Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid

HPLC analysis of sialic acids released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N-acetylneuraminic acid and N-glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz1H-NMR spectroscopy, of the enzymatically released N-linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that alpha 2-3 linked N-glycolylneuraminic acid can occur in different N-acetyllactosamine type antennary structures.     

The expression profile of de-N-acetyl polysialic acid (NeuPSA) in normal and diseased human tissue

Although sialic acids have a key role in many aspects of human biology, the expression of polysialic acid (PSA) in human tissues is thought to be relatively rare. We identified a derivative of PSA called neuraminic acid-containing PSA or NeuPSA that was highly expressed in primary human melanoma tumors and in several cancer cell lines. Moreover, anti-NeuPSA antibodies could induce apoptosis of cancer cells. However, little was known about NeuPSA expression in normal or diseased tissues. In this study we investigated the complete expression profile of NeuPSA in human tissues and a few primary tumors using the anti-NeuPSA monoclonal antibody, SEAM 3. Almost every human tissue tested spanning a representative sample of all organ types was positive for SEAM 3 binding. Specificity of SEAM 3 binding was established by inhibition with NeuPSA but not closely related meningococcal C polysaccharide and loss of SEAM 3 binding when specimens were treated with periodate at high pH, which specifically destroys NeuPSA. Only subsets of cells in each specimen stained positive, and the relative staining between tissues was variable. The distribution and amount of NeuPSA antigen in tissues was correlated with known levels of polysialyltransferase PST or STX expression. The majority of anti-NeuPSA binding occurred intracellularly in the cytoplasm of cells. Tumors generally exhibited considerably increased staining compared with corresponding normal tissues. Identifying the diverse tissue distribution and intracellular location of NeuPSA provides a foundation for investigating the functional role of NeuPSA in human health and disease.     

Expression of herpes simplex virus type 1 glycoproteins in interferon-treated human neuroblastoma cells.

Human alpha interferon (IFN) significantly inhibits the replication of herpes simplex virus type 1 in human neuroblastoma cells. This inhibitory effect can be blocked by pretreatment with antiserum to IFN. We observed no significant differences in the expression of major nucleocapsid proteins, including VP5, between IFN-treated and untreated neuroblastoma cells. Electron micrographs demonstrated that there were distinct viral nucleocapsids within IFN-treated neuroblastoma cells. The expression of glycoproteins B and E was significantly reduced in these IFN-treated cells. On the other hand, glycoprotein D, although reduced in quantity, was expressed after IFN treatment. An immunofluorescence assay of the IFN-treated and virus-infected cells detected glycoprotein D in the Golgi complexes and in the nuclear membranes. Our results indicate that human alpha IFN may be useful in the study of gene expression in IFN-treated cells of neuronal origin.     

Multiple oligosaccharide chains in the voltage-sensitive Na channel from electrophorus electricus: evidence for alpha-2,8-linked polysialic acid

Carbohydrate substituents on the large peptide of the voltage-sensitive Na channel from Electrophorus electricus electroplax have been partially characterized by their sensitivity to endoglycosidases H and F, peptide:N-glycosidase F, Endo-N-acetylneuraminidase, and to neuraminidase. The results suggest the presence of at least two classes of oligosaccharides: neutral, high mannose or hybrid oligosaccharides, and acidic, complex oligosaccharides with a core-structure terminating in an unbranched homopolymer of sialosyl units in alpha-2,8 linkages (much greater than 5 tandem sialic acids). Large decreases in apparent Mr produced by sialidase treatments suggest an extended carbohydrate structure that could inhibit protein-protein interaction. Polysialic acid was formerly proposed to be a unique constituent of neural cell adhesion molecules (N-CAMs) in vertebrates. However, ratios of sialic acid to galactose reported for mammalian brain and muscle Na channels suggest they may also carry this oligosaccharide.     

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.     

Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary

Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing alpha2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two alpha2,8-polysialyltransferases (alpha2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which alpha2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the alpha2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo alpha2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the alpha2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP.     

3,4-Dihydroxyphenylalanine (DOPA) metabolism and retinoic acid induced differentiation in human neuroblastoma

In mature cells of the sympathetic nervous system and the adrenal gland, the activity of dihydroxyphenylalanine decarboxylase (DDC) is higher than that of tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) does not accumulate in the cells. On the other hand, it is known that in some neuroblastoma cells there is a relative deficiency of DDC, resulting in accumulation and secretion of DOPA. Such a relative deficiency of DDC is a characteristic of neural cells at an early stage of neural crest development, suggesting the neuroblastoma are cells arrested in early neural crest development. If this were the case, it is possible that agents such as retinoic acid (RA) could induce neuroblastoma to differentiate into mature cells with respect to their metabolism of catecholamines. We have measured the effect of RA on the metabolism of DOPA and expression of tyrosine hydroxylase and DDC in human neuroblastoma cell lines, CHP-126, CHP-134, IMR-32, NB-59, and LA-N-5. When the cell cultures were treated with RA, they showed wide variations in response as measured by morphological change, growth inhibition, enzyme activities and DDC, but does not increase DDC relative to tyrosine hydroxylase. It is concluded that RA does not induce biochemical differentiation of the neuroblastoma into mature cells even when there are extensive morphological changes and suppression of growth rate.     

聚乙二醇-聚唾液酸嵌段聚合修饰尿酸酶

探索一种综合聚唾液酸(PSA)和聚乙二醇(PEG)优势的蛋白修饰方法。对纯化的聚唾液酸进行两步活化,先在非还原端氧化产生活性醛基,再加入胱胺形成活性巯基;活化的聚唾液酸(相对分子质量为3.4×104)和异基双功能的PEG(相对分子质量为3.5×103)形成嵌段聚合物,然后于4℃修饰尿酸酶。利用凝胶层析(Toyopearl HW-55F)对修饰后的尿酸酶进行纯化,收集相应峰进行化学法和SDS-PAGE电泳鉴定,经多角度激光光散射凝胶系统测定缀合物相对分子质量为5.214×105;相对于原始酶,修饰酶酶活保留率72.4%,体外热失活半衰期由115.5 h提高到231 h,对高温、酸碱、胰蛋白酶的耐受稳定性显著提高。     

Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

Nerve growth factor (NGF) induced differentiation of human neuroblastoma cells

1. Nerve growth factor (NGF) induced differentiation of human neuroblastoma cell line. 2. The differentiated cells had a relatively high activity of adenylate cyclase and cyclic AMP phosphodiesterase, and a high intracellular level of cyclic AMP. 3. These cells synthesized a higher amount of met5-o-enkephalin than undifferentiated cells. 4. Undifferentiated cells bound less met5-enkephalin than differentiated cells. The maximum number of [3H]met5-enkephalin receptor sites per mg of membrane protein increased more in differentiated cells. 5. Previous observations taken together with our results suggests that increased intracellular levels of cyclic AMP after treatment with NGF induced differentiation of human neuroblastoma cells. Reversal of undifferentiated tumor cells into the differentiated changes the capacity of synthesis of met5-enkephalin and its interaction with receptors.     

Composition and distribution of carbohydrate chains in glycoproteins of human erythrocyte membrane

The M-, N-, and MN-glycoproteins obtained from human erythrocytes by phenol-water extraction were purified by gel filtration and digested with Pronase and trypsin. The products of degradation were fractionated by gel filtration on Sephadex G-25 and DEAE-Sephadex A-50 and the fractions were examined by poly(acrylamide)-gel electrophoresis in the presence of dodecyl sodium sulfate, analyzed for carbohydrate and amino acid contents, and tested for M and N blood-group activity. From the results, it is suggested that the glycoprotein chains are composed of a hydrophobic moiety devoid of carbohydrate chains and a hydrophilic moiety containing carbohydrate chains of different compositions, irregularly distributed along the protein chains and linked to L-asparagine, L-serine, or L-threonine residues. The M and N activity typical for the undegraded glycoproteins, and the “basic” or “precursor-type” N activity, were found in different glycopeptide fractions.     

Oscillation of human period 1 (hPER1) reporter gene activity in human neuroblastoma cells in vivo

The mammalian period (Per) genes, which are components of the circadian clock, are mainly regulated via an autoregulatory feedback loop. Here we provide evidence that human Per1 (hPER1) reporter gene activity shows circadian rhythmicity in a human neuroblastoma, but not in a astrocytoma or a hepatoma cell line. Medium change and various pharmacological stimuli differentially induce this behavior. This circadian oscillation was strongly dampened and could be followed over maximally three cycles. It was even possible to phase-shift the course of this oscillation by repeated application of stimuli.     

Microwave radiation-induced calcium ion efflux from human neuroblastoma cells in culture

Monolayer cultures of human neuroblastoma cells were exposed to 915-MHz radiation, with or without sinusoidal amplitude modulation (80%) at 16 Hz, at specific absorption rates (SAR) for the culture medium and cells of 0.00, 0.01, 0.05, 0.075, 0.1, 0.5, 0.75, 1.0, 1.5, 2, or 5 mW/g. A significant increase in the efflux of calcium ions (⁴⁵Ca²⁺) as compared to unexposed control cultures occurred at two SAR values: 0.05 and 1 mW/g. Increased efflux at 0.05 mW/g was dependent on the presence of amplitude modulation at 16 Hz but at the higher value it was not. These results indicate that human neuroblastoma cells are sensitive to extremely low levels of microwave radiation at certain narrow ranges of SAR.     

Oxidative stress induces intracellular accumulation of amyloid beta-protein (Abeta) in human neuroblastoma cells

Several lines of evidence suggest that enhanced oxidative stress is involved in the pathogenesis and/or progression of Alzheimer's disease (AD). Amyloid beta-protein (Abeta) that composes senile plaques, a major neuropathological hallmark of AD, is considered to have a causal role in AD. Thus, we have studied the effect of oxidative stress on Abeta metabolism within the cell. Here, we report that oxidative stress induced by H(2)O(2) (100-250 microM) caused an increase in the levels of intracellular Abeta in human neuroblastoma SH-SY5Y cells. Treatment with 200 microM H(2)O(2) caused significant decreases in the protein levels of full-length beta-amyloid precursor protein (APP) and its COOH-terminal fragment that is generated by beta-cleavage, while the gene expression of APP was not altered under these conditions. A pulse-chase experiment further showed a decrease in the half-life of this amyloidogenic COOH-terminal fragment but not in that of nonamyloidogenic counterpart in the H(2)O(2)-treated cells. These results suggest that oxidative stress promotes intracellular accumulation of Abeta through enhancing the amyloidogenic pathway.