Studies on thyroid cell surface glycoproteins: Isolation of plasma membranes and characterization of carbohydrate units

Plasma membranes were isolated from calf thyroid microsomes and further resolved into two subfractions by sucrose density gradient centrifugations. The lighter and major membrane fraction was obtained in a yield of 10 mg/100 g of thyroid and was enriched 38-fold with respect to 5′-nucleotidase activity compared to the homogenate. It differed from the denser plasma membrane fraction in containing greater amounts of phospholipid and cholesterol but had a similar total carbohydrate content (16 mg/100 mg protein) and monosaccharide composition. The membranes were found to retain most (80%) of their carbohydrate after delipidation. The major protein-bound sugars present in the lighter membrane fraction expressed as micromoles per 100 mg of peptide were: galactose 24, mannose 17, fucose 3, glucosamine 23, galactosamine 4, and sialic acid 9. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the lipid-free membranes revealed at least 18 protein bands and 3 periodic acid-Schiffreactive glycoprotein components. Incubation of the delipidated membranes with Pronase resulted in the solubilization of 95% of the saccharide portion which upon filtration through Bio-Gel P-6 and P-10 columns yielded several glycopeptide fractions. While some of the carbohydrate was found in glycopeptides which appeared to contain the well-known complex and polymannose asparagine-bound oligosaccharides, as well as small O-glycosidically linked units, approximately half was recovered in high molecular weight components which contained galactose and glucosamine as their principal sugar constituents, and which were similar in composition to glycopeptides recently isolated (T. Krusius, J. Finne, and H. Rauvala, 1978, Eur. J. Biochem.92, 289–300) from human erythrocyte membranes.     

Structure of the O-glycosidically linked carbohydrate units of rat brain glycoproteins.

The O-glycosidically linked carbohydrate units of rat brain glycopeptides were released as reduced oligosaccharides with NaOH/NaBH4 treatment. Five oligosaccharides were isolated using gel filtration, ion-exchange chromatography and preparative thin-layer chromatography. Studies employing periodate oxidation, methylation analysis, chromium trixide oxidation and gas-liquid chromatography-mass spectrometry indicated the following structures: (I) alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (II) beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (III) N-acetylneuraminyl-[beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol], (IV) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and (V) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)[N-acetylneuraminyl-(2 leads to 6)]-N-acetylgalactosaminitol.     

Thio-functionalized carbohydrate thiosemicarbazones and evaluation of their anticancer activity

Thiosemicarbazides and their analogs have shown potential medical applications as antiviral, antibacterial and anticancer drugs. We designed, synthesized and evaluated in vitro anticancer activity against ovarian (A2780), cervix (HeLa), colon (LoVo), breast (MCF-7) and brain (MO59J) human cancer cell lines of seven novel compounds –S-glycosylated thiosemicarbazones. We assessed the cyto- and genotoxic properties of all novel compounds using a variety of methods including comet assay, XTT assay, various fluorescent assays and toxicology PathwayFinder expression array. We tried to evaluate their possible mechanism of action with particular attention to induction of DNA damage and repair, apoptosis, oxidative stress analysis and cellular response in terms of changes in gene expression. The most sensitive cell line was human ovarian cancer. The results revealed that the major activity against A2780 cancer cell line displayed by our compounds is induction of DNA damage. This effect is not associated with apoptosis or oxidative stress induction and the resulting damage will not lead to cell cycle arrest. We also observed up-expression of heat shock related genes and NQO1 gene in response to our compounds. The second effect seems to be specific to glycosylated S-bond compounds as we observed it earlier. Upregulation of heat shock protein encoding genes suggest that our compounds induce stressful conditions. The nature of this phenomena (heat shock, pH shift or hypoxia) needs further study.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

The biosynthesis of carbohydrate chains of glycoproteins and their heterogeneity

Current state of research on the mechanism of biosynthesis of carbohydrate chains of N- and O-glycoproteins is reviewed. Functional predetermination of a multistage mechanism of the carbohydrate components' biosynthesis in N-glycosylproteins is suggested. Origin and character of heterogeneity of the carbohydrate chains in these biopolymers are discussed.     

Schistosoma mansoni surface glycoproteins. Analysis of their expression and antigenicity

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin-affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose-binding protein, only in the concanavalin-A-bound fractions were glycoproteins identified. Changes in concanavalin-A-binding glycoproteins were detected after transformation and early maturation of the schistosomula. Some glycoproteins disappeared (Mr 38 000, 29 000 and 25 000), some appeared independently of host molecules (Mr 19 000), others only appeared after culture in human serum (Mr 45 000). Two major glycoproteins of Mr 32 000 and 16 000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3-h schistosomula only the strong Mr-38 000-32 000 complex was found to be antigenic. Thus many major low-molecular-mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr-38 000-32 000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.     

Mutants of Polysphondylium pallidum altered in cell aggregation and in the expression of a carbohydrate epitope on cell surface glycoproteins

Mutants of the cellular slime mold Polysphondylium pallidum have been selected using a cell sorter and a fluorescentlabeled monoclonal antibody, mAb 293. This antibody blocks cell adhesion when applied as Fab, and recognizes a carbohydrate epitope containing L-fucose. This epitope is expressed on the cell surface and is present on >10 membrane glycoproteins of different apparent mol. wts. Twenty mutants were obtained which did not bind mAb 293 when tested at 2 h of starvation. After longer periods of starvation the epitope became detectable in the mutants. In all these mutants aggregation patterns were atypical. Generally streams of cells that were radially orientated around aggregation centers were missing or were much shorter than in wild-type. Genetic analysis demonstrated that aberrant aggregation was linked to the alteration in carbohydrate epitope expression. One mutant was unstable and gave rise to subclones in which almost no antibody binding was observed, even after 24 h of starvation, and only few aggregation centers with no streams or very short ones were formed. These results indicate that the capability of the cells to aggregate is correlated with the exposure on their surfaces of the carbohydrate epitope recognized by mAb 293, whose function in development remains to be established.     

Affinity chromatographic isolation of cell surface glycoproteins from human foetal brains and their interaction with lectins.

The cell surface glycoproteins of foetal human neurons and glial cells were isolated by affinity chromatography on Con A-Sepharose 4B. Dissociation of Con A from the matrix took place independent of buffer composition and the absence of lipids and/or detergents during chromatography. It was apparently related to the nature of glyco proteins. Pretreatment of Con A-Sepharose 4B with urea or guanidine minimized this problem. The elution of glycoproteins from the affinity matrix at 4 degrees C, instead of the usual 25 degrees C, reduced both Con A and glycolipid contamination in the eluate. Dot-enzyme-linked-lectin assay was carried out with horse radish peroxidase conjugated lectins and serotonin. It was observed that total glycoproteins contained high mannose, hybrid and a limited quantity of biantennary complex type oligosaccharide chains. O-linked oligosaccharides were also present. Desialylation and sodium chloride inhibited binding to serotonin and wheat germ agglutinin indicating the presence of sialic acid residues. Fucose was attached to the innermost core GlcNAc residue, as revealed by affinity towards pea lectin.     

Thomsen-Friedenreich (T)-active glycoproteins, blood group N antigen precursor glycoproteins and N antigen precursor glycoproteins with T activity from ascitic fluids of liver cancer patients.

1. Five perchloric acid-soluble fractions (PASFs) obtained from ascitic fluids of three patients with primary hepatocellular carcinoma (HCC), a patient with liver metastatic carcinoma (LUC) from ureteral carcinoma and human normal serum (NS) were subjected to DEAE-cellulose column chromatography to separate seven glycoprotein fractions, respectively. 2. In this chromatography, two HCC-PASFs gave a Thomsen-Friedenreich (T)-active glycoprotein, respectively. 3. Other HCC-PASF gave a T-active glycoprotein, two blood group N antigen precursor glycoproteins and an N antigen precursor glycoprotein with T activity. 4. LUC-PASF gave two T-active glycoproteins and an N antigen precursor glycoprotein with T activity. 5. NS-PASF did not give these serologically active glycoproteins.     

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.     

Isolation and characterization of blood group N antigen precursor glycoproteins with Thomsen-Friedenreich (T) activity, N antigen precursor glycoproteins and T-active glycoproteins from cyst fluids of malignant ovarian clear cell carcinoma

1. Two perchloric acid-soluble glycoprotein fractions from cyst fluids of 2 patients with ovarian clear cell carcinoma in malignant were subjected to a systematic affinity chromatography using Vicia unijuga lectin-Cellulofine column and Arachis hypogaea lectin-Cellulofine column and separated into four glycoproteins, blood group N antigen precursor glycoprotein with Thomsen-Friedenreich (T) activity, N antigen precursor glycoprotein, T-active glycoprotein and serologically inactive glycoprotein, respectively. 2. These serologically active glycoproteins isolated in yields of 0.3-3.3% of PASFs, were proved to be mucin-type glycoproteins with Mw of 509,000-2,772,000 Da and contained 33.9-81.7% carbohydrates.