Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked beta 1-2, beta 1-4, or beta 1-6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.

A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin.

     

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.     

The binding site of chicken hepatic lectin

The binding site of the chicken hepatic lectin involved in the clearance of N-acetylglucosamine-terminated serum glycoproteins was explored by a competitive binding assay using 3H-labeled agalacto-orosomucoid and various glycoproteins, polysaccharides, monosaccharides, and glycosides as inhibitors. The binding site is relatively small, involving a terminal nonreducing DGlcNAc structure with an equatorial N-acetamido group on carbon 2 and an equatorial hydroxyl group on carbon 4. Among the mono- and oligosaccharides tested, benzyl alpha DGlcNAc was the best inhibitor, being three times as effective as DGlcNAc; and in general, all alpha-anomeric glycosides were better than beta-glycosides. All oligosaccharides with terminal nonreducing beta DGlcNAc have almost the same inhibitory power, whereas those with nonreducing DGlc or DGal were relatively inactive. Among the serum and blood group glycoproteins, a Smith degraded human H substance with several exposed terminal nonreducing beta DGlcNAc residues was the most active and twice as effective as agalacto-orosomucoid and an A substance, Hog 75 10% precipitate. Almost all hog preparations, some with A or with H activity, were equally effective. A glycopeptide with terminal DGlcNAc was twice as active as one with terminal nonreducing DMan and DGlcNAc residues and almost three times as potent as one with terminal nonreducing DGal; a glycopeptide with terminal sialic acid was inactive. The slopes of the inhibition lines differed, reflecting the heterogeneity of the various determinant groups on the glycoproteins.     

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens

BackgroundSialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.MethodsNeuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.ResultsClostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.ConclusionsImmobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.General significanceImmobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.     

The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Immunochemical studies on the combining site of the d-galactopyranose and 2-acetamido-2-deoxy-d-galactopyranose specific lectin isolated from Bauhinia purpurea alba seeds

The combining site of the Bauhinia purpurea alba lectin was studied by quantitative precipitin and precipitin inhibition assays. Of 45 blood group substances, glycoproteins, and polysaccharides tested, 35 precipitated over 75% of the lectin. Precursor blood group substances with I activity (Cyst OG 10% from 20% and Cyst OG 20% from 10%), desialized fetuin, and desialized ovine salivary glycoprotein, in which more than 75% of the carbohydrate side chains have dGalN Ac linked through α1 → to the OH group of Ser or Thr of a protein core, completely precipitated the lectin. The poorly reactive blood group substances after mild acid hydrolysis or Smith degradation, as well as sialic acid-containing glycoproteins after removal of sialic acid, had substantially increased activity so that more than 80% of the lectin was precipitated. Precipitability with various blood group substances and glycoproteins is ascribable to the terminal nonreducing dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 3 or 4dGlcNAc, and dGalβ1 → 3 or 4dGlcNAcβ1 → 3dGal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, dGalNAc and its p-nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding dGal compounds but methyl βDGalNAcp was only about 40% more active than methyl βdGalp. The α-anomers of p-nitrophenyl DGalNAcp and dGalp, were twice as active as the corresponding β-anomers. Methyl αDGalNAcp was four times as active as the β-anomer but the inhibitory power of the methyl α- and β-anomers of dGal were about equal. Among the oligosaccharides tested, dGalβ1 → 3dGalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as dGalβ1 → 3dGalNAc, which was somewhat more active than dGalNAcα1 → 6dGal and dGalNAc, and 2.5 and 5 times as active as dGalNAcα1 → 3dGalβ1 → 3dGlcNAc and dGalNAcαl → 3dGa1, respectively (blood group A specific). These findings suggest that a subterminal dGalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that dGalβ1 → 3dGlcNAc and dGalβ1 → 6dGal were poorer inhibitors although dGalβ1 → 3dGlcNAc was two to three times as active as glycosides of dGal. Oligosaccharides with terminal nonreducing dGal and subterminal α-linked dGal were as active or less active than dGal. dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc (lacto-N-tetraose) and dGalβ1 → 3dGlcNAcβ1 → 3dGal-β1-O-(CH₂)₈COOCH₃ were equally active and 1.5 times as potent as dGalβ1 → 3dGlcNAc whereas dGalβ1 → 3dGlcNAcβ1 → 6dGal was only 40% as potent as dGalβ1 → 3dGlcNAc suggesting that a third sugar may be part of the determinant. Substitution of dGalβ1 → 3dGlcNAcβ1 → 3dGalβ1 → 4dGlc on the subterminal dGlcNAc by lFucα1 → 4 in lacto-N-fucopentaose II reduced activity fourfold; if the nonreducing dGal is substituted by lFucα1 → 3 as in lacto-N-fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing dGal as well as subterminal dGlcNAc are contributing to binding. The β → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4dGlcNAc is a poorer inhibitor. Although the available data suggest that the combining site of the lectin Bauhinia purpurea alba may be most complementary to the structure dGalβ1 → 3dGalNAcβ1 → 3dGal, several other possibilities remain to be tested when suitable oligosaccharides become available.     

Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.     

Unaltered catabolism of desialylated low-density lipoprotein in the pig and in cultured rat hepatocytes.

Removal of the terminal sialic acid residues from many serum glycoproteins results in exposure of their penultimate galactose residues and rapid clearance from circulation by the liver. Low-density lipoprotein is a glycoprotein containing 21 galactose and 9 sialic acid residues per particle. Studies in this laboratory and others have shown that both the liver and extrahepatic tissues contribute to the degradation of low-density lipoprotein. This study was undertaken to determine whether desialylation of pig low-density lipoprotein alters its removal from circulation. Low-density lipoprotein was incubated at 37 degrees C with an agarose-bound neuraminidase, proteinase-free, from Clostridium perfringens. After 18 h at pH 5.0, 70% of the sialic acid residues were removed. The desialylated 131I-labelled and native 125I-labelled low-density lipoproteins were simultaneously injected into a pig, and their disappearance from plasma was followed for 96 h. The turnovers of the two were identical. In contrast, neuraminidase-treated fetuin was cleared about 200-fold faster than native fetuin. Studies were also performed in cultured rat hepatocytes. Rates of degradation of native and neuraminidase-treated low-density lipoprotein were similar, whereas asialo-fetuin was degraded at six to ten times the rate of native fetuin. Thus desialylation does not appear to alter low-density-lipoprotein catabolism by hepatic or extrahepatic cells.     

Isolation and characterization of a second lectin (SNA-II) present in elderberry (Sambucus nigra L.) bark

A second lectin (SNA-II) has been isolated from elderberry (Sambucus nigra L.) bark by affinity chromatography on immobilized asialo-glycophorin. This lectin is a blood group nonspecific glycoprotein containing 7.8% carbohydrate and which is rich in asparagine/aspartic acid, glutamine/glutamic acid, glycine, valine, and leucine. Gel filtration on Superose 12 gave a single symmetrical peak corresponding to Mr, 51,000; SDS-acrylamide electrophoresis gave a single polypeptide, Mr, 30,000. Hence SNA-II appears to be a homodimer. The lectin is a Gal/GalNAc-specific lectin which is precipitated by glycoproteins containing GalNAc-terminated oligosaccharide chains (e.g., asialo-ovine submaxillary and hog gastric mucins), and by glycoproteins and polysaccharides having multiple terminal nonreducing D-galactosyl groups as occur in asialoglycophorin, asialo-laminin and Type 14 pneumococcal polysaccharide. The carbohydrate binding specificity of SNA-II was studied by sugar hapten inhibition of the asialo-glycophorin precipitation reaction. The lectin's binding site appears to be most complementary to Gal-NAc linked alpha to the C-2, C-3, or C-6 hydroxyl group of galactose. These disaccharide units are approximately 100 times more potent than melibiose, 60 times more potent than N-acetyllactosamine, and 30 times more potent than lactose. Interestingly, the blood group A-active trisaccharide containing an L-fucosyl group linked alpha 1-2 to galactose was 10-fold poorer as an inhibitor than the parent oligosaccharide (GalNAc alpha 1-3Gal), suggesting steric hindrance to binding by the alpha-L-fucosyl group; this explains the failure of the lectin to exhibit blood group A specificity.     

Anomer specificity of the 14 kDa galactose-binding lectin: A reappraisal

A β-anomer preference among galactosides has been attributed to the S-type 14 kDa galactose binding lectin. Here the anomeric preference of this lectin from bovine brain (BBL) is reexamined using inhibition of lectin-mediated haemagglutination, binding of the lectin to dot-blotted glycoproteins and affinity electrophoresis of the lectin through polysaccharide-containing gels. 1.0-methyl α-D-galactoside was 8 times better inhibitor of BBL than the corresponding ß-anomer. The terminal galactose in bovine thyroglobulin (exclusively. α-linked) were also nearly 8 times more inhibitory than those in asialofetuin (exclusively ß-linked). The terminal α-galactose-containing endogenous glycoproteins of bovine brain were nearly 4 times better inhibitors of BBL than laminin. Removal of terminal α-galactose units by α-galactosidase fully abolished the BBL binding of thyroglobulin and endogenous glycoproteins. BBL was also sugar-specifically retarded by polyacrylamide gel containing guar galactommannan which bears only α-linked galactose. Data indicated that α-galactosides were sometimes better than their β-anomers in binding to BBL. The significance of this observation to the physiological role of galactose-binding lectins is discussed.     

The use of galactosyltransferase to probe nitrocellulose-immobilized glycoproteins for nonreducing terminal N-acetylglucosamine residues

We report the use of UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyl-transferase (EC 2.4.1.38), purified from bovine milk, to detect nonreducing terminal N-acetylglucosamine residues on glycoproteins immobilized on nitrocellulose by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. Soluble galactosyltransferase incorporates radiolabeled galactose from the substrate UDP-[6-3H]galactose into the appropriate immobilized acceptor with high specificity. Incorporation is proportional to substrate amount and is saturable with time. The kinetics of labeling are independent of substrate amount. Half-maximal incorporation occurs by 4 h and saturation occurs by 16 h. We have used galactosyltransferase as a probe (i) to verify the presence of nonreducing terminal N-acetylglucosamine residues in bovine rod outer segment membrane rhodopsin and in several glycoproteins in F9 murine teratocarcinoma cells and (ii) to detect previously reported endo-beta-N-acetylglucosaminidase activity in a commercial preparation of endoglycosidase F.     

Characterization and partial purification of beta-N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Urine from Sd(a+) individuals was found to contain a beta-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3'-sialyllactose and glycoproteins carrying the terminal NeuAc alpha-3Gal beta group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn2+ but not for detergents. The Km for UDP-GalNAc was 66 X 10(-6) M, using fetuin as an acceptor. Like beta-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [3H]GalNAc to 3'-sialyllactose, we propose that the enzyme attaches GalNAc in beta-anomeric configuration to O-4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a-) donor lacking beta-GalNAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the beta-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171-184) the urinary transferase is able to transfer beta-GalNAc to the NeuAc alpha-3Gal beta-3(NeuAc alpha-6)GalNAc chains bound to the native glycophorin.     

Purification and characterization of a human lectin specific for penultimate galactose residues

A novel lectin has been found in human plasma. The lectin was purified by affinity chromatography using an adsorbent in which 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosylhydroxylysine (Glc-Gal-Hyl) was coupled to Sepharose. The molecular weight of the lectin was determined by gradient gel electrophoresis to be approximately 240,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the subunit had the molecular weight of 29,500. Composition analysis has shown the lectin is a glycoprotein in which 12% of the molecule consists of carbohydrate. Native human, horse, calf, sheep, rabbit, and rat erythrocytes were agglutinated by the lectin in the presence of calcium. Glc-Gal-Hyl, N-acetylated Glc-Gal-Hyl, and stachyose inhibited the hemagglutination, whereas monosaccharides, maltose, cellobiose, lactose, raffinose, galactosylhydroxylysine, and N-acetylated galactosylhydroxylysine were not inhibitory. The lectin is strongly inhibited by the desialylated bovine erythrocyte glycoprotein, which contains galactose beta 1-3galactose beta-sequence at the nonreducing termini of the sugar chains, whereas disialylated orosomucoid did not inhibit the lectin. These results indicate that the lectin recognizes the penultimate galactose residue in a hapten molecule in contrast to usual galactose-binding proteins or galactose-specific lectins, which recognize exposed, terminal galactose residues of sugar chains.     

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 mm; N-glycolylneuraminic acid, 0.90 mm; d-glucosamine, 4.9 mm; d-galactosamine, 7.6 mm; N-acetyl-d-glucosamine, 23 mm; and N-acetyl-d-galactosamine, 24 mm. d-Galactose, d-glucose, d-mannose, α-methyl-d-glucoside, α-methyl-d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin

Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.     

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.     

Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

A comparative study on the purification of the Amaranthus leucocarpus syn. hypocondriacus lectin.

Amaranthus leucocarpus lectin is a homodimeric glycoprotein of 35 kDa per sub-unit, which interacts specifically with N-acetyl-galactosamine. In this work, we compared different glycoproteins that contain Galbeta1-3 GalNAcalpha1-3 Ser/Thr or GalNAcalpha1-3 Ser/Thr in their structure as ligands to purify the A. leucocarpus lectin. From the glycoproteins tested, fetuin was the most potent inhibitor of the hemagglutinating activity and the better ligand for lectin purification; however, the use of desialylated stroma from erythrocytes represented the cheapest method to purify this lectin. O-linked glycans released from the glycoproteins used as affinity matrix and those from different erythrocytes were less inhibitory than parental glycoproteins. The NH2-terminal of the lectin is blocked; moreover, this is the only example of a lectin isolated from this genus to be a glycoprotein. Analysis of the glycoprotein sequences with inhibitory activity for the lectin, showed a different pattern in the O-glycosylation, which confirms that A. leucocarpus lectin recognizes conformation and, probably, distances among O-linked glycans moieties.