The role of tryptophan residues in the antigenic activity of carcinoembryonic antigen

Antigenic determinants of carcino-embryonic antigen (CEA) were spatially located using N-bromosuccinimide modification of tryptophan residues both in native (acetate buffer solution) and unfolded (guanidinium chloride solution) molecule of the antigen. Modification of exposed tryptophan residues failed to alter CEA antigenic activity and conformation of its protein portion as shown by CD spectroscopy. On the contrary, modification of buried tryptophan residues induced conformational changes of CEA protein portion connected with a considerable loss of its antigenic activity. It was shown that CEA antigenic activity depends on spatial structure of its protein moiety.     

Structural studies on carcinoembryonic antigen. Periodate oxidation.

Periodate oxidation has been applied to examine the carbohydrate structure of carcinoembryonic antigen (CEA) and the possible role of the carbohydrate residues in its antigenic activity. Sialic acid (N-acetylneuraminic acid) and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate treatment (Smith degradation) destroyed additional amounts of galactose and mannose as well as significant amounts of N-acetylglucosamine. Prior removal of sialic acid by neuraminidase treatment led to increased destruction of galactose by periodate. Antigenic activity persisted indicating that the residues destroyed played little, if any, part in the antigenicity of CEA. These results yield an initial view of the structural arrangement of the carbohydrate residues in the CEA molecule.     

Localization of antigenic determinants on a molecule of trophoblast-specific beta 1-glycoprotein

Spatial localization of antigenic determinants of trophoblast-specific beta I-glycoprotein (TSG) has been elucidated using chemical modifications of the sugar and protein moieties of the molecule. Various deglycosylation procedures of TSG afforded fragments slightly soluble even in the presence of powerful detergents. Treatment of TSG with boric acid and its salts, accompanied with a conformational change of the sugar moiety, failed to alter conformation of the protein portion as evidenced by CD spectral data. This modification was found to increase the antigenic activity of TSG only scarcely. Modification of tryptophane or tyrosine residues of TSG changed spatial structure of the protein portion to can be a considerable loss of the TSG antigenic activity. The data obtained led to the conclusion that antigenic determinants of TSG are localized at the protein portion of the molecule and are topographic. A tryptophane residue is an indispensable constituent of the antigenic determinants.     

Carbohydrate structures of a normal counterpart of the carcinoembryonic antigen produced by colon epithelial cells of normal adults

Normal Faecal antigen-2 (NFA-2) and non-specific crossreactingantigen-2 (NCA-2), cross-reacting with anticarcinoembryonicantigen (CEA) antibodies, were found in normal human faececand meconium, respectively. Because NFA-2, NCA-2 and CEA areconsidered as the same gene products, NFA-2 and NCA-2 shouldbe normal counterparts of CEA produced by colon epithelial cellsof normal adults and fetuses, respectively. Comparison of sugarchain structures of these three antigens is indispensable inorder to unravel the stnaural alteration induced by malignanttransformation and development of colon epithelial cells. Thesugar chain structures of CEA (Yamashita,K. et al., Cancer Res.,47,3451–3459,1987) and NCA-2 (Yarnashita,K. et al., J.Bid Chem, 264,17873-17881,1989) were previously reported. Inthis paper, the structures of the oligosaccharides releasedfrom four NFA-2 samples by hydrazinolysis were studied by meansof lectin-affinity column chromatography, endo- and em-glycosidasedigestion, methylation analysis, hydrazinolys-nitrous acid deaminationand electrospray ionization mass spectrometry. NFA-2 contains24–27 mol of N-linked sugar chains/molecule, which issimilar to NCA-2 (27 mol) and CEA (24–27 mol). In contrastto CEA, which contains {small tilde} 8% high-mannose-type sugarchains all sugar chains of NFA-2 are mono- to tetra-antennarycomplex-type chains having four types of tri-mannosyl cores,with or without bisecting N-acetylglucosamine and fucose residues.The structures of their outer chain moieties comprise Galß1     

Radioimmunochemical evidence for a role of carbohydrate in antigenic determinant(s) on human liver alpha-L-fucosidase.

A competitive-binding radioimmunoassay method was employed to investigate the role of carbohydrate in antigenic determinant(s) of human liver alpha-L-fucosidase. Competition curves were used to quantify the concentrations of competitors needed to cause 30% inhibition of the precipitation of 125I-labelled alpha-L-fucosidase. The isoelectric forms of alpha-L-fucosidase, which are related by sialic acid residues, were separated preparatively and used as competitors in the radioimmunoassay. A pattern of increasing effectiveness as competitors with increasing acidity of the forms was found, suggesting that sialic acid may be involved in the antigenic determinant(s) of alpha-L-fucosidase. Specificity was exhibited when sugar and sugar derivatives were used as competitors in the radioimmunoassay: a 51-fold range of competitive ability was found, and sialic acids (N-acetylneuraminic acid and N-glycollylneuraminic acid) and colominic acid (a polymer of N-acetylneuraminic acid) were the best competitors. The results of our studies suggest that carbohydrate contributes to antigenic determinant(s) of alpha-L-fucosidase and that sialic acid is probably the major sugar involved.     

Nature of the antigenic determinants of T locus antigens

The nature of the antigenic specificities of several antigens associated with the T/t complex in the mouse were analyzed by means of glycosidase and haptene inhibition studies. Results indicate that on testicular cells sugar residues are involved in at least six different T/t antigenic determinants. The immunodominant sugar appears to be different for each of the specificities. The specificity for the following T/t antigens resides predominantly in the sugars indicated: T:sialic acid; t12:beta-D-galactose; tw32:beta-D-galactose; t0:L-fucose; tw1:N-acetyl-D-galactosamine; tw18:L-fucose. It seems probable that these sugars are found at the terminal reducing ends of the carbohydrate portion of T/t-bearing moleculse. These studies imply that at least some of the genes in the T locus code for glycosyltransferases or regulators of glycosyltransferases which modigy oligosaccharide structures and impart specificity to the T/t antigens by alteration of their terminal sugar residues.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

N-linked neutral sugar chains of aminopeptidase N purified from rat small intestinal brush-border membrane.

Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high mannose type sugar chains (7% of the total neutral sugar chains) are also included. The unique feature of the complex-type sugar chains is the most of them contain terminal N-acetylglucosamine residues and blood group H antigenic determinants in their outer chain moieties, and bisecting N-acetylglucosamine residues in their trimannosyl cores. Both the type 1H and type 2H determinants are found, but the former is mainly expressed at the distal portions of the outer chain moieties of the oligosaccharides.     

Purification and characterization of a high molecular weight eosinophil chemotactic factor from Schistosoma japonicum eggs

A high m.w. eosinophil chemotactic factor (ECF-SjE) was isolated and purified from a soluble egg antigen preparation (SEA) of Schistosoma japonicum by gel filtration on Sephacryl S-200, anion-exchange chromatography on DE52, and isoelectric focusing. ECF-SjE had a m.w. of more than 900,000 and an isoelectric point of 4.1. It contained 40% (w/w) sugar residues and bound to concanavalin A (Con A). The chemotactic activity of ECF-SjE was heat stable (100 degrees C, 60 min) and resistant to pronase digestion, but was destroyed by periodate oxidation. IgG antibody to ECF-SjE was detected in the serum of a rabbit infected with S. japonicum, demonstrating the antigenic nature of ECF-SjE. The antigenicity of ECF-SjE was also sensitive to periodate oxidation. Thus, ECF-SjE is a glycoprotein or proteoglycan from the eggs of S. japonicum, and the sugar chain is important for the expression of chemotactic and antigenic activities. However ECF-SjE differs from the major allergenic components of S. japonicum (JEAL) in m.w. and isoelectric point. A low m.w. eosinophil chemotactic factor was also detected in SEA. Together they are proposed to have a role in the direct accumulation of eosinophils in the egg-induced granulomas in S. japonicum infection.     

ABH-blood-group antigens and glycolipids of human saliva

The nature of ABH-blood-group antigens in saliva was investigated. Human saliva was examined serologically for ABH-blood-group activity in its native form and after various treatments. The activity of the native form persisted in the delipidated samples, but was entirely lost after alkaline degradation. The lipid portion of saliva was completely inactive in the ABH hemagglutination inhibition system. The same results were obtained when purified glycolipid fraction of saliva was used instead of whole lipid extract. Neither alkaline treatment nor excessive amounts of salivary lipids effected antigenic activity of A-active glycosphingolipids of hog gastric mucosa admixed to saliva samples before alkaline degradation and/or in presence of large amounts of salivary lipids. The isolated glycolipid fractions contained at least eight glycolipids, each of which was composed of glucose, glyceryl ethers and fatty acids and differed from others with respect to number of glucose residues. Sphingosine and sugar residues involved in formation of ABH antigenic determinants were not detected. These findings together with data on stomach secretion [1,2] led us to the conclusion that ABH-blood-group antigens of saliva are exclusively of glycoprotein nature.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Immunologically Active Heterosaccharides of Carcinoembryonic Antigen of Human Digestive System

THE carcinoembryonic antigen (CEA) of the human digestive system is a glycoprotein with an approximate carbohydrate/ protein ratio of 2–3/1 (refs. 1–3). Indirect evidence suggests the carbohydrate moiety is associated with the antigenic determinant site(s)^1,4. We have therefore prepared and analysed heterosaccharide fragments of CEA which are free of amino-acids but retain anti-CEA binding activity.     

Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen

Exchanging the glycophosphatidylinositol (GPI) anchor signal sequence of neural cell adhesion molecule (NCAM) for the signal sequence of carcinoembryonic antigen (CEA) generates a mature protein with NCAM external domains but CEA-like tumorigenic activity. We hypothesized that this resulted from the presence of a functional specificity signal within this sequence and generated CEA/NCAM chimeras to identify this signal. Replacing the residues (GLSAG) 6-10 amino acids downstream of the CEA anchor addition site with the corresponding NCAM residues resulted in GPI-anchored proteins lacking the CEA-like biological functions of integrin modulation and differentiation blockage. Transferring this region from CEA into NCAM in conjunction with the upstream proline (PGLSAG) was sufficient to specify the addition of the CEA anchor. Therefore, this study identifies a novel specificity signal consisting of six amino acids located within the GPI anchor attachment signal, which is necessary and sufficient to specify the addition of a particular functional GPI anchor and, thereby, the ultimate function of the mature protein.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).     

Multiple Smith-degradations of carcinoembryonic antigen (CEA) and of asialo CEA

Carcinoembryonic antigen (CEA) and asialo CEA were subjected to multiple Smith-degradation (i.e., for each degradation, application in sequence of periodate oxidation, borohydride reduction, and mild hydrolysis with acid; borohydride-t was substituted for unlabelled borohydride). High yields of modified glycoproteins were obtained at each stage. After three complete degradations and a further periodate-borohydride-t treatment, the carbohydrate content of CEA and of asialo CEA had decreased from 45–50% to 11–12% (i.e., 90% removal of carbohydrate). Glycerol was always one of the products obtained after each degradation, but threitol and erythritol were not detected. The second degradation caused a substantial loss of 2-acetamido-2-deoxyglucose, which is consistent with the location of some of this monosaccharide towards the terminal (non-reducing) end of the oligosaccharides. The “core” region of the oligosaccharides is composed of galactose, mannose, and 2-acetamido-2-deoxyglucose. After the fourth oxidation, 2-acetamido-2-deoxyglucose was 50–60% of the total content of residual carbohydrate. After the first degradation, there was a progressive loss in antigenic activity, but this was associated with a small amount of hydrolysis of the protein moiety of CEA.     

Antigenic activity of porcine muscle lactate dehydrogenase fragment 180-214 containing the histidine residue of the isoenzyme active center

Solid phase immunoenzymatic analysis was used to study the antigenic activity of proteolytic degradation products of the porcine muscle lactate dehydrogenase isoform M4. The presence in the enzyme structure of topographic (linear) antigenic determinants was demonstrated. Peptide 180-214 containing histidine-195 in the active center of lactate dehydrogenase was isolated from the tryptic hydrolysate of the carboxymethylated enzyme. This peptide interacts with antibodies against the native enzyme, i.e., antibodies bound to the immunoadsorbent, and causes a 20-25% inhibition of the antigen-antibody complex formation. Protein modification by fluorescein mercuriacetate at Cys-165 essential for the enzyme activity does not result in the synthesis of antibodies that would stimulate the inhibition of the lactate dehydrogenase catalytic activity as compared to antibodies to the native isoenzyme. The putative role of some amino acid residues in the structure of antigenic determinants of porcine muscle lactate dehydrogenase is discussed.     

Chemical modification studies of Artocarpus lakoocha lectin artocarpin.

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.     

Chemical modification studies on the glucose/mannose specific lectins from field and lablab beans.

Seeds of Dolichos lablab var. lignosus (field beans) and variety typicus (lablab beans) contain glucose/mannose specific lectins that have been affinity purified and well characterised (Siva Kumar N., and Rajagopal Rao, D., J.Biosci., 1986, 10, 95-109, (1) Rajasekhar et al., (Biochem.Archives. 1997, 13, 233-240) (2). Purified lectins are glycoproteins with a native molecular mass of 60 kDa and are made of two types of subunits (Gowda et al., 1994, J.Biol.Chem. 269, 18789-18793) (3). Chemical modifications of various groups in purified lectins (using group specific reagents) such as lysine (citraconic anhydride), carboxyl groups (water soluble carbodiimide) tyrosine (N-acetyl imidazole) and tryptophan (2-hydroxy 5-nitro benzylbromide) revealed that 14 out of 21 residues of lysines 7 out of 92 residues of carboxyl groups, 16 out of 24 tyrosine residues and 2 out of 10 tryptophan residues were modified. Lysine and carboxyl group modification led to 95% loss in haemaglutinating activity compared to control while tyrosine and tryptophan modifications led to complete loss of lectin activity. Arginine and histidine modifications led to only 50% loss in activity. The extent of modification and loss in activity was same when the lysine and carboxyl groups were modified in the presence and absence of the inhibitory sugar methyl alpha-D-glucopyranoside at 0.1 M concentration. However protection of modification and lectin activity was observed when the tyrosine and tryptophan residues were modified in the presence of the inhibitory sugar. Earlier CD studies carried out (1) and extensive chemical modification studies reported here substantiate the involvement of tyrosine and tryptophan residues in the sugar binding site of these lectins.     

Role of a carbohydrate component in the formation of immunochemical determinants of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) isolated from metastases of colon adenocarcinoma was subjected to deglycosylation with liquid hydrogen fluoride. The protein fraction obtained (PF CEA) was used for to prepare monospecific antiserum to PF CEA. Comparative studies of CEA, PF CEA, and non-specific cross-reacting antigen (NCA-1) have been carried out using monospecific antisera. Circular dichroism spectra of CEA and PF CEA have been studied. The data obtained suggest that some immunodominant regions of CEA are topographic, and their formation needs a specific conformation of the macromolecule, which is stabilized by the sugar moiety.     

Purification, characterization and serological properties of a glycolipid antigen reactive with a serovar-specific monoclonal antibody against Leptospira interrogans serovar canicola

A glycolipid antigen possessing a serovar-specific antigenic determinant of Leptospira interrogans serovar canicola was purified from a chloroform/methanol extract of the organism. The purification procedures included silicic acid column chromatography and preparative thin-layer chromatography (TLC). Antigenic activity was detected by a TLC-enzyme immunostaining technique using monoclonal antibody CT3, which specifically agglutinates serovar canicola and only weakly serovar sumneri but no other serovars of Leptospira. The purified glycolipid reacted with CT3 antibody, indicating that the glycolipid possessed a serovar-specific antigenic determinant. Infrared spectrum and proton nuclear magnetic resonance analyses showed that the glycolipid contained sugar and lipid moieties, which possessed amide linkages and an acetyl group. Gas-liquid chromatography-mass spectrometry analysis showed that the glycolipid contained two unknown sugars, one of which (unknown sugar II) appeared to be associated with the antigenic determinant specific for canicola. The serovar-specific antigenic determinant was destroyed by mild alkali treatment of the glycolipid. These findings suggested that the antigenic determinant was an alkali-labile moiety which may be related to the unknown sugar II.