O-glycosidic Linkage Research Articles

Location and characterization of the three carbohydrate prosthetic groups of human protein HC

Three different carbohydrate prosthetic groups associated to three chymotryptic peptides, Ql, Q2 and Q3, were isolated from the reduced and carboxymethylated human protein HC. The first oligosaccharide forms an O-glycosidic linkage with a threonine residue at position 5 in the polypeptide chain of protein HC. The second and third carbohydrate prosthetic groups form N-linkages with asparagine residues at positions 17 and 96. Oligosaccharides present in Q1 contain 1 residue of NANA, 2 of Ga1NAc and 1 of Gal corresponding to the following structure: -O-Ga1NAc-Ga1NAc-Gal-NANA. Q2 contains 3 NANA, 9 GlcNAc, 2 Gal and 3 Man, and Q3 contains 2 NANA, 5 GlcNAc, 1 Gal and 2 Man. The sugar compositions of Q2 and Q3 oligosaccharides are compatible with that of the complex kind. The amount of oligosaccharides present in Q1, Q2 and Q3 corresponded respectively to 3.0%, 12.2% and 7.3% of the weight of protein HC. No difference was found between the carbohydrate composition of urinary and plasma protein HC.

Biosynthesis of mannoproteins by cell-free extracts from Phycomyces blakesleeanus

Most of the manosyl transferase activity in Phycomyces blakesleeanus was found associated with a crude membrane fraction sedimenting at 48,400 g ( R av). Triton X-100 and Nonidet NP-40 inhibited 95% of the enzyme activity. Digitonin caused 47% of inhibition and when removed, the membrane-bound enzymatic activity increased by about 35%; no activity was detected in supernatant. The rate of mannosyl transfer increased in the presence of 4 or 8 m M Mg 2+ ions. Several compounds, including glycoproteins, mucoran, and mucoric acid, failed to act as acceptors of mannosyl residues. Guanosine diphosphate and guanosine monophosphate inhibited the transfer of mannosyl residues by 60 and 19%, respectively. Mannosyl transfer involves participation of lipid intermediates.β elimination of the product synthesized in vitro revealed the presence of mannose, mannobiose, and mannotriose, suggesting that they are bound to protein via O-glycosidic linkages. The alkaline-resistant carbohydrate part of the glycoproteins consisted mainly of mannose residues that were probably connected to the protein moiety through N-glycosidic bonds.

Purification and enzymatic properties of an Endo- α- N-Acetyl-Galactosaminidase from Bacillus laterosporus SS-16

A Gram positive bacterium, Bacillus laterosporus, isolated from soil was found to produce an endo- α- N-acetylgalactosaminidase. The optimum pH of the enzyme was found to be 8.0 and the stable pH range was 3.5–9.0. The apparent K m values were 0.52 mM and 0.78 mM with asialofetuin and asialo-κ-casein as substrates, respectively. The molecular weight was 210,000 and the isoelectric point was 5.9. The enzyme hydrolyzed the O-glycosidic linkage between α- N-acetylgalactosamine and the hydroxyl group of serine or threonine residues in mucus and mucin-type glycoproteins.

Cytokeratin 13 Contains O-Glycosidically Linked N-Acetylglucosamine Residues

The glycosylation of human cytokeratins was investigated in cultured human keratinocytes and A431 cells by metabolic labeling with [3H]glucosamine. In the presence of tunicamycin, keratinocytes incorporated [3H]glucosamine into a vitamin A-regulated acidic 53-kDa component of the cytoskeleton which was identified as cytokeratin 13 by one- and two-dimensional immunoblotting with specific monoclonal antibodies. This cytoskeletal component was also labeled with [3H]glucosamine in A431 cells but not in KB cells, which do not express cytokeratin 13. Its labeling was resistant to tunicamycin, suggesting that [3H]glucosamine had not been incorporated into N-linked oligosaccharides. Acid hydrolysis followed by paper and ion-exchange chromatography showed that the radioactivity in electrophoretically purified cytokeratin 13 was still present as glucosamine. Radioactivity was completely removed by treatment with beta-N-acetylglucosaminidase, suggesting that it was present in terminal N-acetylglucosamine residues. The labeled carbohydrate was released by alkaline borohydride treatment and was bound by a phenylboronic acid column, indicating an O-glycosidic linkage. On Bio-Gel P-2 columns, the beta-eliminated carbohydrate co-eluted with authentic N-acetylglucosaminitol. The results indicate that cytokeratin 13 contains single residues of N-acetylglucosamine O-glycosidically linked to the polypeptide chain.

Molecular analysis of a spermatozoal glycoprotein having immuno regulatory properties

A water-soluble fraction of sialoglycoprotein containing sulfate was isolated from the mucosal scrapings of the rat small intestine without prior treatment with proteolytic enzymes. Chromatography of the water-soluble mucin on a DEAE-cellulose column gave three main fractions: a major carbohydrate-rich fraction containing sulfate (IGP-A), one high in protein content, and a third with a composition similar to the starting material. Fraction IGP-A was resolved into two components by ultracentrifugation and disc-gel electrophoresis. The higher molecular-weight species of IGP-A was separated from the second component by Sepharose-4B chromatography. These two glycoprotein fractions designated IGP-A1 and IGP-A2 had the same chemical composition as IGP-A.The carbohydrate component of IGP-A consisted of galactosamine, glucosamine, fucose, galactose, and sialic acid. The molar ratio of glucosamine to galactosamine was 1.1 and that of sulfate to hexosamine was 0.18. The sulfate content was 2.4%, 40% of which was acid labile. Both sialic acid and fucose were rapidly released by acid hydrolysis suggesting that they occupy terminal positions. The monosaccharide components of the isolated glycoprotein were removed by acid hydrolysis in the following order: sialic acid > fucose > glucosamine > galactose > galactosamine. Threonine, serine, and proline made up 69% of the total amino acid residues. The destruction of serine, threonine, and galactosamine with alkaline borohydride treatment was concomitant with the production of alanine, α-amino-butyric acid, and galactosaminitol, respectively. This finding strongly suggests that galactosamine is bound to the mucin through serine and threonine residues involving O-glycosidic linkages.

Isolation and characterization of platelet glycoprotein IV (CD36)

Platelet glycoprotein IV (GPIV, Mr 88,000), which is immunologically related to the leukocyte differentiation antigen CD36, has been isolated from both intact and trypsinized platelet membranes by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on wheat germ agglutinin-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA-44. The homogenous product contained 26% carbohydrate (sialic acid, Gal, Man, GalNAc, GlcNAc), of which approximately two-thirds were in alkali-labile O-glycosidic linkages. A rabbit polyclonal antibody raised against purified GPIV gave a single band on immunoblot and on immunoprecipitation from solubilized, 3H-labeled platelet membranes indicating its monospecificity. The antibody gave a strongly positive reaction with platelets on flow cytofluorimetry further confirming the surface location of GPIV. Immunoblotting and flow cytometry also identified GPIV-like molecules on the surface of U937, HEL, and C32 cells but not on HT1080 fibroblasts. Amino acid analysis showed values comparable with those deduced from the cloning data for GPIb, GPIIb, and GPIIIa. Automated Edman degradation allowed the identification of the sequence of the first 36 residues of the NH2-terminal domain. G-X-D-R-N-X-G-L-I-A-G-A-V-I-G-A-V- L-A-V-F-G-G-I-L-M-P-V-G-D-L-P-X-Q-K-F. There are no identifiable homologies between the NH2-terminal domain and other known protein sequences. Following a hydrophilic hexapeptide, there is a hydrophobic sequence of 23 amino acids (underlined) that is of the size and composition expected for a transmembrane domain. Since the NH2-terminal domain lacks tyrosine, but GPIV may be readily iodinated in intact platelets, this suggests that GPIV may have a configuration expressing other extramembranous domains.

Reassessment of the toxic glycoprotein isolated from Rhynchosporium secalis (Oud.) Davis culture filtrates: Physicochemical properties and evidence of its presence in infected barley plants

A phytotoxic acidic glycoprotein from Rhynchosporium secalis filtrate contained mannose, rhamnose, glucosamine and galactose in the ratio of 1:1:1:2. Serine and threonine are involved in O-glycosidic linkages with the sugar moieties which account for 85% of the total weight. The toxin displays a great stability to heating and mild acid treatments. Mild alkali, protease or periodic acid treatments completely destroyed activity. Biological activity, chromatographic or electrophoretic data have shown the probable presence of the toxin in the infected barley leaves.

Caseins of various origins and biologically active casein peptides and oligosaccharides: structural and physiological aspects.

The first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (alpha s1-, alpha s2-, beta- and kappa). The calcium-sensitive casein fractions are always phosphorylated whereas kappa-caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca2+. In kappa-caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine kappa-casein and up to 10 in human kappa-casein. The structures of the known kappa-casein carbohydrate moieties are described. Finally the milk clotting process (interaction kappa-casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena. The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A 'strategic zone' containing immunostimulating and opioid peptides could be located in cow and human beta-caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the kappa-caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities. In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components. If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.

Synthesis and secretion fo mucous glycoprotein by the gill of Mytilus edulis I. Histochemical and chromatographic analysis of [ 14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [ 14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose at the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [ 14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. “Paul's Scarlet”). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 105M−1 or 9.8 × 105M−1 with a maximum of approximately 108 lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.

Release of oligosaccharides possessing reducing-end N-acetylgalactosamine from mucus glycoprotein in [formula omitted] sp. OH-11242 culture medium through action of endo-type glycosidase

A crude enzyme preparation from a culture medium of Streptomyces sp. OH-11242 contained endo-α-N-acetylgalactosaminidase activity. The activity could be induced by the addition of purified porcine gastric mucin to the culture medium. Oligosaccharides corresponding to approximately 2–14 glucose units were detected in the culture medium and also in an incubated reaction mixture of crude enzyme preparation and mucus glycoprotein. The resulting product with N-acetylgalactosamine at the reducing terminal implied the presence of a new type of endo-glycosidase liberating not only Ga1ß1–3Ga1NAc but also other larger oligosaccharides by hydrolysis of the O-glycosidic linkage between Ga1NAc and Ser (Thr).

Structural concepts of the human blood group A, B, H, Le(a), Le(b), I and i active glycoproteins purified from human ovarian cyst fluid.

Regardless of the A, B, H, Le(a), Le(b), I and i activity, purified water-soluble blood group glycoproteins from human ovarian cyst fluid have a similar overall structure. They are polydisperse macromolecules (Mr 2.0 x 10(5) to several million) of similar composition (75 to 85% carbohydrate, 15 to 20% protein) and consist of multiple heterosaccharide side chains attached by an O-glycosidic linkage at their internal reducing ends to serine or threonine of the polypeptide backbone. About 90% of these carbohydrate side chains range in size from one to less than twenty-four sugar residues (twelve sugars in the internal structure and twelve key sugars specific as blood group determinants). Three-fourths of these side chains contain fewer than twelve sugars. A generalized blood group active carbohydrate chain is shown above. Three disaccharide units-Type I chain (Gal beta 1----3GlcNAc beta 1----3), Type II chain (Gal beta 1----4GlcNAc beta 1----6) and T determinant [Gal beta 1----3GalNAc alpha 1----Ser(Thr)]-are used to elucidate the internal structure of the carbohydrate chains. The complete internal structure is considered to have a core structure with four branches, to which the blood group key sugars are attached at the appropriate locations. The core structure is a tetrasaccharide, composed of one unit of Type I chain at the nonreducing end and the T determinant at the other end, linked to Ser or Thr of the protein moiety. Branch I is Type I chain and Branch II is Type II chain. They are linked to Gal at the nonreducing end of the core structure. Branch III is usually a Type II chain, but may sometimes be a Type I chain, linked to the GalNAc of the reducing end. The length of Branch III can be increased by adding one or more monosaccharides of Type I chain sequence such as Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----6GalNAc alpha 1----Ser(Thr), a combination of Type I and Type II chains. A new Branch IV is made up of Type II chain, which in turn is linked to the Gal end of the T determinant. The Type II chains react with the antibody to the type XIV pneumococcal capsular polysaccharide and with the anti-I(Ma) cold agglutinin.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of endo-.ALPHA.-N-acetylgalactosaminidase from Alcaligenes sp.

A gram negative bacterium isolated from soil was found to produce an endo-α-N-acetylgalactosaminidase in culture. The microorganism was identified as Alcaligenes sp. from various bacteriological characteristics. The enzyme was purified from the culture fluid by fractionation with ammonium sulfate, and column chromatographies on DEAE-Sephadex A-50 and hydroxylapatite. The molecular weight of the enzyme was estimated to be 160,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH was found to be 4.5 and the stable pH range was 4.5 ~ 6.5. The Km values were 3.7 mm and 3.2 mm with asialofetuin and asialo κ-casein glycopeptide as substrates, respectively. The enzyme released the disaccharide, Galβ1→3GalNAc, from glycoproteins possessing serine or threonine O-glycosidic linkages. The enzyme production was highly induced by porcine gastric mucin added to the medium.

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa.

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).

UDP-L-arabinose-hydroxyproline- O-glycosyltransferases in Volvox carteri

Hydroxyproline-containing peptides of different length and amino acid sequence have been used to demonstrate UDP-L-arabinose-hydroxyproline- O-glycosyltransferases in a crude microsomal fraction from the green alga Volvox carteri. The formation of O-glycosidic linkages by transfer of UDP-activated arabinose to the side chain of hydroxyproline was concluded from the resistance of the glycopeptides under the basic conditions of β-elimination and their susceptibility to hydrolysis by trifluoroacetic acid. This treatment yielded arabinose as the only cleavage product. Arabinose transfer to the various peptide substrates was found to be stimulated by low concentrations of detergent, to require divalent cations and to proceed optimally at pH values around 7.0. The smallest arabinose acceptor peptide was the tripeptide Tyr-Hyp-Lys. The glycosyl acceptor electivity increased with increasing numbers of repeated hydroxyproline residues, suggesting that hydroxyproline clusters critically affect substrate recognition by the Volvox transferase(s).

A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride

A novel method is described for the extraction of DNAs from fungi and yeasts. Anhydrous hydrogen fluoride (HF) selectively cleaves their cell walls under mild conditions (for 5 min at 0°C), enabling the effective extraction of DNAs from organisms with a cell wall. A possible mechanism for this method concerning the selective cleavage of O-glycosidic linkages in cell walls has been described previously [(1977) Anal. Biochem. 82, 289–309]. The extracted DNA is intact: in fact, the yeast DNA is directly applicable for restriction analysis and transformation of Escherichia coli.

Structural properties of porcine submaxillary gland apomucin.

Porcine submaxillary gland mucin was deglycosylated with a mixture of pure glycosidases to give apomucin containing less than 1% carbohydrate. The resulting apomucin freed of glycosidases was found to contain nine amino acids: threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, and arginine. Serine, threonine, glycine, and alanine comprise 77% of the composition. The molecular weight of apomucin was 96,500 as determined by gel filtration in guanidine hydrochloride. Its Stokes radius was greater than 68.6 A, a far larger value than expected for a globular protein with Mr = 96,500. Circular dichroism spectroscopy of apomucin suggests that it contains 42% aperiodic or "other" structure, 40% beta-turns, 10% antiparallel pleated sheet, and 8% helical structures. The predicted secondary structure of a 50-residue peptide from ovine submaxillary gland mucin resembles the circular dichroism predictions, being dominated by turns that would lead to an extended nonglobular structure. Analysis for the secondary structure of a 36-residue tryptic peptide derived from porcine submaxillary gland apomucin predicts a similar structure. It is concluded that apomucin is likely devoid of traditional secondary structure and serves as a scaffold upon which oligosaccharides are added in O-glycosidic linkage. When sufficient sialic acid is present in the oligosaccharides, native highly viscous mucin containing about two-thirds carbohydrate by weight is obtained.

O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins.

Glycoproteins bearing a single N-acetylglucosamine (GlcNAc) residue attached by an O-glycosidic linkage to the polypeptide chain (Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057) have been found to be enriched in the nuclear and soluble fractions of rat liver. Our goal was to determine the localization and membrane topography of proteins bearing O-linked GlcNAc using galactosyltransferase and wheat germ agglutinin (WGA) as membrane-impermeant probes. Latency of the enzyme mannose-6-phosphatase was used to quantitatively confirm the intactness of the nuclear envelope during incubations with galactosyltransferase or WGA. The O-linked GlcNAc residues of nuclei were fully accessible to modification by galactosyltransferase under conditions where the nuclear envelope mannose-6-phosphatase was 70% latent. Addition of detergent destroyed the permeability barrier but did not increase galactosylation of the O-linked GlcnAc. The major polypeptides bearing O-linked GlcNAc residues on nuclei were peripheral rather than integral membrane proteins with apparent molecular masses ranging from 210 to 54 kDa. The proteins were also detected on sealed nuclei using conjugates of WGA. WGA-rhodamine labeled intact nuclei when examined by immunofluorescence; WGA-peroxidase was used to identify the nuclear glycoproteins after transfer to nitrocellulose. WGA-ferritin selectively labels the cytoplasmic and nucleoplasmic faces of the nuclear pore complex when examined by electron microscopy. Taken together, these data strongly suggest that proteins bearing cytoplasmically oriented O-linked GlcNAc are components of the nuclear pore complex, thereby raising the possibility that cytoplasmic and nucleoplasmic glycoproteins are involved in the assembly or functioning of the nuclear pore.

D-galactofuranose in the N-linked sugar chain of a glycopeptide from Ascobolus furfuraceus.

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced beta-elimination of a small oligosaccharide. Evidence for the O-glycosidic linkage was provided by increase in absorbance at 240 nm, decrease in threonine and serine content after the alkaline treatment and detection of tritiated oligosaccharide following alkaline NaB3H4 reduction. Mannose is the sugar involved in the O-glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N-glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo-beta-N-acetyl-glucosaminidase. Sequential NaB3H4 reduction and acid hydrolysis gave [3H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis.

Isolation of a novel O-linked, sulfated polysaccharide of high molecular weight from an ovarian cyst glycoprotein having blood group “A” activity

Treatment of a blood group A-active ovarian cyst mucin glycoprotein with alkaline borohydride under conditions expected to cleave O-glycosidic linkages between carbohydrate and peptide releases a sulfated polysaccharide of average molecular weight 20 000. Its peptide and mannose content is less than 1%, and carbohydrate analysis gives Fuc/GaINAc/Gal/GlcNAc in the ratio of 1:1:2.2:2.2. Galactosaminitol is recovered at the level of one residue per 112-residue average polysaccharide chain. The 13C- and 1H-NMR spectra show that the polysaccharide has side chains whose non-reducing terminals have the blood group A structure on a type 1 chain: ▪ Methylation analysis confirms the presence of these blood group A type 1 sidechains as well as 4-substituted GlcNAc, 3-substituted galactose and 3,6-substituted galactose branch points. Periodate oxidation removes all the fucose and GaINAc from the non-reducing terminal but leaves intact the backbone composed of β-linked Gal and GlcNAc, as would be expected for a polylactosamine. Although the native polysaccharide is resistant to endo-β-galactosidase digestion, the product of periodate degradation is partially digested, giving a 30% yield of a trisaccharide shown by 1H-NMR spectroscopy to be: Gal( β1 → 3)GlcNAc( β1 → 3)Gal We conclude that this is a high molecular weight sulfated polysaccharide which is related to the asparagine-linked polylactosamine chains of cell surface glycoproteins which have been implicated in cell differentiation. However, the blood group A polysaccharide from the ovarian cyst mucin is unique in several respects. It is linked to the protein by an O-glycosidic bond rather than the N-asparagine linkage of the previously known polylactosamines which have a trimannosyl core, and its blood group A side chains are on a type 1 core rather than type 2 which is found on other polylactosamines.