Does glycosylation of melanoma cells influence their interactions with fibronectin?

Cell surface integrins, especially those binding to fibronectin (FN), participate in processes of tumor cell invasion and metastasis. Changes in glycosylation of cell surface adhesion proteins are often associated with malignant transformation of cells. In this study we examined the influence of swainsonine (SW) on adhesion, wound healing and haptotactic migration on FN, comparing the responses of different human melanoma cell lines: primary WM35 and metastatic WM9, WM239 and A375. We also examined the role of alpha subunits in adhesion to FN. All of the antibodies inhibited adhesion to FN but with different efficiencies depending on the cell line. Adhesion was mediated mainly by integrin alpha(5)beta(1) (WM9, A375), alpha(3)beta(1) (WM35, A375, WM239). Scratch wound repair was significantly faster on FN-coated wells than on plastic for all cells except for WM9. A375 and WM9 had the greatest migration ability, both expressing the highest level of alpha(5)beta(1) integrin. It seems very likely that adhesion to FN can be accomplished by many different integrins, but for effective migration alpha(5)beta(1) integrin is responsible. Only A375 and WM239 cell lines reacted to SW treatment. In the presence of SW WM239 and A375 cells had 70% and 40% increased adhesion to FN, and their migration was decreased 40% and 50%, respectively. Interestingly, although most of the cell lines share a common profile of integrins, each line interacted with FN differently. They differed mainly in the repertoire of integrins used for adhesion, and in the manner in which glycosylation affected these processes. The influence of SW was observed in two metastatic cell lines indicating the contribution of glycosylation status to the progression of melanoma. The lack of reaction to SW in WM9 cells may suggest that there is a threshold in the expression level of the highly branched N-glycans that may influence the adhesion and migration properties of the cell.

Different adhesion and migration properties of human HCV29 non-malignant urothelial and T24 bladder cancer cells: role of glycosylation.

In tumour cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. This study used cell lines having very different behaviour in vivo: HCV29 non-malignant transitional epithelium and T24 bladder transitional cell carcinoma. These differences in behaviour might be due in part to differences in cellular glycosylation patterns. Glycan chain analysis of their glycoproteins was performed with the use of specific lectins. The functional role of carbohydrates was studied by treating these cells with swainsonine, an inhibitor of Golgi alpha-mannosidase II, and in vitro adhesion and migration assays. The adhesion of swainsonine-treated HCV29 and T24 cells was increased on fibronectin and type IV collagen by 1.5- and 2-fold, respectively, whereas adhesion on laminin was virtually unchanged after swainsonine-treatment in HCV29 cells and was increased in T24 cells. Swainsonine treatment reduced the rate of T24 cell migration by 20%. We concluded that beta1-6 branched tri- and tetraantennary complex-type glycans have an important function in adhesion and migration in the studied cell lines. These data support the view that oligosaccharides are involved in several steps of the metastatic process.

Rat submandibular gland during the maturation process: changes in enzyme activities, protein and lectin-binding profiles.

The total protein glycosylation profile and specific activity of lysosomal enzymes were investigated in rat submandibular glands isolated from very young (1-month), young (1.5-months) and adult rats (3-months) rats. The specific activity of lysosomal hydrolases (i.e. acid phosphatase, arylsulfatases A and B, beta-N-acetyl-D-glucosaminidase, beta-galactosidase and beta-glucuronidase) decreased in parallel to increasing age of the animals. Furthermore, the thermal stability of acid phosphatase and beta-N-acetyl-D-glucosaminidase was influenced by the age of rats. Age-related changes in protein profile regarding the intensity of particular bands as well as the appearance of certain proteins limited to special age groups were also demonstrated as revealed by Coomassie and lectin staining. Moreover, the marked age-related increase in structures Man (alpha1-2, alpha1-3, alpha1-6) Man, Fuc (alpha1-6) GlcNAc as well as Gal (beta1-3) GlcNAc was observed, whereas staining with terminal NeuAc and GlcNAc showed an inverse correlation. The reaction with (beta1-6) branched N-glycans and Gal (beta1-3) Gal structures was limited to 1-month-old rats. No significant changes in a specific reaction with NeuAc (alpha2-3) Gal were observed. We speculate that the observed differences with respect to protein and glycosylation profiles between 1-month-old rats and older ones could be caused by a modification of the diet composition as well as by the functional and morphological maturation of the rat submandibular gland.

Comparison of the lectin-binding pattern in different human melanoma cell lines.

Glycosylation is generally altered in tumour cells in comparison with their normal counterparts. These alterations are thought to be important because they contribute to the abnormal behaviour of cancer cells. Therefore, we have comparatively analysed the glycoproteins in cell extracts from human melanoma (primary site--WM35; metastatic sites-- WM239, WM9 and A375) cell lines using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and lectin staining. The glycoprotein pattern of the WM35 line differed from that of the other cell lines in having less proteins that reacted with Sambucus nigra, Maackia amurensis and Phaseolus vulgaris agglutinins. A glycoprotein of about 70 kDa had a significantly increased reaction with Sambucus nigra agglutinin in all the cell lines from metastatic sites. In the WM9, WM239 and A375 cell lines, additional bands (160-100 kDa) were stained with Phaseolus vulgaris agglutinin, suggesting that cells from metastatic sites contain more glycoproteins with beta1-6 branches. On the other hand, only minor changes in the reaction with Galanthus nivalis agglutinin, a mannose-specific lectin, were detected. Among the proteins showing different lectin staining, one, with an apparent molecular weight of 133 kDa, was recognized by antibodies as N-cadherin. The present results suggest that in human melanoma the expression of branched and sialylated complex type N-oligosaccharides consistently increased in cells from metastatic sites, and support the view that carbohydrates are associated with the acquisition of the metastatic potential of tumour cells.

Affinity chromatography of branched oligosaccharides in rat liver beta-glucuronidase.

Rat liver microsomal and lysosomal beta-glucuronidase-derived glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. These glycopeptides were studied by sequential chromatography on lectin-affinity columns such as concanavalin A, lentil lectin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin I, Triticum vulgaris agglutinin, Glycine max agglutinin and Ulex europaeus agglutinin. Using serial lectin affinity chromatography approach combined with neuraminidase treatment allowed us to show the unexpected presence of complex tri- and/or tetraantennary type glycans (40.8 and 17.0% for microsomal and lysosomal enzyme, respectively). Moreover, the application of neuraminidase treatment revealed that complex biantennary type glycans, present on lysosomal beta-glucuronidase, are almost fully sialylated while the same type of glycans present on microsomal enzyme do not contain sialic acid. Furthermore, the results obtained confirmed that microsomal and lysosomal beta-glucuronidases possess high mannose and/or hybrid type glycans (19.6 and 36.6%, respectively), and complex biantennary type glycans (38.9 and 46.4%, respectively).

Differences of alpha3beta1 integrin glycans from different human bladder cell lines.

Expression as well as properties of integrins are altered upon transformation. Cell adhesion regulated by integrins is modulated by glycosylation, one of the most frequent biochemical alteration associated with tumorogenesis. Characterisation of carbohydrate moieties of alpha3beta1 integrin on the cultured human bladder carcinoma (T-24, Hu456, HCV 29T) and human normal ureter and bladder epithelium (HCV 29, Hu609) cell lines was carried out after an electrophoresis and blotting, followed by immunochemical identification of alpha3 and beta1 integrin chains and analysis of their carbohydrates moieties using highly specific digoxigenin-labelled lectins. In all the studied cell lines alpha3beta1 integrin was glycosylated although in general each subunit differently. Basic structures recognized in beta1 subunit were tri- or tetraantennary complex type glycans in some cases sialylated (T-24, HCV 29, HCV 29T) and fucosylated (Hu609, HCV 29T). Positive reaction with Phaseolus vulgaris agglutinin and Datura stramonium agglutinin suggesting the presence of beta1-6 branched N-linked oligosaccharides was found in cancerous cell lines (T-24, Hu456) as well as in normal bladder epithelium cells (Hu609). High mannose type glycan was found only in beta1 subunit from Hu456 transitional cell cancer line. On the other hand alpha3 subunit was much less glycosylated except the invasive cancer cell line T-24 where high mannose as well as sialylated tri- or tetraantennary complex type glycans were detected. This observation suggests that changes in glycosylation profile attributed to invasive phenotype are rather associated with alpha3 not beta1 subunit.

Human hyaluronidases: electrophoretic multiple forms in somatic tissues and body fluids. Evidence for conserved hyaluronidase potential N-glycosylation sites in different mammalian species.

Some properties of the multiple forms of human hyaluronidases in somatic tissues and in body fluids were investigated. Liver and placenta exhibited seven hyaluronidase forms when analyzed electrophoretically on a polyacrylamide-hyaluronan gel. Ovary, breast, myometrium, endometrium, skin, leukocytes and platelets displayed distinct patterns of enzymatic micropolydispersity. The most acidic forms of hyaluronidase were in synovial fluid and serum, some serum exhibited an additional basic form. Following sialidase treatment, the number of forms decreased to two in placenta, three in liver and to a broad basic form in serum. The native serum and placental hyaluronidases remained fully active after thermal inactivation but desialylated hyaluronidase was inactivated slowly in serum, and quickly in placenta suggesting a higher overall glycosylation of the plasma enzyme. Potential N-glycosylation sites were searched in the amino acid sequences of six human hyaluronidases and several hyaluronidases from different mammalian species using the PROSITE motif database. A potential N-glycosylation site (site 1) with similar tripeptide patterns was observed at the same position in human plasma (HYAL1), human lysosomes (HYAL2) and in two newly reported hyaluronidases (HYAL4 and HYALP1). The same site was also present in mouse plasma (HYAL1) and mouse lysosomes (HYAL2), and in rat lysosomes (HYAL2). This site was absent in human HYAL3 and in all sperm hyaluronidases (PH-20) studied (human, macaque, mouse, guinea pig, rabbit and fox). A second potential N-glycosylation site was observed at a location further in the polypeptide chain. This site is present in all mammalian hyaluronidase isoenzymes reported in the present study whatever the species and organ localization. The pattern at site 2 is NVT for all hyaluronidases except for hyaluronidases of lysosomal origin where it is NVS. Such conserved sites strongly suggest that they may represent actual N-glycosylation sites.

High-mannose-type oligosaccharides from human placental arylsulfatase A are core fucosylated as confirmed by MALDI MS.

Despite numerous studies on arylsulfatase A, the structure of its glycans is not well understood. It has been shown that the concentration of arylsulfatase A increases in the body fluids of patients with some forms of cancer, and the carbohydrate component of arylsulfatase A synthesized in tumor tissues and transformed cells undergoes increased sialylation, phosphorylation and sulfation. To understand the significance of any changes in the glycosylation of arylsulfatase A in cancer, it is important to know the structure of its carbohydrate component in normal tissue. In the present study we have analyzed carbohydrate moieties of human placental arylsylfatase A using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting on Immobilon P and on-blot deglycosylation using PNGase F for glycan release. Profiles of N-glycans were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS and the computer matching of the resulting masses with those derived from a sequence database. Fifty picomoles (6 microg) of arylsulfatase A applied to the gel were sufficient to characterize its oligosaccharide content. The results indicated that human placental arylsulfatase A possesses only high-mannose-type oligosaccharides, of which almost half are core fucosylated. In addition, there was a minor species of high-mannose-type glycan bearing six mannose residues with a core fucose. This structure was not expected since high-mannose-type oligosaccharides basically have not been recognized as a substrate for the alpha1,6-fucosyltransferase.

Changes in glycosylation of rat liver arylsulfatase B in relation to age.

The aim of this study was to determine how glycosylation of the rat liver arylsulfatase B was influenced by the age of the animal. The enzyme was purified from a liver lysosomal fraction obtained from male Wistar rats aged 18 days of gestation, 1 week, and 1, 1.5, 3 and 18 months by an affinity chromatography. Examination of the carbohydrate structures was performed after electrophoresis and blotting, followed by a very sensitive detection system with a set of six highly specific digoxygenin-labelled lectins. After densitometric measurement of the intensity of a digoxigenin-labelled lectin binding to arylsulfatase B, it could be stated that, at least, changes in sialylation are related to the growth and development of rats. Sialylation increases while fucosylation slightly decreases with age of the animal.

Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines.

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.

Does glycosylation of lysosomal proteins show age-related changes in rat liver?

We studied the pattern of lectins binding by liver lysosomal proteins from rats between 18 days of gestation and 72 weeks of age. An analysis of the carbohydrate structure was carried out after an electrophoresis and blotting, followed by a very sensitive detection system with highly specific digoxigenin-labelled lectins. The only age-related differences were observed in the reaction with sialic acid--(MAA; Macckia amurensis, SNA; Sambucus nigra) and fucose--(AAA; Aleuria aurantia) specific lectins. Sialylation increased and fucosylation decreased with age. We also observed a specific reaction with Galanthus nivalis (GNA), Phaseolus vulgaris (PHA-L) and peanut agglutinin (PNA), without any significant changes with age.

Age-related profile of beta-N-acetylhexosaminidase glycosylation in rat liver.

Beta-N-acetylhexosaminidase was prepared from a liver lysosomal fraction obtained from rats between 18 days of gestation (group I) and 72 weeks of age (groups II-VI). A glycan chain analysis was performed after an electrophoresis and blotting, followed by a very sensitive detection system with highly specific digoxigenin-labelled lectins. The presence of high-mannose/hybrid type glycans, as well as their fucosylated forms was shown in all the experimental groups. Complex-type glycans with terminal sialic acid or galactose were present in all the groups except for 1-week-old rats in which only a positive reaction with lectins from Galanthus nivalis and Aleuria aurantia was observed. Thus it may be assumed that age-related changes in the glycosylation pattern occur on the first days after birth.

Arylsulfatase A from human placenta possesses only high mannose-type glycans.

It has been shown that the concentration of arylsulfatase A increases in the body fluids of patients with some forms of cancer and the carbohydrate component of arylsulfatase A synthesized in tumor tissues and transformed cells undergoes increased sialylation, phosphorylation and sulfation. The specificity of changes in the glycosylation of glycoproteins in cancer is still unknown. To understand the significance of any changes in glycosylation of arylsulfatase A in cancer, it is important to know the structure of its carbohydrate component in normal tissue. Here, carbohydrate moieties of human placental arylsulfatase A were studied by sequential lectin affinity chromatography after enzymatic cleavage and labelling with tritiated sodium borohydride. Labelled oligosaccharides were subjected to ion exchange chromatography. The uncharged fraction and the neuraminidase treated charged fraction were further analysed using the lectins: Concanavalin A (Con A), Ricinus communis (RCA I), Triticum vulgaris (L-PHA) and Aleuria aurantia (AAL). The results indicated that 97% of the arylsulfatase A oligosaccharides were low molecular weight high mannose type glycans possessing up to 5 mannose residues. This was supported by the approximately 2.4 kDa decrease in the molecular weight of arylsulfatase. A subunits upon complete peptide N-glycosidase F deglycosylation, as shown using SDS-PAGE. The remaining 3% of the arylsulfatase A oligosaccharides were of the high mannose type, possessing more than 5 mannose residues. Most (97.5%) of the glycans were uncharged, while 2.5% were charged. Neuraminidase treatment of the latter did not remove the charge, suggesting the presence of phosphate or sulfate residues. This study, of arylsulfatase A oligosaccharides separated from the protein part, shows that all glycans of the enzyme from human placenta are of the high mannose type.

Characterization of the oligosaccharide component of arylsulfatase B from rat liver.

A glycan chain analysis of the total oligosaccharide pool derived from rat liver arylsulfatase B was carried out by. P4 Gel Permeation Chromatography and sequential exoglycosidase digestion. It was found that 71% of rat liver arylsulfatase B oligosaccharides were sialylated. The relative contribution of particular structures in the total glycan pool was as follows: sialylated biantennary complex type glycans with terminal galactose--65%, high-mannose type glycans--15%, biantennary complex type glycans with core fucose and terminal N-acetylglucosamine--5%, O-linked oligosaccharides--3.5%.

Tumor cell N-glycans in metastasis.

Metastasis accounts for most of deaths caused by cancer. The increasing body of evidence suggests that changes in N-glycosylation of tumor cell proteins such as increased branching, increased sialylation, polysialylation, decreased fucosylation, enhanced formation of Lewis X and sialyl Lewis X antigens are among important factors determining metastatic potential of tumor cell. Most of the adhesion proteins, e.g., integrins, members of immunoglobulin superfamily, and cadherins are heavily N-glycosylated. The other proteins involved in adhesion, like galectins and type-C selectins, recognize N-glycans as a part of their specific ligands. In this review we focus on recent reports concerning the contribution of N-glycosylation of tumor cell adhesion molecules and some selected membrane proteins in the tumor invasion and metastasis.

Hyaluronidase in human somatic tissues and urine: polymorphism and the activity in diseases.

The polymorphism of hyaluronidase (EC 3.2.1.35) (Hyase) was studied on a hyaluronan-polyacrylamide gel. Liver, placenta, ovary and breast tissue were found to have 7 active isoforms while leukocytes and platelets 5 and fibroblasts displayed no hyaluronidase activity. In serum, synovial fluid and urine soluble the most acidic forms are present. Desialylation showed that most of the hyaluronidase isoforms differ in the content of sialic acid. In patients with rheumatoid arthritis, hyaluronidase activity in the synovial fluid varied from not detectable to very high. A partial deficiency was demonstrated in sera from some patients with dysostosis multiplex without mucopolysacchariduria. In I-cell disease, hyaluronidase activity in serum was as that in controls.

Preliminary characterization of the oligosaccharide component of arylsulfatase B from human placenta.

Isoelectric focusing of homogenous arylsulfatase B from human placenta pointed to the presence of enzymatically active and inactive forms of high pI (pH 9-8) and of lower pI (pH 6.5-5.5). Glycan chain analysis performed with the use of a Glycan Differentiation Kit showed that basic forms of arylsulfatase B from human placenta contained mostly high mannose/hybrid type glycans, with 6-O-L-fucose bound to the innermost N-acetylglucosamine residue, whereas acidic forms of the enzyme contained complex type glycans containing fucose and sialic acid. However, the latter forms constitute a small percentage of the total carbohydrate component. Lectin affinity chromatography of the native enzyme confirmed the presence of a core fucose and a sialic acid.

Characterization of human arylsulfatase A glycans.

Despite numerous studies on arylsulfatase A, the structure of the glycans present in each of its two subunits has not been determined. This is important because the carbohydrate component of human arylsulfatase A synthesized in tumor tissues and transformed cells has been shown to undergo apparent changes. This study elucidates some of their major features. Glycan chain analysis of native and deglycosylated arylsulfatase A as well as its subunits was performed with the use of a Glycan Differentiation Kit and lectin affinity chromatography. Each of the two subunits of arylsulfatase A from placenta, separated electrophoretically on polyacrylamide gel in reducing conditions, reacted with digoxigenin-labelled Galantus nivalis agglutinin and Aleuria aurantia agglutinin, while those from liver enzyme reacted with the former only. The subunits of both enzymes did not react with Sambucus nigra, Maakia amuriensis, Datura stramonium or Peanut agglutinin. Deglycosylation of arylsulfatase A with peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F resulted in complete cleavage of its carbohydrate component from each subunit. Their molecular weights decreased by 3 kDa. Neuraminidase treatment of the enzyme from liver and placenta followed by isoelectrofocusing separation showed the presence of sialylated forms which constituted a small percentage of total enzyme activity. Placental arylsulfatase A became bound to Lens culinaris agglutinin agarose, while no interaction with Ricinus communis or Griffonia simplicifolia agglutinin agarose was observed. The study shows that both subunits of arylsulfatase A from human placenta possess two high mannose/hybrid type glycans as major structures, with at least one 6-O-L-fucose bound to the innermost N-acetylglucosamine on each.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural assessment of beta-glucuronidase carbohydrate chains by lectin affinity chromatography.

Rat liver beta-glucuronidase was studied by sequential lectin affinity chromatography. beta-Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography. Ulex europaeus agglutinin-agarose chromatography revealed the presence of alpha(1-3)linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin, Ricinus communis agglutinin and Phaseolus vulgaris erythroagglutinin.

Purification of lysosomal arylsulfatase B from rat liver and its reactivity with lectins in affinity immunoelectrophoresis.

1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.