Identification of proteins bearing β1–6 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 β1–6-N-acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 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 β1–6 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 β1–6 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.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

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.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM? Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.     

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.     

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.     

Glycosylation profile of integrin alpha 3 beta 1 changes with melanoma progression

Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of alpha(3) and beta(1) subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide-N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). beta(1) integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex(7)HexNAc(6)FucSia(4)) present. In contrast, only alpha(3) subunit from metastatic cells possessed beta1-6 branched structures. Our data indicate that the beta(1) and alpha(3) subunits expressed by the metastatic A375 cell line carry beta1-6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of alpha(3)beta(1) integrin. In direct ligand binding assays, alpha(3)beta(1) integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified alpha(3)beta(1) integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of alpha(3)beta(1) integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.     

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.     

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.     

Characterization of glycosylation and adherent properties of melanoma cell lines

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins—fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases—α2,3(Galβ1,3)- and α2,3(Galβ1,4)-sialyltransferases, α1,2- and α1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing α-2,6- and α-2,3-linked sialic acid. The average number of polypeptides bearing β-1,6-branched tri- and tetra antennary glycans(characteristic of the metastatic phenotype)were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Strict specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra lectin

We have elucidated the carbohydrate-binding profile of a non-monosaccharide-binding lectin named Eucheuma serra lectin (ESA)-2 from the red alga Eucheuma serra using a lectin-immobilized column and a centrifugal ultrafiltration-high performance liquid chromatography method with a variety of fluorescence-labeled oligosaccharides. In both methods, ESA-2 exclusively bound with high-mannose type (HM) N-glycans, but not with any of other N-glycans including complex type, hybrid type and core pentasaccharides, and oligosaccharides from glycolipids. These findings indicate that ESA-2 recognizes the branched oligomannosides of the N-glycans. However, ESA-2 did not bind with any of the free oligomannoses examined that are constituents of the branched oligomannosides implying that the portion of the core N-acetyl-D-glucosamine (GlcNAc) residue(s) of the N-glycans is also essential for binding. Thus, the algal lectin was strictly specific for HM N-glycans and recognized the extended carbohydrate structure with a minimum size of the pentasaccharide, Man(alpha1-3)Man(alpha1-6)Man(beta1-4)GlcNAc(beta1-4) GlcNAc. Kinetic analysis of binding with a HM heptasaccharide (M5) showed that ESA-2 has four carbohydrate-binding sites per polypeptide with a high association constant of 1.6x10(8) M-1. Sequence analysis, by a combination of Edman degradation and mass analyses of the intact protein and of peptides produced by its enzymic digestions, showed that ESA-2 is composed of 268 amino acids (molecular weight 27950) with four tandemly repeated domains of 67 amino acids. The number of repeats coincided with the number of carbohydrate-binding sites in the monomeric molecule. Surprisingly, the marine algal lectin was homologous to hemagglutinin from the soil bacterium Myxococcus xanthus.     

Branched N-glycans regulate the biological functions of integrins and cadherins

Glycosylation is one of the most common post-translational modifications, and approximately 50% of all proteins are presumed to be glycosylated in eukaryotes. Branched N-glycans, such as bisecting GlcNAc, beta-1,6-GlcNAc and core fucose (alpha-1,6-fucose), are enzymatic products of N-acetylglucosaminyltransferase III, N-acetylglucosaminyltransferase V and alpha-1,6-fucosyltransferase, respectively. These branched structures are highly associated with various biological functions of cell adhesion molecules, including cell adhesion and cancer metastasis. E-cadherin and integrins, bearing N-glycans, are representative adhesion molecules. Typically, both are glycosylated by N-acetylglucosaminyltransferase III, which inhibits cell migration. In contrast, integrins glycosylated by N-acetylglucosaminyltransferase V promote cell migration. Core fucosylation is essential for integrin-mediated cell migration and signal transduction. Collectively, N-glycans on adhesion molecules, especially those on E-cadherin and integrins, play key roles in cell-cell and cell-extracellular matrix interactions, thereby affecting cancer metastasis.     

N-linked oligosaccharides of glycoproteins from Ginkgo biloba pollen, an allergenic pollen.

The pollen of Ginkgo biloba is one of the allergens that cause pollen allergy symptoms. The plant complex type N-glycans bearing beta1-2 xylose and/or alpha1-3 fucose residue(s) linked to glycoallergens have been considered to be critical epitopes in various immune reactions. In this report, the structures of N-glycans of total glycoproteins prepared from Ginkgo biloba pollens were analyzed to confirm whether such plant complex type N-glycans occur in the pollen glycoproteins. The glycoproteins were extracted by SDS-Tris buffer. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine and the resulting pyridylaminated (PA-)N-glycans were purified by a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, IS-MS, and MS/MS. The plant complex type structures (GlcNAc2Man3Xyl1Fuc1GlcNAc2 (31%), GlcNAc2Man3Xyl1GlcNAc2 (5%), Man3Xyl1Fuc1GlcNAc2 (13%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (8%), and GlcNAc1Man3Xyl1GlcNAc2 (17%)) have been found among the N-glycans of the glycoproteins of Ginkgo biloba pollen, which might be candidates for the epitopes involved in Ginkgo pollen allergy. The remaining 26% of the total pollen N-glycans have the typical high-mannose type structures: Man8GlcNAc2 (11%) and Man6GlcNAc2 (15%).     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.     

Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 glycan branching influences metastatic melanoma cell migration on fibronectin

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, β1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), β1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc β1,6-branched glycans on the main fibronectin receptors – integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.     

Asparagine-linked oligosaccharides in malignant tumour growth

The expression of beta 1-6-branched complex-type oligosaccharides in several tumour cell models appears to be associated with enhanced metastatic potential. P2B, a major PHA-L-binding glycoprotein was isolated from metastatic MDAY-D2 cells and shown to bind to collagen, fibronectin and laminin with increased affinity after removal of N-linked sialic acid or polylactosamine. Sialylated polylactosamine-containing beta 1-6-branched oligosaccharides on proteins such as P2B and fibronectin may reduce cell adhesion and enhance tumour cell invasion. The loss of branched complex-type oligosaccharides in tumour cells due to somatic mutations or inhibition by swainsonine is also associated with decreased cell proliferation in tissue culture and slower rates of solid tumour growth in mice.     

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).