Effect of brefeldin A on membrane localization of MUC1 mucin and adhesive properties of cancer cells.

Transmembrane glycoproteins play a significant role in cancer cells adhesion and metastatic process, just for that reason the glycosylation inhibitors are used to change the glycan structure and in this way the membrane expression of glycoproteins. The inhibitory effect of brefeldin A (BFA) on the expression of some glycoproteins: MUC1 mucin and alpha2beta1 integrin on cell surface of breast (MCF-7 and MDA-MB-231 lines) and endometrial (Ishikawa line) cancer cells was evaluated in our study. In MCF-7 and MDA-MB-231 cells, a decrease in MUC1 expression depended on brefeldin A concentration and equaled about 40% in cells treated with 1mg% of drug. In Ishikawa cells, a decrease in MUC1 expression was lower and amounted to about 25%. The expression of alpha2beta1 integrin was greatly inhibited in brefeldin-treated MCF-7 and Ishikawa cells, though it was unchanged in MDA-MB-231 cells. A decrease in MUC1 mucin and alpha2beta1 integrin level reduced the adhesive properties of BFA-treated cells. Adhesion to type I collagen was greatly diminished in BFA-treated MCF-7 and Ishikawa cells (above 70%), and to a lesser degree in MDA-MB-231 cells (about 50%); which was mainly caused by the inhibited integrin expression. These findings have proved that brefeldin A, by changing the surface glycoproteins level, can alter carcinoma cells adhesion to extracellular matrix proteins.

The participation of ribosomes in protein glycosylation. Interaction of the ribosome-UDP-N-acetyl-glucosamine complex with dolichol phosphate.

UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.

The participation of ribosome-UDP-GalNAc complex in the initiation of protein glycosylation in vitro.

The gastric epithelial cells ribosome-UDP-GalNAc complex is a donor of UDP-GalNAc as the substrate for N-acetylgalactosaminyltransferase, which catalyse the transfer of GalNAc residue to the polypeptide, existing on polysomes. It was observed that the deglycosylated porcine mucin and synthetic peptide (PTSSPIST) can be also glycosylated with participation of N-acetylgalactosaminyltransferase and ribosome-UDP-GalNAc complex. The probability of the ribosome-UDP-GalNAc complex as an intermediate in the O-glycosylation is considered.

Activity of partially purified UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase with different peptide acceptors.

As part of investigations on the role of the UDP-GalNAc-ribosome complex in the initial O-glycosylation of proteins, we have isolated from porcine gastric mucosa GalNAc-transferase, mucin and apomucin, and its three fractions containing carbohydrate in the amounts: I - 1.6%, II - 0.65% and III - 0.00% (wt/wt) of apomucin mass. Amino acid analysis showed that fractions I and II contained slightly higher amounts of serine and threonine as compared to native mucin and apomucin. The short peptide Pro-Thr-Ser-Ser-Pro-Ile-Ser-Thr was the most effectively glycosylated. Our apomucin preparations are also good acceptors of GalNAc and can be used for testing of O-glycosylation in vitro.

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation.

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.

The substances of plant origin that inhibit protein biosynthesis.

Some plants were used for a long time in folk medicine as sources of anti-tumour remedies. Their effects on protein biosynthesis in vitro have been examined and described. The separate features of the peptide elongation system, isolated from tumoural cells, have been demonstrated. Some elongation factors or ribosomes have been shown to be a target site for the inhibition of protein biosynthesis caused by the substances isolated from various sources. The glycoside and caffeic acid, isolated from Melissa officinalis leaves, inhibited protein biosynthesis by direct influence the elongation factor eEF-2. The activity of this factor was also inhibited by aloin and aloeemodin. Saponin glycoside and its aglycon, isolated from Verbascum thapsiforme flowers, as well as digoxin, emetine and cepheline directly inactivated ribosomes. "Chagi" fraction, isolated from Inonotus obliquus, is responsible for the inhibitory effect caused by the aqueous tannin--less extract from this fungus. The target site for quercetin has been found to be the subunit form EF-1 alpha. It may be supposed that, the plant inhibitors of protein biosynthesis could be utilized for searching specific antitumoural preparations.

Expression of MUC1 mucin in full-term pregnancy human placenta.

PURPOSE: MUC1 mucin is a component of glycocalyx in human endometrium and may play an important role in generation of "receptive window" at embryo implantation. Considering that MUC1 expression in human placenta is changed during pregnancy, and that MUC1 structure and function are not completely known in this organ, we have undertaken isolation of this mucin and detection of glycan epitopes, since they are crucial for its properties. MATERIAL AND METHODS: Samples of human placenta were homogenized and MUC1 was extracted in different conditions with the use of ionic or non-ionic detergents. Identification of this glycoprotein was performed by Western and lectin blotting, after its purification on Sepharose 4B column. RESULTS: The best extraction of MUC1 glycoprotein was achieved with a non-ionic detergent, Triton X-100. Reactions with anti-MUC1 antibody showed a few glycoforms with molecular weights between 116 and 205 kDa, with the most visible glycoform approximating 205 kDa. Reactions with lectins enabled detection of carbohydrate antigens, such as T and Tn with sialic acid linked by alpha2, 3 and to a lesser extent by alpha2, 6 bond. CONCLUSION: MUC1 mucin is present in several glycoforms on the maternal side of human placenta after term delivery. They contain short glycan structures, similar to some tumor carbohydrate antigens.

[Human gastrointestinal tract mucins encoded by the MUC gene family]. / Mucyny ludzkie przewodu pokarmowego jako produkty genów MUC.

Epithelial mucins, MUC gene products, are widely expressed in human organs such as airways, the urogenital and gastrointestinal tracts, and the eyes. MUC-type mucins have very large sizes and complex structures with very extensive O-glycosylation and are regarded as protective molecules. The aim of this review is to discuss mucin glycoproteins structure, biosynthesis and functions in normal and pathologically altered epithelial mucosa of human gastrointestinal tract.

Deglycosylated mucin as a substrate in enzymatic O-glycosylation in vitro.

To obtain the apomucin as a substrate for glycosyltransferases activity testing, the pig gastric mucin deglycosylation by chemical method was carried out. Resulted apomucin, rich in Ser, Thr and Pro residues, was as good carbohydrate acceptor in enzymatic O-glycosylation in vitro, as synthetic peptide, analogue of MUC2 mucin tandem repeats sequence.

The attachment of UDP-hexosamines to the ribosomes isolated from rat liver.

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

The influence of selected potential oncostatics of plant origin on the protein biosynthesis in vitro.

Five potential oncostatics of plant origin (reserpine, amphotericin B, rutoside, digoxin, dry aloe extract), and cyclic AMP were investigated for their effect on protein synthesis. The solutions of digoxin and dry aloe extract inhibited protein biosynthesis in vitro. The direct inhibiting effect of digoxin on the ribosomes suggests that this drug forms an inactive complex with this organelle. Therefore it can be concluded that ribosome is the target site of digoxin action. Aloin and aloeemodin are responsible for the inhibitory effect of the solution of dry aloe extract. They inhibit markedly [14C]-leucine incorporation into proteins. Aloin and aloeemodin do not influence directly the ribosomes, but they inhibit elongation factors and peptidyltransferase activities in the complete elongation system. Some preliminary experiments have shown that direct interaction between these substances and elongation factor EF-2 should be taken in account. This observation is the subject of further experiments, in which the characteristics of the inhibitory effect of the components isolated from dry aloe extract will be performed.

The use of preparative polyacrylamide gel electrophoresis and electroelution for purification of mucus glycoproteins.

This paper describes a novel technique for purifying glycoproteins from porcine gastric mucus by preparative polyacrylamide gel electrophoresis and electroelution. The method is based on the observation that the high-molecular-weight buffer/SDS-soluble mucins do not penetrate through the polyacrylamide gel, but remain on the gel surface. Mucus solution extracted with 6 M urea was fractionated on Sepharose CL-2B column and Vo peak mucin was submitted to purification by preparative polyacrylamide gel electrophoresis (22 h). Nonpenetrated mucin layer was electroeluted from the gel after the reversing of electrode polarity (3 h). A comparison of mucin preparations purified by our method and by CsCl density gradient centrifugation indicated that the GalNAc/protein and GalNAc/DNA ratios were three times higher than those of the first method. The method is a relatively short and efficient procedure and yields pure mucin preparation free of contaminating proteins and nucleic acids.

An improved method for chemical deglycosylation of gastric mucus glycoprotein.

We describe an improved method for chemical deglycosylation of gastric mucin which involves: reduction, alkylation, desialylation, periodate oxidation with beta-elimination and two-steps of TFMSA/anisole treatment. The product was 96% deglycosylated protein with amino acid composition similar to purified mucin with apparent molecular weight of 90 kDa.

Preparation of gastric mucin "STP-domains" using pronase digestion and chemical deglycosylation.

In this work we present a method of mucin protein backbone fragments preparation, which are rich in serine, threonine and proline amino acid residues. The purified native gastric mucin was reduced, the pronase digested and chemically deglycosylated. The obtained apomucin preparations contained slight quantities of residual carbohydrates (0-1%). In O-glycosylation reaction in vitro, with the participation of GalNAc-transferase, the amounts of incorporated [14C]GalNAc to apomucin fractions were about twofold greater in relation to the deglycosylated non-digested mucin subunits. We also demonstrate the usefulness of immunoblotting technique of apomucin preparations detection.

Nucleotide-sugar binding to ribosomes. Comparison of affinity and capacity of gastric mucosa and liver ribosomes to sugar-nucleotides.

Highly purified ribosomes from gastric mucosa and liver were isolated and tested for their affinity and capacity toward UDP- monosaccharide binding. Ribosomes from both tissues bind UDP-N-acetylglucosamine nearly at the same level with similar affinity, but binding capacity of gastric ribosomes to UDP-N-acetylgalactosamine was twice higher than in case of liver ribosomes, and with higher affinity than in case of UDP-N-acetylglucosamine. The binding of UDP-N-acetylgalactosamine to mucosa ribosomes requires the whole sugar-nucleotide structure.

Biosynthesis of MUC1 mucin in human endometrial adenocarcinoma is modulated by estradiol and tamoxifen.

Polymorphic epithelial mucin MUC1 is expressed by most epithelial cancers, although free natural MUC1 antibodies are present in the circulation of healthy subjects as well as in that of cancer patients. The role of MUC1 mucin molecules in cancer cells of endometrium is not precisely known. The results reported here demonstrate that MUC1 biosynthesis in human endometrial adenocarcinoma cells (Ishikawa line) is stimulated by estradiol hormone and inhibited by tamoxifen, which was measured by [(14)C]threonine or [(3)H]glucosamine incorporation into MUC1 protein. Tamoxifen applied in combination with estradiol also inhibited this process, but pre-incubation of cells with estradiol resulted in a decrease in the inhibitory effect of tamoxifen. Electroblotting and reactions with antibodies against MUC1 core protein epitopes confirmed the presence of MUC1 in cell lysates and culture media of Ishikawa cells. Reactions with lectins showed the presence of oligosaccharide structures demonstrating antigen-T activity and the presence of sialic acid residues. The results confirm that there is downregulation of MUC1 expression in cancer culture cells treated with selective estrogen receptor modulators, which may be essential for reducing the migration of cancer cells and the metastatic properties of tumor cells.

Chromatographic method for the isolation of active 40S and 60S subunits from rat liver polyribosomes.

A rapid and simple procedure for isolation of 40S and 60S ribosomal subunits by ion-exchange column chromatography is described. The dissociated ribosomes can be separated and non-ribosomal proteins and low-molecular-weight substances removed. An assessment by physicochemical and functional criteria showed that the ribosomal subunits obtained are active and sufficiently homogeneous.