Substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4 towards galactose, glucose, and their derivatives

Galactokinases (GalKs) have attracted significant research attention for their potential applications in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 presents a remarkably broad substrate range including 14 diverse natural and unnatural sugars. TLC and MS studies revealed that GalKSpe4 had relaxed activity towards galactose derivatives with modifications on the C-6, 4- or 2-positions. Additionally, GalKSpe4 can also tolerate glucose while glucose derivatives with modifications on the C-6, 4- or 2-positions were unacceptable. More interestingly, GalKSpe4 can phosphorylate L-mannose in moderate yield (43%), while other L-sugars such as L-Gal cannot be recognized by this enzyme. These results are very significant because there is rarely enzyme reported that can phosphorylate such uncommon substrates as l-mannose.     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

The molecular architecture of human N-acetylgalactosamine kinase

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.     

The Philadelphia variant of galactokinase.

We have previously reported the existence of a polymorphism that causes black populations to have lower mean RBC galactokinase activity than comparable white populations. We have designated this allele the Philadelphia variant, GALKP, and have suggested that it is common in blacks and rare in whites. GALKP individuals have normal WBC GALK activity, in contrast to the half normal WBC GALK activities of heterozygotes for the allele (GALKG) that causes the galactokinase-deficient form of galactosemia. In one family, we have presented evidence for the existence of two sisters heterozygous for both GALKG and GALKP alleles. These individuals have 50% normal WBC GALK activity and less than 50% normal red cell activity. The latter finding indicates that the two variant GALK alleles additively affect RBC activity. The WBC results suggest that the low activity of GALK in RBC of individuals with the GALKP allele is due to its relative instability. We could obtain no evidence for such instability from studies of high reticulocyte bloods or RBC fractionation. Furthermore, we could not demonstrate that the GALK in WBC from GALKP individuals has altered electrophoretic migration.     

Specificity ofAmaranthus leucocarpus lectin

We have demonstrated thatAmaranthus leucocarpus lectin hemagglutinating activity was powerfully inhibited by the T-antigen, containing Gal(1–3)GalNAc(1–3)Ser/Thr, and the T_n-antigen, which contains GalNAc(1–3)Ser/Thr. This suggests that the acetamido group at C-2 and the axial -OH at C-4 of theN-acetyl-D-galactopyranosylamine ring are important for lectin binding. The hemagglutination assays also established that desialylated and Pronase-treated human typeO erythrocytes with an M phenotype were better recognized than erythrocytes from all other blood groups. The recognition was dependent on pH and ionic strength.     

Human erythrocyte galactokinase and galactose-1-phosphate uridylyltransferase: a population survey.

Erythrocyte (RBC) galactokinase (GALK) and galactose-1-phosphate uridylyl-transferase (GALT) activities were measured in a random sample of 1,700 (1.082 black and 618 white) pregnant women from the Philadelphia area to estimate the frequency of the genes GALKG and GALTG responsible for the two biochemically distinct forms of galactosemia. Blacks have significantly lower mean RBC GALK activities than whites (P less than .0005). The distribution of individual GALK activities for blacks differs from a normal distribution (X227=43.0, P less than .03) whereas that for whites does not (X224=25.5, P approximately equal to .30). These results are consistent with the thesis that reduced RBC GALK activity in blacks is due to the Philadelphia variant (GALKP), which is common in blacks and rare in whites. The frequency of heterozygotes (GALKG/GALKA, GALKG/GALKP) for GALK galactosemia observed in this sample is 1/340 for the total, 1/347 for blacks, and 1/309 for whites. The existence of the GALKP variant allele has been considered in this determination. However, because a method for distinguishing the GALKP and GALKG alleles became available only in the latter part of the study, the frequency of the GALK G allele in the black population may be underestimated. The mean RBC GALT activity for blacks is higher than that for whites, a difference that may be due to a higher frequency of the Duarte variant allele GALTD in whites. Heterozygotes (GALTG/GALTA) for GALT galactosemia were distinguished by family studies and starch gel electrophoresis from individuals who have half-normal RBC GALT activity due to the GALTD allele. The GALTG/GALTA frequency is 1/212 for the total, 1/217 for blacks, and 1/206 for whites. Of the 1,700 individuals surveyed three had atypically high RBC GALK activity, similar to that found in red blood cells of newborns.     

Culture conditions for a new phytase-producing fungus

Extracellular phytase produced by Aspergillus sp. 5990 showed a 5-fold higher activity in liquid culture when compared with cultures of Aspergillus ficuum NRRL 3135. The optimum fermentation conditions were determined to be 35 °C, neutral pH, and 4 days incubation. The phytase had a higher optimum temperature for its activity than the commercial enzyme, Natuphos, from Aspergillus ficuum NRRL 3135.     

Characterization of β-1,3-galactosyl-<Emphasis Type="Italic">N</Emphasis>-acetylhexosamine phosphorylase from <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>

Homologs of the β-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) gene (gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto-N-biose (Galβ1→3GalNAc, GNB) than that on lacto-N-biose I (Galβ1→3GlcNAc). K _m value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto-N-biose phosphorylase. The large k _cat value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB.     

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galβ1→3GlcNAc, LNB) and galacto-N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBP_Bl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k _cat/K _m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBP_Bl showed similar k _cat/K _m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.     

Characterization and partial purification of beta-N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Urine from Sd(a+) individuals was found to contain a beta-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3'-sialyllactose and glycoproteins carrying the terminal NeuAc alpha-3Gal beta group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn2+ but not for detergents. The Km for UDP-GalNAc was 66 X 10(-6) M, using fetuin as an acceptor. Like beta-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [3H]GalNAc to 3'-sialyllactose, we propose that the enzyme attaches GalNAc in beta-anomeric configuration to O-4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a-) donor lacking beta-GalNAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the beta-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171-184) the urinary transferase is able to transfer beta-GalNAc to the NeuAc alpha-3Gal beta-3(NeuAc alpha-6)GalNAc chains bound to the native glycophorin.     

Identification of a novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase in the connective tissue of the snail Lymnaea stagnalis

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in beta 1-3 linkage to terminal GalNAc of GalNAc beta 1-4GlcNAc-R [R = beta 1-2Man alpha 1-O(CH2)8COOMe, beta 1-OMe, or alpha,beta 1-OH]. Using GalNAc beta 1-4GlcNAc beta 1-2Man alpha-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe (Vmax 140 nmol.h-1.mg protein-1; Km 1.02 mM), GalNAc beta 1-4GlcNAc (Vmax 105 nmol.h-1.mg protein-1; Km 0.99 mM), and GalNAc beta 1-4GlcNAc beta 1-OMe (Vmax 108 nmol.h-1.mg protein-1; Km 1.33 mM). The products formed from GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe and GalNAc beta 1-4GlcNAc beta 1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz 1H-NMR spectroscopy to be Gal beta 1-3GalNAc beta 1-4GlcNAc 1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNac beta 1-3 GalNAc alpha 1-OC6H5, GalNAc alpha 1--ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.     

One-pot enzymatic production of 2-acetamido-2-deoxy-d-galactose (GalNAc) from 2-acetamido-2-deoxy-d-glucose (GlcNAc)

2-Acetamido-2-deoxy-d-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-d-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.     

Molecular diversity of the two sugar-binding sites of the β-trefoil lectin HA33/C (HA1) from Clostridium botulinum type C neurotoxin

A critical role in internalizing the Clostridium botulinum neurotoxin into gastrointestinal cells is played by nontoxic components complexed with the toxin. One of the components, a β-trefoil lectin has been known as HA33 or HA1. The HA33 from C. botulinum type A (HA33/A) has been predicted to have a single sugar-binding site, while type C HA33 (HA33/C) has two sites. Here we constructed HA33/C mutants and evaluated the binding capacities of the individual sites through mucin-assay and isothermal titration calorimetry. The mutant W176A (site I knockout) had a K_d value of 31.5 mM for galactose (Gal) and 61.3 mM for N-acetylgalactosamine (GalNAc), while the K_d value for N-acetylneuraminic acid (Neu5Ac) was too high to be determined. In contrast, the double mutant N278A/Q279A (site II knockout) had a K_d value of 11.8 mM for Neu5Ac. We also determined the crystal structures of wild-type and the F179I mutant in complex with GalNAc at site II. The results suggest that site I of HA33/C is quite unique in that it mainly recognizes Neu5Ac, and site II seems less important for the lectin specificity. The architectures and the properties of the sugar-binding sites of HA33/C and HA33/A were shown to be drastically different.     

Glk: A locus controlling galactokinase activity in the mouse

Inbred strains of mice exhibit significant variation in whole blood galactokinase (GALK) activity. Activities tend to cluster into two classes, one class having approximately twice the activity of the other. Hybrids (F₁) between high and low strains have activity intermediate between the parental activities. Two sets of recombinant inbred (RI) lines were developed by brother-sister mating, beginning with F₂ generations of crosses between different pairs of high and low GALK activity strains. The RI lines segregated in terms of GALK activity, indicating single gene inheritance; this galactokinase locus has been designated Glk. The strain distribution patterns of both RI series agreed closely with esterase-3 (Es-3) alleles of the respective parental strains (31/34 independently derived strains were concordant for Es-3 and Glk genotypes), a finding consistent with a map distance between loci of 2.5 cM. Es-3 has been located on the distal end of chromosome 11. Glk, with its alleles Glk ^a (lower activity) and Glk ^b (higher activity), is therefore assigned to the same region.This project was supported by NIH Grants GM-02138 and GM-18684 from the National Institute of General Medical Sciences and HD-04861 and HD-00588 from the National Institute of Child Health and Human Development, and by a grant from the Clark Foundation, New York City. The Jackson Laboratory is fully certified by the American Association for Accreditation of Laboratory Animal Care.This work was carried out while J. D. M. was enrolled in the Summer Program for College, Graduate, and Medical Students at the Jackson Laboratory.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

A genetic factor for age-related cataract: identification and characterization of a novel galactokinase variant, "Osaka," in Asians

Galactokinase (GALK) deficiency is an autosomal recessive disorder characterized by hypergalactosemia and cataract formation. Through mass screening of newborn infants, we identified a novel and prevalent GALK variant (designated here as the "Osaka" variant) associated with an A198V mutation in three infants with mild GALK deficiency. GALK activity and the amount of immunoreactive protein in the mutant were both 20% of normal construct in expression analysis. The K(m) values for galactose and ATP-Mg(2+) in erythrocytes with homozygous A198V were similar to those of the healthy adult control subjects. A population study for A198V revealed prevalences of 4.1% in Japanese and 2.8% in Koreans, lower incidence in Taiwanese and Chinese, no incidence in blacks and whites from the United States, and a significantly high frequency (7.8%; P < .023) in Japanese individuals with bilateral cataract. This variant probably originated in Japanese and Korean ancestors and is one of the genetic factors that causes cataract in elderly individuals.     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: DeltaUA(beta1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(beta1-4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: DeltaUA(beta1-3)GalNAc4S(beta1-4)GlcA-ol and DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S-ol and GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.