Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean).

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I >> Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.

Carbohydrate specificity of a lectin isolated from the fungus Sclerotium rolfsii.

In order to investigate the functional roles of a phytopathogenic fungal lectin (SRL) isolated from the bodies of Sclerotium rolfsii, the binding properties of SRL were studied by enzyme linked lectinosorbent assay and by inhibition of SRL-glycan interaction. Among glycoproteins (gp) tested for binding, SRL reacted strongly with GalNAc alpha1-->4Ser/Thr (Tn) and/or Gal beta1-->3GalNAc alpha1-->(T(alpha)) containing gps: human T(alpha) and Tn glycophorin, asialo salivary gps, and asialofetuin, but its reactivity toward sialylated glycoproteins was reduced significantly. Of the sugar ligands tested for inhibition of SRL-asialofetuin binding, Thomsen-Friedenreich residue (T(alpha)) was the best, being 22.4 and 2.24 x 10(3) more active than GalNAc and Gal beta1--> residues, respectively. Other ligands tested were inactive. When the glycans used as inhibitors, T(alpha), and/or Tn containing gps, especially asialo PSM, asialo BSM, asialo OSM, active antifreeze gp, asialo glycophorin and Tn-glycophorin were very active, and 1.0 x 10(4) times more potent than GalNAc. From these results, it is clear that the combining site of SRL should be of a cavity type and recognizes only Tn and T(alpha) residues of glycans; it is suggested that T(alpha) and Tn glycotopes, which are present only in abnormal carbohydrate sequences of higher orders of mammal, are the most likely sites for phytopathogenic fungal attachment as an initial step of infection. The affinity of SRL for ligands can be ranked in decreasing order as follows: multivalent T(alpha) and Tn >> monomeric T(alpha) and Tn > GalNAc >>> II (Gal beta1-->4GlcNAc), L (Gal beta1-->4Glc), and Gal.

Carbohydrate specificity of an agglutinin isolated from the root of Trichosanthes kirilowii.

The root of Trichosanthes kirilowii, which has been used as Chinese folk medicine for more than two thousand years, contains a Gal specific lectin (TKA). In order to elucidate its binding roles, the carbohydrate specificities of TKA were studied by enzyme linked lectinosorbent assay (ELLSA) and by inhibition of lectin-glycoform binding. Among glycoproteins (gp) tested, TKA reacted strongly with complex carbohydrates with Galbeta1-->4GlcNAc clusters as internal or core structures (human blood group ABH active glycoproteins from human ovarian cyst fluids, hog gastric mucin, and fetuin), porcine salivary glycoprotein and its asialo product, but it was inactive with heparin and mannan (negative control). Of the sugar inhibitors tested for inhibition of binding, Neu5Ac alpha2-->3/6Galbeta1-->4Glc was the best and about 4, 14.6 and 27.7 times more active than Galbeta1-->4GlcNAc(II), Galbeta1-->3GalNAc(T) and Gal, respectively. From these results, it is suggested that this agglutinin is specific for terminal or internal polyvalent Galbeta1-->4GlcNAcbeta1-->, terminal Neu5Ac alpha2-->3/6Galbeta1-->4Glc and cluster forms of Galbeta1-->3GalNAc alpha residues. The unusual affinity of TKA for terminal and internal Galbeta1-->glycotopes may be used to explain the possible attachment roles of this agglutinin in this folk medicine to target cells.

Lectinochemical characterization of a GalNAc and multi-Galbeta1-->4GlcNAc reactive lectin from Wistaria sinensis seeds.

An agglutinin that has high affinity for GalNAcbeta1-->, was isolated from seeds of Wistaria sinensis by adsorption to immobilized mild acid-treated hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 M lactose. The binding property of this lectin was characterized by quantitative precipitin assay (QPA) and by inhibition of biotinylated lectin-glycan interaction. Of the 37 glycoforms tested by QPA, this agglutinin reacted best with a GalNAcbeta1-->4 containing glycoprotein (GP) [Tamm-Horsfall Sd(a+) GP]; a Galbeta1-->4GlcNAc containing GP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human alpha1-acid GP) and a GalNAcalpha1-->3GalNAc containing GP (asialo bird nest GP), but poorly or not at all with most sialic acid containing glycoproteins. Among the oligosaccharides tested, GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Fp) was the most active ligand. It was as active as GalNAc and two to 11 times more active than Tn cluster mixtures, Galbeta1--> 3/4GlcNAc (I/II), GalNAcalpha1-->3(L-Fucalpha1-->2)Gal (Ah), Galbeta1-->4Glc (L), Galbeta1-->3GalNAc (T) and Galalpha1--> 3Galalpha-->methyl (B). Of the monosaccharides and their glycosides tested, p-nitrophenyl betaGalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding alpha anomer and GalNAc (or Fp), respectively. GalNAc was 53.3 times more active than Gal. From the present observations, it can be concluded that the Wistaria agglutinin (WSA) binds to the C-3, C-4 and C-6 positions of GalNAc and Gal residues; the N-acetyl group at C-2 enhances its binding dramatically. The combining site of WSA for GalNAc related ligands is most likely of a shallow type, able to recognize both alpha and beta anomers of GalNAc. Gal ligands must be Galbeta1-->3/4GlcNAc related, in which subterminal beta1-->3/4 GlcNAc contributes significantly to binding; hydrophobicity is important for binding of the beta anomer of Gal. The decreasing order of the affinity of WSA for mammalian structural carbohydrate units is Fp >/= multi-II > monomeric II >/= Tn, I and Ah >/= E and L > T > Gal.

Forssman pentasaccharide and polyvalent Galbeta1-->4GlcNAc as major ligands with affinity for Caragana arborescens agglutinin.

The binding properties of Caragana arborescens agglutinin (CAA, pea tree agglutinin) were studied by enzyme linked lectinosorbent assay (ELLSA) and by inhibition of CAA-glycan interaction. Among glycoproteins (gps) tested, CAA reacted strongly with asialo bird nest gp, asialo rat sublingual gp, human Tamm-Horsfall Sd(a(+)) urinary gp (THGP) and asialo THGP that are rich in GalNAcalpha1-->, GalNAcbeta1--> and/or Galbeta1-->4GlcNAc residues. CAA also bound tightly with multi-valent Galbeta1-->4GlcNAc (mII) containing glycoproteins (human blood group precursor gps, asialo fetuin) and asialo ovine salivary glycoprotein (Tn, GalNAcalpha1-->Ser/Thr), but CAA reacted poorly or not at all with sialylated glycoproteins tested. Of the sugars tested for inhibition of binding, Forssman pentasaccharide (F(p), GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc) was the best. It was about 2.3, 9.5 and 52.6 times more active than Galbeta1-->4GlcNAc, GalNAc and Gal, respectively, and about 1.9 times more active than tri-antennary Galbeta1-->4GlcNAc (Tri-II). These results suggest that this agglutinin is mainly specific for F(p), mII and Tn clusters. This property can be used to detect human abnormal glycotopes related to F(p) and unmasked mII/Tn clusters and to study cell growth and differentiation given the lack of toxicity of this lectin toward mouse fibroblast cells.

Studies on the binding of wheat germ agglutinin (Triticum vulgaris) to O-glycans.

The binding profile of Triticum vulgaris (WGA, wheat germ) agglutinin to 23 O-glycans (GalNAc alpha1-->Ser/Thr containing glycoproteins, GPs) was quantitated by the precipitin assay and its specific interactions with O-glycans were confirmed by the precipitin inhibition assay. Of the 28 glycoforms tested, six complex O-glycans (hog gastric mucins, one human blood group A active and two precursor cyst GPs) reacted strongly with WGA and completely precipitated the lectin added. All of the other human blood group A active O-glycans and human blood group precursor GPs also reacted well with the lectin and precipitated over two-thirds of the agglutinin used. They reacted 4-50 times stronger than N-glycans (asialo-fetuin and asialo-human alpha1 acid GP). The binding of WGA to O-glycans was inhibited by either p-NO2-phenyl alpha,betaGlcNAc or GalNAc. From these results, it is highly possible that cluster (multivalent) effects through the high density of weak inhibitory determinants on glycans, such as GalNAc alpha1-->Ser/Thr (Tn), GalNAc at the nonreducing terminal, GlcNAc beta1--> at the non-reducing end and/or as an internal residue, play important roles in precipitation, while the GlcNAc beta1-->4GlcNAc disaccharide may play a minor role in the precipitation of mammalian glycan-WGA complexes.

Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.

Native and asialo-Tamm-Horsfall glycoproteins as important ligands for the detection of GalNAc beta 1-->and Gal beta 1-->4GlcNAc active lectins.

The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein(THGP) and asialo-THGP with various applied lectins was investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins completely precipitated Abrus precatorius agglutinin(APA). They also reacted well with Wistaria floribunda (WFA), Glycine max (soybean, SBA), and Ricinus communis agglutinins and precipitated over 78% of the lectin nitrogen added, but reacted poorly or weakly with all alpha-anomeric GalNAc specific lectins, such as Helix pomatia (HPA), Phaseolus lunatus (lima bean, LBL), and Maclura pomifera (MPL) lectins. The glycoprotein-lectin interaction was inhibited by GalNAc beta 1-->, Gal beta 1-->4GlcNAc, or by both. The findings suggest that Sd (a+) THGP and asialo-THGP are among the best water-soluble glycoprotein ligands for GalNAc beta 1-->and Gal beta 1-->4GlcNAc active lectins.

Fraction A of armadillo submandibular glycoprotein and its desialylated product as sialyl-Tn and Tn receptors for lectins.

Fraction A of the armadillo submandibular glycoprotein (ASG-A) is one of the simplest glycoproteins among mammalian salivary mucins. The carbohydrate side chains of this mucous glycoprotein have one-third of the NeuAc alpha 2-->6GalNAc (sialyl-Tn) sequence and two thirds of Tn (GalNAc alpha-->Ser/Thr) residues. Those of the desialylated product (ASG-Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinant). When the binding properties of these glycoproteins were tested by a precipitin assay with Gal, GalNAc and GlcNAc specific lectins, it was found that ASG-Tn reacted strongly with all of the Tn-active lectins and completely precipitated Vicia villosa (VVL both B4 and mixture of A and B), Maclura pomifera (MPA), and Artocarpus integrifolia (jacalin) lectins. However, it precipitated poorly or negligibly with Ricinus communis (RCA1); Dolichos biflorus (DBA); Viscum album, ML-I; Arachis hypogaea (PNA), and Triticum vulgaris (WGA). The reactivity of ASG-A (sialyl-Tn) was as active as that of ASG-Tn with MPA and less or slightly less active than that of ASG-Tn with VVL-A+B, VVL-B4, HPA, WFA, and jacalin, as one-third of its Tn was sialylated. These findings indicate that ASG-A and its desialylated product (ASG-Tn) are highly useful reagents for the differentiation of Tn, T (Gal beta 1-->3GalNAc), A (GalNAc alpha 1-->3Gal) or Gal specific lectins and monoclonal antibodies against such epitopes.

Interaction of native and asialo rat sublingual glycoproteins with lectins.

The binding properties of the rat sublingual glycoprotein (RSL) and its asialo product with lectins were characterized by quantitative precipitin(QPA) and precipitin inhibition(QPIA) assays. Among twenty lectins tested for QPA, native RSL reacted well only with Artocarpus integrifolia (jacalin), but weakly or not at all with the other lectins. However, its asialo product (asialo-RSL) reacted strongly with many Gal and GalNAc specific lectins-it bound best to three of the GalNAc alpha 1-->Ser/Thr (Tn) and/or Gal beta 1-->4GlcNAc (II) active lectins [jacalin, Wistaria floribunda and Ricinus communis agglutinins] and completely precipitated each of these three lectins. Asialo-RSL also reacted well with Abrus precatorius, Glycine max, Bauhinia purpurea alba, and Maclura pomifera agglutinins, and abrin-a, but not with Arachis hypogeae and Dolichos biflorus agglutinins. The interaction between asialo-RSL and lectins were inhibited by either Gal beta 1-->4GlcNAc, p-NO2-phenyl alpha-GalNAc or both. The mapping of the precipitation and inhibition profiles leads to the conclusion that the asialo rat sublingual glycoprotein provides important ligands for II (Gal beta 1-->4GlcNAc beta 1-->) and Tn (GalNAc alpha 1-->Ser/Thr) active lectins.

Structure, biosynthesis, and function of salivary mucins.

The glandular secretions of the oral cavity lining the underlying buccal mucosa are highly specialized fluids which provide lubrication, prevent mechanical damage, protect efficiently against viral and bacterial infections, and promote the clearance of external pollutants. This mucus blanket contains large glycoproteins termed mucins which contribute greatly to the viscoelastic nature of saliva and affect its complex physiological activity. The protein core of mucins consists of repetitive sequences, rich in O-glycosylated serine and threonine, and containing many helix-breaking proline residues. These features account for the extended, somewhat rigid structure of the molecule, a high hydrodynamic volume, its high buoyant density, and high viscosity. The oligosaccharide moiety of salivary mucins accounts for up to 85% of their weight. The oligosaccharide side chains exhibit an astonishing structural diversity. The isolation, composition, structure, molecular characteristics, and functional relevance of salivary mucins and their constituents is discussed in relation to recent advancements in biochemistry and molecular biology.

Interaction of hamster submaxillary sialyl-Tn and Tn glycoproteins with Gal, GalNAc and GlcNAc specific lectins.

Hamster submaxillary glycoprotein (HSM), one of the simplest glycoproteins among mammalian salivary mucins, is composed of approximately equivalent amounts of protein, hexosamine and sialic acid. The Thr and Ser residues in the protein core account for more than half of all of the amino acid residues, while Lys, Glu, Pro and Ala are the major components of the remaining portion of amino acids. The carbohydrate side chains of this mucous glycoprotein have mainly the NeuAc-GalNAc-(sialyl-Tn) sequence (HSM), and those of the desialylated product (HSM-Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinants). The binding properties of sialyl-Tn (HSM) and asialo-HSM (HSM-Tn) glycoproteins were tested by precipitin assay with Gal, GalNAc and GlcNAc specific lectins. The HSM-Tn completely precipitated Vicia villosa (VVL both B4 and mixture of A and B), Maclura pomifera (MPL), and Artocarpus integrifolia (Jacalin) lectins; less than 2 micrograms of HSM-Tn were required for precipitating 50% of 5.0-6.3 micrograms lectin nitrogen added. HSM-Tn also reacted well with Helix pomatia lectin (HPL), Wistaria floribunda lectin (WFL) and Abrus precatorius agglutinin (APA) and precipitated in each case over 81% of the lectin nitrogen added. The reactivity of HSM-Tn with other lectins (Ricinus communis, RCA1; Dolichol biflorus, DBL; Viscum album, ML-I; Arachis hypogaea, PNA, and Triticum vulgaris, WGA) was weak or negligible. The activity of sialyl-Tn (HSM) was more restricted; HSM reacted well with Jacalin, moderately with MPL and VVL-B4, but was inactive or only weakly with the other lectins used. These findings indicate that HSM and its desialylated product (HSM-Tn) are highly useful reagents for the differentiation of Tn and T/Gal specific lectins and for anti-T, Tn and Af monoclonal antibodies.

Biochemical analysis of thyroid cyst fluid obtained by fine-needle aspiration.

Despite the relatively ready availability of thyroid cyst fluid specimens, little has been published on their biochemical composition. We measured the concentrations of 18 analytes in thyroid cyst fluid specimens from benign (n = 17) and malignant (n = 3) lesions and in homogenates of normal thyroid tissue (n = 5). The concentrations of an additional five analytes were measured in selected cyst fluid specimens only. Compared with normal human serum specimens, we found that in thyroid cyst fluid specimens the activities of acid phosphatase, aspartate aminotransferase, amylase, and lactate dehydrogenase, and the concentrations of iron and total bilirubin were highly increased. The concentration of glucose was low. The gross appearance of the fluids and the presence of certain analytes were consistent with a hemorrhagic origin of most of the benign and malignant cyst fluid specimens. Other biochemical markers, however, indicated colloidlike features and/or an admixture of thyroid tissue components to the cyst fluid. Although we have limited data for cyst fluid specimens from malignant thyroid lesions, we found no evidence that the results of any of the common biochemical tests would distinguish benign from malignant lesions.

Defining carbohydrate specificity of Ricinus communis agglutinin as Gal beta 1-->4GlcNAc (II) > Gal beta 1-->3GlcNAc (I) > Gal alpha 1-->3Gal (B) > Gal beta 1-->3GalNAc (T).

To define carbohydrate specificity of Ricinus communis agglutinin (RCA1), the combining site of RCA1 was further characterized by quantitative precipitin (QPA) and precipitin-inhibition assays (QPIA). Among the oligosaccharides tested for QPIA, Gal beta 1-->4GlcNAc (II, human blood group type II precursor sequence) was found to be 7.1 times more active than Gal beta 1-->3GalNAc (T, Thomsen-Friedenreich sequence) and about 1.7 times more active than the other three disaccharides tested--Gal beta 1-->4Man, Gal beta 1-->3DAra and Gal beta 1-->6GalNAc. Gal alpha 1-->4Gal, the receptor of the uropathogenic E. coli ligand was 3.6 times less active than the II sequence. These results indicate that the beta 1-->4 linkage of the terminal Gal to subterminal GlcNAc is important as this beta 1-->4GlcNAc sequence is at least 1.6 times more active than other types of disaccharides. Among the glycoproteins examined for QPA, native and desialized bovine submandibular glycoproteins, native and desialized human plasma alpha 1-acid glycoproteins, as well as crude hog stomach mucin and its three mild acid hydrolyzed products reacted well with the lectin. These glycoproteins precipitated over 75% of the lectin nitrogen added indicating that RCA1 has the ability to recognize Gal beta 1-->4/3GlcNAc and/or the related residues at the non-reducing ends and at positions in the interior of the chains. However, Tn (GalNAc alpha 1-->Ser/Thr sequence) rich glycoproteins such as desialized ovine submandibular glycoprotein and desialized armadillo salivary glycoprotein, in which over 90% of the carbohydrate side chains are Tn determinants with none or only a trace of I/II or T determinants, precipitated poorly with RCA1. From the present and previous results obtained, the carbohydrate specificity of RCA1 can be constructed and summarized in decreasing order by lectin determinants as follows: II (Gal beta 1-->4GlcNAc) > I (Gal beta 1-->3GlcNAc) > E (Gal alpha 1-->4Gal) and B (Gal alpha 1-->3Gal) > T (Gal beta 1-->3GalNAc), while Tn (GalNAc alpha 1-->Ser/Thr) is a poor inhibitor.

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.

Some properties of a partially purified inhibitor of protein synthesis isolated from bovine cornea.

Bovine cornea extracted with 0.154 M NaCl yielded a protein fraction which (i) inhibited protein synthesis in rabbit reticulocyte lysates, and (ii) reduced the incorporation of formyl-methionine from f[35S]Met-tRNA(f) into polypeptides. The inhibition was reversed by millimolar concentrations of glucose 6-phosphate or cAMP and partially reversed by the addition of initiation factor eIF-2. Thus, the corneal inhibitor may act by directly interfering with the activity of eIF-2.

Studies on the isolation and composition of human ocular mucin.

A method for the isolation and purification of human ocular mucin from the brief saline extract of human ocular mucus is reported. Initial purification of ocular mucin was achieved by sequential chromatography of the saline-soluble mucus extract from an individual donor's mucus pool on columns of Sephadex G-50 and Sepharose CL-4B. A portion of such mucin isolate was subjected to quantitative analysis of the O-seryl (threonyl)-N-acetylgalactosaminyl linkage, characteristic of mucins, by alkaline beta-elimination and tritiated borohydride reduction. Following Bio-Gel P-2 filtration, the mucin isolate whose cleaved oligosaccharides contained tritiated galactosaminitol greater than 0.5 microCi mg-1, a value that represents at least 64% of that observed for bovine and ovine submaxillary reference mucins, was considered to be mucin-rich. These isolates were subjected to further purification on Sephacryl S-500 and DEAE-Trisacryl M column chromatographies. The purified mucin had a minimum molecular weight of 120 kDa. It consisted of 25-30% protein and 54-55% carbohydrate. Its amino acid and carbohydrate compositions are characteristic of a mucin structure. The purity of the mucin was verified by SDS-gradient PAGE. Upon isoelectric focusing, polydispersity/microheterogeneity were exhibited in the pI range 5.0-6.6.

Biochemistry and lectin binding properties of mammalian salivary mucous glycoproteins.

The molecules responsible for the highly viscous properties of mucus are secretory glycoproteins referred to as mucins. Salivary mucins are characterized by a high sugar to protein ratio and are of a broad range of molecular weight from 7 x 10(4) to millions. With a few exceptions, they contain up to 30% of hexosamine (galactosamine and glucosamine), 8-33% of sialic acid, trace to 15% of galactose or fucose and little or no mannose. The size of carbohydrate side chains of these glycoproteins ranges from one to about fifteen units of sugar. These carbohydrate side chains are usually O-glycosidically linked through N-acetylgalactosamine to a peptidyl serine or threonine. In some instances, ester sulfate groups, mainly on N-acetylglucosamine, are also a structural feature. In many of these glycoproteins, the saccharide sequence is the same as that which determines the specificity of blood groups. Carbohydrate sequence analysis shows that salivary mucins exhibit considerable polydispersity, great diversity and remarkable structural flexibility not only among animal species but also within the same mucin molecule. Based on their lectin-binding ability, they can be used for purification of lectins, and lectins coupled to resin may be useful for the isolation of mucin-type glycoproteins. The epithelial mucous secretions modulate oral microbial flora; many secretory components serve as lectin-receptors for the attachment of microbes. The judicious use of lectins with widely differing binding characteristics has already been valuable in the in situ localization of salivary glycoproteins, in elucidating structural details, recording sugar density within a given tissue section, and defining host-parasite interactions. It is hoped that their use, together with monoclonal antibody (158) and tissue culture techniques (159, 160) will further clarify the roles of individual secretory mucous glycoproteins in health and disease.