Biomedical subjects

I J Goldstein

Publications and source records attributed to I J Goldstein.

At least 55 records · Page 3Linked to original sources

PubMed1994

Wild-type and cultured Ehrlich ascites tumour cells differ in tumorigenicity, lectin binding patterns and binding to basement membranes.

Three different variants of the Ehrlich ascites tumour (EAT) cell were derived and the lectin surface reactivities, as well as the malignant characteristics of each variant, were studied. Wild-type cells (EAT-wt) were selected for growth on basement membranes and tissue culture plastic to give EAT-c cells. The EAT-c were passaged in mice by i.p. injection, giving rise to a third variant (EAT-c/m). Each of these three cell variants was characterized for: (i) specific lectin agglutinability patterns; (ii) the ability to produce ascites tumours in mice; (iii) the ability to produce solid tumours; and (iv) the attachment to and growth on basement membranes and purified extracellular matrix molecules. Analysis of the total protein and carbohydrate content of each cell line showed that there was an increase in the glycosylation of the EAT-c cells compared to EAT-wt cells. After repeated passage of the EAT-c/m cells in mice, the glycosylation level of the EAT-c/m cells returned to that of the EAT-wt cell line. In addition, the EAT-c cells displayed an increase in the number of terminal non-reducing sugars which could indicate either an increase degree of branching or the presence of additional N- and/or O-linked oligosaccharide chains of the cellular glycoproteins. This phenotype was retained by the EAT-c/m cells which had been passaged repeatedly in mice. The most significant increase was in the content of sialic acid-containing glycoproteins found in the EAT-c cells. The sialic acid-binding lectin Maackia amurensis leukoagglutinin (MAL) agglutinated all three EAT cell variants, while the sialic acid-binding Sambucus nigra (elderberry bark) lectin (SNA) agglutinated only the EAT-c and early-passage EAT-c/m cells. These findings indicate the presence of alpha 2,3-linked sialic acid on all three variants, but only the cultured cells and early-passage EAT-c/m cells possess the Neu5Ac alpha 2,6 linkage. The EAT-c cells attached avidly to wells coated with either laminin or fibronectin, as well as extracellular matrix produced by cultured bovine endothelial cells, but the EAT-wt and EAT-c/m cells did not. Paradoxically, the EAT-c cells were incapable of producing solid tumours when injected into a basement membrane-rich skeletal muscle bed, whereas the EAT-wt and EAT-c/m cells produced rapidly growing tumours when injected into the same environment. Lectin agglutination patterns established that ascitic tumour cells within the peritoneal cavity were derived from injected EAT-c cells.

Animals↗

PubMed1993

Differences in cell surface carbohydrates, and in laminin and fibronectin synthesis, between adherent and non-adherent Ehrlich ascites tumor cells.

Differences in cell surface carbohydrates and in laminin and fibronectin synthesis between 2 Ehrlich ascites tumor (EAT) cell lines, the adherent and non-adherent EAT cells, have been studied. The adherent EAT (a-EAT) cells grow in monolayer in vitro in the presence of fetal bovine serum. The classical, or non-adherent EAT (na-EAT), cells grow in suspension in ascites form in the peritoneal cavity of mice, and they do not adhere when cultured in vitro. Both EAT cell lines express surface glycoproteins reactive with Maackia amurensis lectin (MAL) and Griffonia simplicifolia isolectin GS I-B4. However, only a-EAT cells react with elderberry (Sambucus nigra) bark lectin (SNA), suggesting that there are some differences in the sialylation of cell surface carbohydrate moieties between these 2 EAT cell lines. Removal of cell surface sialic acid by treating a-EAT cells with Vibrio cholerae neuraminidase did not abolish the ability of these cells to adhere to laminin- or fibronectin-coated plates, indicating that the sialic acid of the cell surface glycoproteins is not essential for their adhesion to these extracellular matrices. Therefore, the difference in sialylation of cell surface glycoproteins is not responsible for the difference in cell adhesion between these 2 lines of EAT cells. Both EAT cell lines express detectable amounts of laminin but not fibronectin on their surfaces; they both secrete fibronectin and entactin into the medium. The na-EAT cells (but not the a-EAT cells) also secrete laminin A chain into the culture medium; however, no B chain was detected in the culture medium of either cell line. The laminin isolated from the cell surface of na-EAT cells reacts with GS I and MAL lectins, but not with SNA, whereas the laminin isolated from a-EAT cells reacts with SNA, as well as GS I and MAL.

Animals↗

PubMed1993

Purification and characterization of a mannose-specific lectin from Shallot (Allium ascalonicum) bulbs.

A new mannose-binding lectin was isolated from shallot (Allium ascalonicum) bulbs by affinity chromatography on an immobilized D-mannose column. The lectin (A. ascalonicum agglutinin, AAA) appeared homogeneous by polyacrylamide gel electrophoresis at pH 4.3 and gave a single protein band with an apparent M(r) of 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single symmetrical peak of 11 kDa by gel filtration on a Sephacryl S-200 HR column, indicating that AAA exists as a monomeric protein at neutral pH under the gel filtration condition employed. However, chemical cross-linking studies revealed that some degree of self-association of the lectin molecules occurs and that the lectin exists in solution as a mixture of monomers and oligomers. Scatchard analysis of equilibrium dialysis data showed the presence of one carbohydrate binding site for Man (alpha 1-3) Man-alpha-O-Me per monomer, with Ka = 1.62 x 10(4) M-1. The carbohydrate-binding properties of the purified AAA were investigated by quantitative precipitation and hapten inhibition assays. Purified AAA precipitated asialofetuin, asialotransferrin, asialothyroglobulin, asialoorosomucoid, as well as their agalacto derivatives, but did not precipitate either sialylated glycoproteins or mucins. AAA also reacted strongly with the highly branched yeast mannan obtained from Saccharomyces cerevisiae. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the AAA-asialofetuin precipitation system, whereas D-glucose, D-altrose, D-talose, N-acetyl-D-mannosamine, and derivatives of D-mannose, including 2-deoxy-, 2-deoxy-2-fluoro-, 3-deoxy-, and 6-deoxy-D-mannose were noninhibitors. These results suggest that the presence of equatorial hydroxyl groups at the C-3 and C-4 positions, an axial hydroxyl group at the C-2 position, and a free hydroxyl group at the C-6 position of the pyranose ring are the most important loci for the binding of D-mannose to AAA. Of the oligosaccharides tested, the best inhibitors were oligosaccharides containing terminal Man(alpha 1-6) [Man(alpha 1-3)]Man groups. Oligosaccharides containing either Man(alpha 1-3)Man or Man(alpha 1-6)Man units were also moderately good inhibitors of the AAA-asialofetuin precipitation system. These results indicate that AAA has an extended carbohydrate-binding site, which is most complementary to a branched mannotriosyl residue, i.e., Man(alpha 1-6)[Man(alpha 1-3)]Man.(ABSTRACT TRUNCATED AT 400 WORDS)

Allium↗

PubMed1993

The alpha-mannosyl-binding lectin from leaves of the orchid twayblade (Listera ovata). Application to separation of alpha-D-mannans from alpha-D-glucans.

The carbohydrate-binding specificity of an alpha-D-mannose-specific lectin isolated from leaves of the orchid twayblade (Listera ovata) was elucidated by quantitative precipitation of mannose-containing polysaccharides and glycoproteins, hapten inhibition, and affinity chromatography on the immobilized lectin. L. ovata agglutinin (LOA) interacted with various alpha-mannans and galactomannans of yeasts, fungi and bacteria, but not with alpha-glucans, e.g., dextran and glycogen, as do mannose/glucose-binding lectins. This lectin, LOA, appears to be highly specific for alpha 1-3 mannosidic linkages. It reacted with a linear alpha 1-3-mannan (D. P. 15) and, surprisingly, even with a linear alpha 1-3-mannoheptasaccharide. The LOA/C. tropicalis mannan precipitation reaction was inhibited by alpha-linked mannooligosaccharides, in the order, alpha 1-3 > alpha 1-6 > alpha 1-2 linkages; alpha 1-3 [Man]4 and [Man]5 were the best inhibitors among various mannooligosaccharides tested, having 7-times greater potency than alpha 1-3 [Man]2, and 18-times that of methyl, alpha-mannoside. LOA/mannan interaction was also inhibited by periodate-oxidized and reduced alpha 1-3 [Man]5 which had an inhibitory potency similar to that of alpha 1-3 [Man]3, confirming that LOA also recognizes the internal alpha 1-3-mannosidic linkages of carbohydrate chains. Complete resolution of mannan and glycogen from yeast cells, by affinity chromatography on an immobilized LOA column, and retention of several high-mannose-glycoproteins suggest this lectin to be a useful tool for purification and structural investigation of alpha-mannosyl-containing polysaccharides and glycoconjugates.

Binding Sites↗

PubMed1993

Binding determinants of the sialic acid-specific lectin from the slug Limax flavus.

The specific structural features of 24 N-acetylneuraminic acid derivatives required for the high affinity interaction of sialoglycoconjugates with the sialic acid-specific lectin from the slug Limax flavus were studied by hapten inhibition of precipitation. These results provide insight regarding the structure of the binding pocket for N-acetylneuraminic acid that exists in L. flavus agglutinin (LFA). The alpha-anomer of sialic acid is a very important factor in binding to the slug lectin. The carboxylic acid group makes only a moderate contribution to binding, since modifications of the carboxylic group decrease binding approximately 5-fold. Modification or removal of the hydroxyl group on carbon 4 does not affect binding. However, when the C4 epimer was tested, there was a dramatic decrease in binding, suggesting that whereas the equatorial hydroxyl at C4 does not contribute to binding, the introduction of an axial hydroxyl group at C4 sterically hinders the binding interaction. The substituent on the 5-amino group occupies an important role in binding of Neu5Ac to LFA as well. When the acetyl is modified by the addition of a hydroxyl group to yield the N-glycolyl derivative, we observed a 20-fold decrease, while the removal of the methyl to form the N-formyl derivative resulted in a 50-fold decrease. The 5-amino derivative was the poorest inhibitor of all compounds examined, indicating a critical role for the N-acetyl group in high affinity binding to LFA. The glyceryl tail also appears to be critical for binding inasmuch as acetylation of the C9 hydroxyl group or periodate cleavage of carbons 8 and 9 resulted in a 20- to 50-fold decrease in binding. The equilibrium constant (K(a)) for binding of Neu5Ac to LFA is 3.8 x 10(4) M-1, with a single binding site (n = 0.85) per monomer.

Acetylation↗

PubMed1993

Carbohydrate-binding protein 35. II. Analysis of the interaction of the recombinant polypeptide with saccharides.

The carbohydrate binding specificity of recombinant carbohydrate-binding protein 35 (rCBP35) has been investigated by quantitative precipitation using a series of glycoproteins and carbohydrate-protein conjugates and by inhibition of precipitation using well defined carbohydrate haptens. Synthetic glycoconjugates and glycoproteins containing terminal nonreducing galactosyl units in beta-linkage were capable of forming a precipitate with rCBP35. If the glycoprotein or glycoconjugate contained terminal Neu5Ac, or galactose in alpha-linkage, precipitate formation was not observed. We also found that murine laminin, which contains polylactosamine structures, reacted more strongly than did bovine fetuin. Using carbohydrate-bovine serum albumin (BSA) glycoconjugates, we found that the tetrasaccharide Gal beta 1, 4GlcNAc beta 1, 3Gal beta 1,4-GlcNAc-BSA reacted more strongly than the disaccharide Gal beta 1, 4GlcNAc-BSA conjugate, suggesting that the binding site accommodates carbohydrate ligands greater in size than a disaccharide. Equilibrium dialysis experiments using [3H]lactose showed that rCBP35 binds 1 mol (n = 0.84) of lactose/30,000 g atoms of protein, with an affinity constant of 2.07 x 10(4) M-1. The binding site on the polypeptide appears to contain four subsites that recognize the sequence Gal beta 1,4GlcNAc beta 1, Gal beta 1,X-. All disaccharides tested that contain a nonreducing beta-galactosyl unit behaved as inhibitors of precipitation at approximately the same concentration, suggesting that the reducing position of the tetrasaccharide does not play an important role in the specific binding to the fourth subsite. The reducing sugar may serve to hold the saccharide in a tunnel like binding pocket since methyl-beta-D-galactoside itself is an extremely poor inhibitor.

Animals↗

PubMed1993

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry.

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.

Amino Acids↗

PubMed1993

Cultured Ehrlich ascites tumor cells show increased N-linked alpha 2,6-sialyltransferase activity.

Ehrlich ascites tumor cells (EAT cells) are routinely grown in the peritoneal cavity of mice. These cells, EAT-wt, grow in suspension and exhibit a high level of alpha-2,3-O-linked sialyltransferase activity with benzyl-T-antigen (Gal beta 1,3Ga1NAc-alpha-O-CH2C6H5) as acceptor. These cells also contain a very low level of alpha-2,6-O-linked and alpha-2,6-N-linked sialyltransferase activity. A variant of these cells, EAT-c, has been selected to grow in cell culture, attached to the surface of culture flasks. EAT-c cells exhibit a selective increase of two- to fivefold in the activity of alpha-2,6-N-linked sialyltransferase activity, using asialo-alpha 1-acid glycoprotein as acceptor. Since a similar selective increase has been previously observed in metastatic human colorectal cancer tissues, the EAT-wt/EAT-c cell system may serve as a good experimental model for the investigation of sialyltransferases and their cell surface sialylated products in relation to cancer, metastasis, and cell-cell interaction.

Animals↗

PubMed1993

Cloning and characterization of the lectin cDNA clones from onion, shallot and leek.

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells. cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level. Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5-13 kDa) after post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.

Allium↗

PubMed1993

Monoclonal antibodies that recognize the trisaccharide epitope Gal alpha 1-3Gal beta 1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins.

Monoclonal antibodies were prepared against the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc, a sequence which occurs on the surface of Ehrlich ascites tumor cells as well as in thyroglobulin, laminin and a variety of other proteins. This was accomplished by immunizing BALB/c mice with the fraction of Ehrlich cell membrane glycoproteins obtained by affinity chromatography on a Griffonia simplicifolia I (GS I) column which selectively binds alpha-D-galactosyl-terminated structures. Detection of Gal alpha 1-3Gal beta 1-4GlcNAc-specific antibodies was accomplished by employing glycoproteins containing the trisaccharide sequence; fusion with spleen cells from an immunized mouse was accomplished in the presence of polyethylene glycol (PEG1500). An enzyme-linked immunosorbent assay (ELISA) system was used to identify two clones (2.10G and 6.8E), which recognized the desired trisaccharide conjugate. These clones also recognized a thyroglobulin fraction isolated by GS I affinity chromatography and murine laminin, both of which possess the Gal alpha 1-3Gal beta 1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Gal alpha 1-3Gal beta 1-4GlcNAc/Glc, were the best inhibitory haptens; Gal beta 1-4GlcNAc (LacNAc), Gal alpha 1-3Gal and Gal beta 1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4 degrees C revealed fluorescence over the entire cell surface. Indirect immunogold labeling of semithin and ultrathin sections of aldehyde fixed and Lowicryl K4M-embedded Ehrlich cells resulted in specific labeling of the cell surface and internal structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Peptide ligands for the carbohydrate-binding protein concanavalin A (Con A) have been identified by screening a large, diverse peptide library expressed on the surface of filamentous phage. A dodecapeptide containing the consensus sequence Tyr-Pro-Tyr was found to bind Con A with an affinity (dissociation constant, Kd) of 46 microM, comparable to that of a known carbohydrate ligand, methyl alpha-D-mannopyranoside (Kd of 89 microM). In addition the peptide inhibited precipitation of the alpha-glucan dextran 1355 by Con A. Given the complexity of oligosaccharide synthesis, the prospect of finding peptides that competitively inhibit carbohydrate-specific receptors may simplify the development of new therapeutic agents.

Amino Acid Sequence↗

PubMed1992

A family of concanavalin A-binding peptides from a hexapeptide epitope library.

The lectin concanavalin A (Con A) binds methyl alpha-D-mannopyranoside (Me alpha Man) as well as alpha-D-mannosyl groups at the nonreducing terminus of oligosaccharides. Ligand peptides that mimic the binding of Me alpha Man to Con A were identified from screening an epitope library composed of filamentous phage displaying random hexapeptides. A consensus sequence was identified among affinity-purified phage; Con A binds phage bearing this sequence and is inhibited from doing so by Me alpha Man. When tested for binding against a panel of lectins, phage bearing this sequence bind only weakly to a closely related D-mannose-binding lectin, indicating that binding to Con A is highly selective. A synthetic peptide bearing the consensus sequence blocks the precipitation of Con A by dextran with an inhibition strength equivalent to that of methyl alpha-D-glucopyranoside. These results demonstrate that the specificity of Con A is not limited to carbohydrates and that highly selective sugar-mimics for lectins of plant, animal, or bacterial origin may be identified from epitope libraries.

Amino Acid Sequence↗

PubMed1992

The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.

Amino Acid Sequence↗

PubMed1992

Interaction of linear manno-oligosaccharides with three mannose-specific bulb lectins. Comparison with mannose/glucose-binding lectins.

Three new mannose-binding lectins, isolated from daffodil (NPA), amaryllis (HHA), and snowdrop (GNA) bulbs, are capable of precipitating with a linear mannopentaose (Man alpha 1-3Man alpha 1-3Man alpha 1-3Man alpha 1-2Man). NPA and HHA reacted strongly with the mannopentaose whereas GNA gave a precipitate only at concentrations greater than 500 microM. A phosphate group at C-6 of the nonreducing terminal mannosyl group prevented precipitation in all three cases. The reduced (NaBH4) mannopentaose, Man4Man-ol, did not precipitate with GNA or NPA, but was active with HHA. This activity was lost when Man4Man-ol was converted (NaIO4 then NaBH4; mild acid hydrolysis of the reduced product) into trisaccharide derivatives. With alpha-D-Manp-OMe the three lectins gave UV difference spectra having large positive peaks at 292-293 and 283-284 nm, and a small positive peak at 275 nm, characteristic of tryptophanyl and tyrosyl residues. The association constants for the interaction with alpha-D-Manp-OMe were very low (NPA, 86; HHA, 66; and GNA, 41 M-1), but the lectins bound methyl (1----3)-alpha-mannobioside with increased affinity (K for NPA 540, for HHA 2400, and for GNA 200 M-1). The bulb lectins lack binding sites for hydrophobic ligands, as judged by their failure to interact with the fluorescent probes 8-anilino-1-napthalenesulfonic acid (ANS) and 6-p-toluidino-2-naphthalenesulfonic acid (TNS).

Carbohydrate Sequence↗

PubMed1992

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.

Allium↗

PubMed1992

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.

Animals↗

PubMed1992

Lectin reactivities as intermediate biomarkers in premalignant colorectal epithelium.

Normal colonic epithelial cells undergo maturation as they traverse the crypt to the lumenal surface. The binding of lectins to goblet cell mucins and other glycoconjugates changes as the cells migrate and differentiate. Additional stepwise modifications in glycoconjugate expression occur in premalignant and malignant neoplasms that may be detected by lectin binding studies. The lectins Dolichos biflorus agglutinin (DBA) and soybean agglutinin (SBA) have been developed as markers of differentiation in normal-appearing colonic epithelium. Using a quantitative biometric system to score tissues, reduced levels of lectin binding have been found in rectal tissue from patients with familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer. The lectin Amaranthus caudatus agglutinin (ACA) binds to a cytoplasmic glycoconjugate expressed at the base of the colonic crypt and serves as a possible proliferation marker in the distal, but not proximal, colon. ACA binding increases in tandem with increased levels of proliferation (using BrdU incorporation) in neoplastic tissues. Binding by the peanut lectin (PNA) occurs late in the adenoma-to-carcinoma sequence--in larger adenomas and in cancers--and serves as a marker of advancing neoplasia. Lectins identify the stepwise changes that occur during normal differentiation, proliferation and in advancing neoplasia. By selecting the appropriate probe, biomarkers may be developed for early, intermediate, and late events in colorectal cancer.

Biomarkers↗

PubMed1992

Synthesis of high-affinity, hydrophobic monosaccharide derivatives and study of their interaction with concanavalin A, the pea, the lentil, and fava bean lectins.

Concanavalin A (Con A) and agglutinins from the pea (PSA), lentil (LCH), and fava bean (VFA) constitute a group of D-mannose/D-glucose binding legume lectins. In addition to their sugar binding specificity, these lectins also contain sites that bind hydrophobic ligands. The present study explores a class of nonpolar binding sites reportedly present adjacent to the carbohydrate binding site in PSA, LCH, and VFA. A series of 2-O- and 3-O-substituted nitrobenzoyl and nitrobenzyl derivatives of methyl alpha-D-glucopyranoside and methyl alpha-D-mannopyranoside were synthesized. Evaluation of their binding to Con A, PSA, LCH, and VFA was carried out by the technique of hapten inhibition of precipitation reaction. The hapten inhibition assay results reveal that the presence of a methyl or methylene group at the O-2 or O-3 position of the sugar is essential for hydrophobic interaction with PSA, LCH, and VFA. The substitution of methyl by nitrobenzyl leads to enhanced binding (1.7-16.7 times for the 2-O-substituted compounds and 7.9-40.5 times for the 3-O-substituted compounds) with the m-nitrobenzyl group contributing to maximum binding. A hydrophobic interaction is also involved between Con A and 2-O-nitrobenzyl derivatives, resulting in enhanced binding, but the corresponding 3-O-isomers bind poorly due probably to steric reasons. These results may be rationalized on the basis of the recently published X-ray data of Con A and VFA. The nitrobenzyl derivatives, after transformation to their azido analogs, have potential applications in the photoaffinity labeling of these lectins.

Chemical Phenomena↗