The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient's serum

MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionization-mass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di- or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid alpha2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material.     

Analysis of the oligosaccharides on androgen-binding proteins: Implications concerning their role in structure/function relationships

Rat androgen-binding protein (rABP), human testosterone-binding globulin (hTeBG) and rabbit (rb) TeBG are heterodimeric proteins. The source of the heterogeneity arises from the differential glycosylation of a common protein core. This glycosylation results in a heavy subunit (more glycosylation) and a light subunit (less glycosylation). Glycosylation is one factor responsible for multiple charged species seen when rABP, hTeBG, and rbTeBG are analyzed by two-dimensional gel electrophoresis. Enzymatic digestion with the endoglycosidase, peptide: N-glycosidase F indicated that all three proteins have asparagine (Asn)-linked oligosaccharides as their major glycan substituent. Treatment with exoglycosidases provided evidence for terminal sialic acid, galactose and mannose and N-acetylglucosamine residues. About 16–22% of the mass of the heavy subunit and about 8–14% of the mass of the light subunit is contributed by carbohydrate.

Serial lectin chromatography indicated that rABP is glycosylated differently from hTeBG and rbTeBG. About 40% of the rABP contains tri and tetraantennary complex oligosaccharides, while only about 20% of the hTeBG and TeBG from pregnant rabbits contains these types of glycans. About 9% of the TeBG from male rabbits bears these types of oligosaccharides. All of the biantennary complex oligosaccharides on rABP are fucosylated on the chitobiose core, but only 8% of those on hTeBG and none of those on rbTeBG are fucosylated in this manner. All three proteins are glycosylated at more than one site. The data indicate that the proteins may have more than one type of oligosaccharide on them. It is likely that differences in glycosylation are responsible for different physiological roles of the proteins.     

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.     

Characterization of Fucosyl Oligosaccharides Associated with Synaptic Membrane and Synaptic Junctional Glycoproteins

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.     

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)     

Core fucosylation of N-linked glycans in leukocyte adhesion deficiency/congenital disorder of glycosylation IIc fibroblasts

Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a genetic disease characterized by a decreased expression of fucose in glycoconjugates, resulting in leukocyte adhesion deficiency and severe morphological and neurological abnormalities. The biochemical defect is a reduced transport of guanosine diphosphate-L-fucose (GDP-L-fucose) from cytosol into the Golgi compartment, which reduces its availability as substrate for fucosyltransferases. The aim of this study was to determine the effects of a limited supply of GDP-L-fucose inside the Golgi on core fucosylation (alpha1,6-fucose linked to core N-acetylglucosamine [GlcNAc]) of N-linked glycans in LAD II fibroblasts. The results showed that, although [3H]fucose incorporation was generally reduced in LAD II cells, core fucosylation was affected to a greater extent compared with other types of fucosylation of N-linked oligosaccharides. In particular, core fucosylation was found to be nearly absent in biantennary negatively charged oligosaccharides, whereas other types of structures, in particular triantennary neutral species, were less affected by the reduction. Expression and activity of alpha1,6-fucosyltransferase (FUT8) in control and LAD II fibroblasts were comparable, thus excluding the possibility of a decreased activity of the transferase. The data obtained confirm that the concentration of GDP-L-fucose inside the Golgi can differentially affect the various types of fucosylation in vivo and also indicate that core fucosylation is not dependent only on the availability of GDP-L-fucose, but it is significantly influenced by the type of oligosaccharide structure. The relevant reduction in core fucosylation observed in some species of oligosaccharides could also provide clues for the identification of glycans involved in the severe developmental abnormalities observed in LAD II.     

A combined defect in the biosynthesis of N- and O-glycans in patients with cutis laxa and neurological involvement: the biochemical characteristics

Based on our preliminary observation of abnormal glycosylation in a cutis laxa patient, nine cutis laxa patients were analyzed for congenital defects of glycosylation (CDG). Isoelectric focusing of plasma transferrin and apolipoproteinC-III showed that three out of nine patients had a defect in the biosynthesis of N-glycans and core 1 mucin type O-glycans, respectively. Mass spectrometric N-glycan analyses revealed a relative increase of glycans lacking sialic acid and glycans lacking sialic acid and galactose residues. Mutation analysis of the fibulin-5 gene (FBLN5), which has been reported in cases of autosomal recessive cutis laxa, revealed no mutations in the patients' DNA. Evidence is presented that extracellular matrix (ECM) proteins of skin are likely to be highly glycosylated with N- and/or mucin type O-glycans by using algorithms for predicting glycosylation. The conclusions in this study were that the clinical phenotype of autosomal recessive cutis laxa seen in three patients is not caused by mutations in the FBLN5 gene. Our findings define a novel form of CDG with cutis laxa and neurological involvement due to a defect in the sialylation and/or galactosylation of N- and O-glycans. Improper glycosylation of ECM proteins of skin may form the pathophysiological basis for the cutis laxa phenotype.     

Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.     

Oligosaccharides of the Hazelhurst vesicular stomatitis virus glycoprotein are more extensively processed in Rous sarcoma virus-transformed baby hamster kidney cells

Because of the extensive oligosaccharide heterogeneity of the membrane glycoprotein (G) from the Hazelhurst strain of vesicular stomatitis virus, this virus has been used as a specific intracellular probe of altered protein glycosylation in Rous sarcoma virus-transformed versus normal baby hamster kidney cells. Over 70% of G protein from virus released from the transformed cells had acidic-type oligosaccharides at both glycosylation sites, compared to less than 50% from the corresponding normal host cells. The remaining G protein contained an acidic-type oligosaccharide at one site and an endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide at the other. The major endoglycosidase-sensitive species were sialylated hybrid-type (NeuNAc-Gal-GlcNAc-Man5GlcNAc2-Asn) from the transformed and neutral-type (Man5-6GlcNAc2-Asn) from the normal host cells. The degree of branching of the acidic-type oligosaccharides was not increased in the transformed cells (approx. 80% biantennary for viral G protein from both cell types). At a reduced growth temperature (24 versus 37 degrees C), the G protein oligosaccharides were more extensively processed in both cell types (approximately 85-95% of G protein contained acidic-type structures at both sites), even though the level of viral protein synthesis and virus release was decreased. Essentially all of the minor, endoglycosidase-sensitive oligosaccharides on mature viral G protein were sialic acid-containing hybrid-type structures. At 24 degrees C the branching of the acidic-type oligosaccharides was increased in the virus released from the transformed cells versus normal cells.     

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.     

Site-specific N-glycan characterization of human complement factor H

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS). CFH is a 155-kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). A 17.9-kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H (Endo H) deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. MS analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 and 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified. Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein.     

Changes in glycosylation of human bile-salt-stimulated lipase during lactation

Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.     

Asparagine-linked oligosaccharide processing in lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells

Previous studies from this laboratory have established that lepidopteran insect cells possess the glycosylation machinery needed to assemble N-linked complex-type oligosaccharides on Asn289 of recombinant human plasminogen (r-HPg). In the present paper, we show that the nature of N289-linked glycosylation of [R561E]r-HPg expressed in Spodoptera frugiperda (IPLB-SF-21AE) cells is dependent upon the length of time of infection of the cells with the recombinant baculovirus/HPg-cDNA construct. At the earliest postinfection (p.i.) time period studied, i.e., 0-20 h, virtually all (96%) of the oligosaccharides released with glycopeptidase F from N289 of the expressed r-HPg were of the high-mannose type and comprised nearly the full range of such structures, containing 3-9 mannose units. At a time window of 60-96 h, p.i., essentially all of the oligosaccharides (92% of the total) assembled on N289 of rHPg were of the biantennary, triantennary, and tetraantennary complex classes, with varying extents of outer arm completion. At an intermediate time period window, of 20-60 h, p.i., a mixture of complex-type oligosaccharides, totaling approximately 77% of the glycans, with various levels of branching and outer arm completion, and high-mannose type of oligosaccharides, totaling approximately 23% of the glycans, was assembled on N289 of the r-HPg produced. These studies demonstrate that lepidopteran insect cells contain the glycosyltransferase genes required for assembly of N-linked complex oligosaccharide and that these transferases are utilized under proper conditions. The time dependency of the assembly of complex-type oligosaccharides on r-HPg indicates that an activation of the appropriate glycosyl transferases and/or transferase genes can take place. Thus, one consequence of the infective process with the recombinant baculovirus/HPg-cDNA construct is to alter the normal glycosylation characteristics of insect cells and to allow complex-type oligosaccharide processing to occur.     

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.     

N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

N-linked oligosaccharide structures in the diamine oxidase from porcine kidney

Structures of the N-linked glycans released from porcine kidney diamine oxidase (DAO) were characterized utilizing various analytical techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), high-performance capillary electrophoresis (HPCE), and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide sequences present in DAO were conclusively determined using specific exoglycosidases in conjunction with MALDI/TOF-MS. The structures found in the glycoprotein are primarily linear, di-, or tribranched fucosylated complex type. MS analysis of the esterified N-glycan pool derived from DAO indicated the presence of several di- and trisialylated structures.     

Glycosylation Provides Both Stimulatory and Inhibitory Effects on Cell Surface and Soluble CD44 Binding to Hyaluronan

Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (K_d = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.     

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.     

Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chromatography

Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.     

A multi-mode chromatographic method for the comparison of the N-glycosylation of a recombinant HIV envelope glycoprotein (gp160s-MN/LAI) purified by two different processes

The glycosylation pattern of a recombinant gp160s-MN/LAI variant of human immunodeficiency virus type 1 (HIV-1) was studied in relation to two alternative purification techniques one of which involves an immunoprecipitation step. For analysis a multi-mode high performance liquid chromatography (HPLC) method which combines gel permeation chromatography on the RAAM 2000 GlycoSequencer, weak anion exchange chromatography and normal phase chromatography was developed and profiles were obtained for the fluorescently-labelled glycans released from the two gp160s-MN/LAI preparations. Charged glycans accounted for 77 and 80% of the total glycans for the IAP- and SP-purified samples, respectively. The acidic character of these glycans was mainly due to the presence of sialic acids. However, following sialidase treatment, residual charged glycans were still found. No differences were found in the glycan distributions of the two gp160s-MN/LAI preparations either in their degree of sialylation or in their relative proportion of each separated structure. Although this did not reach statistical significance, a lower proportion of large glycan structures regardless of their charge status was found on the gp160s-MN/LAI prepared by the procedure which involved an immunoaffinity chromatography step.