Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid

Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8–9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.     

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal{beta}1-3GlcNAc

The monoclonal antibody LU-BCRU-G7, that was generated by invitro immunization, shows clinical value as a prognostic markerin early stage breast carcinoma. It has now been characterizedwith regard to its binding epitope. Using a recently describedmethod based on the construction of N-substituted polyacrylamide(PAA) derivatives of carbohydrates (pseudopolysaccharides),the structure of the epitope for the monoclonal antibody LU-BCRU-G7has been determined. Competitive binding assays and inhibitoryenzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharideshave shown the LU-BCRU-G7 epitope to be a disaccharide Galß1-3GlcNAc(Le^c; where Gal is D-galactose, Glc is D-glucose and GlcNAcis N-acetyl-D-glucosainine). Both galactose and N-acetyl glucosaminemoieties are essential for binding. Substitution on C-2 or C-3of the terminal galactose abolished binding, as did galactose-terminated oligosaccharides. The galactose moiety alone, asexpressed by the Galß-PAA conjugate, appeared to hea more important feature of the epitope than the GlcNAc-PAAconjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody.In the N-acetyl glucosamine moiety, binding was decreased butnot eliminated by fucose substitution, as in Le^a, or changein configuration of C-4, as in Galß1-3GlcNAc. Omissionof the NAc group resulted in complete loss of activity. Thetetrasaccharide lacto-N-tetraose, although containing the terminalLe^c disaccharide, does not react with the antibody, suggestingconformational interference of the binding site. These findingsshow that the monoclonal antibody LU-BCRU-G7 recognizes a terminalisolactosamine fragment on a tumour-associated glycoprotein,which we have previously shown to be inversely related to survivalin breast cancer. breast cancer Galß1-3GlcNAc LU-BCRU-G7 monoclonal antibody pseudopolysaccharides     

Non-Reducing Terminal Fucose within N-Linked Glycan Plays a Significant Role in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.     

Presence of a xyloglucan in the cell wall of Phaseolus aureus hypocotyls

The non-cellulosic ß-glucan₁ in the cell wall of Phaseolusaureus hypocotyb was studied. Evidence that xyloglucan is presentin a hemicellulose fraction was obtained by its isolation fromcell wall preparations. This polysaccharide was homogeneouson zone electrophoresis and ultracentrifugation. On acid hydrolysis,it gave glucose, xylose, galactose, and fucose in the approximatemolar ratio of 10 : 7 : 2.5 : 1. Its solution gave a reddishviolet color with iodine-staining solution. The results of partialacid hydrolysis and cellulase treatment suggest a structurein which xylose, galactose, and fucose attached as side chainsto a sequenceof ß-l,4-linked glucose. The xyloglucanisolated accounted for 13.9% of the total non-cellulosic fractions. (Received May 10, 1976; )     

The periodate oxidation of bovine bone sialoprotein, and some observations on its structure

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.     

Involvement of Both the N-Glycan-relevant and N-Glycan-irrelevant Structural Elements in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

Monoclonal antibody 1CF11 has been suggested to specifically recognize a certain carbohydrate epitope shared by glycoproteins in human external secretions. We examined the effect of cleaving the polypeptide backbone and removing N-linked oligosaccharides on the reactivity with 1CF11 of human milk lactoferrin (hLf) to elucidate the structural features of the 1CF11 epitope. We reveal by treating hLF with trypsin and/or N-glycosidase that both the N-glycan-relevant and N-glycan-irrelevant structural elements were involved in the recognition of hLf by 1CF11.     

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans

The prestalk and prespore cells from the Dictyostelium discoideummulticellular slug stage of development differ in assembly ofglycoconjugates. Prespore cells are 2- to 3-fold more activethan prestalk cells in the assembly of N-linked glycans and20-fold more active in their fucosylation. Only prespore cellssynthesize an O-linked glycan consisting in part of Fuc -linkedto N-acetylglucosamine. Incorporation of fucose, glucosamine,mannose and galactose into large pronase-resistant glycoconjugateswas almost exclusively into prespore cells. Such glucosamine-labelledglycoconjugates resist fragmentation by ß-eliminationand include a glycoantigen dependent on the modB genetic locus.In contrast, large fucose-labelled glycoconjugates consistedof multiple, small, O-linked oligosaccharides on carrier peptides.The spore coat protein SP96 has several fucosylated O-linkedoligosaccharides, one of which correlates with a fucose epitopepreviously shown to localize in prespore vesicles and the outerlayer of the spore coat. Dictyostelium discoideum glycoconjugates glycoproteins prespore prestalk     

Terminal {beta}-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens

Indirect evidence that the immunodominant N-glycans of the parasite,Trichinella spiralis are capped by novel ß-linked3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelase, Tyv)was obtained from immunochemical assays employing monoclonalantibodies and synthetic oligosaccharides. Three of four previouslycharacterized monoclonal antibodies generated from the lymphocytesof T.spiralis infected rats bind BSA glycoconjugates bearingthe synthetic epitope ß-D-Tyvp(1     

Chemical characterisation of the released polysaccharide from the cyanobacterium Aphanothece halophytica GR02

The released polysaccharide from the halophilic cyanobacterium Aphanothece halophytica GR02 was separated into two main fractions byanion-exchange chromatography. The major fraction consisted of glucose,fucose, mannose, arabinose and glucuronic acid. Judging from thechromatography on Sepharose 2B, the major fraction was not furtherfractionated, and its apparent molecular weight was above 2.0 × 10⁶ Da.The minor fraction consisted of rhamnose, mannose, fucose,glucose, galactose and glucuronic acid, with traces of arabinose.Methylation and GC-MS spectrometry analyses of the major fractionrevealed the presence of 1-linked glucose, 1,3-linked glucose, 1,3-linkedfucose, 1,4-linked fucose, 1,3-linked arabinose, 1,2,4-linked mannose,1,3,6-linked mannose, 1-linked glucuronic acid and 1,3-linked glucuronicacid residues. The major fraction was thought to originate from capsularpolysaccharide. The released polysaccharides, obtained from cultures atdifferent age of culture, showed no striking variations in themonosaccharide composition and the relative proportions of themonosaccharides. However, the proportions of galactose and rhamnose inthe released polysaccharides, obtained from cultures under different salinity,were significantly different. The released polysaccharide also exhibitedgelling properties and strong affinity for metal ions.     

Isolation and Characterization of Golgi Bodies from Vegetative Tissue 1Fucus serratus

Cell fractions consisting primarily of functional and morphologicallyrecognizable Golgi bodies were prepared from the brown algaFucus serratus. Several enzyme activities were found to be associatedwith these fractions: thiamine pyrophosphatase, inosine diphosphatase,and galactosyl-transferases. The fractions catalysed the transferof galactose from UDP-galactose to endogenous acceptors, N-acetylglucosamine,ovalbumin, fucose, and fucoidan. These activities are latent,being activated by detergents (Triton X-100 or sodium deoxycholate).The chemical composition of the isolated fractions was examinedchromatographically and electrophoretically.     

Drosophila sperm surface alpha-l-fucosidase interacts with the egg coats through its core fucose residues

Sperm–oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein–carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α-l-fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α-l-fucosidases have fundamental function(s) during gamete interactions. An α-l-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-l-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α-l-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. The ability of sperm α-l-fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α-l-fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α-l-fucosidase was pre-incubated with fucoidan, a polymer of α-l-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-l-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm–egg interactions in Drosophila.     

Identification of a GDP-L-fucose:polypeptide fucosyltransferase and enzymatic addition of O-linked fucose to EGF domains

An assay for GDP-fucose:polypeptide fucosyltransferase has beenestablished. The enzyme catalyzes the reaction that attachesfucose through an O-glycosidic linkage to a conserved serineor threonine residue in EGF domains. The assay uses recombinanthuman factor VII EGF-1 domain as acceptor substrate and GDP-fucoseas donor substrate. Synthetic peptides with sequences takenfrom five proteins previously shown to contain O-linked fucose(Harris and Spellman, 1993; Glycobiology 3, 219–224) didnot serve as efficient acceptor substrates. These syntheticpeptides did not comprise complete EGF domains and did not containall six cysteine residues that define the EGF structure. Therefore,the enzyme appears to require more than just a consensus primarysequence and likely requires that the EGF domain disulfide bondsbe properly formed. The enzymatic reaction showed linear dependencyof its activity on time, amount of enzyme, and substrates. Althoughthe enzyme did not exhibit an absolute requirement for Mn²⁺enzymatic activity did increase ten fold in the presence of50 mM MnCl₂. The in vitro glycosylation reaction resulted incomplete conversion of the acceptor substrate to glycosylatedproduct, and characterization of the purified product by electrospraymass spectrometry revealed that one fucose was added onto thepolypeptide. Most of the enzymatic activity was found to bein the soluble fraction of CHO cell homogenates. However, whenenzyme was prepared from rat liver in the presence of proteaseinhibitors, 37% of the activity was recovered by Triton X-100extraction of the membrane particles after extensive aqueouswashes. The result suggests that the enzyme is probably a membraneprotein and, by analogy with other glycosyl transferases, probablyhas a ‘stem’ region that is very susceptible toproteolysis. fucosyltransferase O-linked fucose EGF domain glycosylation     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

Observations on the carbohydrate moiety of bovine pancreatic deoxyribonuclease A

The carbohydrate side chain of bovine pancreatic deoxyribonuclease A, which is attached to asparagine residue 18, contains two residues of N-acetylglucosamine proximal to the peptide chain followed by a variable number of mannose residues (4–10). The oligosaccharide structure bears a similarity to that in bovine pancreatic ribonuclease B. The present sequence studies have made use of α-mannosidase chromatographically purified from jack bean meal.     

Studies on by-products from the industrial extraction of alginate

The chemical composition of fucans isolated from leach-water, an industrial alginate extraction by-product, was investigated. Several fractions were obtained by anion exchange and gel permeation chromatography. They all contained fucose, but differed in the uronic acid, sulfate, xylose and galactose contents. They distributed as a continuum between uronic acid rich and sulfate poor to sulfate rich and uronic acid poor molecules. Two highly sulfated fractions were studied in particularly by chemical means (methylation, carboxy reduction, desulfation, controlled acid hydrolysis) and by¹³C nuclear magnetic resonance spectroscopy. One fraction consisted of a highly branched fucan (43.8% fucose) composed mostly of 1,2,3,4- and 1,2,4-linked fucose with some 1,4-,1,3,4- and 1,3-linkages and sulfate (23.9%) occurring on O₂ and/or O₃ and/or O₄. The other was composed mainly of fucose (31.6%), galactose (24.7%) and sulfate (23.7%). It consisted primarily of 1,6-, 1,4,6-, 1,3- and 1,3,6-linked galactose 6-and/or 4- and/or 3-sulfate on which are linked essentially terminal fucose or 1,4-linked with sulfate on O₂ and/or O₃ and/or O₄. None of these highly branched fractions contained sufficiently regular segments to yield series of homologous oligosaccharides on partial acid hydrolysis or interpretable¹³C NMR spectra.author for correspondence     

Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

ABSTRACT

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen–deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs.

Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-β-N-acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen–deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance–solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.     

Glycoprotein Biosynthesis in Chlamydomonas II. No Evidence for Lipid Intermediates in Protein Galactosylation

A crude membrane fraction from Chlamydomonas reinhardii transferredradioactivity from UDP-[¹⁴C]galactose to endogenous lipids.Most of the radioactivity was detected in digalactosyl diacylglycerolmoieties in which only the distal galactose residue (-linkedto monogalactosyl diacylglycerol) was labeled. A second compoundwas identified as monogalactosyl diacylglycerol labeled in thegalactose moiety ß-linked to glycerol. In additionto these two galactolipid species, trace amounts of radioactiveglucose were detected in the aqueous phase after mild acid treatmentof the total lipid fraction. This demonstrates the presenceof a 4-epimerase and provides indirect evidence for the presenceof a small amount of polyprenyl-monophosphate-glucose whichwas presumably not detectable per se because of the bulk ofneutral galactolipids. The failure to detect polyprenyl-phosphate-galactoseor mild acid labile galactose at any time during the incubationsuggests that galactosylation of Chlamydomonas proteins mightoccur without the involvement of lipid intermediates. (Received May 10, 1982; Accepted August 21, 1982)     

Carrageenan from Sarconema scinaioides (Gigartinales, Rhodophyta) of Indian waters

Carrageenan was extracted from the red seaweed Sarconema scinaioides of Indian waters and was characterized. The crude carrageenan as well as its alkali modified derivative was composed of 3,6-anhydro galactose, 6-O-methyl galactose as well as galactose moieties in various proportions. Linkage analysis exhibited that these two carrageenan samples consisted of 4-linked 3,6-anhydrogalactose residue sulphated at position 2, and 3-linked galactose residue sulphated at position 4. The physicochemical and rheological data along with molecular weight data, FT-IR, 1D and 2D NMR (¹H, ¹³C, COSY and HSQC) spectrometry suggested that the polysaccharide was composed predominantly of iota- along with a small amount of its precursor nu (ν)-carrageenan, unlike the hybrid carrageenans (iota-, pyruvated- and kappa-carrageenans) from this seaweed reported in the literature. This Indian seaweed species would be a potentially important source of iota-carrageenan.     

Structural characterization of novel oligosaccharides of cell-surface glycoproteins of Trypanosoma cruzi

Affinity-purified glycopeptides were prepared from Trypanosomacruzi using the carbohydrate-specific monoclonal antibody WIC29.26.These glycopeptides contain rhamnose, fucose, xylose, and galactose,in the ratio 1:1:2:3. A series of oligosaccharides was releasedfrom the glycopeptides by mild acid hydrolysis, while, in contrast,no oligosaccharides were released by either peptide N-glycosidaseF or conventional base-catalyzed ß-elimination andreduction. This suggested the presence of a phosphodiester linkagebetween the carbohydrate and peptide, which was further supportedby the detection of phosphothreonine in the glycopeptides. Themild acid liberated (MAL) fraction was resolved into two majoracidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutraloligosaccharides (MAL P1b and MAL-P2b) and a neutral fraction(MAL-N1), consisting of Gal and Xyl monosaccharides. The MAL-P1and MAL-P2 oligosaccharides proved to be hexa- and heptasaccharidesthat shared a common xylose reducing terminus, but differedby one galactofuranose residue, and their negative charge wasshown to be due to the presence of cyclic-phosphate attachedto nonreducing terminal galactofuranose residues. The MAL-P1band MAL-P2b oligosaccharides appeared to be nonphosphorylatedversions of MAL-P1 and MAL-P2. Partial structures of MAL-P1and MAL-P2 are suggested, based on compositional analyses, electrospraymass spectrometry, and tandem mass spectrometry before and afterpermethylation. The origin and significance of these uniquetrypanosomatid glycoconjugates is discussed. glycoprotein monoclonal antibody oligosaccharide structure Trapanosoma cruzi     

Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells

Recombinant human erythropoietin (rHuEPO) is used abundantlyin the clinic to stimulate red blood cell growth in anaemicpatients. The efficacy of the drug depends strongly on the extentof sialylation of its carbohydrate moiety. Prompted by conflictingliterature reports on the issue, we reinvestigated the structuresof the intact sialylated carbohydrate chains of rHuEPO expressedin Chinese hamster ovary (CHO) cells. The asparagine-linkedoligosaccharides were released from rHuEPO with N-glycanaseand fractionated by anion-exchange chromatography. The O-linkedoligosaccharides were released under alkaline borohydride conditions.The primary structures of the major sialylated N- and O-typeoligosaccharides were identified by 500-MHz ¹H-NMR spectroscopy,supported by data from composition analysis, methylation analysis,low- and high-pH anion-exchange chromatography, and fast atombombardment-mass spectrometry. The mod abundant N-linked oligosaccharidesin CHO cell-derived rHuEPO were found to be di-antennary, 2,4-branchedtri-antennary, 2,6-branched tri-antennary and tetra-antennarychains (in the ratio of 7:6:5:82), with the latter containingbetween zero and three repeating N-acetyllactosamine units,in well-defined branches. The major (>95%) di-, tri- andtetra-antennary structures are fully sialylated, i.e. they havetwo, three and four sialic acid residues, respectively, Linkedexclusively (23) to galactose residues. The majority (>95%)of N-Linked structures contain (16)-linked fucose at the proximalGlcNAc residue. The O-type mono- and disialyl oligosaccharideswere characterized as a linear tri- and a branched tetra-saccharide,respectively. erythropoietin FAB-MS ¹H-NMR recombinant glycoprotein sialic acid