Active fragments obtained by cyanogen bromide cleavage of ovomucoid.

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.     

Immunochemical and chemical studies on streptococcal group-specific carbohydrates.

Structural studies on the carbohydrates of Groups A, C, and A-variant (AV) streptococci have utilized periodate oxidation, permethylation analysis, and immunochemical comparison of intact and periodate-oxidized polysaccharides. The data indicate that a similar 1,2- and 1,3-linked rhamnose chain is present in both the A and AV carbohydrates. The group A carbohydrate contains in addition N-acetylglucosamine residues at nonreducing terminals, whereas the AV is a homopolymer of rhamnose. There is some evidence that Group Ccarbohydrate contains the same rhamnose chain, but structural comparisons to the A and AV carbohydrates are complicated by the presence of intrachain N-acetylgalactosamine residues. Periodate oxidation and permethylation analysis show that while approximately 50% of the N-acetylgalactosamine of the Group C carbohydrate occupies terminal positions, the remainder is present as 1,3-linked units. Removal of the nonreducing terminal hexosamine units from the Group A carbohydrate by periodate treatment significantly enhanced its cross-reactivity with AV antiserum, whereas no enhancement was observed after similar treatment of the Group C carbohydrate. The data indicate the presence of an alpha-1,3-linked N-acetylgalactosamine disaccharide at the nonreducing terminal of the Group C carbohydrate.     

Phosphorylation of chondroitin sulfate in proteoglycans from the swarm rat chondrosarcoma

Proteoglycans isolated from the Swarm rat chondrosarcoma were shown to contain 35 mol of phosphate/mol of proteoglycan. While 20% of this phosphate was released by digestion with dilute alkali in the presence of sodium borohydride and is presumably of the phosphoserine/phosphothreonine type, 78% of the phosphate copurified with the peptide-free chondroitin sulfate chains. When chondroitin sulfate chains purified by ethanol precipitation or Sephacryl S200 column chromatography were digested with chondroitinase AC and the digests chromatographed on Bio-Gel P-4, the phosphate co-migrated with a carbohydrate fragment that contained 2 glucuronic acid (one as delta 4,5-unsaturated sugar), 1-galactosamine, 2-galactose, and 1-phosphate residue/xylitol. A second fragment of similar composition but lacking phosphate was also recovered in a ratio of about 3 to 1 relative to the phosphorylated fragment. The phosphate in the chondroitin sulfate linkage region fragment had the alkaline phosphatase sensitivity as well as 31P NMR spectra of a monophosphate esterified to a secondary sugar alcohol. The phosphate was localized on the C-2 of the chain initiating xylose since these residues as xylitol showed a delayed release during acid hydrolysis and the xylitol was recovered intact after periodate oxidation. In the chondrosarcoma, 2-phosphoxylose appears to be a normal synthetic product since [32P]phosphate was readily incorporated into the proteoglycan and the incorporated isotope had similar biochemical properties as the unlabeled phosphate.     

Structural studies on carcinoembryonic antigen. Periodate oxidation.

Periodate oxidation has been applied to examine the carbohydrate structure of carcinoembryonic antigen (CEA) and the possible role of the carbohydrate residues in its antigenic activity. Sialic acid (N-acetylneuraminic acid) and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate treatment (Smith degradation) destroyed additional amounts of galactose and mannose as well as significant amounts of N-acetylglucosamine. Prior removal of sialic acid by neuraminidase treatment led to increased destruction of galactose by periodate. Antigenic activity persisted indicating that the residues destroyed played little, if any, part in the antigenicity of CEA. These results yield an initial view of the structural arrangement of the carbohydrate residues in the CEA molecule.     

The co-polymeric structure of pig skin dermatan sulphate. Distribution of L-iduronic acid sulphate residues in co-polymeric chains.

1. Pig skin dermatan sulphate was degraded by periodate oxidation followed by alkaline elimination or by chondroitinase-ABC to quantify irregular repeating units, i.e. those containing D-GlcUA (D-glucuronic acid) and L-IdUA-SO4 (sulphated iduronic acid). 2. Previous results of periodate oxidation (Fransson, 1974) indicated repeating sequences in pig skin dermatan sulphate containing, on average, 3D-GlcUA, 9 L-IdUA-SO4 or 28 L-IdUA units in addition to N-acetylgalactosamine sulphate. However, complete digestion with chondroitinase-ABC yielded, at the most, 3-4 disulphated disaccharides/chain. Consequently, more than one-half of the L-IdUA-SO4 residues were present in monosulphated periods, i.e. IdUA-(SO4)-GalNAc. 3. To determine the location of L-IdUA-SO4 residues along the copolymeric chain dermatan sulphate was digested with testicular hyaluronidase. (This enzyme cleaves GalNAc-GlcUA bonds within block regions containing D-GlcUA.) By NaB3H4 reduction GalNAc residues located in the reducing end of the fragments were converted into [3H]GalNAcOH (N-acetylgalactosaminitol). Finally, the radioactive product was fragmented by periodate oxidation followed by alkaline elimination. The bulk of the radioactivity was associated with periodate-resistant oligosaccharides indicating that clusters of GlcUA-GalNAc-SO4 periods are often adjacent to a varying number of (n = 1-4) of L-IdUA-SO4-containing periods. 4. To study the distribution of L-IdUA-SO4-containing periods in relation to blocks of IdUA-GalNAc-SO4 periods different fractions of hyaluronidase-degraded dermatan sulphate were degraded separately. In all types of fragments (mol. wts. 1,500-10,000) L-IdUA-SO4-containing periods were demonstrated. In short fragments reducing terminal GalNAc-6-SO4 (6-sulphated N-acetylgalactosamine) was found confirming that these sequences were joined to relatively long D-GlcUA-containing block sequences via GalNAc-6-SO4. Moreover, low-molecular-weight oligosaccharides composed of alternating sequences were encountered. An octasaccharide derived from the carbohydrate sequence -GalNAc---GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc-IdUA-GalNAc---GlcUA-GalNAc (--- indicates the position of cleavage by hyaluronidase) was identified.     

Quantitative determination of the protein and carbohydrate polymers in the cell wall of Streptococcus group A

The content of protein and carbohydrate polymers was estimated in the cell wall of Streptococcus, group A, type 29. A method was developed for analysing peptidoglycane in a polysaccharide-peptidoglycane complex after the prior oxidation by sodium periodate. It was found that the cell wall peptidoglycane bears two carbohydrate and three amino acid residues, i. e. N-acetylglucosamin, muramic acid, glutamic acid, alanine and lysine, in the ratio 1:1:1:4:1, respectively. The data on the cell wall composition prior to and after its oxidation with sodium periodate are given, and the ratio between the main structural components is determined: proteins (60% mol), polysaccharide (23% mol), peptidoglycane (17% mol).     

Studies on the metabolism of the protozoa: The molecular structure of the reserve polysaccharide from Astasia ocellata

1. The flagellate Astasia ocellata synthesizes as reserve carbohydrate a water-insoluble polysaccharide (paramylon) containing chains of beta-(1-->3)-linked d-glucose residues. 2. The average chain length, determined by methylation analysis, is about 43, and the minimum degree of polymerization from periodate oxidation data is 50-55. Most of the molecules are therefore essentially linear.     

Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.     

A Model System for Convenient Fluorescent Labeling of Sugar Chain in Taka-amylase A

A convenient detection of sugar chains in Taka-amylase A (TAA) was done by using 40 μg of enzyme, where a decrease in the UV absorption of NaIO₄ during the periodate oxidation reaction was monitored. The periodate-oxidized sugar chain was labeled with a fluorescent reagent, N-1-ethylenediaminonaphthalene (EDAN), by incubation at pH 9.5 and 30°C for 1 h. The excess EDAN was removed by either quenching with o-phthaladehyde or Bio-Gel P-2 gel adsorption. Among the peptide fragments prepared from the EDAN-labeled TAA, a fluorescent peptide corresponding to the sugar chain was distinguished by the ODS column. These results suggest that periodate oxidation and subsequent fluorescent labeling were useful for the sensitive analysis of various glycoprotein samples.     

Asparagine-linked carbohydrate chains of inducible rat parotid proline-rich glycoprotein contain terminalβ-linked N-acetylgalactosamine

Rats treated with daily injection of DL-isoproterenol for 10 consecutive days (25 mg kg1 body weight) showed marked induction of a proline-rich glycoprotein (GPRP) of 220 kDa. Proteinase K digestion of GPRP produced a homogeneous glycopeptide with an average chemical composition as follows (residues per mol): Pro4, Glx3, Asx2, Gly1, His1, Thr1, Arg1, GlcNAc5, GalNac1, Man3, Gal2–3, and Fuc1. The structural analysis of the asparagine-linked carbohydrate unit was performed by methylation, periodate oxidation and enzymatic degradation. Methylation studies indicated that the three mannosyl residues were substituted at 1,2-, 1,2,4-, and 1,3,6-positions. Fucose, N-acetylgalactosamine, 1.5 residues of galactose and 0.35 residues of N-acetylglucosamine were terminally located and one galactose residue was 1,4-substituted. Approximately four of the 5 N-acetylglucosamine residues were substituted at 1,4-position and approximately 1 residue of N-acetylglucosamine was substituted at 1,4,6-positions. Periodate oxidation studies and exoglycosidase results were consistent with the methylation data. Based on the results of Smith degradation, methylation and sequential exoglycosidase digestions a triantennary oligosaccharide structure having terminal N-acetylgalactosamine in one of the branches is proposed for the major Asn-linked carbohydrate moiety of GPRP. This revised version was published online in August 2006 with corrections to the Cover Date.     

The structure of a sulfated glycoprotein of chick allantoic fluid: methylation and periodate oxidation.

The structure of an antigenic, sulfated glycoprotein from chick chorioallantoic fluid has been investigated by exogalactosidase digestion, methylation and mass spectral analyses, periodate oxidation, and Smith degradation. The main carbohydrate chains are composed of D-galactosyl residues linked at C-3 and 2-acetamido-2-deoxyglucose residues linked at C-4. Fucose and N-acetylneuraminic acid residues are nonreducing terminal groups, and the N-acetylneuraminic acid groups are linked to the D-galactose residues at C-3. Most of the sulfate groups (91% of the sulfate) are located on C-6 of the 2-acetamido-2-deoxyglucose residues, and the rest on C-6 of the D-galactose residues. A large number of the D-galactose residues (36.9% of the total) are present as nonreducing terminal groups and another 21.7% of the D-galactose residues are in penultimate position to the nonreducing terminal N-acetylneuraminic acid residues. Although mild periodate oxidation indicates the presence of D-galactose in furanoside form (5.5% of total D-galactose), no 5-O-methyl derivative of D-galactose was observed on methylation.     

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.     

Periodate oxidation of sperm-whale myoglobin and the role of the methionine residues in the antigen–antibody reaction

1. Oxidation of sperm-whale metmyoglobin and its apoprotein with periodate has been investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH6.8 and 22 degrees consumption of periodate ceased in 3(1/2)hr. at 43 moles of periodate/mole of myoglobin. The two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues were oxidized; serine increased in the hydrolysates from 6 to 9 residues/mol. 3. At pH5.0 and 22 degrees , consumption levelled off in 4(1/2)hr. at 26 moles of periodate/mole of myoglobin and resulted in the modification of the two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues; serine increased from 6 to 7 residues/mol. and, also, ferrihaem suffered considerable oxidation. 4. Oxidation at pH5.0 and 0 degrees resulted at completion (4hr.) in the consumption of 22 moles of periodate/mole of myoglobin and in the modification of the methionine, tyrosine and tryptophan residues. Spectral studies indicated oxidation of the haem group. This derivative reacted very poorly with rabbit antisera to MbX (the major component no. 10 obtained by CM-cellulose chromatography; Atassi, 1964). 5. Oxidation of apomyoglobin at pH5.0 and 0 degrees was complete in 4hr. with the consumption of 7.23 moles of periodate/mole of apoprotein. The rate of oxidation in decreasing order was: methionine; tryptophan; tyrosine; and after 7hr. of reaction the following residues/mol. were oxidized: methionine, 2.0; tryptophan, 1.6; tyrosine, 0.99. No peptide bonds were cleaved. Metmyoglobin prepared from the 7hr.-oxidized apoprotein showed that the reactivity with antisera to MbX had diminished considerably. 6. Milder oxidation of apoprotein (2 molar excess of periodate, pH5.0, 0 degrees , 2hr.) resulted in the modification of 1.66 residues of methionine/mol. Metmyoglobin prepared from this apoprotein was identical with native MbX spectrally, electrophoretically and immunochemically. It was concluded that the methionine residues at positions 55 and 131 were not essential parts of the antigenic sites of metmyoglobin.     

1-Deoxyglycitolation of protein amino groups and their regeneration by periodate oxidation

Reductive alkylation of lysyl epsilon-amino groups with sugars (1-deoxyglycitolation) using pyridine borane as the reducing agent has been recently described [Wong, W.S.D., Osuga, D.T. & Feeney, R.E. (1984) Anal. Biochem. 139, 58-67]. The regeneration of the lysines has now been achieved by oxidation with approximately 10 mM periodate. In experiments with glycitolated bovine serum albumin, reactions using 5, 20, and 80 mM periodate were essentially complete in the first 10 min. Oxidations of methionine to the sulfoxide were evident even at this lower concentration of periodate, while higher concentrations and prolonged reaction times only caused unnecessary destruction of amino acids. The removal was shown to occur in a wide pH range with little variation in the recovery of the lysines. The degree of glycitolation, or the nature of the attached carbohydrate residues, had no effect on the yield of products. Reductive 1-deoxygalactitolation of approximately 55% of the lysyl epsilon-amino groups of lysozyme caused no loss in enzymatic activity; when 5 mM periodate was used to remove the 1-deoxygalactitol moiety, approximately 95% of the amino groups were regenerated, concomitant with destruction of approximately 16% of the activity. On reductive 1-deoxygalactitolation of the amino groups of turkey ovomucoid, 65% of the lysyl epsilon-amino groups were modified with 70% loss of the inhibitory activity against trypsin. On treatment with 5 mM periodate, approximately 80% of the amino groups were regained with a similar recovery of the inhibitory activity.     

The effect of periodate oxidation and α-mannosidase treatment of Dolichos biflorus lectin

The effects of periodate and α-mannosidase treatment of the Dolichos biflorus lectin were determined. Destruction by periodate of 16% of the mannose residues of the lactin had no effect on its ability to agglutinate type A erythrocytes, precipitate blood group A + H substance or to be precipitated by concanavalin A. Removal of up to 40% of the mannose by either periodate or α-mannosidase rendered the lecton nonprecipitable by concanavalin A. The lectrin treated by α-mannosidase retained its ability to agglutinate erythrocytes and precipitate blood group A + H substance, but the lectin treated with periodate lost most of its activity.The results suggest that the complete integrity of the carbohydrate unit of the lectin is not necessary for its activity and that the periodate may be affecting the protein portion of the molecule as well as its carbohydrate residues. No conversion of form A to form B of the lectin was observed with either periodate oxidation or α-mannosidase treatment.     

Influence of soil type,crop and air drying on residual carbohydrate content and aggregate stability after treatment with periodate and tetraborate

Summary The relationship between the water stability of microaggregates and the residual carbohydrate content of soil was examined in 15 soils from 7 soil series under various cultivations. The carbohydrate was progressively removed by increasing the time of treatment with 0.02M periodate and 0.1M tetraborate. The resulting decrease in reducing sugar content was significantly correlated with an increased disruption of microaggregates (>45 m) as determined by a turbidimetric method. The most effective treatment removed about 80% of the soil carbohydrate and caused an increase of about 75% in the fraction of microaggregates (<45 m) compared to untreated soil.15–20 percent of the soil carbohydrate was resistant to oxidation by periodate, even after prolonged reaction times and contained a higher relative proportion of glucose, arabinose, and xylose than the oxidised material. Sugars typical of microbial sources, mannose, galactose, rhamnose and fucose, were therefore preferentially oxidised by the periodate treatment.The grassland soils generally had higher carbohydrate contents than the arable soils and initially had a greater degree of aggregation. However, periodate oxidation affected each soil in its own characteristic manner. A significant inverse linear relationship between the degree of disruption and the residual sugar content was found with 13 of the 15 soils. Over the range measured aggregate stability was therefore related to the presence of carbohydrate predominantly from microbial sources.     

Structural studies of the carbohydrate moieties of rat kidney gamma-glutamyltranspeptidase. An extremely heterogeneous pattern enriched with nonreducing terminal N-acetylglucosamine residues

The carbohydrate moieties of gamma-glutamyltranspeptidase purified from rat kidney were released as oligosaccharides by hydrazinolysis. Fractionation of the oligosaccharide mixture by paper electrophoresis and Bi-Gel P-4 column chromatography and structural study of each component by sequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation have revealed that it is composed of 23 neutral oligosaccharides, monosialyl derivatives of 67 oligosaccharides, disialyl derivatives of 62 oligosaccharides, and trisialyl derivatives of 5 oligosaccharides. The neutral oligosaccharides are either high mannose type or biantennary complex type, and the acidic oligosaccharides are bi-, tri-, and tetranntennary complex type sugar chains. Most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the beta-mannosyl residue of their trimannosyl core. Another characteristic feature of these complex type sugar chains is that they are enriched with nonreducing terminal beta-N-acetylglucosamine residues.     

Location of the carbohydrate groups of ovomucoid.

Tryptic glycopeptides were purified from the sialic acid-free variant of ovomucoid, O1, and its CNBr fragments. The amino acid sequences adjacent to the four major sites of carbohydrate (Carb.) attachment were: (1), Phe-Pro-Asn(Carb.)-Ala-Thr-Asp-Lys-Glu-Gly-Lys; (2), Ala-Try-Ser-Ile-Glu-Phe-Gly-Thr-Asn (Carb.)-Ile-Ser-Lys; (3), Glu, Thr-Val-Pro-Met-Asn(Carb.)-cys-Ser; (4), Ser-Ser-Tyr-Ala-Asn (Carb.)-Thr-Thr-Ser-Glu-Asp-Gly-Lys, Glycosylated Asn residues were located at position 10, between residues 49 and 60, and at positions 69 and 75, in the primary sequence. All of these carbohydrate groups contained GlcNAc, Man and Gal in the approximate molar proprotions 5:3:0.5. A further glycopeptide containing His was isolated in low yield, suggesting that some carbohydrate is attached at a fifth site. Two of the carbohydrate-attachment sites (Asn-10 and Asn-75) occur in sequences that show internal homologies. These are presumed to have evolved as a consequence of partial gene duplication. Three of the carbohydrate-attachment sites occur in similar positions to the carbohydrate groups in quail ovomucoid [Laskowski (1976) Protides Biol. Fluids Proc. Colloq. 23, in the press]. Prediction of peptide conformation from the sequence data by the method of Chou & Fasman [(1974) Biochemistry 13, 222-225] indicated that four glycosylated Asn residues in hen ovomucoid are very close to groups of amino acids that occur with high frequency in beta-turns. The possible significance of peptide-chain conformation in the attachment of carbohydrate to glycoproteins is briefly discussed.     

Polysaccharides of algae: 60. Fucoidan from the Pacific brown alga Analipus japonicus (Harv.) Winne (Ectocarpales, Scytosiphonaceae)

A fucoidan containing L-fucose, sulfate, and O-acetyl groups at a molar ratio of 3 : 2 : 1, as well as minor amounts of xylose, galactose, and uronic acids was isolated from the brown alga Analipus japonicus collected in the Sea of Japan. The structures of the native polysaccharide and the products of its desulfation and deacetylation were studied by the methods of methylation, periodate oxidation, and NMR spectroscopy. It was shown that the polysaccharide molecule mainly consists of a linear carbohydrate chain of (1-->3)-linked alpha-L-fucopyranose residues, which bear numerous branches in the form of single alpha-L-fucopyranose residues (three branches at position 4 and one branch at position 2 per each ten residues of the main chain). Sulfate groups occupy positions 2 and (to a lesser extent) 4, most of the terminal nonreducing fucose residues being sulfated twice. The acetyl groups are located predominantly at positions 4. The structural role of minor monosaccharides was not established.     

Temporal changes in the carbohydrates expressed on BG01 human embryonic stem cells during differentiation as embryoid bodies

Cell surface carbohydrates present on BG01 human embryonic stem cells after 28 days of differentiation were examined using two classes of carbohydrate binding proteins: lectins and antibodies specific for carbohydrate epitopes. Specificity of lectin staining was verified using carbohydrate ligands to block lectin interaction, glycohydrolases to cleave specific sugar residues that are receptors for these proteins, and periodate oxidation to destroy susceptible sugar residues. Specific antibodies were used to identify various tissue types and germ layers present in the 12- and 28-day differentiating embryoid bodies. Results from 12 and 28-day differentiated embryoid bodies were compared to determine changes over time. A slight increase in the sialylation of α-GalNAc was seen between 12 and 28 days of differentiation due to the presence of sialyl Tn and/or other sialylated α-GalNAc residues. Increases were also observed in GalNAc, the T antigen (Gal β1,3 GalNAc), and difucosylated LacNAc residues during this time interval. Additionally, some distinct differences in the pattern of lectin staining between 12 and 28 days were observed. Not unexpectedly, the presence of most differentiated cell-types increased during this time period with the exception of neural progenitors, which decreased. Undifferentiated cells, which were prevalent in the 12-day EBs, were undetectable after 28 days. We conclude that several changes in glycosylation occurred during the differentiation of embryonic stem cells, and that these changes may play a role in embryonic development. Lectin abbreviations can be found in Table 1.