Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.     

Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.     

The carbohydrate moieties of human urinary ribonuclease UL

Ribonuclease UL purified from pooled human urine contains approximately 20.7% of neutral sugar and 7.8% of aminosugar. All sugars were quantitatively released as oligosaccharides on hydrazinolysis. The oligosaccharides were converted to tritium-labeled oligosaccharides on reduction with NaB3H4. The radioactive oligosaccharide fraction was separated into a neutral and an acidic fraction on paper electrophoresis. All oligosaccharides in the acidic fraction could be converted to neutral oligosaccharides with the release of one sialic acid residue by sialidase digestion. Both fractions were shown to be mixtures of more than fourteen oligosaccharides by gel permeation chromatography. Structural studies on these oligosaccharides involving sequential exoglycosidase digestion in combination with methylation analysis revealed that ribonuclease UL contains sialylated and non-sialylated mono, bi-, tri-, and tetraantennary complex type sugar chains with N-acetyllactosamine outer chains, and tri- and tetraantennary complex type sugar chains with various numbers of Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains. An important finding was that all sialic acid residues in the acidic oligosaccharides only occur as the Sia alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 group. Both fucosylated and non-fucosylated trimannosyl cores were found among the asparagine-linked sugar chains of ribonuclease UL.     

Structures of sugar chains of human kidney gamma-glutamyltranspeptidase

gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.     

The asparagine-linked sugar chains of human follicle-stimulating hormone

The asparagine-linked sugar chains of human follicle-stimulating hormone (hFSH) were liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Ninety-five percent of the oligosaccharides were acidic and all were converted to a mixture of neutral oligosaccharides on sialidase treatment. The mixture of neutral oligosaccharides was subjected to sequential immobilized lectin column chromatography on E-PHA-agarose, AAL-Sepharose, and Con A-Sepharose, and six fractions were obtained. The results of sequential exoglycosidase digestion of each oligosaccharide and methylation analysis led us to propose that the asparagine-linked sugar chains of hFSH are a mixture of complex-type bi-, tri-, and tetraantennary sialylated sugar chains with and without a fucose residue linked at the C-6 position of the proximal N-acetylglucosamine. Some of these sugar chains contain bisecting N-acetylglucosamine residue.     

The structures of the asparagine-linked sugar chains of human apolipoprotein B-100

The asparagine-linked sugar chains of human apolipoprotein B-100 were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Their structures were elucidated by sequential exoglycosidase digestion in combination with methylation analysis after fractionation by paper electrophoresis and gel permeation chromatography. One neutral and two acidic fractions were obtained by paper electrophoresis in a molar ratio of 7:8:5. The neutral fraction contained high-mannose type oligosaccharides consisting of Man5GlcNAc2 to Man9GlcNAc2. The acidic fractions contained monosialylated and disialylated biantennary complex type oligosaccharides. As minor components in the monosialylated fraction, biantennary complex-type oligosaccharides which were absent one terminal galactose residue, monoantennary complex type, and hybrid type oligosaccharides were detected. Apolipoprotein B-100 was calculated to contain 5-6 mol of high-mannose type and 8-10 mol of complex type oligosaccharides per mole protein.     

Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (HL-60) during monocytoid differentiation and myeloid differentiation. Appearance of high mannose-type oligosaccharides in neutral fraction

Structural changes in the asparagine-linked sugar chains of plasma membrane glycoproteins during myeloid and monocytoid differentiation were investigated by the use of an in vitro differentiation system for human promyelocytic leukemic cells (HL-60), which can be induced to more mature myeloid cells by exposure to dimethyl sulfoxide and to macrophage-like cells by a phorbol ester. The asparagine-linked sugar chains released from their plasma membranes by hydrazinolysis were separated into a neutral fraction and an acidic fraction composed of over ten components. The content of neutral oligosaccharides, which accounted for 8% of the total asparagine-linked sugar chains in HL-60 cells, increased slightly to 13% in dimethyl sulfoxide-induced cells and markedly to 33% in phorbol ester-induced cells. Structural analyses revealed that the neutral oligosaccharides of HL-60 cells are all of the complex type with a variety of Gal beta 1----4GlcNAc beta 1----units in their outer chain moieties and the following core structure: (sequence; see text) After myeloid and monocytoid differentiation, the total amount of neutral complex-type sugar chains did not change significantly, but newly found high mannose-type sugar chains contributed up to 3% and 24% of the total sugar chains, respectively.     

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

Characterization of oligosaccharide units of p-N-collagen type III from dermatosparactice bovine skin

p-N-collagen type III was extracted from dermatosparactic and normal fetal bovine skin and purified by ion-exchange chromatography using DEAE- and CM-cellulose. Asparagine-linked sugar chains were fractionated by high voltage paper chromatography from the products obtained after hydrazinolysis and reduction with NaB³H₄. These oligosaccharides composed of neutral and acidic components are mannose-containing oligosugars of the complex type. Their abundance is much higher in dermatosparactic p-N-collagen type III.     

N-linked neutral sugar chains of aminopeptidase N purified from rat small intestinal brush-border membrane.

Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high mannose type sugar chains (7% of the total neutral sugar chains) are also included. The unique feature of the complex-type sugar chains is the most of them contain terminal N-acetylglucosamine residues and blood group H antigenic determinants in their outer chain moieties, and bisecting N-acetylglucosamine residues in their trimannosyl cores. Both the type 1H and type 2H determinants are found, but the former is mainly expressed at the distal portions of the outer chain moieties of the oligosaccharides.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

Carbohydrates of lysosomal enzymes secreted by Tetrahymena pyriformis

The carbohydrate structures of acid phosphatase and alpha-glucosidase secreted into culture medium by Tetrahymena pyriformis strain W were studied. Their asparagine-linked sugar chains were quantitatively liberated as radioactive oligosaccharides from their polypeptide moieties by controlled hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The approximate amounts of total sugar chains liberated from 1 mol each of acid phosphatase and alpha-glucosidase were 6 and 4 mol, respectively. Paper electrophoresis revealed that only neutral oligosaccharides were obtained from both enzymes. The oligosaccharide fraction from acid phosphatase was separated into seven components by Bio-Gel P-4 column chromatography while that from alpha-glucosidase was resolved into three components. The structures of these oligosaccharides were determined by sequential glycosidase digestion in combination with methylation analysis. The sugar chains of the two enzymes can be primarily classified as high mannose-type oligosaccharides. However, they have the following characteristic features: 1) their common core is not the usual Man5 . GlcNAc2 structure, it is Man3 . GlcNAc2; 2) some of the sugar chains of acid phosphatase have 1 approximately 3 glucose residues linked to the nonreducing terminal Man alpha 1----2 residue. The structural characteristics of the sugar moieties of the two enzymes indicate that they might be produced by the so-called "alternate pathway," in which lipid-linked Glc3 . Man5 . GlcNAc2 functions as an oligosaccharide donor.     

Comparative studies of the sugar chains of aminopeptidase N and dipeptidylpeptidase IV purified from rat kidney brush-border membrane

Asparagine-linked sugar chains of rat kidney aminopeptidase N and dipeptidylpeptidase IV were investigated comparatively. Oligosaccharides released from the two enzymes by hydrazinolysis were fractionated by paper electrophoresis, serial chromatographies on columns of immobilized Aleuria aurantia lectin, concanavalin A, phytohemagglutinin E4, and Datura stramonium agglutinin, and Bio-Gel P-4 (less than 400 mesh) column chromatography. Structures of oligosaccharides in each fraction were assumed by their effective molecular sizes and behaviors on the four lectin columns and then confirmed by sequential exoglycosidase digestion and methylation analysis. The sugar chains of aminopeptidase N and dipeptidylpeptidase IV are almost the same as those of rat kidney gamma-glutamyltranspeptidase reported previously [Yamashita, K., Hitoi, A., Matsuda, Y., Tsuji, A., Katunuma, N., & Kobata, A. (1983) J. Biol. Chem. 258, 1098-1107]. They are a mixture of 5 high mannose type sugar chains and 32 (for aminopeptidase N) or 26 (for dipeptidylpeptidase IV) mono-, bi-, tri-, and tetraantennary complex type sugar chains. The unique feature of the complex-type sugar chains of both enzymes is that they all contain the bisecting N-acetylglucosamine residue and are incompletely galactosylated in their outer-chain moieties.     

Structural study on the carbohydrate moiety of human placental alkaline phosphatase

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.