Sialic Acid-rich Glycoproteins Research Articles

Sialic acid concentration changes in acute lymphocytic leukemia and acute mylogenous leukemia patients after chemotherapy treatment

Sialic acid—rich glycoprotein binds selectively in human and other organisms cancer cells that can metastasize .This helps these late stage cancer cells enter the blood stream. Biological markers can be used to monitor cancer elevation in the serum. Total sialic acid (TSA) concentration was observed in a number of cancer types, the chemotherapy is used as the only cancer treatment. The present study included 5l patients age (4—69 year) with leukemia, they were included 13 patients with acute lymphoblastic leukemia (ALL), 13 with acute myelogenous leukemia (AML) and 25 with chronic lymphocytic leukemia (CLL). This study included also 50 healthy subjects as normal control, all the patients had chemotherapy. The study included measurements of fasting serum total sialic acid (STSA) level, which is significantly increased in post sampling of leukemia patients compared with normal control subjects. A higher significant difference of STSA at the first week during chemotherapy in the three type of the leukemia patients, also there were significant difference of STSA level at the third week during chemotherapy in the ALL and AML patients. On the other hand, there were no significant differences in STSA level after five weeks of chemotherapy treatment for ALL and AML patients. According the duration of chemotherapy dose there were significant differences in STSA level between the first dose week and third dose of chemotherapy. Also, there were significant differences between the first weeks and 5th weeks of chemotherapy treatment for ALL and AML patients.

An impedimetric biosensor for the diagnosis of renal cell carcinoma based on the interaction between 3-aminophenyl boronic acid and sialic acid

Renal cell carcinoma (RCC) often expresses a high density of sialic acid-rich glycoproteins which helps these late-stage cancer cells to enter the blood stream or urine. Blood diagnosis is a complex and time-consuming process. In this study, we developed a facile and non-invasive electrochemical cytosensor for early detection of RCC in urine samples based on specific recognition by 3-aminophenyl boronic acid (APBA). Polypyrrole (PPy) and bovine serum albumin (BSA)-incorporated Ag submicron particles (Ag@BSA) were co-deposited on a gold electrode (GE) to take advantages of the excellent properties of these biomaterials, including good biocompatibility, low cytotoxicity and excellent electro-conductivity. To further increase the biosensor's sensitivity, APBA molecules were integrated to recognize sialic acid (SA) on the cell surface. Under optimal conditions, the impedimetric cytosensor exhibited a good linear relationship with the logarithm of the cell concentration from 17 to 1.7×106 cellsmL−1, and the low detection limit was 6 cellsmL−1 (S/N=3). Therefore, the electrochemical impedimetric biosensor offers a potential approach to bedside rapid detection of RCC in clinical applications.

Evaluation of Sialic acid Levels in Patients with Type 2 Diabetes Mellitus

Sialic acid-rich glycoprotein is found mainly in cell membranes, and elevated levels may indicate excessive cell membrane damage, but more specifically for cells of vascular tissue. Serum total Sialic acid is a marker of the acute phase response. Elevated levels have also been associated with cardiovascular disease especially in Type 2 diabetic subjects. The relation between type2 diabetes mellitus compared to the nondiabetics is not clear. The current study represents association between Sialic acid and cardiovascular disease is stronger in diabetics than in non-diabetic subjects.

Preparation of Functionalized Alkynyl Magnetic Microspheres for the Selective Enrichment of Cell Glycoproteins Based on Click Chemistry

Functionalized alkynyl polyvinyl alcohol magnetic microspheres (PVA MMs) were developed for the specific enrichment of sialic acid-rich glycoproteins by click chemistry. The capture capability for proteins was evaluated through a novel dual-labeled bovine serum albumin (BSA) that utilizes fluorescence resonance energy transfer (FRET). The PVA MM parameters, including the size and coverage of functionalized groups, were optimized by response surface methodology. The optimal parameters obtained were 1.25-6.31 μm in size and 48.53-73.05% in coverage. Then, the optimal PVA MMs were synthesized, and the morphology and surface chemical properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). To capture glycoproteins from the cell surface, a bioorthogonal chemical method was applied to metabolically label them with an azide group. The functionalized alkynyl PVA MMs showed a high specificity and strong binding capability for glycoproteins through a [3 + 2] cycloaddition reaction. The results indicated that the functionalized alkynyl PVA MMs could be applied to the enrichment of cell glycoproteins, and the merits of the MMs suggested an attractive and potential way to facilitate glycoprotein research.

Extracellular matrix proteins of dentine.

Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell types. The transient expression in pre-ameloblasts suggests a role of epithelial-mesenchymal interactions in the formation of the tooth.

Interaction of vitronectin with Haemophilus influenzae.

Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.

Interaction of vitronectin with Haemophilus influenzae

Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.

Interaction between Aeromonas veronii and epithelial cells of spotted sand bass (Paralabrax maculatofasciatus) in culture.

An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms). Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease. Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer. veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates. The three primary-culture cells are susceptible to Aer. veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial cells. Aer. veronii adherence was dependent on bacterial load and incubation time. The effect of glycoconjugates on Aer. veronii adhesion was investigated by pre-incubating Aer. veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides. In addition, the participation of a 48-kDa Aer. veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer. veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer. veronii MCBP. Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer. veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.

Serum sialic acid and sialoglycoproteins in asymptomatic carotid artery atherosclerosis

Serum total sialic acid (S-TSA) is a recently identified risk marker for atherosclerosis and cardiovascular mortality. The purpose of this study was to evaluate the influence of three sialic acid rich glycoproteins (orosomucoid, haptoglobin, and α1-antitrypsin) on the relationship between S-TSA and carotid atherosclerosis. The mean S-TSA was 0.045 g/l higher among cases than controls ( P<0.001) in 310 45–64 year-old male and female pairs of carotid atherosclerosis cases and disease-free controls from the Atherosclerosis Risk in Communities (ARIC) Study. Also mean serum levels of the glycoproteins were significantly higher in cases compared to controls. In a conditional multiple logistic regression model with the glycoproteins as independent variables, orosomucoid was correlated most strongly with case control status. However, when incorporated into the mathematical model, S-TSA not only contributed additional information as to the risk of atherosclerosis; none of the three glycoproteins contributed further once S-TSA had been accounted for. Thus, some other source of serum sialic acid or variations in the degree of sialylation of glycoproteins may be essential for understanding the relation between S-TSA and atherosclerosis.

The hatching gland cells of trout embryos: characterisation of N- and O-linked oligosaccharides.

A histochemical, light and electron microscopy study of the hatching gland cells (HGCs) in incubated 50-d-old trout embryos is reported. The distribution of carbohydrate residues in the glycoconjugates of these cells was studied by means of a battery of 13 different lectins conjugated with horseradish peroxidase (PNA, ConA, LCA, WGA, SBA, UEA-I, HPA, DBA) or digoxigenin (DSA, MAA, AAA, SNA, GNA). Identification of N- and O-linked oligosaccharides in HGCs was performed by application of both chemical and enzymatic treatments. Present results suggest that HGCs are seromucous cells which store both high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), and that their cytoplasmic granules, endoplasmic reticulum and Golgi complex contain additional sialic acid-rich glycoproteins. The negative charge of these glycoproteins might be responsible for the rapid expansion of mucin to form a highly hydrated gel, which would facilite the action of these enzymes in programmed cell death and might play a major role during the morphogenic events.

The hatching gland cells of trout embryos: characterisation of N- and O-linked oligosaccharides

A histochemical, light and electron microscopy study of the hatching gland cells (HGCs) in incubated 50-d-old trout embryos is reported. The distribution of carbohydrate residues in the glycoconjugates of these cells was studied by means of a battery of 13 different lectins conjugated with horseradish peroxidase (PNA, ConA, LCA, WGA, SBA, UEA-I, HPA, DBA) or digoxigenin (DSA, MAA, AAA, SNA, GNA). Identification of N- and O-linked oligosaccharides in HGCs was performed by application of both chemical and enzymatic treatments. Present results suggest that HGCs are seromucous cells which store both high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), and that their cytoplasmic granules, endoplasmic reticulum and Golgi complex contain additional sialic acid-rich glycoproteins. The negative charge of these glycoproteins might be responsible for the rapid expansion of mucin to form a highly hydrated gel, which would facilite the action of these enzymes in programmed cell death and might play a major role during the morphogenic events.

Blood group antigens defined by the amino acid sequences of red cell surface proteins

The antigens of 18 blood group systems are expressed on proteins that are intrinsic to the red cell. The proteins which carry the antigens of these systems have been identified and primary sequence information is available for all but two (SC, DO). Several different functional groups are evident. Antigens of the DI, CO, RH, XK and JK systems are located on proteins which have the structure of membrane transport proteins. The FY antigens mark a cytokine receptor. The IN, LW, XG antigens are associated with molecules which have adhesion functions and the LU glycoprotein also has a structure which suggests a role in adhesion. YT and KEL antigens are located on cell surface enzymes and the CR and KN antigen on molecules involved in complement regulation. Finally, the MN and GE antigens are located on sialic acid-rich glycoproteins (glycophorins A, B and C/D respectively), a group of molecules which do not, as yet, have a clearly defined function. The molecular basis of antigens in several blood group systems have been defined and shown to depend upon the amino acid sequence.

Differential Reactivity of Two Types of N-Glycolylneuraminic Acid Dimers toward Enzymatic and Nonenzymatic Hydrolysis of Their Interketosidic Linkages

The kinetics of acid- and sialidase-catalyzed hydrolysis of the interketosidic linkages of two different disialic acids, Neu5Gcα2→5-OglycolylNeu5Gc and Neu5Gcα2→8Neu5Gc, were studied. The former sequence was recently identified in the polysialic acid chains of a sialic acid-rich glycoprotein isolated from the egg jelly coat of two different species of sea urchins, and the latter was previously found in the cortical alveolar-derived polysialoglycoprotein from rainbow trout eggs. At pH values < 3.8, the rate of hydrolysis of Neu5Gcα2→5-OglycolylNeu5Gc was greater than that of Neu5Gcα2→8Neu5Gc. Paradoxically, however, Neu5Gcα2→5-OglycolylNeu5Gc was more stable than Neu5Gcα2→8Neu5Gc at pH values > 3.8. These findings indicate a greater contribution of intramolecular general acid catalysis to the lability of the α2→5-ketosidic linkage. Neu5Gcα2→5-OglycolylNeu5Gc was a poor substrate for Arthrobacter ureafaciens, Clostridium perfringens, and Vibrio cholerae sialidases, in contrast to Neu5Gcα2→8Neu5Gc. Neu5Gcα2→5-OglycolylNeu5Gc was essentially resistant to hydrolysis by A. ureafaciens sialidase.

Identification of polysialic acid-containing glycoprotein in the jelly coat of sea urchin eggs. Occurrence of a novel type of polysialic acid structure.

Sea urchin eggs are surrounded by a gelatinous layer (called the jelly coat) that consists of mixture of fucoserich polysaccharides and sialic acid-rich glycoproteins. Chemical and 500 MHz 1H NMR spectroscopic studies revealed for the first time the presence of a novel polysialic acid (polySia) structure in the jelly coat glycoproteins isolated from Hemicentrotus pulcherrimus (designated polySia-gp(H)). The structure of the polySia chains was thoroughly characterized as (-->5-Oglycolyl-Neu5Gc alpha 2-->)n, where n ranges from 4 to more than 40 Neu5Gc residues. The polyNeu5Gc chains were attached to core oligosaccharides that were O-glycosidically linked to threonine residues on a core polypeptide. Each polypeptide contained about 17 O-linked polysialylglycan chains. The apparent molecular weight of polySia-gp(H) was 180,000. The expression of this new polySia structure in place of alpha 2-->8-linked polySia is the main structural feature that distinguishes polySia-gp from other known polysialylated glycoproteins. The (-->5-Ogly-colyl-Neu5Gc alpha 2-->)n chains were resistant to exo- and endosialidases from Arthrobacter ureafaciens and bacteriophage K1F, respectively. Discovery of these (-->5-Ogly-colyl-Neu5Gc alpha 2-->)n chains adds a new class of naturally occurring polySia to the structurally diverse family of polysialylated glycoproteins. The structure of a poly-Sia-gp from a different sea urchin species, Stronglyocentrotus purpuratus (designated polySia-gp(S)), was also determined to ascertain if there were any species-specific differences. The 500 MHz 1H NMR spectra of the two polySia-gps were identical, indicating that at this level of molecular detail the structures were the same. The molecular weight of polySiagp(S) was larger, however (250,000), and it contained about 25 polySia chains O-glycosidically linked to both threonine (two-thirds) and serine (one-third) residues.

Binding of vitronectin and plasminogen to Helicobacter pylori.

We have studied how some extracellular matrix proteins, fibronectin, fibrinogen, collagen type I and type IV, plasminogen and vitronectin bind to Helicobacter pylori. Radiolabelled vitronectin and plasminogen bound to the haemagglutinating H. pylori strain 17874 at a high level (53% and 32%, respectively), type IV collagen showed an intermediate level of binding (16%), while binding by 125I-labelled fibrinogen, fibronectin and collagen type I remained at a low level (5-7%). Both 125I-vitronectin and plasminogen showed a dose-dependent binding to cells of H. pylori 17874. Plasminogen binding by this strain was specific since the binding was inhibited by nonlabelled plasminogen, but not by highly glycosylated glycoproteins such as fetuin and orosomucoid or by a variety of monosaccharides. We have previously shown that 125I-vitronectin shows a specific and saturable binding to H. pylori 17874, and that sialic acid-rich glycoproteins such as fetuin and orosomucoid drastically reduced binding. We now report that a simultaneous incubation of 125I-vitronectin and 125I-plasminogen with cells of H. pylori 17874 showed a total binding approximately similar to the level of binding when either 125I-plasminogen, or 125I-vitronectin only were incubated with the bacterial cells. Nonlabelled vitronectin inhibited the binding of 125I-plasminogen by H. pylori, but nonlabelled plasminogen had no effect on the binding of 125I-vitronectin. Our findings suggest that there are different but probably closely localized binding sites for vitronectin and plasminogen on H. pylori 17874.

Serum concentrations of total sialic acid and sialoglycoproteins in relation to coronary heart disease risk markers

This study investigated the relationship between serum sialic acid concentration and cardiovascular mortality. Correlations were determined between lifestyle-related coronary heart disease risk markers (cigarette consumption, alcohol consumption, and leisure time physical activity), biological risk markers (apolipoprotein Al, apolipoprotein B, lipoprotein(a), and diastolic blood pressure) on the one hand and the concentration of sialic acid as well as sialic acid-rich acute phase proteins (orosomucoid, haptoglobin, and al-antitrypsin) on the other. A total of 145 men aged 21–46 years and with a C-reactive protein concentration below 5 mg/l were included. Total sialic acid concentration correlated significantly with apolipoprotein B ( r = 0.48), number of cigarettes smoked daily ( r = 0.32), and leisure time physical activity ( r = −0.23) after adjustment for age and other cardiovascular risk markers. No significant partial correlations were found between serum total sialic acid concentration on the one hand and alcohol consumption, apolipoprotein Al, lipoprotein(a), and diastolic blood pressure on the other. Of the sialic acid-rich glycoproteins, orosomucoid correlated with apolipoprotein B ( r = 0.38), haptoglobin with cigarette consumption ( r = 0.35) and leisure time physical activity ( r = −0.26) and al-antitrypsin with cigarette consumption ( r = 0.18), leisure time physical activity ( r = 0.17), alcohol consumption ( r = −0.18), and apolipoprotein B ( r = 0.21) after adjustment for age and other cardiovascular risk markers.

Particle agglutination assays to identify fibronectin and collagen cell surface receptors and lectins in Aeromonas and Vibrio species

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.

Inhibition of herpes simplex virus infection by tannins and related compounds

Several chemically defined plant extracts were investigated for their antiviral action on herpes simplex virus (HSV-1, HSV-2)-infected African green monkey kidney cells and human adenocarcinoma cells, using a plaque formation assay. Among them, the monomeric hydrolyzable tannins, oligomeric ellagitannins and condensed tannins, having galloyl groups or hexahydroxydiphenoyl groups, had the most potent anti-HSV activity. Their 50% effective doses (0.03–0.1 μg/ml) were by two-three orders of magnitude lower than their 50% cytotoxic doses (> 10 μg/ml). On the other hand, gallic acid, neutral polysaccharides, chemically modified ( N, N-dimethylaminoethyl-, carboxymethyl-, and sulfated-) glucans, sialic acid-rich glycoproteins, and uronic acid-rich pine cone polysaccharide showed little or no activity. Using radiolabeled virus particles, we demonstrated that the anti-HSV effect of the tannins is due to inhibition of virus adsorption to the cells.

Proteins of muscle subcellular fractions in Duchenne progressive muscular dystrophy stained with "stains-all" cationic carbocyanine dye and with Coomassie Blue.

The protein compositions of subcellular fractions of muscle obtained from 17 Duchenne dystrophy patients, 15 disease controls (10 different primary myopathies, 5 spinal muscular atrophy patients), and 10 normals were examined by polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue and with "Stains-all," which stains calcium-binding proteins, sialic acid-rich glycoproteins, and phosphoproteins. In muscle membrane fractions of Duchenne dystrophy patients there was a marked reduction in the concentrations of calsequestrin and a 39 kDa protein that stained blue with "Stains-all." There were changes in the proteins of all subcellular fractions of Duchenne's patients; some of these changes appear to be specific for Duchenne dystrophy (DD). There was no apparent correlation between the protein changes observed on acrylamide gels and the age of the patients, the duration of the disease, the degree of disability, or activity of creatine kinase. A decreased level of calsequestrin in DD sarcoplasmic reticulum may contribute to an increased level of free calcium seen in muscle from these patients.

Pathogenetic Implications of Ultrastructural Findings inCampylobacter pyloriRelated Gastroduodenal Disease

There is now substantial evidence that Campylobacter pylori (Cp) is able to colonize the gastroduodenal mucosa and is responsible for active chronic gastritis, its role in duodenitis, gastric ulceration and duodenal ulceration is still under debate. Cp has a lot of characteristics which are prerequisites for a pathogen: the typical S-shape, the corkscrew-like movement and the powerful urease and protease enzymes. These features allow a rapid movement through the mucous layer to permit access to the apical membranes of the surface mucous cells. There they adhere directly to the membranes and induce several ultrastructural alterations: degeneration of microvilli, depletion of mucous granules and an increase in sialic-acid rich glycoproteins in the apical part of the cytoplasma. Cp weakens the tight-junction complex and is found between the cells and sometimes intracellularly. Cp is phagocytized by invading polymorphonuclear leukocytes and causes an intense inflammatory response. These observations clearly demonstrate pathological alterations which in the cellular level induced by Cp with the result of a disrupted mucosal barrier of the stomach and the duodenum.