Glycan residues of N- and O-linked oligosaccharides in the premeiotic spermatogenetic cells of the urodele amphibian Pleurodeles waltl characterize by means of lectin histochemistry

The aim of this work was the characterization of the glycoconjugates of the premeiotic spermatogenetic cells of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. In the cytoplasm of the primordial germ cells, primary and secondary spermatogonia and primary spermatocytes, a granular structure can be observed close to the nucleus. These granules contain four types of sugar chains according to their appearance during the differentiation process: 1. some oligosaccharides that are identified in all the four cell types above mentioned, which include N-linked oligosaccharides with Fuc, Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc and O-linked oligosaccharides with Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc; 2. other glycan chains that are not present in the primary spermatocytes (N-linked oligosaccharides with DBA-positive GalNAc, GlcNAc, and a slight amount of Neu5Ac alpha2,6Gal/GalNAc and O-linked oligosaccharides with WGA-positive GlcNAc); 3. the sugar chains that are not in the earliest step of spermatogenesis (formed by both N-linked and O-linked oligosaccharides with Glc); and 4. other that appear at the earliest and latest stages, but not in the intermediate ones, (N-linked oligosaccharides with Man and O-linked oligosaccharides with SBA- and HPA-positive GalNAc and PNA-positive Gal beta1,3GalNAc). This structure could be related with the Drosophila spectrosome and fusome, unusual cytoplasmic organelles implicated in cystic germ cell development. Data from the present work, as compared with those from mammals and other vertebrates, suggest that, although no dramatic changes in the glycosylation pattern are observed, some cell glycoconjugates are modified in a predetermined way during the early steps of the spermatogenetic differentiation process.     

Mucin biosynthesis revisited. The enzymatic transfer of Gal in beta 1,3 linkage to the GalNAc moiety of the core structure R1-GlcNAc beta 1,6GalNAc alpha-O-R2.

Synthetic glycosides containing the core, -Glc-NAc beta 1,6GalNAc alpha-, acted as acceptors for beta-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the alpha-benzylglycoside of LacNAc beta 1,6GalNAc (LGBn) (25.1 nmol of Gal) and the alpha-ortho-nitrophenylglycosides of LacNAc beta 1,6GalNAc (22.0 nmol of Gal), GlcNAc beta 1,6GalNAc (15.5 nmol of Gal), and Fuc alpha 1,3GlcNAc beta 1,6GalNAc (25.9 nmol of Gal); LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the alpha-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. The alpha GalNAc:beta 1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum----1.4; ascitic fluid----0.9; tumor----17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37 degrees C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37 degrees C was continued for 40 h at 4 degrees C, indicating the high affinity of LGBn for the enzyme at lower temp. Km for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; Km for ACGM (M(r) 200,000) in presence of LGBn was 16.1 microM and Ki for ACGM (as the inhibitor) was 41.7 microM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fuc alpha 1,3GlcNAc beta 1,6GalNAc alpha-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of beta 1,3Gal attachment on alpha GalNAc, which has already been substituted by beta 1,6GlcNAc, then elongated by beta 1,4Gal and also terminated by alpha 1,3Fuc.     

The structures of the serine-linked sugar chains on human chorionic gonadotropin

The human chorionic gonadotropin beta-subunit tryptic COOH-terminal peptide (residues 123-145) which contains 3 serine-linked sugar chains was isolated. The sugar chains were cleaved by beta-elimination and then separated by gel filtration. The peaks were pooled and their compositions determined. The products of serial glycosidase digestion and periodate oxidation of the intact glycopeptide were also characterized. Of the serine-linked sugar chains, 13% were the hexasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,3 Gal beta 1,4 GlcNAc beta 1,6) GalNAc, 34% the tetrasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,6) GalNAc, 43% the trisaccharide NeuAc alpha 2,3 Gal beta 1,3 GalNAc and 10% the disaccharide NeuAc alpha 2,6 GalNAc.     

Endo-beta-galactosidase of Escherichia freundii. Hydrolysis of pig colonic mucin and milk oligosaccharides by endoglycosidic action.

The purified keratansulfate degrading enzyme from $\text{Eschericia freundii}$ could hydrolyze desialyzed pig colonic mucin and milk oligosaccharides. Desialyzed pig colonic mucin was digested to produce GlcNAcβ(1→3)Gal, GlcNAc-6Sβ(1→3)Gal and resistant polymer. Lacto-N-tetraose and lacto-N-tetraitol were hydrolyzed endoglycosidically to release glucose and sorbitol, respectively. Therefore, this enzyme was found to be an endo-β-galactosidase of rather wide specificity.     

Human ovarian cancer, lymphoma spleen, and bovine milk GlcNAc:beta1,4Gal/GalNAc transferases: two molecular species in ovarian tumor and induction of GalNAcbeta1,4Glc synthesis by alpha-lactalbumin

Affinity Gel-UDP was utilized to purify GlcNAc:beta1,4Gal/GalNAc transferases (Ts) from human lymphoma spleen, ovarian tumor, and ovarian cancer sera. Mn(2+) was found to be an absolute requirement for activity. Two molecular species containing both beta1,4Gal/GalNAc-T activities were discernible when the purified ovarian tumor microsomal enzyme was subjected to Sephacryl S-100 HR column chromatography as well as native polyacylamide gel-electrophoresis. Acceptor specificity studies of the affinity-purified lymphoma spleen and ovarian tumor microsomal enzymes and the conventionally purified, as well as the cloned, bovine milk GlcNAc:beta1,4Gal-Ts using a number of synthetic acceptors showed that the beta(1,6)-linked GlcNAc moiety to alpha-GalNAc was the most efficient acceptor. As compared to the purified milk enzyme, the recombinant form exhibited sixfold GlcNAc:beta1,4 GalNAc-T activity and up to eightfold GlcNAc6SO3beta-:beta1,4Gal-T activity. Further, the recombinant enzyme catalyzed the transfer of GalNAc to the terminal beta-linked GlcNAc6SO3 moiety. Alpha-lactalbumin (alpha-LA) inhibited up to 85%, the transfer of Gal to the GlcNAc moiety linked either to Man or GlcNAc. On the contrary, alpha-LA had no significant influence on the transfer of GalNAc to the above acceptors. alpha-LA had no appreciable effect on the recombinant enzyme, except for the transfer of Gal or GalNAc to Glc. Both alpha- and beta-glucosides, as well as alpha-N-acetylglucosaminide, did not serve as acceptors.     

Engineering of the glycan-binding specificity of Agrocybe cylindracea galectin towards α(2,3)-linked sialic acid by saturation mutagenesis

Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Distribution of sialoglycoconjugates in the oviductal isthmus of the horse during anoestrus, oestrus and pregnancy: a lectin histochemistry study

The distribution of sialic acid residues as well as other glycosidic sugars has been investigated in the horse oviductal isthmus during anoestrus, oestrus and pregnancy by means of lectin and pre-lectin methods. Ciliated cells and non-ciliated (secretory) cells exhibited different lectin binding profiles that were found to change during the investigated stages. Ciliated cells did not show any reactivity in the basal cytoplasm, while the supra-nuclear cytoplasm displayed a few of oligosaccharides with terminal and internal alphamannose (Man) and/or alphaglucose (Glc) during oestrus and pregnancy and a moderate presence of oligosaccharides terminating in alphafucose (Fuc) during oestrus; cilia exhibited a more complex glycoconjugate pattern for the presence of oligosaccharides terminating in N-acetylgalactosamine (GalNAc), GalNAcalpha1,3 GalNAcalpha1,3galactose(Gal)beta1,4Galbeta1,4N-acetylglucosamine(GlcNAc), Fuc, sialic acid (Neu5Ac)-aGalNAc belonging or not to the GalNAca1,3GalNAca1,3 Galb1,4 Galb1, 4GlcNAc sequence, and. alphaGalNAc and Neu5Aca 2,6Gal/GalNAc increased during oestrus. Cilia displayed terminal Galbeta1,3 GalNAc in pregnancy, terminal alphaGal in anoestrus and pregnancy and terminal or internal D-GlcNAc during anoestrus and pregnancy, respectively. The whole cytoplasm of non-ciliated cells showed oligosaccharides terminating with alphaGalNAc, Neu5Aca2,6Gal/GalNAc, Neu5Ac GalNAca 1,3GalNAcalpha1,3Galbeta1,4Galbeta1,4GlcNAc during the investigated stages, as well as GlcNAc in anoestrus and pregnancy. The supra-nuclear zone of non-ciliated cells exhibited oligosaccharides with terminal Galbeta1,4GlcNAc and internal Man during oestrus and pregnancy as well as terminal alphaGal and Fuc in oestrus and Neu5Ac-Galbeta1,3GalNAc in pregnancy. The luminal surface of non-ciliated cells showed glycans terminating with alphaGalNAc and/or Neu5Ac GalNAcalpha1,3 GalNAcalpha1,3Galbeta1,4Galbeta1,4GlcNAc in all specimens, oligosaccharides with terminal Galbeta1,4GlcNAc and internal Man during oestrus and pregnancy, Neu5Ac alpha2,6Gal/GalNAc in anoestrus and oestrus, and glycans terminating with Galbeta1,3GalNAc, Neu5A acalpha2,3 Galbeta1, 4GlcNac, Neu5ac-Galbeta1,3GalNAc, Neu5Ac-Galbeta1,4 GlcNAc in pregnancy. These findings show the presence of sialoglycoconjugates in the oviductal isthmus of the mare as well as the existence of great modifications in the glycoconjugates linked to different physiological conditions.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Characterization of distinct Gal : 3-O-sulfotransferase activities in human tumor epithelial cell lines and of calf lymph node GlcNAc : 6-O-sulfotransferase activity

We found earlier in human breast and colon tumors, an augmented level of Gal : 3-O-sulfotransferase activities showing, respectively, an acceptor preference to blood group T-hapten (Group A enzymes) or Galbeta1,4GlcNAc (Group B enzymes) on the mucin Core 2 structure [Chandrasekaran EV, Jain RK, Vig R, and Matta KL (1997) Glycobiology 7: 753-68]. The present study reports these enzyme activities in human tumor cell lines and additional tumor specimens. The human colon tumor epithelial cell lines, akin to their parent tumors, express Group B enzyme activity. The acceptor specificity and kinetic properties, such as divalent metal ion activation and pH dependent activity profile, of the colon cancer line LS180 enzyme activity are identical to those of colon tissue specimens. Consistent with breast tumor specimens, the Group A enzyme activity is present in human breast tumor epithelial cell lines, with some exceptions. The Gal : 3-O-sulfotransferases show specific binding to Aleuria aurantia lectin, suggesting the presence of asparagine linked carbohydrate chains containing an inner core alpha1,6-fucosyl residue on these enzymes. Calf lymph nodes contain GlcNAc : 6-O-sulfotransferase as well as Group A Gal : 3-O-sulfotransferase activities, which differ in pH dependent profiles, pH optima (7.6 and 7.0, respectively) and the influence of Mn2+.     

Purification and structures of oligosaccharide chains in swine trachea and Cowper's gland mucin glycoproteins

Mucin glycoproteins were purified from extracts of swine trachea mucosa and Cowper's gland. The gelatinous extracts were solubilized by reduction and carboxymethylation and then purified by chromatography on Sepharose CL-6B and DEAE-Sepharose. The structure of some of the carbohydrate units in these glycoproteins were determined and compared. Alkaline borohydride treatment indicated that more than 85% of the carbohydrate chains in these glycoproteins were linked to serine or threonine residues in the polypeptide chain through O-glycosidic bonds with N-acetylgalactosamine. Reduced oligosaccharides released by treatment with alkaline borohydride were isolated by gel filtration on Bio-Gel P-6 and chromatography on DEAE-cellulose and paper. The structures of the oligosaccharides were established by methylation analysis, gas chromatography, and sequential hydrolysis with specific exoglycosidases. The major oligosaccharides in Cowper's gland mucin glycoproteins were sialylated short chains: NeuAc alpha 2,6GalNAcol and NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAcol. In marked contrast, branched chains containing a Gal beta 1,3(GlcNAc beta 1,6)GalNAc core unit were the major components of trachea mucin glycoprotein. Ten of these chains had the following structures: (Formula: see text).     

Histochemical study of glycoconjugates in the toadfish Halobatrachus didactylus oesophagus epithelium

The carbohydrate expression in the epithelium lining the oesophagus of the toadfish Halobatrachus didactylus was studied by means of conventional and lectin histochemistry. The stratified epithelium was constituted by basal cells, polymorphous cells in the intermediate layer, pyramidal and flattened cells in the outer layer and contained two types of large secretory cells: goblet cells and sacciform cells. PAS, Alcian blue pH 2.5 and pH 1.0 stained very strongly the goblet cells, weakly the surface of the other epithelial cells but did not stain the sacciform cells. The goblet cells cytoplasm contained oligosaccharides with terminal Galbeta1,3GalNAc, alpha/betaGalNAc, Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc residues (PNA, SBA, RCA120, UEA I, LTA and KOH-sialidase-WGA affinity). Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc were also found in the glycocalyx. The sacciform cells expressed sialyloligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acbeta2,6Gal/GalNAc, Neu5AcForssman pentasaccharide (MAL II, SNA, KOH-sialidase-DBA staining) as well as asialo-glycoconjugates with terminal/internal alphaMan (Con A affinity) and with terminal Galbeta1,3GalNAc, Forssman pentasaccharide, Galbeta1,4GlcNAc, GalNAc (HPA and SBA reactivity), alphaGal (GSA I-B4 reactivity), D-GlcNAc (GSA II labelling), alphaL-Fuc. The basal cells cytoplasm exhibited terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc. Intermediate cells showed oligosaccharides with terminal/internal alphaMan and/or terminating with Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc in the cytoplasm and with Neu5Acalpha2,3Galbeta1,4GlcNac, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc in the glycocalyx. The pyramidal cells expressed terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalbeta1,4NAc, alphaGal, alphaL-Fuc in the entire cytoplasm, terminal Neu5Acalpha2,3Galbeta1,4GlcNac and Forssman pentasaccharide in the apical extension, internal betaGlcNAc and/or terminal alphaL-Fuc in the luminal surface, Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alphaGal in the basolateral surface. The flattened cells displayed glycans with terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalNAc, alphaGal, D-GlcNAc in the entire cytoplasm, glycans terminating with Galbeta1,3GalNAc and/or internal betaGlcNAc in the sub-nuclear cytoplasm.     

Ovarian cancer alpha 1,3-L-fucosyltransferase. Differentiation of distinct catalytic species with the unique substrate, 3'-sulfo-N-acetyllactosamine in conjunction with other synthetic acceptors.

Several N-acetyllactosamine (LacNAc) derivatives were tested as acceptors for alpha 1,3-L-fucosyltransferase present in human ovarian cancer sera and ovarian tumor. The enzyme of the soluble fraction of tumor was purified to apparent homogeneity by chromatography on bovine IgG glycopeptide-Sepharose followed by Sephacryl S-200 (M(r) < 67,000). As compared with 2'-methyl LacNAc, 3'-sulfo LacNAc was about 5-fold more sensitive in measuring alpha 1,3-fucosyltransferase in sera (Km, 3'-sulfo LacNAc, 0.12 mM; 2'-methyl LacNAc, 6.67 mM). When ovarian cancer serum was the enzyme source, either the sulfate group or a sialyl moiety at C-3' of LacNAc enhanced the acceptor ability (341 and 242%, respectively), whereas the sulfate group at C-2' or C-6' reduced the activity (22-36%); sulfate at C-6 or fucose at C-2' increased the activity (172 and 253%). The beta-benzylation of the reducing end, in general, increased the activity 2-3-fold. The enzyme of the soluble fraction of tumor exhibited more activity toward 3'-sulfo LacNAc (447%), 2'-fucosyl-LacNAc (436%), and 6-sulfo LacNAc (272%). Very low activity was observed with 3'-sialyl LacNAc (12.4%), 2'-sulfo LacNAc (33%), and 6'-sulfo LacNAc (5%); Fuc alpha 1,2Gal beta 1,3GlcNAc beta-O-p-nitrophenyl (166%), 2-methyl Gal beta 1,3GlcNAc beta-O-benzyl (204%), and 3-sulfo Gal beta 1,3GlcNAc (415%) also acted as acceptors, indicating the coexistence of alpha 1,3- and alpha 1,4-fucosyltransferase. The tumor particulate enzyme behaved entirely different, exhibiting low activity with 3'-sulfo LacNAc (39%) and 2'-fucosyl-LacNAc (148%); 3'-sialyl, 6'-sulfo, 6-sulfo, or 2'-sulfo LacNAc were 3, 43, 53, and 10% active, respectively. Thus, the ovarian cancer serum alpha 1,3-fucosyltransferase acts equally well on H-type 2,3'-sialyl LacNAc and 3'-sulfo LacNAc, but not on H-type 1. The enzyme of soluble tumor fraction acts on H-type 2,3'-sulfo LacNAc as well as H-type 1 but poorly on 3'-sialyl LacNAc. The tumor particulate enzyme acts on H-type 2 but poorly on 3'-sulfo or 3'-sialyl LacNAc and is inactive with H-type 1. When normal serum was examined with synthetic acceptors, > 80% activity was found as alpha 1,2-fucosyltransferase and the rest as alpha 1,3-fucosyltransferase. A screening of 21 ovarian cancer and 3 normal sera (3'-sulfo LacNAc as acceptor) showed 17-572% increase (average increase, 188%) of alpha 1,3-fucosyltransferase activity in cancer.(ABSTRACT TRUNCATED AT 400 WORDS)     

A small region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to alpha 2,8-disialyl and branched alpha 2,6-sialyl residues. A comparison with Siglec-9.

Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells. Here we characterize the sugar-binding specificity of Siglec-7 expressed on Chinese hamster ovary cells using polyvalent streptavidin-based glyco-probes. Glyco-probes carrying unique oligosaccharide structures such as GD3 (NeuAc alpha 2,8NeuAc alpha 2,3Gal beta 1,4Glc) and LSTb (Gal beta 1,3[NeuAc alpha 2,6]GlcNAc beta 1,3Gal beta 1,4Glc) oligosaccharides bound to Siglec-7 better than those carrying LSTc (NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal beta 1,4Glc) or GD1a (NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,4[NeuAc alpha 2,3]Gal beta 1,4Glc) oligosaccharides. In contrast, Siglec-9, which is 84% identical to Siglec-7, did not bind to the GD3 and LSTb probes but did bind to the LSTc and GD1a probes. To identify a region(s) responsible for their difference in binding specificity, we prepared a series of V-set domain chimeras between Siglecs-7 and -9. Substitution of a small region, Asn(70)-Lys(75), of Siglec-7 with the equivalent region of Siglec-9 resulted in loss of Siglec-7-like binding specificity and acquisition of Siglec-9-like binding properties. In comparison, a Siglec-9-based chimera, which contains Asn(70)-Lys(75) with additional amino acids derived from Siglec-7, exhibited Siglec-7-like specificity. These results, combined with molecular modeling, suggest that the C-C' loop in the sugar-binding domain plays a major role in determining the binding specificities of Siglecs-7 and -9.     

Identification of physiologically relevant substrates for cloned Gal: 3-O-sulfotransferases (Gal3STs): distinct high affinity of Gal3ST-2 and LS180 sulfotransferase for the globo H backbone, Gal3ST-3 for N-glycan multiterminal Galbeta1, 4GlcNAcbeta units and 6-sulfoGalbeta1, 4GlcNAcbeta, and Gal3ST-4 for the mucin core-2 trisaccharide

Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galbeta1,4GlcNAcbeta1,6(3-O-MeGalbeta1,3)GalNAcalpha-O-Bn (where Bn is benzyl) and the disaccharide Galbeta1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalbeta1,4GlcNAcbeta1,-6(Galbeta1,3)GalNAcalpha-O-Bn and Galbeta1,3GalNAcalpha-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galbeta1,3GalNAcbeta1,3Galalpha-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galbeta1,4GlcNAcbeta unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galbeta1,4GlcNAcbeta1,6(3-O-sulfoGalbeta1,3)GalNAcalpha-O-Bn but not 3-O-sulfoGalbeta1,-4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galbeta1,4(6-O-sulfo)-GlcNAcbeta-O-Al as acceptor, Gal3ST-2 acted efficiently on Galbeta1,3(6-O-sulfo)GlcNAcbeta-O-Al, and Gal3ST-4 acted efficiently on Galbeta1,3(6-O-sulfo)GalNAcalpha-O-Al. Mg(2+), Mn(2+), and Ca(2+) stimulated the activities of Gal3ST-2, whereas only Mg(2+) augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn(2+) concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

Tissue-specific expression of sialyltransferases

Three sialyltransferases which attach terminal sialic acids to glycoprotein sugar chains are shown to exhibit striking differential expression in seven tissues of the rat. Using cDNA probes for the Gal beta 1,4GlcNAc alpha-2,6-sialytransferase which forms a NeuAc alpha 2-6Gal beta 1-4GlcNAc sequence on N-linked sugar chains, three different sized mRNAs are detected, two of which (4.7 and 4.3 kilobases (kb] have high homology along the full length, and a third (3.6 kb, in kidney) which is missing the 5' region corresponding to 45% of the NH2-terminal coding sequence. The 4.7- and 4.3-kb mRNAs exhibit differential expression of over 50-fold with the highest levels in liver and lowest in brain and heart. Assays for enzyme activity in tissue homogenates show high correspondence to the levels of mRNA. Evidence of tissue-specific expression was also obtained for two other sialyltransferases which form the NeuAc alpha 2-3Gal beta 1-4/3GlcNAc and NeuAc alpha 2-3Gal beta 1-3GalNAc sequences on N-linked and O-linked sugar chains, respectively. Comparison of the ratios of the three enzymes in several tissues suggests that they are expressed independently. The results are discussed for their relevance to cellular control of terminal glycosylation sequences on glycoproteins and glycolipids.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.     

Neutral oligosaccharides of bovine submaxillary mucin. A combined mass spectrometry and 1H-NMR study.

Twenty-two neutral O-linked oligosaccharides ranging from monosaccharides to octasaccharides were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3-linked to N-acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(beta 1-6) [Gal(beta 1-3)]GalNAcol or GlcNAc(beta 1-6)[GlcNAc(beta 1-3)]GalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAc alpha 1-6GalNAcol (GalNAc, N-acetylgalactosamine and the hexasaccharide GlcNAc(beta 1-6) [GalNAc(alpha 1-3)( Fuc (alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-3)]GalNAcol (Fuc, L-fucose). Oligosaccharides of the blood-group-A type have not been detected previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric-mucosal glycoproteins has been established by classical immunochemical studies.