeptides, human glycophorin A, and serum IgA1. The structures of the fluorescently labeled sugars were determined from a combination of HPLC data, mass spectrometric composition and mass fragmentation data, and exoglycosidase digestions. This approach was then applied to human neutrophil gelatinase B and secretory IgA, where 18 and 25 O-glycans were identified, respectively, and the parameters of these glycans were added to the database. This approach provides a basis for the analysis of subpicomole quantities of O-glycans from normal levels of natural glycoproteins.

Reducing-End Modification of N-Linked Oligosaccharides with Tyrosine

The N-linked oligosaccharides from bovine fetuin were purified using newly developed preparative purification methodology. N-linked oligosaccharides were released from tryptic glycopeptides utilizing N-glycosidase F on the 5-g scale. Selective desialylation with neuraminidase from Clostridium perfringens resulted in the formation of a mono-sialyl-oligosaccharide and asialo-oligosaccharides. The reducing ends of the oligosaccharides were converted to the glycosylamine and reacted with the N-hydroxysuccinimide ester of Boctyrosine. The tyrosinated oligosaccharides were resolved into individual peaks on RP-HPLC and then characterized by proton NMR and FAB-MS. A single asialo-triantennary, an asialo-biantennary, and a mono-sialyl-triantennary oligosaccharide were recovered in good yield. Each product contained a single BocTyr residue attached to the reducing-end GlcNAc residue through a β-glycosylamide linkage. The procedure was utilized to isolate multi-micromole quantities of oligosaccharides from gram quantities of glycoprotein, thus providing a new route to purify large quantities of N-linked oligosaccharides which contain a terminal tyrosine residue. The tyrosinated oligosaccharides are valuable glycoconjugate ligands which contain a chromophore that absorbs at 280 nm and has sufficient hydrophobicity to facilitate RP-HPLC separations. Furthermore, this group can be deprotected by removal of Hoc to reveal a primary amine suitable for further derivatization and can also be radioiodinated for tracking during biological experiments.

The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2-aminoacridone.

A method is described for the electrophoretic analysis of fluorophore-labeled acidic glycans. Various glycoproteins were treated with the enzyme, peptide N-glycosidase F, to release mixtures of asparagine-linked glycans that were then labeled with the fluorophore 2-aminoacridone, and the resulting derivatives electrophoresed in high-density polyacrylamide gels. The acidic glycans were separated with high resolution and distinct, well-resolved fluorescent band patterns were produced. Neutral saccharides did not electrophorese in the system. Information on the structure of the glycans was revealed by the changes in the band patterns observed after the mixtures of the glycan fluorophore derivatives were treated with either of the enzymes neuraminidase or beta-galactosidase. Quantities of glycoprotein as little as 10 micrograms were analysed without difficulty. The electrofluorograms were viewed using a digital imaging system based on a cooled charge-coupled device. The method was also demonstrated using purified acidic glycans of known structure. The method was easy to use, enabled the sensitive analysis of multiple samples in parallel and should be a useful addition to the range of glycan analytical techniques.

Baculovirus-expressed herpes simplex virus type 2 glycoprotein D is immunogenic and protective against lethal HSV challenge

Herpes simplex virus type 2 glycoprotein D (gD2) was cloned and expressed in the baculovirus— Spodoptera frugiperda system. Milligram quantities of glycoprotein were recovered from suspension culture and subjected to purification by ion-exchange and immunoaffinity chromatography. The resultant purified gD existed as a homogeneous 57 500 M W monomeric species demonstrating reactivity with anti-gD monoclonal antibodies including those directed at a non-sequential neutralizing epitope of gD. Immunization of Balb/c mice with doses of 0.1–10.0μg of AlPO 4-absorbed gD resulted in elicitation of humoral and cellular responses to both HSV1 and HSV2 as well as to purified gD1 and gD2. Immunized mice receiving an infectious dose of 1 × 10 6 p.f.u. of HSV2 via the footpad route were significantly protected against infection at all doses tested when compared with unimmunized AlPO 4 and uninoculated control animals.

Developmental heterogeneity of mesenchymal glycosaminoglycans (GAG) distribution in chick embryo lung anlagen.

The presence and distribution of mesenchymal components in the extracellular matrix during lung development in the chick embryo (from 5 1/2/6 to 18 incubation days) has been examined histochemically. Attention is focused mainly on glycosaminoglycans (GAG). Morphological reconstructions show three main stages: first (5 1/2/6-8 days), formation of 2nd-order branching; second (9-12 days), proliferation of parabronchi and third (from 13th day on), formation of air capillaries. In the first phase, hyaluronic acid (HA) prevails around the mesobronchus, but chondroitin sulfate (CS) dominates the 2nd-order branches. Basement membranes of 2nd-order branches are strongly positive for sulphated GAG. In the second phase, CSA increases in the ground substance of mesenchyme. This increase is irregular, being smaller in older areas (mesobronchus, branches of 2nd order) and larger in the more recent parabronchi, which extend into the lateral and dorsal areas of the rudiment. An increase in both sulfated GAG and glycoprotein (GP) occurs in basement membranes. In the third phase, GAGs are uniformly distributed in the mesenchymal septa and around the interlobular vascular network. This concentration decreases while the GP concentration increases. Basement membranes around every branch of the 1st, 2nd, and 3rd orders possess large quantities of GP. Mesenchymal GAG occurs in every stage of lung development, temporally correlating with the morphogenesis and differentiation of epithelium. Our results provide necessary information, which has not been available so far. Experimental studies specifically designed to clarify the developmental significance of such a heterogeneous distribution may be interpreted in the light of this information.

Changes in exudate composition during the development of the ovary glands in Aptenia cordifolia

Summary Constituent monomers of the polysaccharide fraction of ovary exudates obtained from flowering Aptenia cordifolia-plants were separated (after hydrolysis) using anion exchange chromatography of their borate complexes and were analysed subsequently by colorimetric detection. The polysaccharide composition of the exudates shows a demonstrable correlation to the developmental stages of the ovary glands. There is no significant variation in polysaccharide composition during the pre-degenerative stages (in total about 10 days), with exception of a moderate quantitative increase of glucose, xylose, galactose, arabinose, glucosamin, and galactosamin and of a moderate decrease of galacturonic and glucuronic acids. In contrast, during the transition period from the pre-degenerative to the degenerative stage, there is a dramatic quantitative increase of xylose, galactose and arabinose; a strong decrease of glucose and glucosamin; and a complete disappearance of the uronic acids. This significant change in the polysaccharide composition is probably based in large part upon an enzymatic digestion of the gland cell walls. From the polysaccharide composition of the different phases can be concluded that pectic substances are initially secreted. Subsequently, other non-cellulosic polysaccharides which contain a large portion of glucose, galactose and arabinose are secreted, as well as small quantities of glycoproteins. During the degeneration of the ovary glands, presumably xylan-based hemicelluloses are accumulated in the ovary exudate. By comparison of the polysaccharide composition with the gland ultrastructure at different stages, it is shown that the transport and the extrusion of pectic substances, of glycoproteins, and of other non-cellulosic polysaccharides probably proceeds via the Golgi apparatus. Also demonstrated is that the hemicelluloses are contributed to the exudate during cell wall digestion. The identification of gentiobiose and melibiose indicates the existence of gentianose and raffinose, or of their oligomers in the polysaccharide fraction.

The effect of ascorbic acid on the nature and production of collagen and elastin by rat smooth-muscle cells

1. The effects of various concentrations of ascorbic acid on the quality and quantity of the insoluble extracellular matrices produced by two strains of cultured rat smooth-muscle cells were studied. 2. Ascorbic acid was necessary for the appearance of insoluble collagen in the extracellular matrix. 3. Secretion of soluble collagen continued in the absence of ascorbic acid, but this soluble collagen was markedly underhydroxylated. 4. The amount of insoluble collagen present in the matrix was directly related to the ascorbic acid concentration. 5. The insoluble collagen that appeared in the matrix under conditions where ascorbic acid was limiting was no more than 7% underhydroxylated. 6. In contrast, the amount of insoluble elastin produced was inversely proportional to the ascorbic acid concentration. 7. The elastin produced in the absence of ascorbic acid had the expected amino acid composition, but hydroxyproline was absent. 8. The hydroxyproline content of elastin was also directly dependent on the ascorbic acid concentration. 9. Ascorbic acid had variable effects on the quantity of glycoprotein(s) present in the matrix. 10. The appearance of insoluble collagen in the extracellular matrices produced by cultured human fibroblasts and calf endothelial cells was also completely dependent on the presence of ascorbic acid.

Simple inexpensive apparatus for the rapid DEAE-cellulose chromatography of small quantities of glycoproteins

Simple inexpensive apparatus for the rapid DEAE-cellulose chromatography of small quantities of glycoproteins

Isolation of an abnormally phosphorylated erythrocyte membrane band 3 glycoprotein from patients with myotonic muscular dystrophy

A fraction of erythrocyte Band 3 (Mr, 93,000) glycoprotein that demonstrates decreased autophosphorylation in membranes from myotonic muscular dystrophy patients is demonstrated. Sequential affinity chromatography of Triton X-100 solubilized erythrocyte membrane proteins separated three specifically retained glycoprotein fractions on a Ricin Communis I-Sepharose 4B column. One fraction contains a portion of the major sialoglycoprotein (apparent Mr, 78,000) and is specifically eluted from the column by 10 mM NaCl and 100 mM D-galactose (10/100). The two other glycoprotein fractions are eluted by 100 mM NaCl, 10 mM D-galactose (100/10) and 100 mM NaCl, 100 mM D-galactose (100/100). The composition of both fractions contains greater than 95% Band 3 (apparent Mr, 93,000 glycoprotein. The quantities of glycoprotein in each fraction obtained from erythrocytes of myotonic dystrophy patients did not differ from the quantities obtained from control erythrocytes. Following endogenous protein kinase incubations of ghosts with [gamma-32P]ATP, the specific [32P] phosphorylation of the 10/100 and 100/10 fractions are identical. The 100/100 fraction, which makes up approximately 3% of the total erythrocyte membrane protein, demonstrates a different pattern for myotonic dystrophy patients; specific phosphorylation was reduced by 50% relative to activity in control experiments. These findings are consistent with previous experiments that demonstrated decreased autophosphorylation of the glycoprotein portion of Band 3 (Roses & Appel, 1975, J. Membrane Biol 20:51) and are consistent with the autosomal dominant mode of inheritance in this disease.