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Query: UNIPROT:P06889 (Mol)
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The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3 degrees and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures. The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous. The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Cell Biochem 1991 Mar 27
PMID:Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins. 205 1

Lectin histochemical studies were performed on paraffin embedded sections of the olfactory system of the eel to identify specific glycoconjugates on the surface of primary olfactory neurons. The olfactory receptors, the olfactory nerve fibres and their terminals in the bulbs were labelled with the lectins (SBA, BSA-I, BSA-I-B4 and DBA) HRP-conjugated or biotinylated. The lectin staining patterns indicate that the membrane of olfactory neurons of the eel had oligosaccharides with alpha-galactose and alpha-N-acetyl-D-galactosamine residues. These findings represent the demonstration of a molecular probe that recognizes specific sets of neurons. The identical histochemical features previously described in the olfactory neurons in amphibians suggest that these carbohydrate moieties might to related to modulation of the cell-cell interactions in the olfactory system of vertebrates.
Cell Mol Biol 1991
PMID:Lectin histochemical study of olfactory neurons in the eel. 205 86

The discovery of jacalin, a group of lectins from jackfruit seeds (Artocarpus heterophyllus), has attracted considerable attention due to its numerous interesting immunological properties as well as its usefulness in the isolation of various serum proteins. We have further identified a similar lectin from the seeds of Champedak (Artocarpus integer) which we refer to as lectin-C and performed comparative studies with two types of jacalin isolated from different batches of the Malaysian jackfruit seeds (jacalin-M1 and jacalin-M2). The three purified lectins demonstrated equivalent apparent Mr of about 52,500, each of which comprised of a combination of two types of non-covalently-linked subunits with apparent Mr of approximately 13,300 and 16,000. The lectins demonstrated equal haemagglutinating activity against human erythrocytes of blood groups A, B, AB and O. Our data also demonstrated that lectin-C, jacalin-M1 and jacalin-M2 are similar by selectively precipitating human serum IgA1 and colostral sIgA but not IgA2, IgD, IgG and IgM. When immunoelectrophoresis was performed on normal human sera and reacted with the lectins, single precipitin arcs corresponding to IgA immunoprecipitates were detected with lectin-C and jacalin-MI. Jacalin-M2, however, exhibited two closely associated precipitin arcs. The binding of these lectins with IgA was pronouncedly inhibited in the presence of p-nitrophenyl-beta-D-galactopyranoside, 1-o-methyl-alpha-D-galactopyranoside, D-melibiose, N-acetyl-D-galactosamine and D-galactose. The data therefore provide evidence on the differential specificity of IgA binding lectins isolated from seeds of similar as well as distinct Artocarpus species.
Mol Immunol
PMID:IgA binding lectins isolated from distinct Artocarpus species demonstrate differential specificity. 206 19

One of the mouse sperm surface binding sites for zona pellucida ligands exhibits galactosyltransferase (GT) enzyme activity. The present study was undertaken to ascertain whether the GT site behaves as a noncatalytic binding site in its physiological capacity, with no glycosylation of zona ligands, or whether glycosylation of zona ligands is an integral part of sperm-zona binding. The effects of Mn2+, the obligatory cation for GT catalysis, on enzyme activity and sperm-zona binding were examined. With uridine-5'-diphosphogalactose (UDPgal) as galactose donor, and N-acetylglucosamine (GlcNAc) as galactose acceptor, increasing concentrations of Mn2+ in the range of 0.1-10 mM increased GT enzyme activity, with half-maximal activation at 0.65 mM Mn2+ (Vmax = 20 pmol/hr/10(6) cells). In the presence of 0-2 mM Mn2+, sperm-zona binding was inhibited in a concentration-dependent manner; 50% inhibition occurred at 1.25 mM Mn2+. At this concentration, GT enzyme activity was at 65% Vmax. To determine the specificity of the GT site for glycoprotein terminal carbohydrate residues, spermatozoa were incubated with, asialo-ovine submaxillary mucin (N-acetylgalactosamine residues), asialo-, -alpha 1-acid glycoprotein (beta 1-4 galactose residues) ovalbumin (Ov; GlcNAc residues), and asialo-agalacto-/alpha 1-acid glycoprotein (AsAgAGP; GlcN-Ac residues). Only Ov and AsAgAGP acted as acceptors for galactose in the enzyme assay and inhibitors in the sperm-zona binding assay. The kinetics of the interaction of AsAgAGP with the GT site were determined: the Km was 3.6 mg/ml, with Vmax of 33 pmol/hr/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1990 Apr
PMID:Further characterization of the mouse sperm surface zona-binding site with galactosyltransferase activity. 210 19

Previously, on the basis of lectin binding and glycosidase digestion assays, we have suggested that N-acetyl-D-glucosamine residues (GlcNAc) are major structural components of both trophozoites and in vivo cysts of the intestinal parasite Giardia lamblia. In this report we confirm that GlcNAc is present both in trophozoites and in vitro cysts as assessed by lectin binding and glycosidase digestion assays, galactosyltransferase labeling, immunochemical analysis using antibodies specific for GlcNAc and its beta 1-4 oligomers, and by gas chromatography/mass spectrometry (GC/MS). The results show that wheatgerm agglutinin (WGA) binds specifically to intact trophozoites and in vitro cysts as well as to SDS-PAGE separated proteins. WGA binding to the separated proteins was markedly reduced after their digestion with N-acetyl-beta-D-glucosaminidase, supporting the conclusion that WGA is reacting with terminal beta-linked GlcNAc residues. Labeling of trophozoites and cysts by 3H-exogalactosylation with galactosyltransferase further confirmed the presence of terminal GlcNAc in both surface and intracellular glycoproteins. The presence of GlcNAc is also supported by microfluorometric analysis using antibodies to (GlcNAc)1, (GlcNAc)2, and (GlcNAc)3, which revealed a sugar-inhibitable binding of the antibody to live trophozoites. Finally, the presence of GlcNAc in both cysts and trophozoites was unequivocally confirmed by GC/MS analysis of detergent-extracted membranes and of glycoproteins isolated by affinity chromatography on WGA-agarose. GC/MS analysis also revealed mannose (Man), N-acetyl-D-galactosamine (GalNAc), fucose (Fuc), galactose (Gal), glucose (Glc) and N-acetylneuraminic acid (NANA) to be present in cysts. All these sugars were also present in trophozoites, except for GalNAc. The glycoproteins isolated by WGA affinity chromatography were 5- to 40-fold enriched in GlcNAc, further supporting the conclusion that WGA reacts with GlcNAc in Giardia. In summary, the data presented here provide biological and chemical evidence for GlcNAc in both cysts and trophozoites of G. lamblia and are consistent with previously published results from this and other laboratories.
Mol Biochem Parasitol 1990 Dec
PMID:N-acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lamblia and serves as receptor for wheatgerm agglutinin. 212 47

Crotalarin, the N-acetyl-D-galactosamine-binding blood group A-specific lectin from the seeds of Crotalaria striata was subjected to various chemical modifications in order to ascertain the amino acid residues responsible for its carbohydrate-binding property. Modification of lysine, cysteine and arginine residues did not affect the carbohydrate-binding activity of the lectin. However, modification of tyrosine residue and carboxy group of the acidic amino acids led to a complete loss of its activity, indicating the involvement of tyrosine and aspartic and glutamic acid in the saccharide-binding respectively. The hemagglutinating activity of the lectin was completely/almost completely lost by modification of tryptophan residues. The relative loss in hemagglutinating activity on modification of tryptophan residues indicate that one residue/molecule is required for the carbohydrate-binding activity of the lectin. Modification was not effective in the presence of D-galactose (0.2 M). A marked decrease in the fluorescence emission was found as the tryptophan residues of crotalarin were modified. The c.d. spectra showed the presence of an identical pattern of conformation in the native and modified lectins which confirms that the loss in activity was due to modification only. The effect of periodate oxidation on crotalarin showed loss of activity whereas action of enzymes retained most of the activity.
Mol Cell Biochem 1990 Aug 10
PMID:Chemical modification studies on a blood group A-specific lectin, crotalarin (Crotalaria striata) and its effect on hemagglutinating activity. 217 41

In cells isolated from guinea-pig or rat ventricular muscle occurrence and distribution of carbohydrate components of the surface coat were monitored using fluorochrome-coupled lectins. Fluorescence of membrane-bound lectins was assayed by an image analysis system. The lectins ConA, WGA, sWGA, LFA and RCA-I showed specific binding to the whole myocyte surface, indicating a homogeneous distribution of alpha-mannosyl, alpha-glycosyl, N-acetylglucosaminyl, N-acetylneuraminate and beta-galactosyl residues. Binding of DBA and SBA, with specific affinity for N-acetylgalactosaminyl residues, to guinea-pig cardiac myocytes was mainly at the cell poles corresponding to intercalated discs in intact tissue. Both lectins failed to interact with rat myocytes. UEA-I, specific for alpha-L-fucose, bound slightly to rat and not to guinea-pig myocytes. Binding of PNA to guinea-pig myocytes was observed only after cleaving off sialic acids from cell surface, suggesting that sialic acids mask galactosyl-beta(1,3)-N-acetylgalactosamine residues. Specificity of lectin-cell interaction was tested by an inhibition assay where free sugars were tested for their capacity to inhibit lectin binding to the myocytes. When comparing different isolation procedures based on different proteolytic enzymes, the myocytes' affinity to any lectin was found to be qualitatively unchanged. Investigation of lectin-decorated myocytes by means of confocal laser scan microscopy showed that lectin binding sites are not confined to the cell surface but are also present in sarcolemmal invaginations, i.e. transverse tubules. This suggests that the tubular system is lined with a carbohydrate layer similar to, and continuous with, that of the peripheral cell surface.
J Mol Cell Cardiol 1990 Jul
PMID:The cell surface of isolated cardiac myocytes--a light microscope study with use of fluorochrome-coupled lectins. 223 45

An air-liquid interface (biphasic) primary culture system in which guinea pig tracheal epithelial cells maintain morphologic characteristics of differentiated epithelium has been developed in this laboratory. In this report, we compared quantitatively cell populations of 8-day cultures to those of epithelial mucosa in intact trachea. In addition, high molecular weight glycoconjugates released by the cultured cells were isolated and characterized. Quantitative morphometric analysis revealed similar volume densities of ciliated, secretory, basal, and "other" cells in cultures and in intact tracheal surface epithelium, although the cultures tended to have smaller cells and contained fewer basal cells. High molecular weight glycoconjugates released apically by cell cultures and excluded from Sepharose CL-4B columns contained approximately 5% hyaluronic acid but undetectable amounts of other proteoglycans, such as chondroitin sulfate, heparan sulfate, and dermatan sulfate. The hyaluronidase-resistant glycoconjugates exhibited a peak buoyant density at 1.49 g/ml on cesium chloride density gradient centrifugation and were shown to contain mucin-type carbohydrate to peptide linkages (i.e., GalNAc to ser/thr) and an amino acid composition typical of respiratory mucins. The results indicate that this organotypic cell culture system mimics quite closely morphology of mucosal epithelium in intact airways and that the cells release high molecular weight glycoconjugates with biochemical properties of mucin-type glycoproteins. Thus, this in vitro system appears well-suited for studies of mucin secretion and other functions of respiratory epithelial cells.
Am J Respir Cell Mol Biol 1990 Feb
PMID:Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: cellular composition and biochemical analysis of released glycoconjugates. 230 71

Guinea pig erythrocytes desialated by treatment with neuraminidase from Vibrio cholerae were lyzed in autologous serum through a natural-antibody-dependent activation of the classical complement pathway. Lysis was inhibited when a mannose, glucose, galactose or N-acetyl-glucosamine was added to the incubation mixture. Methyl-alpha- or -beta-D-galactopyranosides were poorly effective and N-acetyl-D-galactosamine was not effective at all. Inhibition of lysis by the carbohydrates was due neither to an anti-complementary effect nor to a modification of the osmotic pressure since: (a) they did not alter the total complement haemolytic activity of guinea pig serum, and (b) they did not inhibit lysis of desialated guinea pig erythrocytes in human serum through activation of the alternative complement pathway. The presence of mannose, glucose, galactose or N-acetyl-glucosamine in the incubation mixture resulted in an impaired fixation of natural auto-antibodies on antigenic sites, namely the T-antigen (Thomsen-Friedenreich), which were unmasked following membrane sialic acid removal. When tested under the same conditions, only small percentage of the normal human population showed the phenomenon of lysis of desialated erythrocytes in autologous serum. Lysis was not due to a particular susceptibility of erythrocytes from these individuals to complement-mediated lysis but to the presence in their serum of complement-activating anti-T antibodies. As expected, the activity of human anti-T antibodies was inhibited by galactose and N-acetyl-galactosamine, which are the immunodominant sugars of the human T-antigen. Mannose and glucose had no effect, and methyl- alpha- or - beta-D-galactopyranosides were almost as effective as galactose. The heterogeneity of the human population with regard to the complement-activating capacity of anti-T antibodies could be of significance for the individual response of the host to an infection by a neuraminidase-producing microorganism. That the immunodominant sugars of the T-antigen were different between humans and guinea pigs was further assessed by absorption experiments. We have demonstrated that guinea pig anti-T antibodies were not removed during contact with desialated human red cells which do not have the mannose specificity, whereas human antibodies were almost entirely retained on desialated guinea pig red cells which, beside mannose, express galactose. These results also suggest that guinea pig antibodies are mostly directed towards mannose and glucose.
Mol Immunol 1985 Sep
PMID:Differences in carbohydrate specificities and complement-activating capacity of guinea pig and human antibodies to neuraminidase-treated autologous erythrocytes. 241 14

Hybridoma generation, using specifically, maximally desialylated human blood group O erythrocytes (T RBC) as immunogen, and biochemical studies suggested the presence of immunogenic Tn epitopes. GalNAc alpha-O, on T RBC. We therefore investigated by immunochemical means whether or not Tn-specific epitopes immunoreactive with anti-Tn antibodies present in ordinary human sera occur on T RBC and on Thomsen-Friedenreich (T) antigen prepared from them. We did detect the Tn epitope with such antibodies, in addition to the T epitope, on isolated T antigen. T RBC absorbed specifically, under standard conditions, 25-60% of the heterogeneous anti-Tn antibody populations in ordinary human sera of appropriately adjusted titer score. The anti-Tn eluted from T RBC had scores ranging from 6.5 to 35% of those of the unabsorbed parent sera. The varying fine specificities of eluted anti-Tn were demonstrated by inhibition of Tn RBC agglutination with putative haptens and antigens. Tn-specific haptens and antigens were the most powerful inhibitors. Depending on the serum used to prepare the anti-Tn eluates, the antibodies could be divided into those that were inhibited well exclusively by GalNAc alpha-O derivatives and those that were also inhibited by Gal, notably by Gal alpha-O derivatives and more strongly by GalNAc and Me-alpha-GalNAc. In the two reciprocal hemagglutination inhibition systems used, Tn-specific haptens were considerably more active than the T-hapten Gal beta 1----3GalNAc alpha-O, and desialylated ovine submaxillary mucin (AS-OSM) had higher activity than T antigen. Inhibition of Tn RBC agglutination by haptens was uniformly more efficient than that of T RBC; this is, at least in part, due to the much higher negative charge of Tn as opposed to T RBC. In microprecipitin tests, Helix pomatia lectin was nearly as powerful a precipitin of T antigen as of AS-OSM. The importance of the terminal GalNAc alpha of T antigen for its precipitation with the Helix lectin was demonstrated by the very high and virtually exclusive inhibitory activity of Me-alpha-GalNAc and GalNAc. Our findings may contribute to comprehension of the significance of uncovered Tn in most carcinomas, and the role of anti-Tn as a "natural" anti-carcinoma antibody. They may also help illuminate the rare heterozygous, autosomal, apparently premalignant spot mutation that leads to Tn RBC in vivo.
Mol Immunol 1985 Nov
PMID:Tn epitopes, immunoreactive with ordinary anti-Tn antibodies, on normal, desialylated human erythrocytes and on Thomsen-Friedenreich antigen isolated therefrom. 241 12


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