Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.     

The identification and characterization of xenoantigenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney

The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the alpha-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.     

Male-specific modification of human CD52.

CD52 is an unusually short, bipolar glycopeptide bearing a highly charged N-linked carbohydrate moiety and a glycosylphosphatidylinositol membrane anchor. It is exclusively expressed on lymphocytes and in the male genital tract where it is shed into the seminal plasma and inserts into the sperm membrane. The sperm surface molecule has potential significance as a target for antibodies that inhibit sperm function and gamete interaction. Western blot analyses suggested cell type-specific modifications of the antigen. It was purified from seminal plasma and a detailed structural analysis performed. The majority of anchor structures in male genital tract CD52 showed 2-inositol palmitoylation, rendering molecules insensitive toward phospholipase C, and a sn-1-alkyl-2-lyso-glycerol structure in place of the diacylated anchor described by Treumann et al. (Treumann, A., Lifely, M. R., Schneider, P., and Ferguson, M. A. (1995) J. Biol. Chem. 270, 6088-6099). N-Glycans of the male genital tract product were based on bi-, tri-, and tetraantennary structures of highly charged (up to -7), terminally sialylated complex-type sugars. A substantial proportion carried varying numbers of lactosamine repeats of which nearly 30% were branched. Different from lymphocytes, 10-15% of all N-glycans of the male genital tract antigen also contained peripheral fucose. These data confirm that male genital tract CD52 is distinct from the lymphocyte form by both N-linked glycans and COOH-terminal attached lipid anchor.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

Structures of the asparagine-linked sugar chains of laminin

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.     

Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3 glycoproteins from mouse zona pellucida

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [SH]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing alpha-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/ exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.     

N-linked sugar chains of mouse B16 melanoma cells and their low-metastasizing variant selected by wheat germ agglutinin.

The N-linked sugar chains of metastasizing mouse B16 melanoma cells (F1) and their wheat germ agglutinin-resistant variant (Wa4-b1) showing a dramatic decrease in metastasizing and tumorigenic potentials were liberated from their membrane glycoproteins by hydrazinolysis, and their structures were comparatively analysed. The results indicated that both cell lines contain high-mannose-type and bi-, tri- and tetraantennary complex-type oligosaccharides, and their ratios are similar. However, outer chain moieties of tri- and tetraantennary oligosaccharides of the variant are extensively fucosylated, resulting in the formation of X-antigenic determinants, Gal beta 1----4(Fuc alpha 1----3)GlcNAc. Oligosaccharides containing X-antigenic determinants amounted to 71% of the total complex-type oligosaccharides. Fucosylation occurs on every N-acetyllactosamine unit and the number of the determinants ranges from one to three in triantennary oligosaccharides and one to four in tetraantennary oligosaccharides. The determinants occur predominantly in non-sialylated forms, although some are in sialylated forms. Oligosaccharides containing non-sialylated X-antigenic determinants and those containing sialylated X-antigenic determinants are approximately in the ratio 6:1. Since no significant difference was found in the extent of branching of complex-type oligosaccharides of the two cell lines, it is suggested that non-fucosylated outer chains are important for the expression of metastasizing potential by the tumour cells.     

Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode

This paper reports the first rigorous evidence for the existence of N-linked oligosaccharides in Dictyocaulus viviparus, an economically important nematode that parasitises cattle. Structural strategies based upon fast atom bombardment mass spectrometry were employed to examine detergent extracts of homogenised adult D.viviparus for their N-glycan content. These revealed that detergent-soluble material is rich in high mannose, truncated and complex-type families of N-linked oligosaccharides. Importantly, the most abundant antennae in the complex-type structures were shown to carry the Lewis(x)epitope (Galbeta1-4(Fucalpha1-3)GlcNAc). Although the Lewis(x)moiety occurs in other helminths such as schistosomes, nematodes have previously been thought to lack this epitope. The Lewis(x)epitopes in D.viviparus are carried on bi-, tri-, and tetraantennary glycans and are therefore candidates for recognition events requiring multivalent ligands. There is compelling evidence from schistosome research that glycoconjugates containing Lewis(x)structures are immunomodulators. We propose that the Lewis(x)-rich glycans identified in this study might similarly be involved in D.viviparus host interactions.     

Glycosylation of three molecular forms of human alpha 1-acid glycoprotein having different interactions with concanavalin A. Variations in the occurrence of di-, tri-, and tetraantennary glycans and the degree of sialylation

Human alpha 1-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 15%) using concanavalin A (ConA)-Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively. The occurrence of N-linked di-, tri- and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties. The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri- and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA). With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri- and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP. The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHA-agarose and Ricinus communis agglutinin-I--agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri- and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri- and 65% of the tetraantennary chains. It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed.     

Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture

Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell clone (EPO-81) grown in serum-free medium. Our previous work showed that there was a significant increase in the heterogeneity of the glycoforms of EPO and a reduction of the sialylation at 20 mM NH(4)Cl. In the work presented here, the effects of ammonia on EPO N-linked oligosaccharides were analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC. The FACE N-linked oligosaccharide profile showed that the sialylated glycans contain one prominent band at a position corresponding to eight glucose units. The density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The proportion of tetraantennary structures was reduced by 60%, while the tri- and biantennary structures were increased proportionally in the presence of ammonia. Glycan analysis by HPLC using a weak anion exchange column showed that the most significant characteristic effect of ammonia was a reduction of the proportion of glycans with four sialic acids from 46% in control cultures to 29% in ammonia-treated cultures. Analysis of the desialylated glycans by normal phase chromatography indicated a distribution of tetra-, tri-, and biantennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion followed by FACE. The results indicated a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraantennary and tetrasialylated oligosaccharide structures. However, the presence of ammonia in the cultures did not change the oligosaccharide sequence.     

Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line

The oligosaccharide structures linked to Asn289 of a recombinant (r) variant (R561S) human plasminogen (HPg) expressed in Chinese hamster ovary (CHO) cells, after transfection of these cells with a plasmid containing the cDNA coding for the variant HPg, have been determined. Employing high-performance anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the protein by glycopeptidase F, compared with elution positions of standard oligosaccharides, coupled with monosaccharide compositional determinations and analyses of sequential exoglycosidase digestions and specific lectin binding, we find that considerable microheterogeneity in oligosaccharide structure exists at this sole potential N-linked glycosylation site on HPg. A variety of high-mannose structures, as well as bi-, tri-, and tetraantennary complex-type carbohydrate, has been found, in relative amounts of 1-25% of the total oligosaccharides. The complex-type structures contain variable amounts of sialic acid (Sia), ranging from 0 to 5 mol/mol of oligosaccharide in the different glycan structures. Neither hybrid-type molecules, N-acetylglucosamine bisecting oligosaccharides, nor N-acetyllactosaminyl-repeat structures were found to be present in the complex-type carbohydrate pool in observable amounts. Of interest, a significant portion of the Sia exists an outer arm structures in an (alpha 2,6) linkage to the penultimate galactose, a novel finding in CHO cell-directed glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prion glycoprotein: structure, dynamics, and roles for the sugars

The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP(Sc) from the brains of Syrian hamsters contains the same set of glycans as PrP(C), but a higher proportion of tri- and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.     

H (0) blood group determinant is present on soluble human L-selectin expressed in BHK-cells.

In the present study we show that the H (0) blood group determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1-R is present on N-linked glycans of soluble human L-selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1 was detected for bi-, 2,4 branched tri- and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.     

Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states

Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma.     

Galactose-specific elimination of human asialotransferrin by the bone marrow in the rabbit

Rabbit bone marrow accretes and degrades human asialotransferrin in vivo through a mechanism that recognizes the exposed galactose groups in this glycoprotein. After the liver, rabbit bone marrow is the second mammalian tissue now being identified as possessing a galactose-specific pathway for the elimination of a plasma protein. However, comparative studies with desialylated glycoproteins containing bi-, tri-, and tetraantennary glycans (asialofetuin, asialoorosomucoid, chicken α₁-acid glycoprotein, and rabbit transferrin) indicate that the bone marrow recognizes fewer glycan structures than does the liver. Optimal uptake and degradation of an asialoglycoprotein by the bone marrow requires the presence of biantennary glycans.     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator

Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N-glycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules. Three glycopeptides were isolated and characterized by 1H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary N-acetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.     

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

Characterization of the oligosaccharide component of alpha3beta1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin alpha3beta1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, alpha3beta1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the alpha3 and beta1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In alpha3beta1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of alpha3beta1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex7HexNAc6FucSia4 was present. In a direct ligand binding assay, desialylated alpha3beta1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, alpha3beta1 integrin was shown to take part in T24 cell migration on fibronectin: anti-alpha3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of beta1,6 branched complex type glycans in this process. Our data show that alpha3beta1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.