Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes

Purified lactotetraosylceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc1-Cer) was tested for its ability to accept [14C]sialic acid from CMP-[14C]sialic into monosialoganglioside fractions in the presence of membrane fractions purified from human colorectal carcinoma cells (SW1116). Membrane fractions were isolated by three different methods: sucrose density centrifugation, CMP-agarose gel column chromatography, and LcOse4 gel chromatography. We optimized the incubation conditions for detergent dependency (taurocholate), pH (6.3), and acceptor concentration. The sialyltransferase activity was dependent on membrane protein and linear for time up to at least 4 h. The LcOse4 affinity chromatography of the crude microsomal membrane pellet from these cells yielded a membrane fraction that was 136-fold enriched in LcOse4 acceptor specific activity compared to cell homogenates. The apparent Km for the sialyltransferase activity with LcOse4Cer acceptor in the presence of affinity-purified membranes was 20 microM and the Vmax was 7 pmol h-1 (100 micrograms of protein)-1. Acceptor capabilities of other core structures were 5-20-fold lower: LcOse4Cer much greater than GgOse4Cer greater than nLcOse4Cer much greater than GbOse4Cer. The enzymatic activity was purified further (900-fold) by a combination of LcOse4 and CMP affinity gels. SDS-PAGE electrophoresis of this material showed a major set of closely migrating bands of Mr 58,000-54,000 compared to authentic proteins, as well as a minor band at 27,000. We analyzed picomole quantities of the radioactive product by convenient controlled short-term hydrolyses with an endoglycoceramidase and sialidases (from four different sources) in comparison to sialylated tetrasaccharides of known structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of polyuronides inMucor rouxii: Partial characterization of fucosyl transferase

Most of the fucosyl transferase activity fromMucor rouxii was detected in a crude membrane fraction. The enzyme transferredl-fucose from GDP-fucose to endogenous and exogenous acceptors. When crude membrane fractions were treated with neutral detergents such as Trition X-100 or Brij 36 T enzyme activity became dependent on exogenous acceptors such as mucoric acid or mucoran. Brij-treated membrane fractions showed maximum fucosyl transferase activity at pH 6.5, and at a temperature between 22 and 28°C. The cations Mn²⁺, Mg²⁺, Co²⁺, Zn²⁺, Fe²⁺, and Ca²⁺ activated the enzyme about twofold. The former was slightly more stimulatory at 4 mM. Km for GDP-fucose was 10 μM. Evidence was obtained that mucoric acid serves as acceptor for fucosyl moieties. Acid hydrolysis of the product synthesized from GDP-fuc by Brij-treated membrane fractions revealed fucose as the major radioactive sugar.     

Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewisa secretion in colorectal carcinoma cultures

The rate of polarised secretion of sialosyl Lewis^a(19-9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 μM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewis^a levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine- to twentyfold increase in sialosyl Lewis^a epitope levels in a light membrane subfraction from RA-treated cells. Antigenic molecules of < 200,000 Mr on acrylamide gradient gels were concentrated in two doublets in the apparent Mr range 106,000–152,000 on Western blots. Carbohydrates analyses of oligosaccharides from SiaLeams of membrane subfraction and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewis^a, fucose/total CHO, and (³H) fucose incorporation in control samples than RA samples. Western blots of samples from membranes subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewis^a epitope, RA treatment did not augment cysteine-rich, PDTRP, blood group H-2, blood group A, and EGF receptor-like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewis^a induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Galβ1-3 moieties at oligosaccharide termini destined for export from the Golgi.     

Biosynthesis of sialosyllactotetraosylceramide in human colorectal carcinoma cells

A monosialoganglioside, IV³-NeuNAcLcOse₄Cer, has recently been detected in colorectal carcinoma cells, small cell lung carcinoma cells, embryonal carcinoma cells and in human brain extracts. We report here the presence of a CMP-sialic: LcOse₄Cer sialyl transferase activity in subcellular membrane fractions of the human colorectal carcinoma, SW1116, which recognizes the non-reducing terminal galactosyl moiety of lactotetraosylceramide. A convenient method for structural analysis of picomolar quantities of the radioactive enzymatic product(s) using bacterial endoglycoceramidase, sialidase and a viral sialidase is presented.     

III6NeuAcLc4Cer in human SW1116 colorectal carcinoma cells: a possible oncofetal antigen that is not dependent on Lewis gene expression

Monospecific rabbit antibodies directed against the human milk sialyloligosaccharides III6NeuAcLcOse4 (sialyltetrasaccharide b), IV3NeuAcLcOse4 (sialyltetrasaccharide a), and IV6NeuAcnLc4Ose (sialyltetrasaccharide c) were used to detect their homologous haptens as gangliosides or ganglioside-derived sialyloligosaccharides from the human colorectal carcinoma cell line SW1116. III6NeuAcLc4Cer was first detected in human meconium [P. A. Prieto and D. F. Smith (1985) Arch. Biochem. Biophys. 241, 281-289], and its presence in a total ganglioside fraction of SW1116 cells together with its absence from a total lipid extract of normal human intestinal mucosa are consistent with III6NeuAcLc4Cer being a tumor-associated oncofetal antigen. IV3NeuAcLc4Cer, a ganglioside in human meconium [P. A. Prieto and D. F. Smith (1986) Arch. Biochem. Biophys. 249, 243-253], was also detected in SW1116 cells; an observation that is consistent with its being the immediate precursor to the sialyl-Lea ganglioside in SW1116 cells. Specific antisera against sialylated type 1 oligosaccharide chains whose expression is independent of the Lewis gene fucosyltransferase may be useful diagnostic reagents for oncofetal, carbohydrate antigens.     

Molecular mechanisms of expression of Lewis b antigen and other type I Lewis antigens in human colorectal cancer.

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Lebamounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with alpha2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.     

Human Lewis {alpha}1->3/4fucosyltransferase: specificity of fucose transfer to GlcNAc{beta}1->3Gal{beta}1->4Glc{beta}1->1Cer (LcOse3Cer)

Biosynthesis of the Le^x series of carbohydrate antigens proceedsby fucose transfer in 13-linkage to the penultimate GlcNAc residueof a neolacto-series oligosaccharide acceptor, a reaction catalysedby multiple enzymes expressed in human tissues. Particularlybroad acceptor specificity, including the ability to catalysefucose transfer to both lacto- and neolacto-series acceptorsas well as the precursor Lc₃ structure (where Lc₃ lactotriaosylceramide,is GlcNAcß13Galß14Glcß1Cer), existsfor one human fucosyltransferase form, the Lewis 13/4fucosyltransferase(FucT-III). To determine if fucose transfer to Lc₃may representan alternate early step in Le^xor Le^a antigen biosynthesis withthis enzyme, the chemical structure of the fucosylated Lc₃ reactionproduct formed by the Lewis 13/4fucosyltransferase from Colo205 cells has been defined. Transfer of [¹⁴C]fucose to Lc₃ yieldeda labelled product migrating as a tetrasaccharide on thin layerchromatography plates. This product remained an acceptor forboth ß13- and ß14-galactosyl transfer onthe terminal GlcNAc residue. The product was degraded to a fucosylatedtrisaccharide derivative by bovine kidney ß-N-acetylglucosaminidase.Fast atom bombardment mass spectrometry and methylation analysisconfirmed that the product was composed exclusively of the followingstructure containing a fucose linked to the 3-position of theinternal Glc residue: GlcNAcß13Galß14Glcß11Cer Such a structure does not represent an intermdiate in Le^xorLe^a antigen biosynthesis. Thus, the evidence suggests that Le^xor Le^a antigen synthesis results exclusively from fucosylationof complete core chains. fucosyltransferase lacto-series LcOse₃Cer Lewis antigen transfer specificity     

Studies of human liver bilirubin-glycosyl transferase. Bilirubin UDP-xylosyl and UDP-glucuronyl transferase activities in diseased human livers.

The activity of bilirubin UDP-xylosyl transferase as well as UDP-glucuronyl transferase in liver biopsy specimens of 3 control subjects, 42 cases with liver disease and 5 cases with Gilbert's syndrome was measured. Normal values of these enzyme levels were determined to be 142--302 U/kg protein for the former and 260--400 U/kg protein for the latter. Both enzyme levels in acute hepatitis in convalescence and chronic hepatitis were nearly in the normal range. In the cirrhotic liver they tended to a small decrease and patients with Gilbert's syndrome demonstrated significantly decreased enzyme levels. These enzyme levels were only correlated with serum unconjugated bilirubin concentration, but not with the other liver function tests. Finally, both enzyme activities were exactly correlated with each other.     

Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes

The sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose-1-phosphate uridyl transferase and uridine diphospho-galactose-4-epimerase.

• 1 Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells.
• 2 Added extracellular Gal-1-P, G-1-P, UDPGal and UPDG do not affect enzyme activities in whole cells.
• 3 The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells.
• 4 Galactokinase activity is inhibited by G-6-P and Gal-1-P, but not by glucose, G-1-P, UDPG, UDPGal, UTP or NAD⁺. It is inhibited by ATP and ADP in high concentration.
• 5 Galactose-1-phosphate uridyl transferase activity is inhibited by G-1-P, G-6-P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD⁺, or galactose.
• 6 Uridine diphospho-galactose-4-epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD⁺ and possibly by Gal-1-P. It is unaffected by G-1-P, G-6-P.
• 7 The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly.
• 8 Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes.
• 9 Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal-u-¹⁴C.
• 10 ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.

     

Biosynthesis of alpha-N-acetylglucosaminidase in cultured human kidney carcinoma cells

The biosynthesis of alpha-N-acetylglucosaminidase was studied in cultured human kidney carcinoma cells by labeling cells with 35S-methionine, isolation of the enzyme by immunoprecipitation and analysis on gel electrophoresis of the denatured polypeptide(s) and analysis of the native enzyme on linear sucrose gradient centrifugation. The enzyme is synthesized as precursor forms of apparent molecular weight 82,000-86,000. Processing of these precursors yields a polypeptide of apparent molecular weight of 80,000. The precursor-product relationship was indicated by pulse-chase as well as endocytosis experiments. Sucrose gradient centrifugation of the native enzyme shows that, extracellularly, the molecule is present with a molecular weight of 80,000; intracellularly, 80-90% of the enzyme is present with an apparent molecular weight of 240,000. We suggest that this is a polymeric form and that polymerization of alpha-N-acetylglucosaminidase is a late event of the maturation process.     

Partial purification and characterization of mold antigens commonly found in foods.

Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.     

Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes.

On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.     

Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase.

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.     

Biological activities of human mannose-binding lectin bound to two different ligand sugar structures, Lewis A and Lewis B antigens and high-mannose type oligosaccharides.

The biological activities of mannose-binding lectin (MBL) which binds to different ligands on mammalian cells were examined using two types of Colo205 cells, a human colon adenocarcinoma cell line: one naturally expressing Lewis A and Lewis B antigens as ligands for MBL (NT-Colo205), and the other modified to express high-mannose type oligosaccharides by treatment with benzyl-2-acetamide-2-deoxy-alpha-galactopyranoside and 1-deoxymannojirimycin (Bz+dMM-Colo205). Although the final lysis was not observed, the deposition of C4 and C3 was observed on both types of Colo205 cells after treatment with MBL and complements as a result of complement activation by MBL. MBL bound to Bz+dMM-Colo205 could also activate human peripheral blood leukocytes and induce superoxide production; however, MBL bound to NT-Colo205 could not. This may be explained by the lower affinity of MBL to Lewis A and Lewis B antigens than to high-mannose type oligosaccharides under physiological conditions, since MBL bound to NT-Colo205 was more easily released from the cell surface than that bound to Bz+dMM-Colo205 at 37 degrees C. These findings suggest that the difference in the affinity of MBL to its ligands could influence the expression of some biological activities of MBL.     

Partial purification and characterization of a recombinase from human cells

We describe the partial purification and characterization of a human recombinase activity from RPMI 1788 B lymphoblasts. Stoichiometric amounts of recombinase carry out a strand transfer reaction between linear duplex DNA and homologous circular single-strand DNA. The product of strand transfer by the recombinase is a joint molecule composed of a single-strand circle joined to one end of the linear duplex molecule by a region of DNA heteroduplex at least 150 bp long. Formation of DNA heteroduplexes is accompanied by strand displacement. Strand invasion initiates at the ends of the linear duplex. Finally, strand displacement by human recombinase exhibits polarity and proceeds in a 3' to 5' direction. This is the first demonstration of a strand transfer activity from a high eukaryote. We discuss similarities between our recombinase and the RecA and rec1 recombination proteins from E. coli and Ustilago maydis, respectively.     

Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.     

Partial purification and characterization of the soluble glutathione transferase isoenzymes from cultured Hep G2 cells

Hep G2 cells, an established cell line derived from a human hepatoma, were mass cultured in a cell factory for the isolation of glutathione transferase isoenzymes. These were enriched by affinity chromatography and separated in an anionic and a cationic fraction. They were partially characterized by different kinetic and inhibition parameters. Three different subunits were observed. The results were compared with human liver data. It is concluded that Hep G2 cells can be considered as a valuable alternative tool for in vitro research of human liver phenomena, especially when toxicological interactions are investigated.     

Partial characterization of steroid sulfohydrolase and steroid sulfotransferase activities in purified porcine Leydig cells

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.