Decreased Immunoglobulin G Core Fucosylation, A Player in Antibody-dependent Cell-mediated Cytotoxicity, is Associated with Autoimmune Thyroid Diseases.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

An Insight into the Proteome of Uveal Melanoma-Derived Ectosomes Reveals the Presence of Potentially Useful Biomarkers.

Cancer cells are known to release extracellular vesicles that often promote disease development and progression. The present study investigated the protein content and glycosylation pattern of ectosomes released in vitro by a human primary uveal melanoma Mel202 cell line. Ectosomes released by Mel202 cells were isolated from conditioned media using sequential centrifugation, and a nano-LC-MS/MS approach was used to determine their protein content. Subsequently, proteins from ectosomes, the whole cell extracts, and the membrane fractions were probed with a panel of lectins using Western blotting and flow cytometry to reveal characteristic glycan structures. As many as 2527 unique proteins were identified, and many of them are known to be involved in cancer cell proliferation and altered metabolism, tumor invasion, metastasis, or drug resistance. Lectin-based studies revealed a distinct glycosylation pattern between Mel202-derived ectosomes and the parental cell membranes. Selective enrichment of ectosomal proteins with bisected complex type N-glycans and α2,6-linked sialic acids may be significant for ectosome formation and sequestration. Differences in the surface glycosylation of Mel202 cells and ectosomes supports recent findings that the budding of ectosomes occurs within strictly determined fragments of the plasma membrane, and thus ectosomes contain a unique protein and glycan composition.

The glycomic effect of N-acetylglucosaminyltransferase III overexpression in metastatic melanoma cells. GnT-III modifies highly branched N-glycans.

N-acetylglucosaminyltransferase III (GnT-III) is known to catalyze N-glycan "bisection" and thereby modulate the formation of highly branched complex structures within the Golgi apparatus. While active, it inhibits the action of other GlcNAc transferases such as GnT-IV and GnT-V. Moreover, GnT-III is considered as an inhibitor of the metastatic potential of cancer cells both in vitro and in vivo. However, the effects of GnT-III may be more diverse and depend on the cellular context. We describe the detailed glycomic analysis of the effect of GnT-III overexpression in WM266-4-GnT-III metastatic melanoma cells. We used MALDI-TOF and ESI-ion-trap-MS/MS together with HILIC-HPLC of 2-AA labeled N-glycans to study the N-glycome of membrane-attached and secreted proteins. We found that the overexpression of GnT-III in melanoma leads to the modification of a broad range of N-glycan types by the introduction of the "bisecting" GlcNAc residue with highly branched complex structures among them. The presence of these unusual complex N-glycans resulted in stronger interactions of cellular glycoproteins with the PHA-L. Based on the data presented here we conclude that elevated activity of GnT-III in cancer cells does not necessarily lead to a total abrogation of the formation of highly branched glycans. In addition, the modification of pre-existing N-glycans by the introduction of "bisecting" GlcNAc can modulate their capacity to interact with carbohydrate-binding proteins such as plant lectins. Our results suggest further studies on the biological function of "bisected" oligosaccharides in cancer cell biology and their interactions with carbohydrate-binding proteins.

ß1,6-branched complex-type N-glycans affect FAK signaling in metastatic melanoma cells.

Integrin-dependent binding of the cell to extracellular matrix (ECM) is a key activator of the focal adhesion kinase (FAK) signaling pathway. N-glycosylation of integrins affects their interactions with ECM proteins. Using WM266-4 cells with overexpression of ß1,6-acetylglucosaminyltransferase V, we showed that ß1,6-branched N-glycans increased tyrosine phosphorylation of FAK in metastatic melanoma cells, resulting in enhanced migration on vitronectin (VN). The co-localization of αvß3 integrin and FAK in focal adhesions of melanoma cells growing on VN indicates their interaction in signal transduction. Melanoma cell migration on VN was mediated by αvß3 caring overexpressed ß1,6-branched structures, important for FAK upregulation.

Gal-3 does not suppress cisplatin-induced apoptosis in A-375 melanoma cells.

Melanoma is the most aggressive of all skin cancers and is exceptionally resistant to therapies. During melanoma progression, cancer cells reprogram their proliferation and survival pathways and achieve resistance to treatment-induced apoptosis. Galectin-3 (gal-3) is a member of the lectin family and is involved in such biological processes as cell adhesion, growth and differentiation, the cell cycle, and apoptosis. Gal-3 also plays an important role in tumor development and metastasis. The relationship between gal-3 expression and these processes is specific to the tumor type and the stage of cancer progression. The biological functions of gal-3 depend on its localization in the cell. In the present study, human metastatic melanoma A-375 cells, characterized by weak endogenous expression of gal-3, were transfected with gal-3 cDNA and cisplatin-induced apoptosis was measured. Data from AnnexinV and mitochondrial membrane potential analysis revealed that gal-3 did not protect the A-375 melanoma cells against cisplatin. This result probably is associated with its nuclear localization in the cells.

Expression of Caveolin-1 in Human Cutaneous and Uveal Melanoma Cells.

Caveolin-1 can act as a tumour promoter or suppressor depending on the cancer type and stage. In melanoma, information available concerning its expression is ambiguous. In this study, we investigated caveolin-1 mRNA and protein expression levels in human melanoma cell lines of different origin and progression stages. Metastatic cutaneous (WM-266-4, A375), primary cutaneous (WM- 115, IGR-39) and primary uveal (mel-202, 92-1) cells were used for quantitative RT-PCR, Western blotting and confocal microscopy. We observed significantly higher expression of caveolin-I mRNA in cutaneous than in uveal melanoma cells. In accordance, immunostaining of caveolin-I was stronger in cutaneous cell extracts, while protein bands of uveal origin displayed weak signals. Finally, we detected differences in the caveolin-I subcellular pattern of distribution between primary and metastatic cells. Overall, this is the first demonstration of caveolin-1 expression in human primary uveal melanoma cell lines and observation that the origin of cells (uveal/cutaneous) has an impact when considering the utility of caveolin-I as a melanoma cell marker.

Studies on primary uveal and cutaneous melanoma cell interaction with vitronectin.

We have examined the diversity between primary uveal (92-1 and Mel202) and cutaneous (FM55P and IGR-39) melanoma cells in their interaction with vitronectin, and established the effect of integrins and ß1,6-branched N-oligosaccharides on this process. The adhesion level of uveal melanoma cells to vitronectin was at least twice lower than that of cutaneous ones, but all cells tested repaired scratch wounds on vitronectin-coated surfaces with similar speed. Swainsonine treatment, by reducing the amount of ß1,6-branches, significantly decreased cell attachment in all cases, but reduction of wound healing efficiency was compromised only in cutaneous melanoma cell. Functional blocking antibodies used in adhesion and migration assays revealed that integrin αvß3 was strongly involved in adhesion and migration only in cutaneous melanoma cells, but its role here was less pronounced than that of integrin αvß5. However, in uveal melanoma the specific anti-αvß5 integrin antibody had no impact on migration speed. Therefore, the anti-α3ß1 integrin antibody was used in order to explain the nature of uveal melanoma interaction with vitronectin, which caused a mild decrease in adhesion efficiency and reduced their motility. Expression of αvß5 integrin differed between the cell lines, but there was no distinct pattern to distinguish uveal melanoma from cutaneous melanoma. In conclusion, αvß5, but not αvß3 integrin is heavily involved in uveal melanoma cell interaction with vitronectin. The role of ß1,6-branched N-glycans in the adhesion, but not during migration, of all cells to vitronectin has been confirmed.

The lectin-binding pattern of nucleolin and its interaction with endogenous galectin-3.

Unlike nuclear nucleolin, surface-expressed and cytoplasmic nucleolin exhibit Tn antigen. Here, we show localization-dependent differences in the glycosylation and proteolysis patterns of nucleolin. Our results provide evidence for different paths of nucleolin proteolysis in the nucleus, in the cytoplasm, and on the cell surface. We found that full-length nucleolin and some proteolytic fragments coexist within live cells and are not solely the result of the preparation procedure. Extranuclear nucleolin undergoes N- and O-glycosylation, and unlike cytoplasmic nucleolin, membrane-associated nucleolin is not fucosylated. Here, we show for the first time that nucleolin and endogenous galectin-3 exist in the same complexes in the nucleolus, the cytoplasm, and on the cell surface of melanoma cells. Assessments of the interaction of nucleolin with galectin-3 revealed nucleolar co-localization in interphase, suggesting that galectin-3 may be involved in DNA organization and ribosome biogenesis.

L1CAM from human melanoma carries a novel type of N-glycan with Galß1-4Galß1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells.

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose ß1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and ß1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galß1-4Galß1- epitope capped with sialic acid residues A1[3]G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.

Cell migration-the role of integrin glycosylation.

BACKGROUND: Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell-ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding. SCOPE OF REVIEW: The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation. GENERAL SIGNIFICANCE: A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized.

Glycans in melanoma screening. Part 1. The role of ß1,6-branched N-linked oligosaccharides in melanoma.

Melanoma, which is one of the most aggressive human tumours, originates from melanin-producing melanocytes. As no effective systemic therapy exists for advanced-stage melanoma, the best chance of recovery remains surgical removal of thin early-stage melanoma. Aberrant glycosylation is a hallmark of malignancy and a well-studied class of ß1,6-branched oligosaccharides is associated with malignant transformation of rodent and human cells, and poor prognosis in cancer patients. It is evident that increased ß1,6 branching significantly contributes to the phenotype of melanoma cells, influencing the adhesion to extracellular matrix components and motility as well as invasive and metastatic potential. Despite the considerable success in establishing the role of ß1,6-branched N-linked oligosaccharides in melanoma biology, there is virtually no progress in using these glycans as a screening tool for the early diagnosis of the disease, or a target-specific therapeutic agent.

Glycans in melanoma screening. Part 2. Towards the understanding of integrin N-glycosylation in melanoma.

Although melanoma is one of the most studied malignancies, it still remains challenging for biomedicine. Since aberrant glycosylation has been considered as an important hallmark of cancer for many years, melanoma glycomic studies give a chance of better understanding the biology of the disease. The multistage nature of melanoma development, which is accompanied by changes in the expression of adhesion receptors from the integrin family, provides a chance for searching for neoglycoforms of proteins that can be considered as future sensitive melanoma biomarkers. The ß1,6-branching, sialylation and fucosylation seem to be important modifications of integrin N-glycans in the case of malignant melanoma progression.

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines.

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.

The new face of nucleolin in human melanoma.

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes.

Contribution of sialic acids to integrin α5ß1 functioning in melanoma cells.

PURPOSE: To establish the relationship between sialylation of integrin α5ß1 and possible alteration in the function of α5ß1 receptor in melanoma cells. MATERIALS AND METHODS: Integrin α5ß1 was isolated from primary WM115 (RGP/VGP-like phenotype) and metastatic WM266-4 (lymph node metastasis) cells via affinity chromatography. Integrin α5ß1 sialylation and the shift in relative masses of the enzymatically desialylated subunits were confirmed by confocal microscopy and SDS-PAGE, respectively. The ELISA assay was performed to evaluate sialic acid (SA) influence on integrin α5ß1 binding to fibronectin (FN). Cell invasion was investigated by the Transwell invasion assay. The effect of neuraminidases treatment on melanoma cells was assessed by flow cytometry using Maackia amurensis and Sambucus nigra lectins. RESULTS: Both subunits of integrin α5ß1 were found to be more abundantly sialylated in primary than in metastatic cells. The removal of SA had no effect on the purified integrin α5ß1 binding to FN. Although metastatic cells underwent more pronounced desialylation than primary cells, invasion of primary WM115 cells was more dependent on the presence of α2-3 linked SA than it was in the case of metastatic WM266-4 cells. In both melanoma cell lines not only integrin α5ß1 was involved in invasion, however simultaneous desialylation and usage of anti-integrin α5ß1 antibodies resulted in lower invasion abilities of primary WM115 cells. CONCLUSIONS: Our data suggest that in primary melanoma cells integrin α5ß1 action is more likely dependent on its glycosylation profile, i.e. the presence of SA residues, which influence (decreased) their invasion properties and may facilitate malignant melanoma progression.

Identification of proteins bearing beta1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis.

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.

Beta1-6 branching of cell surface glycoproteins may contribute to uveal melanoma progression by up-regulating cell motility.

PURPOSE: This study investigated the influence of integrin expression as well as the oligosaccharide structure of surface N-glycoproteins on cell behavior of two primary uveal (92-1 and Mel202) and two primary cutaneous (FM55P and IGR-39) melanoma cell lines. METHODS: Cell adhesion to fibronectin and cell migration on fibronectin (wound healing) were selected as the studied cell behavior parameters. The percentage of cells positive for expression of selected integrins was estimated by flow cytometric analysis. The influence of beta1-6 branched complex-type N-oligosaccharides on wound healing on fibronectin was investigated. Cell surface beta1-6 branched N-oligosaccharides were measured by their specific binding to PHA-L followed by flow cytometry, and the fibronectin receptors bearing beta1-6 GlcNAc branched N-linked glycans were identified. In addition, the transcript of GnT-V (the enzyme that catalyzes the addition of N-acetylglucosamine to the core mannose of di- and tri-antennary N-glycans through a beta1-6 linkage) was analyzed by semiquantitative RT-PCR. RESULTS: Unlike the two examined cutaneous melanoma cell lines, neither of the uveal melanoma cells adhered to fibronectin. The adhesion efficiency of IGR-39 cells was twice that of FM55P cells. In contrast, uveal melanoma cells repaired scratch wounds on fibronectin-coated surfaces twice as fast as cutaneous melanoma cells did. The expression of alpha(3)beta(1), alpha(4)beta(1), alpha(5)beta(1), and alpha(v)beta(3) integrins, acting as fibronectin receptors, differed between the tested cell lines, and no distinct pattern distinguished uveal melanoma from cutaneous melanoma except for high expression of alpha(4)beta(1) integrin on both FM55P and IGR-39 cells. The results also demonstrated that the high levels of alpha(3)beta(1), alpha(4)beta(1), and alpha(5)beta(1) integrin expression on IGR-39 cells promoted their strong attachment to fibronectin-coated surfaces. In addition, 92-1, Mel202, and FM55P cells showed no or low adhesion to fibronectin, perhaps the result of low expression of fibronectin receptors excluding high expression of alpha(4)beta(1) integrin in FM55P cells. Cell migration was significantly decreased in three out of four PHA-L-treated cell lines, suggesting that beta1-6 branched complex type N-oligosaccharides are critical for 92-1, Mel202, and FM55P cell motility. Semiquantitative RT-PCR analysis showed that the tested cells did not differ in mRNA levels of beta1-6 -N-acetylglucosaminyltransferase V. However, FACS analysis showed that 92-1, Mel202 and IGR-39 cells expressed significantly higher amounts of beta1-6 branched N-oligosaccharides on the cell surface than FM55P cells did. All examined alpha(3), alpha(5), alpha(v), and beta(1) integrin subunits were shown to bear beta1-6 branched N-linked glycans. CONCLUSIONS: The role of integrins and their N-glycosylation in the regulation of uveal melanoma growth and progression is largely unknown. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1-6 branches are important factors determining the migration of primary uveal melanoma cells on fibronectin.

Proteomic analysis of Tn-bearing glycoproteins from different stages of melanoma cells reveals new biomarkers.

Cutaneous melanoma, the most aggressive form of skin cancer, responds poorly to conventional therapy. The appearance of Tn antigen-modified proteins in cancer is correlated with metastasis and poor prognoses. The Tn determinant has been recognized as a powerful diagnostic and therapeutic target, and as an object for the development of anti-tumor vaccine strategies. This study was designed to identify Tn-carrying proteins and reveal their influence on cutaneous melanoma progression. We used a lectin-based strategy to purify Tn antigen-enriched cellular glycoproteome, the LC-MS/MS method to identify isolated glycoproteins, and the DAVID bioinformatics tool to classify the identified proteins. We identified 146 different Tn-bearing glycoproteins, 88% of which are new. The Tn-glycoproteome was generally enriched in proteins involved in the control of ribosome biogenesis, CDR-mediated mRNA stabilization, cell-cell adhesion and extracellular vesicle formation. The differential expression patterns of Tn-modified proteins for cutaneous primary and metastatic melanoma cells supported nonmetastatic and metastatic cell phenotypes, respectively. To our knowledge, this study is the first large-scale proteomic analysis of Tn-bearing proteins in human melanoma cells. The identified Tn-modified proteins are related to the biological and molecular nature of cutaneous melanoma and may be valuable biomarkers and therapeutic targets.

Age effect on human aortic valvular glycoproteins.

BACKGROUND: The aortic valve has been the subject of many hemodynamic studies but, to our knowledge, posttranslational modification of human valve proteins has not yet been studied. Thus, the aim of this study was to determine whether any age-related changes in the protein composition of normal human aortic valves and their glycosylation pattern could be observed. METHODS: Aortic valves harvested from male cadaveric donors free of cardiovascular diseases were divided into four age groups: I, mean age 21 years; II, 30 years; III, 41 years; IV, 51 years. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Identification of monosaccharide moieties or oligosaccharide units was performed with the use of eight lectins of narrow specificity: Galantus nivalis agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin, Datura stramonium agglutinin, Aleuria aurantia agglutinin, Arachis hypogeae agglutinin, Phaseolus vulgaris agglutinin, and Lycopersicon esculentum agglutinin. RESULTS: Isolated proteins showed no age-related changes in SDS-PAGE protein profile, contrary to their glycosylation. Protein sialylation, number of tri/tetraantennary complex glycans, proteins having terminal galactose and polylactosaminyl units increased with age, whereas protein fucosylation showed the opposite relationship. Moreover, groups III and IV possessed a larger number of proteins bearing high-mannose and/or hybrid-type glycans, and the quantity of these structures seemed to change, in particular proteins, with the age of donors. CONCLUSIONS: Our results clearly demonstrate that glycosylation profile in human aortic proteins is associated with the age of the donor.

Diversified ß-2-adrenergic Receptor Expression and Action in Melanoma Cells.

BACKGROUND/AIM: Growing evidence links stress hormones with development and progression of various cancer types. The aim of this study was to assess susceptibility of cutaneous and uveal melanoma cells to adrenaline (AD). MATERIALS AND METHODS: The expression of ß-2-adrenergic receptor in primary cutaneous (FM-55-P), primary uveal (92-1, Mel202) and metastatic cutaneous (A375) melanoma cells was estimated at mRNA, protein and cell surface levels. The impact of AD on cell proliferation and migration was also studied. RESULTS: The expression of ß-2-adrenergic receptor was cell line-dependent. Adrenaline treatment caused a slight stimulation of melanoma cell proliferation and activation of matrix metalloproteinases. Adrenaline-treated uveal melanoma cells showed an increased migration rate, whereas, in cutaneous melanoma cells, no changes or even lower migration speed were observed. CONCLUSION: Melanoma cell susceptibility to AD varies depending on origin and progression stage. Metastatic cutaneous melanoma cells were found to be less responsive to AD than primary cutaneous and uveal melanoma cells.