Purification and structures of oligosaccharide chains in swine trachea and Cowper's gland mucin glycoproteins

Mucin glycoproteins were purified from extracts of swine trachea mucosa and Cowper's gland. The gelatinous extracts were solubilized by reduction and carboxymethylation and then purified by chromatography on Sepharose CL-6B and DEAE-Sepharose. The structure of some of the carbohydrate units in these glycoproteins were determined and compared. Alkaline borohydride treatment indicated that more than 85% of the carbohydrate chains in these glycoproteins were linked to serine or threonine residues in the polypeptide chain through O-glycosidic bonds with N-acetylgalactosamine. Reduced oligosaccharides released by treatment with alkaline borohydride were isolated by gel filtration on Bio-Gel P-6 and chromatography on DEAE-cellulose and paper. The structures of the oligosaccharides were established by methylation analysis, gas chromatography, and sequential hydrolysis with specific exoglycosidases. The major oligosaccharides in Cowper's gland mucin glycoproteins were sialylated short chains: NeuAc alpha 2,6GalNAcol and NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAcol. In marked contrast, branched chains containing a Gal beta 1,3(GlcNAc beta 1,6)GalNAc core unit were the major components of trachea mucin glycoprotein. Ten of these chains had the following structures: (Formula: see text).     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.     

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.     

A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.     

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).     

Enzymes that hydrolyze fungal cell wall polysaccharides. The carbonhydrate constitution of mycodextranse, an endo-alpha (1 yields 4)-D-glucanase from Pencillium melinii.

The chemical constitution of the carbohydrate portion of mycodextranase, an exocellular endo-alpha(1 yields 4) D-glucanase of Penicillium melinii, has been investigated. At least 80% of the carbohydrate, consisting exclusively of mannose and glucose, is released from protein by treatment of the enzyme with 0.05 M potassium hydroxide plus 1 M sodium borohydride or 0.5 M sodium hydroxide at 50 degrees. There is concomitant destruction of 60% of the threonine and 15% of the serine of the treated enzyme and an increase in absorption, at 241 nm, of the treated protein's spectrum, indicative of an O-glycosidic beta-hydroxyamino acyl linkage between untreated protein and its associated carbohydrate. Mannose is the monosaccharide involved in this linkage. Smith degradation, methylation, and glycosidase digestions of the carbohydrate indicate that it is present in mycodextranase as side chains of mannose, glucosyl alpha(1 yields 2)-mannose, and mannosyl alpha(1 yields 2)-glucosyl alpha(1 yields 2)-mannose units with each enzyme molecule bearing a calculated average of 25 side chains. Separation of pronase glycopeptides by gel filtration on Sephadex G-25 revealed that 96% of the carbohydrate is present in the highest molecular weight fraction which contains 60% of the threonine of mycodextranase but only 3.5% of the aromatic acids judged by its absorbance at 280 nm. Further fractionation of this glycopeptide component on Sephadex G-75 indicates carbohydrate is restricted to two fractions, one containing 71% by weight of the threonine and serine of mycodextranase and 56% of its carbohydrate. These results suggest carbohydrate chains of mycodextranase are clustered in a few threonin-rich regions along the polypeptide chain rather than being separated from each other by nonglycosylated areas.     

Structural characterization of a glycoprotein cellulase, 1,4-beta-D-glucan cellobiohydrolase C from Trichoderma viride.

A glycoprotein enzyme, 1,4-beta-D-glucan cellobiohycrolase (EC 3.2.1.91) form C, was purified to electrophoretic homogeneity by a procedure which permitted isolation of gram quantities from a commercial Trichoderma viride culture filtrate preparation. Purified cellobiohydrolase C has an E1%/280 nm = 14.2 and degrades both microcrystalline and phosphoric acid-swollen cellulose to cellobiose. The cellobiohydrolase C contains 26.4, 4.8, 2.4 and 3.4 mol of mannose, glucose, galactose and glucosamine, respectively, per mol of enzyme (molecular weight, 48 400). Methylation analysis of cellobiohydrolase glycopeptides indicates an average carbohydrate chain length of two residues. Alkaline borohydride treatment of cellobiohydrolase C released neutral carbohydrate which is bound through an average of 16.7 O-glycosidic linkages to serine and threonine per molecule of enzyme. Glucosamine was not released from the protein by alkaline treatment. Analysis of alkaline borohydride-released carbohydrate by high pressure liquid chromatography demonstrated that an average enzyme molecule contains 8.8 mono-, 1.8 di-, 4.6 tri-, 1.2 tetra-, and 0.4 pentasaccharide chains. The linkages between the neutral monosaccharides are (1 leads to 6) as shown by gas chromatography - mass spectrometry of partially methylated residues. The (1 leads to 6) linkage is consistent with the stability of the linkages to alkaline conditions and the destruction of all neutral carbohydrate by periodate. Action of alpha-mannosidase indicates that some oligosaccharide chains contain alpha-mannose as the terminal residue.     

Resistance to deglycosylation by ammonia of IgA1 O-glycopeptides: implications for the beta-elimination of O-glycans linked to serine and threonine

Pools of O-glycopeptides (and their deglycosylated analogues) derived from trypsin-digested normal human serum IgA1 have been treated with ammonia under conditions reported to result in complete liberation of O-glycans linked to serine and threonine residues in glycopeptides and glycoproteins. MALDI-TOF MS analysis has revealed that only one of the six glycosylated sites is susceptible to beta-elimination under these conditions. It is likely that resistance to beta-elimination is due to very close proximity of proline to the glycosylated serine or threonine residues. Preliminary results using 0.1M NaOH (instead of ammonia) to perform beta-elimination indicated that there was also selective de-O-glycosylation with this reagent, however, these results were complicated by the concomitant hydrolysis of the peptide bonds. These findings may have implications for similarly O-glycosylated peptides and proteins and possibly for other chemical methods that are used to carry out beta-eliminations of O-glycans.     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I. Chemical composition and structural studies.

The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety.     

Characterization of blood group active glycopeptides derived from porcine kidneys

Sialoglycopeptide fractions were prepared from the pronase digest of porcine kidneys by DEAE-Sephadex A-25 column chromatography and gel-filtration through Sephadex G-100. Their chemical compositions and large molecular size suggested that these glycopeptides were derived from mucin-type glycoprotein(s). The results of the beta-elimination reaction indicated that they have the O-glycosidic linkages between N-acetylgalactosamine and serine/threonine. The glycopeptides exhibited blood group A and H activities. The present study revealed that the porcine kidney contains the blood group antigens of glycoprotein nature.     

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/GalNAc/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: (Formula: see text). 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 GalNAc from the non-reducing terminal but leaves intact the backbone composed of beta-linked Gal and GlcNAc, as would be expected for a polylactosamine. Although the native polysaccharide is resistant to endo-beta-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(beta 1----3)GlcNAc(beta 1----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.     

Studying O-linked protein glycosylations in human plasma

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.     

Synthesis and secretion of 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 as 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.     

Isolation and characterization of a blood group active glycopeptide from porcine aorta

A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and alpha-amylase digestion. This glycopeptide was electrophoretically homogeneous. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the beta-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serine/threonine. This glycopeptide exhibited blood group A and H activities. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature.     

Modifications of substituted seryl and threonyl residues in phosphopeptides and a polysialoglycoprotein by beta-elimination and nucleophile additions

The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.     

Structure of the cell wall proteogalactomannan from Neurospora crassa. II. Structural analysis of the polysaccharide part

The native proteoheteroglycan (PHG) from mycelia of Neurospora crassa contain two kinds of carbohydrate chains differing structure. The oligosaccharides containing mannose and galactofuranose are attached by O-glycosidic linkages to serine or threonine residues in the protein (J. Biochem. 96, 1005-1011, 1984). The second kind of carbohydrate chain is a polysaccharide containing mannose and galactofuranose as the main sugar components. The results of structural studies with methylation and NMR analyses on the native PHG and some of its specifically degraded products obtained on partial acid hydrolysis and acetolysis indicate that the polysaccharide moiety of the PHG has an (alpha 1-6) linked mannan backbone with mainly (alpha 1-2) linked side chains, each of which consists of 2 to 5 mannose units, and most of the mannosyl side chains bear beta-galactofuranosyl residues linked to the 2 positions of the mannosyl nonreducing terminals. The galactofuranose residues are linked with each other by (beta 1-5) bonds.     

The structure of chordin: characteristics of protein and carbohydrate components and their role in antigenicity

Using immobilized monoclonal antibodies, a tissue-specific antigen, chordin, was isolated from cell extracts of giant sturgeon (beluga) notochord. The antigen was further purified by gel filtration through SP-Sephadex (pH 2.1) and gel chromatography on TSK Toyopearl HW-60. Purified chordin preparations contained 40% of protein and 60% of carbohydrates. The predominant polar amino acids were threonine, serine, glycine, asparagine and glutamine (or aspartic and glutamic amino acids). The carbohydrate moiety comprised mannose, fucose, galactose, galactosamine and glucosamine. Treatment of chordin with three enroglycosidases specifically hydrolyzing the carbohydrate chains of proteoglycans did not affect the antigenic properties of chordin or its behaviour on gel filtration. These findings and the fact that 75% of galactosamine was converted to galactosaminite after treatment with alkaline NaBH4 permitted to relate chordin to glycoproteins carrying O-glycosidic carbohydrate-peptide bonds between the N-acetyl-galactosamine and beta-hydroxyamino acid residues. Besides, chordin seems to contain a N-glycosylamide carbohydrate-peptide bond as can be judged from glucosaminite formation after treatment of the antigen with alkaline LiHB4. The changes in the antigenic properties of chordin after its treatment with neuraminidase, pronase, sodium periodate, alkali, alkaline NaBH4 or LiBH4 suggest that the polypeptide moiety of the chordin molecule and, perhaps, the N-acetylgalactosamine within the composition of the carbohydrate-peptide bond are involved in the construction of its most immunogenic determinants (P-determinants).     

Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae.

An enzyme that hydrolyzes the O-glycosidic linkage between alpha-N-acetyl-D-galactosamine and serine or threonine in mucins and mucin-type glycoproteins was purified by chromatography on an Affi-Gel 202 column or isoelectric focusing from filtrates of Diplococcus pneumoniae cultures. The final preparations were free of protease and a wide range of other glycosidase activities. The preparation obtained by isoelectric focusing was shown to consist of a single protein by gel filtration and sodium dodecyl sulfate-gel electrophoresis. This preparation had an apparent molecular weight of about 160,000, determined by gel filtration, an optimum pH of 7.6, and an isoelectric point in the range pH 8 to 9. The enzyme releases the disaccharide Gal-GalNAc from a variety of glycopeptide and glycoprotein substrates and appears to have a specific requirement for an unsubstituted galactose in the nonreducing terminus and an alpha linkage between N-acetylgalactosamine and the aglycone. This is the only endoenzyme known capable of cleaving the linkage between a carbohydrate and serine or threonine residues in glycoproteins. The ability of this enzyme to act on macromolecular substrates and its pH optimum makes it ideally suited to explore the distribution and function of mucin-type glycoproteins on normal and cancer cell surfaces.     

CARBOHYDRATE-PEPTIDE LINKAGES IN GLYCOPROTEINS AND MUCOPOLYSACCHARIDES FROM BRAIN

Abstract— Treatment of glycopeptides, prepared from glycoproteins of rat and rabbit brain, with NaOH-NaBH₄ leads to the destruction of a portion of the serine, threonine and galactosamine present, and the appearance in acid hydrolysates of alanine, α-aminobutyric acid and galactosaminitol. These results indicate that N-acetylgalactosamine at the reducing end of oligosaccharide chains in brain glycoproteins is linked O-glycosidically to the hydroxyl groups of serine and threonine residues. 2-acetamido-1-(L-β-aspartamido)-l,2-dideoxy-β-D-glucose was also detected after partial acid hydrolysis of the alkali-stable glycopeptides, and most of the carbohydrate in brain glycoproteins appears to be linked by N-acetylglucosaminylasparagine linkages. The results of the treatment of the sulphated mucopolysaccharides from bovine brain with alkaline-borohydride indicate that the polysaccharide chains in chondroitin sulphate and heparan sulphate are linked exclusively to serine.