Purification of acetyllactosamine-specific tomato lectin by erythroglycan-sepharose affinity chromatography

Tomato lectin is specific for oligomers of poly-N-acetyllactosamine containing 3 repeating Gal(beta 1-4)GlcNAc (beta 1-3)-disaccharides. As such it is highly useful for purifying oligosaccharides or glycopeptides with poly-N-acetyllactosamine character. We have found the lectin very useful as an affinity reagent for isolating glycoproteins or glycoprotein domains having poly-N-acetyllactosamine glycosylation. Conventional preparation of tomato lectin by ovomucoid-Sepharose affinity chromatography was found to be unsatisfactory due to instability of column and bleeding of ovomucoid into eluents requiring the necessity for additional purification steps following affinity chromatography. We prepared a column of human erythrocyte band 3 carbohydrate glycopeptide (erythroglycan) attached to Sepharose as an affinity matrix. The purification of tomato lectin to homogeneity in one step on this column matrix is described in this report.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

LAMP-1 in CHO cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin

Recent studies indicate that some mammalian S-type lectins bind preferentially to oligosaccharides containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. We report here our investigation on the distribution of these sequences in glycoproteins in Chinese hamster ovary (CHO) cells and the interaction of glycoproteins containing PL chains with an immobilized S-type lectin (L14) from calf heart tissue. Our results demonstrate that PL chains are carried by a few high molecular weight glycoproteins which are bound by tomato-lectin Sepharose and one of these was precipitated by antibody to LAMP-1 (a lysosomal-associated membrane glycoprotein). More importantly, these high molecular weight glycoproteins, including LAMP-1, were bound with high affinity by L14. These results indicate that mammalian S-type lectins are highly specific in their interactions with glycoproteins and that LAMPs carry important recognition sequences for these lectins.     

Alpha 1- and beta 2-adrenergic receptors co-expressed on cloned MDCK cells are distinct glycoproteins

We have explored the molecular differences between alpha 1- and beta 2-adrenergic receptors that are co-expressed by a clonally-derived cell line, Madin-Darby canine kidney clone D (MDCK-D). MDCK-D membranes were pre-labeled with selective alpha 1- and beta-adrenergic radioligands and were then solubilized with the non-ionic detergent digitonin. Solubilized alpha 1- and beta 2-adrenergic receptors were retained by immobilized wheat germ agglutinin and were eluted following addition of N-acetyl-D-glucosamine or sialic acid. Both receptors were also retained by immobilized Limax flavus lectin, a sialic acid-binding lectin. Lectins that were specific for N-acetyl-D-glucosamine residues did not bind to these receptors. These results indicate that both alpha 1 and beta 2 receptors are sialylated glycoproteins. The solubilized alpha 1- and beta 2-adrenergic receptors migrated with different elution profiles from an Ultragel AcA 34 column. The apparent molecular sizes of the digitonin-receptor complexes were 68A for the alpha 1 receptor and 55A for the beta 2 receptor. These results show that alpha 1- and beta 2-adrenergic receptors can be present on the same cell as distinct sialic acid-containing glycoproteins.     

Occurrence of secretory glycoprotein-specific GalNAc beta 1-->4GlcNAc sequence in N-glycans in MDCK cells

Many reports show that N-glycans of glycoproteins play important roles in vectorial transport in MDCK cells. To assess whether structural differences in N-glycans exist between secretory glycoproteins and membrane glycoproteins, we studied the N-glycan structures of the glycoproteins isolated from MDCK cells. Polarized MDCK cells were metabolically labeled with [3H]glucosamine, and (3)H-labeled N-glycans of four glycoprotein fractions, secretory glycoproteins in apical and basolateral media, and apical and basolateral membrane glycoproteins, were released by glycopeptidase F. The structures of the free N-glycans were comparatively analyzed using various lectin column chromatographies and sequential glycosidase digestion. The four samples commonly contained high-mannose-type glycans and bi- and tri-antennary glycans with a bisected or non-bisected trimannosyl core. However, secretory glycoproteins in both media predominantly contained (sialyl)LacdiNAc sequences, +/-Sia alpha 2-->6GalNAc beta 1-->4GlcNAc beta 1-->R, which linked only to a non-bisected trimannosyl core. beta1-->4N-acetylgalactosaminyltransferase (beta 4GalNAc-T) activity in MDCK cells preferred non-bisected glycans to bisected ones in accordance with the proposed N-glycan structures. This secretory glycoprotein-predominant LacdiNAc sequence was also found in the case of human embryonic kidney 293 cells. These results suggest that the secretory glycoprotein-specific (sialyl)LacdiNAc sequence and the corresponding beta 4GalNAc-T are involved in transport of secretory glycoproteins.     

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.     

Wheat germ agglutinin chromatography of GlcNac beta 1-3(GlcNAc beta 1-6)Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactosaminoglycans

GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.     

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing Terminal N-Acetylneuraminic Acid alpha 2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans. Comparison with other sialic acid-specific lectins

A lectin from the fruiting body of the Psathyrella velutina mushroom (PVL) was found to bind specifically to N-acetylneuraminic acid, as well as to GlcNAc (Ueda, H., Kojima, K., Saitoh, T., and Ogawa, H. (1999) FEBS Lett. 448, 75-80). In this study, the glycan sequences that PVL recognizes with high affinity on sialoglycoproteins were revealed. Among sialic acid-specific lectins only PVL could reveal the sialylated N-acetyllactosamine structure of glycoproteins in blotting studies, based on the dual specificity. The affinity of PVL to fetuin was measured by surface plasmon resonance to be 10(7) m(-1), which is an order of magnitude higher than those of Sambucus nigra agglutinin and Maackia amurensis mitogen, whereas affinity to asialofetuin was approximately 0 and to asialo-agalactofetuin was 10(8) m(-1), suggesting that PVL exhibits remarkably high affinities toward glycoproteins possessing trisialo- or GlcNAc-exposed glycans. Transferrin was separated into fractions that correspond to the sialylation states on an immobilized PVL column. Transferrin-possessing trisialoglycans containing alpha2,3-linked N-acetylneuraminic acid on the beta1,4-linked GlcNAc branch bound to the PVL column and eluted with GlcNAc; those containing only alpha2,6-linked sialic acids were retarded, whereas other transferrin fractions passed through the column. These results indicate that PVL is a lectin with potential for separation and detection of sialoglycoproteins because of its dual specificity toward sialoglycans and GlcNAc exposed glycans.     

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.     

Isolation, purification, and amino acid composition of the tunicate hemocyte Thy-1 homolog

A serologic cross-reacting homolog to rodent Thy-1 glycoproteins has been isolated from hemocyte cell surfaces of the advanced invertebrate group of tunicates. The Thy-1.1 cross-reacting antigenic activity was followed during purification by inhibiting the binding of MRC OX7 monoclonal antibody to pure rat brain Thy-1 in a soluble phase radioimmunoassay. After solubilization in deoxycholate, tunicate hemocyte Thy-1.1 antigenic activity was purified by affinity chromatography using an MRC OX7 monoclonal antibody affinity column, followed by gel filtration. A 602-fold enrichment in the Thy-1.1 antigenic activity, with a yield of 55.6% compared to the starting crude membrane fraction, was obtained. The antigenic activity was associated with a single glycoprotein of molecular size of 3.1 nm and molecular weight estimated at 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% gels). Amino acid composition of the purified molecule was compared by the S delta Q index of differences in composition to mammalian and non-mammalian Thy-1 glycoproteins, Ig, major histocompatibility complex-encoded polypeptides, beta 2-microglobulin, and other recognition molecules. With this parameter, the tunicate hemocyte Thy-1 homology revealed significant relatedness to avian and mammalian Thy-1 molecules and was interestingly more related to mu chains of primitive vertebrates and to HLA class I and II encoded polypeptides than to Thy-1 molecules of higher vertebrates. Based upon these observations, the tunicate hemocyte Thy-1 homolog seems to represent an ancestral Thy-1 molecule which, in structural terms, may represent an invertebrate member of the Ig superfamily.     

Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing

Stimulus-induced posttranslational processing of human monocyte interleukin-1beta (IL-1beta) is accompanied by major changes to the intracellular ionic environment, activation of caspase-1, and cell death. Certain diarylsulfonylureas inhibit this response, and are designated cytokine release inhibitory drugs (CRIDs). CRIDs arrest activated monocytes so that caspase-1 remains inactive and plasma membrane latency is preserved. Affinity labeling with [(14)C]CRIDs and affinity chromatography on immobilized CRID were used in seeking potential protein targets of their action. Following treatment of intact human monocytes with an epoxide-bearing [(14)C]CRID, glutathione S-transferase (GST) Omega 1-1 was identified as a preferred target. Moreover, labeling of this polypeptide correlated with irreversible inhibition of ATP-induced IL-1beta posttranslational processing. When extracts of human monocytic cells were chromatographed on a CRID affinity column, GST Omega 1-1 bound selectively to the affinity matrix and was eluted by soluble CRID. Recombinant GST Omega 1-1 readily incorporated [(14)C]CRID epoxides, but labeling was negated by co-incubation with S-substituted glutathiones or by mutagenesis of the catalytic center Cys(32) to alanine. Peptide mapping by high performance liquid chromatography-mass spectrometry also demonstrated that Cys(32) was the site of modification. Although S-alkylglutathiones did not arrest ATP-induced IL-1beta posttranslational processing or inhibit [(14)C]CRID incorporation into cell-associated GST Omega 1-1, a glutathione-CRID adduct effectively demonstrated these attributes. Therefore, the ability of CRIDs to arrest stimulus-induced IL-1beta posttranslational processing may be attributable to their interaction with GST Omega 1-1.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

Separation and biochemical characterization of blood group N antigen precursor glycoproteins with Thomsen-Friedenreich (T) activity, T-active glycoproteins and N antigen precursor glycoproteins from ascites of primary ovarian cancer patients

1. Three perchloric acid-soluble fractions from ascites of three primary ovarian cancer patients were subjected to Sephacryl S-300 gel filtration, respectively, and three Fr. 1 which were eluted in the vicinity of void volume as minor fractions, were then separated by a systematic affinity chromatography using Vicia unijuga lectin-Sepharose CL-4B column and Arachis hypogaea lectin-Sepharose CL-4B column into three glycoproteins, blood group N antigen precursor glycoprotein with Thomsen-Friedenreich (T) activity, T-active glycoprotein and N antigen precursor glycoprotein, respectively. 2. These nine glycoproteins separated in yields of 0.1-1.3 mg per 100 ml of ascites, were demonstrated to be mucin-type glycoproteins with Mw of 1,791,000-4,921,000 and contained 33.8-56.1% carbohydrates.     

Mice infected with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAc beta 1-->4GlcNAc) determinants.

Schistosoma mansoni is a parasitic trematode infecting humans and animals. We reported previously that adult S. mansoni synthesizes complex type biantennary N-glycans bearing the terminal sequence GalNAc beta 1-->4GlcNAc-R (lacdiNAc or LDN). We now report that mice infected with S. mansoni generate antibodies to LDN, as assessed by ELISA using a synthetic neoglycoconjugate containing LDN sequences. Sera of infected mice, but not uninfected mice, contained primarily IgM and low levels of IgG toward LDN. Interestingly, these antibodies also recognize bovine milk glycoproteins, which are known to express LDN sequences. The anti-LDN in sera of infected mice were affinity purified on immobilized bovine milk glycoproteins and shown to specifically bind LDN. An IgM monoclonal antibody (SMLDN1.1) was derived from the spleens of S. mansoni infected mice and shown to specifically bind LDN determinants. Immunoblots with affinity purified anti-LDN and SMLDN1.1 demonstrate that LDN sequences occur primarily on N-glycans of numerous glycoproteins of adult S. mansoni. LDN sequences are also expressed in many glycoproteins from S. japonicum and S. haematobium. The availability of antibody to LDN determinants should aid in defining the roles of these glycans in helminth and vertebrate biology.     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

Sialidase treatment exposes the beta1-integrin active ligand binding site on HL60 cells and increases binding to fibronectin

The migration of neutrophils from the circulation to areas of inflammation is the result of the sequential activation of multiple cellular adhesion molecules. beta1-Integrins are cell surface glycoproteins and the class of adhesion molecules responsible for binding to the extracellular matrix. The goal of this study was to determine the contribution of glycosylation, specifically the presence of sialic acid, to beta1-integrin adhesion in a neutrophil model. beta1-Integrins on differentiated HL60 cells were remodeled by treatment with the exoglycosidases, sialidase and beta-galactosidase. beta1-Integrin activity was determined by measuring adherence to the extracellular matrix protein fibronectin. The expression of beta1-integrins, beta2-integrins and activated beta1-integrins was determined by flow cytometry. Remodeling of beta1-integrins by treatment with sialidase increased adhesion by greater than 1,000%. Flow cytometric analysis of remodeled beta1-integrins demonstrated an increased expression of the activated beta1-integrin, but only minor increases in the expression of total beta1- and beta2-integrins. We postulate that glycosidase treatment increases adhesion and expression of activated beta1-integrins by exposure of the normally hidden ligand-binding site. The glycosylation of beta1-integrins on neutrophils may act to hide the ligand-binding site in unstimulated cells thereby contributing to the affinity modulation observed in neutrophil beta1-integrin function.     

Human Neuroectoderm-Derived Cell Line Secretes Fibronectin that Shares the HNK-1/10C5 Carbohydrate Epitope with Neural Cell Adhesion Molecules

A human malignant melanoma cell line, Melur, secretes several glycoproteins that contain a unique carbohydrate epitope shared by neural cell adhesion molecules and recognized by the monoclonal antibodies HNK-1, L2, and 10C5. In this report, we present evidence that one of the major melanoma glycoproteins containing the HNK-1/10C5 epitope is the cell adhesion molecule, fibronectin, or a fibronectin-like molecule. Melanoma-derived fibronectin was isolated from serum-free conditioned medium by gelatin-Sepharose affinity adsorption and shown to react with monoclonal antibodies HNK-1 and 10C5 in Western blot analysis. HNK-1-containing fibronectin was purified on a gelatin-Sepharose column followed by an affinity column using a monoclonal antibody against the HNK-1 carbohydrate. The purified HNK-1-fibronectin then could be incorporated into the extracellular matrix of hamster fibroblasts in vitro, and such a matrix was detectable using the HNK-1 monoclonal antibody in an immunofluorescence assay. Of the seven neuroectoderm-derived tumor cell lines tested, only the Melur melanoma cell secreted fibronectin containing the HNK-1 carbohydrate. Identification of human neuroectoderm-derived fibronectin as a potential carrier of the HNK-1 carbohydrate suggests a new role for fibronectin in neural development and regeneration, and represents a new model for studying the function of this carbohydrate domain in neural cell adhesion.     

Identification of a surface protein of the rabbit blood platelet with high affinity for collagen

Platelet membrane components adhering with high affinity to collagen fibers were studied by means of an affinity column in which fibrillar type I collagen was physically immobilized. Intact rabbit platelets in 1 mM EGTA adhered to the column but did not aggregate. Adhesion was dependent on the collagen concentration and on the number of platelets applied. Passage through the column without adhesion did not affect the potential for subsequent platelet binding. Surface-labelled whole platelets were passaged through this column, lysed in Triton and in SDS and labelled components adhering to the collagen were analysed on SDS-polyacrylamide gels. It was found that Triton lysis removed most of the major surface glycoproteins but left the cytoskeleton on the column. Subsequent SDS elution removed the cytoskeletal proteins along with the remaining major surface glycoproteins. The label left on the column could not be eluted with 8 M urea or up to 4 M NaCl. Collagenase digestion of the column collagen released a single surface glycoprotein of Mr 80,000. Limited chymotryptic digestion of the labelled platelets prior to their application to the column did not affect their binding. A radiolabelled band of the same molecular weight (MW) became bound to the collagen following passage of the chymotrypsin-treated platelets. This band was trypsin-sensitive following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These results, along with other published evidence, suggest that at least one platelet membrane component, expressed on the surface of the unstimulated platelet, binds with high affinity to fibrillar type I collagen and is probably involved in platelet collagen recognition.     

Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.