Incompatible blood-group A determinants in tumoral mucins. Isolation of oligosaccharides having a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group at the non-reducing end

Two glycopeptide fractions were obtained from pseudomyxomatous mucins secreted by an ovarian cystadenocarcinoma from a female having blood-group B, and by an appendix tumor from a male having blood-group O. The carbohydrate and amino acid content of these fractions suggests the presence of numerous carbohydrate side-chains linked through O-glycosyl bonds to a peptide core rich in threonine and proline. The two glycopeptide fractions exhibit compatible B- and H-blood-group activities. They are reactive towards Dolichos biflorus lectin and human anti-A agglutinins, and so exhibit an incompatible A activity. Alkali-borohydride degradation of Pronase-digested glycopeptides gave dialyzable oligosaccharides that were purified and shown to possess 2-acetamido-2-deoxygalactitol at the terminal reducing-end. 2-Acetamido-2-deoxyglucose, galactose, fucose, and neuraminic acid were absent, or present, in variable proportions. Four oligosaccharides containing 2-acetamido-2-deoxy-D-galactose residues were reactive towards Dolichos biflorus lectin and human anti-A agglutinins, indicating the presence, at the nonreducing end, of a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, responsible for blood-group A activity.     

Structural analysis of hexa- to octa-saccharide fractions isolated from sheep gastric-glycoproteins having blood-group I and i activities

Structural data are presented on six oligosaccharide-fractions (hexa- to octa-saccharides) released from sheep -gastric-glycoproteins having blood-group I and i activity by degradation with alkaline borohydride. Previous data on two of the oligosaccharides are included for comparison. The fractions were analysed, before and after treatment with exo-β- -glycosidases and an endo-β- -galactosidase, on Bio-Gel P4 and by p.c., by direct-insertion m.s. (after methylation), and by g.l.c.—m.s. of the derived, partially O-methylated alditol acetates. Each fraction contained 1–3 oligosaccharides, each of which had 2-acetamido-2-deoxy- -galactitol (GalNAc-ol) at the reduced end and involved one of the structures

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The majority of the oligosaccharides contained the unsubstituted “type 2” blood group precuisor-chain sequences, β- -Gal-(1→4)-β- -GlcNAc-(1→6) and single or repeating β- -Gal-(1→4)-β- -GlcNAc-(1→3), which are recognised by various anti-blood-group I and i cold agglutinins. The “type 1” sequence, β- -Gal-(1→3)-β-blood-group Ii activities, the following structural model can be proposed, which consists of (a) a core region; (b) a backbone region having (1→3)- and (1→6)-linked N-acetyl-lactosamine [β-Gal-(1→4)-GlcNAc] branches with I activities, and linear, repeating, (1→3)-linked N-acetyl-lactosamine units with i activities; and (c) a peripheral region with blood-group isotype activities.

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Biosynthesis of a blood-group-I determinant reacting with anti-I Ma serum (group 1).

A blood-group-I determinant reacting specifically with anti-I Ma serum (group 1) has been produced biosynthetically by the action of a beta-galactosyl transferase isolated from human milk on a precursor glycoprotein produced by formic acid hydrolysis and beta-D-galactosidase action on blood-group H substance prepared from hog gastric mucosa.     

Structures of sulfated oligosaccharides in human trachea mucin glycoproteins

The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Importance of size and sulfation of heparin in release of basic fibroblast growth factor from the vascular endothelium and extracellular matrix.

We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

Preparation and in vitro antioxidant activity of kappa-carrageenan oligosaccharides and their oversulfated, acetylated, and phosphorylated derivatives

In order to study the relationship between chemical structure and properties of modified carrageenans versus antioxidant activity in vitro, kappa-carrageenan oligosaccharides were prepared through mild hydrochloric acid hydrolysis of the polysaccharide, and these were used as starting materials for the partial synthesis of their oversulfated, acetylated, and phosphorylated derivatives. The structure and substitution pattern of the oligosaccharides and their derivatives were studied using FTIR and (13)C NMR spectroscopy, and their in vitro antioxidant activities were investigated. Certain derivatives of the carrageenan oligosaccharides exhibited higher antioxidant activity than the polysaccharides and oligosaccharides in certain antioxidant systems. The oversulfated and acetylated derivatives, which scavenge superoxide radicals, the phosphorylated and low-DS acetylated derivatives, which scavenge hydroxyl radicals, and the phosphorylated derivatives, which scavenge DPPH radicals, all exhibited significant antioxidant activities in the systems examined. The effect of the molecular weight of the carrageenan on antioxidant activities, however, is not obvious from these studies.     

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.     

Fucose containing oligosaccharides from human milk: I. Separation and identification of new constituents

Neutral oligosaccharides isolated from pooled human milk were subjected to fractionation on high-performance thin-layer chromatography (HPTLC) plates, Iatrobeads, and reverse-phase chromatography after borohydride reduction and peracetylation. By the combined HPLC and HPTLC separation a mixture of pooled human milk oligosaccharides was separated into 101 fractions. These fractions were characterized by field desorption or fast atom bombardment (FAB)-mass spectrometry. Each of the carbohydrate constituents, the peracetylated glucitol, the galactose, the glucosamine, and the fucose contribute specific mass increments to the molecular weight of the oligosaccharide. Therefore, the exact carbohydrate composition can be calculated from the molecular weight determined by mass spectrometry. Among the fractions obtained one trifucosyl-lacto-N-tetraose, five monofucosyl-, eleven difucosyl-, and nine trifucosyl-lacto-N-hexaoses, one monofucosyl-, eight difucosyl-, seven trifucosyl-, four tetrafucosyl-, and two pentafucosyl-lacto-N-octaoses, one trifucosyl-, and two difucosyl-lacto-N-decaoses could be identified. FAB spectra furnished additional data on structural features of the isolated oligosaccharides.     

Oligosaccharide alterations of rat glioblast membrane-bound glycoproteins during differentiation induced by glia maturation factor

Differentiation of normal glioblasts was induced by glia maturation factor (GMF), and the structural change in the oligosaccharide chains of the plasmalemmal glycoproteins was investigated. After the glycopeptides obtained by trypsin treatment of the intact cells had been digested with pronase, the resulting glycopeptides were separated into 4 fractions by gel filtration. The first 2 fractions were found to contain mainly N-glycosidically linked glycopeptides, and the last 2, O-linked oligosaccharides. There were a variety of N-linked oligosaccharides whose apparent molecular weights were greater than that of isomaltoheptaose. As compared to those, O-linked oligosaccharides were fewer in type and lower in molecular weight. The N-linked oligosaccharides corresponding to isomaltohepta- decaose and larger saccharide chains augmented in differentiated glioblasts, whereas the N-linked oligosaccharides smaller than isomaltoheptade- caose decreased. The turnover rate of the high molecular weight oligosaccharides was faster than that of other membrane oligosaccharides, and was accelerated by GMF treatment. The content of an O-linked oligosaccharide fraction increased after GMF treatment.     

Immunochemical studies on blood groups: Heterogeneity of oligosaccharides liberated by degradation with alkaline borohydride of two human ovarian cyst fractions differing in B,I, and i activities and in reactivity toward concanavalin A

Two fractions obtained by phenol-ethanol fractionation of blood group substance from fluid of a human ovarian cyst and differing in immunological activities and in composition, Tij phenol insoluble, with high B and I-Ma activity and very weak reactivities toward anti-I Step, anti-i Den, and concanavalin (Con A), and Tij 20% 2X, with lower fucose and galactose and with weaker B and I-Ma activity and high reactivity toward anti-I Step, anti-i Den, and Con A, were subjected to degradation with alkaline borohydride under conditions which release carbohydrate from the protein backbone without further peeling from the reducing end. Destruction of serine, threonine and dGalNAc and production of alanine, α-aminobutyric acid, and N-acetyl-d-galactosaminitol occurred. The products of the alkaline degradation were dialyzed. The proportion of cleaved chains remaining undialyzable was much larger for Tij phenol insoluble, in accord with its higher proportion of larger carbohydrate chains. The dialysates of both fractions were chromatographed on Bio-Gel P-2. More fucose was seen in the phenolinsoluble peaks, and dextrorotatory peaks were present in the 20% 2X. Amino acid composition of the undegraded fractions was similar to that of other blood group substances, with serine, threonine, proline, and alanine making up about two-thirds of the total amino acids.     

Determination of Leb-active oligosaccharides in urine of pregnant and lactating women by radioimmunoassay.

Antiserum obtained by immunizing a goat with lacto-N-difucohexaose I, a Leb blood-group hapten, coupled to poly-L-lysine was used in a radioimmunoassay to detect Leb-active oligosaccharides (chiefly lacto-N-difucohexaose I) in urine of 138 pregnant and lactating women of different ABO and Lewis blood groups. Specificity of the method was determined by comparing inhibitory activities of 18 oligosaccharides. Only women who belonged to the Leb blood group excreted Leb-active oligosaccharides in urine. Leb-active oligosaccharides increase during pregnancy, reaching levels up to approximately 70 mumol lacto-N-difucohexaose I equivalents per pmol creatinine in the third trimester and early post-partum period. Excretion varies considerably but tends to be highest in those individuals who strongly express the Leb antigen on their red blood cells or who belong to blood group O. Leb-active oligosaccharides were detected in plasma of a few individuals but at concentrations 1000-fold lower than in urine.     

Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

The xylanase system of Coniophora cerebella

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal-Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.     

Fractionation of the Leuconostoc mesenteroides NRRL B-1299 Dextran and Preliminary Characterization of the Fractions

The extracellular dextran elaborated by Leuconostoc mesenteroides NRRL B-1299, which was shown to be heterogeneous, was separated into five fractions by assorted fractionation methods. Each fraction turned out to be homogeneous by ultracentrifugation and/or electro-phoretic analysis. Gel filtration analysis suggested that these fractions were divided into two groups on the basis of their average molecular weights, one of which had comparatively low molecular weight of 150,000~200,000 (BPS and CPS) and the other had high molecular weight of about 2,000,000 (BPP, CPP and CS). From the results of periodate oxidation, each fraction was shown to contain 41~46% of 1,6-glucosidic linkage (including non-reducing terminal group) as well as 1,4- like (1,4- and/or 1,2-) and 1,3-like linkages. Partial acid hydrolysis of each fraction yielded a series of α-1,6-linked oligosaccharides and acetolysis gave koji-biose and nigerose. Moreover, these fractions gave characteristic precipitation patterns, when incubated with concanavalin A. On the basis of these results, the structural features of the fractions were discussed.     

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by siman virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines.The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of ³⁵SO₄ were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons.These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

Exoenzymic activity of alpha-amylase immobilized on a phenol-formaldehyde resin

Amylose and amylopectin from two starch sources were partially degraded by alpha-amylase immobilized on a phenol-formaldehyde resin. The degradation products were fractionated by gel-permeation chromatography and high-pressure, liquid chromatography. Two distinct fractions were obtained from tapioca amylose. One is a fragment having a molecular weight exceeding 200,000, and the other consists of oligosaccharides of low molecular weight with a degree of polymerization of 1–8. In contrast, treatment of tapioca amylose with soluble alpha-amylase produces a single fraction, nearly all of which has a molecular weight of <35,000, with only traces of small oligosaccharides detectable by high-pressure, liquid chromatography. Even wider differences were observed in degradation products from tapioca amylopectin. Similar activity-patterns were obtained with immobilized and soluble enzymes, using corn amylose and corn amylopectin as substrates. Immobilization of alpha-amylase on the resin apparently restricts the activity of the enzyme to the ends of the starch molecules, making it appear to be limited to exoenzymic activity.     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.