RESUMEN

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.

Asunto(s)

RESUMEN

Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of chronic and life-threatening diseases. Although these molecules are extraordinarily efficacious, many diseases have high dosage requirements of several hundred milligrams of protein for each administration. Multiple doses at this level are often required for treatment. One of the major challenges currently facing the biotechnology industry is the development of large-scale, cost-effective production and manufacturing processes of these biologically synthesized molecules. Metabolic engineering of animal cell expression hosts promises to address this challenge by substantially enhancing recombinant protein quality, productivity, and biological activity. In this report, we describe a novel approach to metabolic engineering in Chinese hamster ovary cells by control of endogenous gene expression. Analysis of the advantages and limitations of using antisense DNA and gene targeting as a means of control are discussed and several gene candidates for regulation with these techniques are identified. Practical considerations for using these technologies to reduce the levels of the CHO cell sialidase (Warner et al., Glycobiology, 3, 455-463, 1993) as a model gene system for regulation are also presented.

Asunto(s)

RESUMEN

The incorporation of sialic acid into therapeutic recombinant glycoprotein expressed in Chinese hamster ovary (CHO) cells during growth in large bioreactors (10 l) has been monitored under high productivity conditions induced by the presence of sodium butyrate. Samples of the bioreactor culture (approximately 4 x 10(6) cells) were labeled with 3H-N-acetylmannosamine, a metabolic precursor of sialic acid. After 24 h, the recombinant glycoprotein, an immunoadhesion chimeric molecule, was purified and the amount of sialic acid incorporated was determined as radioactive counts. The labeling profile of the protein over the course of the culture was compared with the sialic acid content of the molecule as determined by direct chemical analysis. Early in the culture, the two methods of analysis gave a similar sialylation profile. However, after sodium butyrate was included in the culture, the metabolically incorporated sialic acid rapidly and dramatically decreased to near undetectable levels. In contrast, sialic acid content of the protein, as determined by chemical analysis, decreased only moderately and gradually over the culture period, from a maximum of 6.1 to about 5. 0 mol sialic acid/mole of protein after 10 days in culture. These results suggest that butyrate may enhance reutilization of existing glycoproteins in the culture, generating sialic acid for biosynthesis through lysosomal degradation and thereby bypassing de novo biosynthesis.

Asunto(s)

RESUMEN

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.

Asunto(s)

Asunto(s)

RESUMEN

Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996). A soluble sialidase of CHO cell origin degrades the expressed recombinant protein and has been shown to be released into the culture fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA. Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values. To test if this level of sialidase would lead to increased sialic acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the antisense cultures was compared with material made in the wild-type parental cell line. About 20-37% increase in sialic acid content, or 0.6-1.1 mole of additional sialic acid out of a total of 3.0 mole on the product, was found on the DNase made in the antisense cell lines.

Asunto(s)

RESUMEN

The stereochemical course of enzymatic hydrolysis by the soluble sialidase from Chinese hamster ovary cells, expressed as a recombinant protein in insect Sf9 cells, was determined using proton nuclear magnetic resonance spectroscopy. 4-Methyl umbelliferyl-N-acetyl neuraminic acid was employed as substrate, and the stereoselectivity of the enzyme catalysis was ascertained by monitoring the H3 axial and equatorial protons of the sialic acid product over the reaction course. At both high (3 U) and low concentrations (1 U) of the enzyme, the alpha anomer of the sialic acid was clearly observed as the initial reaction product. The corresponding beta anomer of sialic acid appeared much later in the reaction, arising from mutarotation of the alpha anomer. Similar studies were also carried out using the Salmonella typhimurium LT 2 sialidase, a protein of similar size and substrate specificity. Both enzymes apparently cleave the alpha linked sialoside substrate with retention of configuration. Based on the observations of a wide variety of other glycohydrolytic enzymes that have shown a strong correlation of the stereoselectivity of catalysis with active site topology (Gebler et al., J. Biol. Chem. 267, 12559-12561, 1992), the results obtained here suggest that the microbial and mammalian sialidases have a homologous active site architecture even though the molecules do not share significant primary sequence similarities.

Asunto(s)

RESUMEN

A deaminoneuraminosyl-glycohydrolase (KDNase), which catalyses the hydrolysis of alpha-ketosidic 2-keto-3-deoxy-D-glycero-D-galacto- nononic acid (or naturally occurring deaminated neuraminic acid; KDN) linkages in KDN-glycoconjugates, is required for their structural and functional studies since KDN residues are usually resistant to the action of known sialidases. A search for KDNase was initiated by examining various cells and tissues of rainbow trout because KDN-glycoconjugates were first found in this animal species. Tissue localization studies of KDNase activity showed it to be present in kidney, spleen and ovary. The highest KDNase activity was found in ovarian post-ovulatory follicles obtained from female fish at the time when the reproductive organ was undergoing natural effacement. Little if any activity was found in brain, heart, liver, muscle, mature eggs and testis. Developmentally, higher levels of KDNase were usually expressed 3-4 months before ovulation or spermiation. An exception to this was in the ovary (or ovarian follicles) where the most striking increase in KDNase occurred 1-2 months after the maturation of gamete cells. Enzyme extracts containing KDNase activity also contained sialidase activity. From the data based on a kinetic study using mixed substrates, both KDNase and sialidase activities were indicated to reside on a single enzyme protein. The KDN-sialidase displayed broad specificity, which could possibly limit its usefulness as a probe for KDN-glycoconjugates. Nevertheless, unlike sialidases, KDNase can selectively remove KDN residues, thus making it an important new reagent to identify KDN-glycoconjugates in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

RESUMEN

A cDNA encoding a soluble sialidase from Chinese hamster ovary (CHO) cells has been cloned and expressed. Completely degenerate oligonucleotide primers, which were based on the amino acid sequence of peptides obtained from the purified sialidase (Warner et al., Glycobiology, 3, 455-463, 1993), and the polymerase chain reaction, with single-stranded cDNA template, were employed to generate a unique oligonucleotide probe. The unique probe of 93 bp was used for screening a lambda gt 10 CHO cell cDNA library. A single clone, which contained a 1.4 kb insert, was isolated after screening 450,000 recombinants. The complete coding region of the protein, 1137 nucleotides, was contained in the isolated clone and it predicted a protein of 379 amino acids. The insert had a 186 bp 5' non-coding leader sequence and a 40 bp 3' non-coding region. No signal peptide was identified in the insert, suggesting a cytosolic localization for the protein. No significant primary sequence identities were observed when the deduced amino acid sequence of the CHO cell sialidase was compared with other mammalian proteins or microbial sialidases. However, the protein had significant sequence alignment similarity with several bacterial sialidases. Two 'Asp box' motifs in the CHO cell sialidase had a remarkable alignment positioning in the protein sequence with the similar motifs of the Salmonella LT2 and Clostridium perfringens sialidases. High levels of the enzyme were expressed in Spodoptera frugiperda cells infected with a modified Autographa californica nuclear polyhedrosis virus harbouring the sialidase cDNA.

Asunto(s)

RESUMEN

A soluble sialidase that can degrade recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells has been isolated and purified to near homogeneity from the cell culture fluid of this host. Purification of approximately 34,000-fold was carried out using conventional purification techniques including sequential DEAE-Sepharose and S-Sepharose ion-exchange chromatography, followed by hydrophobic interaction chromatography with Phenyl-Toyopearl. Final purification was achieved by heparin-agarose and chromatofocusing chromatography. The minimum molecular weight of the sialidase on SDS-PAGE was approximately 43,000 Da. When the final preparation was examined under non-denaturing conditions, two major (pI = 6.8 and 7.0) and five minor electrophoretic forms with different isoelectric points were identified. The basis for the electrophoretic heterogeneity is not known, but it was not due to carbohydrate diversity since no carbohydrates were detected on the purified protein. The enzyme degraded a variety of sialyl-conjugate substrates, at a pH optimum of 5.9, including intact glycoproteins, oligosaccharides and gangliosides with a 4-fold preference for 2,3- versus 2,6-linked sialic acid residues. With ganglioside substrates, internally linked sialic acid residues were not cleaved by the enzyme. Delineation of this enzyme from the lysosomal and plasma membrane sialidases was made using inhibition studies with C-9 substituted 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2- enonic acid derivatives. The enzyme was identified in several CHO cell lines by immunoblotting using antiserum raised against a synthetic peptide based on amino acid sequence of a fragment derived by trypsin digestion of the purified sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

The sialidase from Salmonella typhimurium LT2 was characterized by using photoaffinity-labelling techniques. The well-known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non- 2-enonic acid (Neu5Ac2en) was modified to contain an amino group at C-9, which permitted the incorporation of 4-azidosalicylic acid in amide linkage at this position. Labelling of the purified protein with the radioactive (125I) photoprobe was determined to be highly specific for a region within the active-site cavity. This conclusion was based on the observation that the competitive inhibitor Neu5Ac2en in the photolysis mixture prevented labelling of the protein. In contrast, compounds with structural and chemical features similar to the probe and Neu5Ac2en, but which were not competitive enzyme inhibitors, did not affect the photolabelling of the protein. The peptide interacting with the probe was identified by CNBr treatment of the labelled protein, followed by N-terminal sequence analysis. Inspection of the primary structure of the protein, predicted from the cloned structural gene for the sialidase [Hoyer, Hamilton, Steenbergen & Vimr (1992) Mol. Microbiol. 6, 873-884] revealed that the label was incorporated into a 9.6 kDa fragment situated within the terminal third of the molecule near the C-terminal end. Secondary-structural predictions using the Garnier-Robson algorithm [Garnier, Osguthorpe & Robson (1978) J. Mol. Biol. 120, 97-120] of the labelled peptide revealed a structural similarity to the active site of influenza-A- and Sendai-HN-virus sialidases with a repetitive series of alternating beta-sheets connected with loops.

Asunto(s)

RESUMEN

The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using 13C-labeled N-acetylneuraminic acid (NeuAc). Consumption of the beta-anomer of [2-13C]N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid. Inhibition by substrate analogues the anomeric oxygen was determined in a similar manner using [2-13C,(50 atom %)18O]N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the [13C]NeuAc substrate and CMP-[13C]NeuAc product was observed due to the 18O substitution. This result implies conservation of the NeuAc oxygen. Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate. Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP.

Asunto(s)

RESUMEN

The intracellular localization of Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been examined by immunofluorescence using four monoclonal anti-Cu,Zn superoxide dismutase antibodies raised against a recombinant human Cu,Zn superoxide dismutase derivative produced and purified from Escherichia coli. Colocalization with catalase, a peroxisomal matrix enzyme, was used to demonstrate the peroxisomal localization of Cu,Zn superoxide dismutase in human fibroblasts and hepatoma cells. In the fibroblasts of Zellweger syndrome patients, the enzyme is not transported to the peroxisomal ghosts but, like catalase, remains in the cytoplasm. In addition, immunocryoelectron microscopy of yeast cells expressing human Cu,Zn superoxide dismutase showed that the enzyme is translocated to the peroxisomes.

Asunto(s)

RESUMEN

Kinetic analysis of mammalian sialidases was carried out using analogs of the potent sialidase inhibitor, 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic+ ++ acid (1). Substitutents at C-9 in place of the terminal hydroxyl group included a, 4-azido-2-nitrophenylthio group to give 5-acetamido-2,6-anhydro-9-S-(4-azido-2-nitrophenyl)-3,5, 9-trideoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (2), and an azide group to give 5-acetamido-2,6-anhydro-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-non-2 -enonic acid (3). Competitive inhibition kinetics were observed when 1,2, and 3 were tested with the lysosomal sialidase (cultured fibroblasts) and the plasma membrane sialidase (adenovirus DNA-transformed, human embryonic kidney cells), giving a Ki of about 10 microM for both enzymes with all three compounds. In contrast, only 1 was a potent inhibitor of the microsomal sialidase (rat muscle).

Asunto(s)

RESUMEN

Photolabeling of the alpha-neuraminidase/beta-galactosidase complex in human placenta (Verheijen, F.W. et al (1987) Eur. J. Biochem. 162, 63-67) was carried out using the radioactive photoprobe, 9-S-(4-azido-3,5-3H-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9- tetradeoxy-9- thio-D-glycero-D-galacto-non-2-enonic acid. Two intensely labeled bands at 61 and 46 kD were detected with autoradiography. Labeling of the 46 kD protein was blocked with the inclusion of the surfactant Triton X-100 in the photolysis mixture, indicating a nonspecific, hydrophobic interaction. The 61 kD protein was protected from labeling only when the neuraminidase inhibitor 2,3 dehydro N-acetyl neuraminic acid (1 mM) was present during photolysis. These results suggest that the neuraminidase activity resides among the proteins in the 61 kD molecular weight range comigrating with the lysosomal beta-galactosidase, under denaturing conditions.

Asunto(s)

RESUMEN

Photolysis of the lysosomal neuraminidase in crude homogenates of cultured human skin fibroblasts was carried out using the potent competitive enzyme inhibitor, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-tetradeoxy-9 -thio-D - glycero-D-galacto-non-2-enonic acid (9-PANP-2,3-D-NANA). Irradiation of the homogenate and the inhibitor (2 min, pH 4.3, 10 degrees C) with a medium pressure mercury lamp resulted in about a 24% reduction of enzyme activity compared to irradiated controls that did not contain additives. No significant loss of activity was observed with homogenate that contained a photoreactive thioglycoside of sialic acid that was not an inhibitor of the enzyme. Similarly, the enzyme activity was not affected when 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid was photolyzed with the homogenate. The latter is a potent competitive inhibitor but it is not photoreactive. Also, the products obtained by prephotolyzing 9-PANP-2,3-D-NANA gave similar enzyme levels under standard assay conditions when compared with the nonirradiated material. Together, these results demonstrate that the photoinactivation is highly specific and both the aryl azide and the unsaturated pyran portion of the molecule are required for inactivation. The title compound may be useful as a potential photolabeling reagent which may facilitate purification of the enzyme and permit further characterization of the mutation in sialidosis patients.

Asunto(s)

RESUMEN

An azidoaryl thioglycoside of sialic acid was prepared, as a potential photoaffinity probe reagent for the analysis of sialidases and sialic acid-binding proteins, by treatment of the glycosyl chloride of N-acetylneuraminic acid methyl ester with potassium thioacetate to give, in 70% yield, methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-2,3,5-trideoxy-2-thio-alph a-D- glycero-D-galacto-2-nonulopyranosonate. Selective hydrolysis of the thioacetate ester, followed by condensation with 4-fluoro-3-nitrophenyl azide, O-deacetylation, and hydrolysis gave (4-azido-2-nitrophenyl)- 5-acetamido-2,3,5-trideoxy-2-thio-alpha-D-glycero-D-galacto-2- nonulopyranosidonic acid.

Asunto(s)

RESUMEN

A photoreactive, potent, competitive inhibitor of the human lysosomal neuraminidase in cultured skin fibroblasts has been prepared. The starting material, 2,3 dehydro-N-acetyl neuraminic acid methyl ester, was selectively tosylated at the C-9 position with tosyl chloride and subsequently peracetylated with acetic anhydride. The tosyl group was displaced with potassium thio acetate in dimethylformamide at 60 degrees C for 80 min. 4-fluoro-3-nitrophenylazide was incorporated by reaction with the thio acetate product and equimolar sodium methoxide in methanol followed by reacetylation. Base hydrolysis gave the final product, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9-tetradeoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (W5). The yields at each step were 50-70%. Competitive inhibition kinetics were observed when W5 was tested with the fibroblast neuraminidase using 4-methylumbelliferyl-N-acetyl-neuraminic acid as substrate giving an apparent Ki of about 10 microM. These results suggest that the terminal hydroxyl group at C-9 may not be important in the recognition and binding of the substrate by the enzyme. Also, the compounds prepared here may be useful as photoaffinity probes or ligands for affinity chromatography for purification.

Asunto(s)

RESUMEN

Prenatal diagnosis of Sandhoff disease (infantile onset) at 16 weeks gestation has been made by detection and analysis of N-acetylglucosaminyl-oligosaccharides in amniotic fluid using high performance liquid chromatography. The elution profile for the branched chain oligosaccharides was identical with that obtained with neonatal and infantile Sandhoff urine. The concentration of the oligosaccharides in the fluid was 1/100th that of urine but when calculated relative to creatinine the levels were similar. No oligosaccharides were detected in normal control amniotic fluids (10 patients) at a similar gestational age. Based on the levels of the amniotic fluid oligosaccharides and the sensitivity limits of the assay, prenatal diagnosis of patients with the juvenile onset form of the disease may also be possible with this technique.

Asunto(s)

RESUMEN

The N-acetyl-glucosaminyl oligosaccharides excreted in urine and accumulating in tissues of Sandhoff disease patients have been analyzed and characterized using a combination of high performance liquid chromatography and 500 MHz proton magnetic resonance spectroscopy. Delineation between infantile and juvenile onset forms of the disease was possible, as the latter forms had 6- to 13-fold lower levels of urinary oligosaccharides. Patients from a geographically isolated population deme in the La Rioja region of Argentina had urinary oligosaccharides similar to unrelated non-Argentinean patients with identical clinical phenotype. Together, these results indicate that the urinary oligosaccharides serve as useful indicators of the mutation differences or clinical heterogeneity within this disease only in cases of markedly differing clinical presentation. Analysis of the accumulating metabolites in liver, kidney, pancreas, lung and spleen, showed a similar oligosaccharide pattern which differed dramatically from brain. These results suggest the possibility of tissue specific regulation of oligosaccharide biosynthesis since there are notable differences between neural and visceral tissues.

Asunto(s)