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.     

Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB₅ toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.     

Influence of the spxB gene on competence in Streptococcus pneumoniae

In Streptococcus pneumoniae expression of pyruvate oxidase (SpxB) peaks during the early growth phase, coincident with the time of natural competence. This study investigated whether SpxB influences parameters of competence, such as spontaneous transformation frequency, expression of competence genes, and DNA release. Knockout of the spxB gene in strain D39 abolished spontaneous transformation (compared to a frequency of 6.3 × 10⁻⁶ in the parent strain [P < 0.01]). It also reduced expression levels of comC and recA as well as DNA release from bacterial cells significantly during the early growth phase, coincident with the time of spontaneous competence in the parent strain. In the spxB mutant, supplementation with competence-stimulating peptide 1 (CSP-1) restored transformation (rate, 1.8 × 10⁻²). This speaks against the role of SpxB as a necessary source of energy for competence. Neither supplementation with CSP-1 nor supplementation with the SpxB products H₂O₂ and acetate altered DNA release. Supplementation of the parent strain with catalase did not reduce DNA release significantly. In conclusion, the pneumococcal spxB gene influences competence; however, the mechanism remains elusive.     

Role of the pyruvate metabolic network on carbohydrate metabolism and virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD⁺ and by SpxB and PDHc to generate acetyl-CoA. In galactose, pyruvate metabolism was shunted toward acetyl-CoA production. The majority of acetyl-CoA generated by PFL was used to regenerate NAD⁺ with a subset used in capsule production, while the acetyl-CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

Identification and biochemical characterization of WbwB,a novel UDP-Gal: Neu5Ac-R α1,4-galactosyltransferase from the intestinal pathogen <Emphasis Type="Italic">Escherichia coli</Emphasis> serotype O104

The intestinal pathogen Escherichia coli serotype O104:H4 (ECO104) can cause bloody diarrhea and haemolytic uremic syndrome. The ECO104 O antigen has the unique repeating unit structure [4Galα1–4Neu5,7,9Ac₃α2–3Galβ1–3GalNAcβ1-], which includes the mammalian sialyl-T antigen as an internal structure. Previously, we identified WbwC from ECO104 as the β3Gal-transferase that synthesizes the T antigen, and showed that α3-sialyl-transferase WbwA transfers sialic acid to the T antigen. Here we identify the wbwB gene product as a unique α1,4-Gal-transferase WbwB that transfers Gal from UDP-Gal to the terminal sialic acid residue of Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid acceptor. NMR analysis of the WbwB enzyme reaction product indicated that Galα1-4Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid was synthesized. WbwB from ECO104 has a unique acceptor specificity for terminal sialic acid as well as the diphosphate group in the acceptor. The characterization studies showed that WbwB does not require divalent metal ion as a cofactor. Mutagenesis identified Lys243 within an RKR motif and both Glu315 and Glu323 of the fourth EX₇E motif as essential for the activity. WbwB is the final glycosyltransferase in the biosynthesis pathway of the ECO104 antigen repeating unit. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.     

Metabolic rewiring of synthetic pyruvate dehydrogenase bypasses for acetone production in cyanobacteria

Designing synthetic pathways for efficient CO₂ fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate‐acetyl‐CoA/malonyl‐CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl‐CoA carboxyltransferase. We demonstrated direct conversion of CO₂ into acetyl‐CoA‐derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl‐CoA pools. The engineered cyanobacterial strain with the AAM‐bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM‐bypass) is a key factor for the high‐level production of an acetyl‐CoA‐derived chemical in photosynthetic organisms.     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H₂O₂. Streptococcus pneumoniae produces exceptionally high levels of H₂O₂, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H₂O₂ inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H₂O₂ production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H₂O₂. In vitro exposure of the spxB mutant to various H₂O₂ concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H₂O₂ or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H₂O₂ produced by S. pneumoniae.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human-specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Galα4Gal-containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Galα4Gal moiety, but parts of the GalNAcβ and glucose residues, which flank the Galα4Gal in globoside (GbO₄), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Galα4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO₄ that dominates In the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG-GbO₄ interaction will make it possible to design antiadherence therapeutics.     

Specificity analysis of the C-type lectin from rattlesnake venom,and its selectivity towards Gal- or GalNAc-terminated glycoproteins

The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Galβ1-4GlcNAc (LacNAc), α-Gal or α-GalNAc non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans, terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a K_D for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with a ten-fold better K_D for GalNAc.     

ON THE STRUCTURE OF A NEW, FUCOSE CONTAINING GANGLIOSIDE FROM PIG CEREBELLUM

A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer. Fucose is α-glycosidically linked to the 2-position of external galactose and one N-acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC-IUB Commission on Biochemical Nomenclature is indicated as II³α(NeuAc)₂ IV²αFuc-GgOse₄Cer.     

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo-GM1 and asialo-GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM₁ (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo-GM₁ and asialo-GM₂ proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal-NAc(1–4)βGal-Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc-(1–4)βGal-BSA was inhibited by PAK pili, Ac-KCTSDQDEOFIPKGCSK-OH (AcPAK(128–144)_ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128–144)_ox-OH) peptides. (In these peptides Ac denotes N^α -acetylation of the N-terminus, -OH means a peptide with a free a-carboxyl group at the C-terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal-biotin to the corresponding BSA-Peptide(128–144)_ox-OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc-biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C-termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo-GM₁ receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.     

Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples

The Human Oral Microbiome Database (HOMD) provides an extensive collection of genome sequences from oral bacteria. The sequence information is a static snapshot of the microbial potential of the so far sequenced species. A major challenge is to connect the microbial potential encoded in the metagenome to an actual function in the in vivo oral biofilm. In the present study we took a reductionist approach and identified a considerably conserved metabolic gene, spxB to be encoded by a majority of oral streptococci using the HOMD metagenome information. spxB encodes the pyruvate oxidase responsible for the production of growth inhibiting amounts of hydrogen peroxide (H₂O₂) and has previously been shown as important in the interspecies competition in the oral biofilm. Here we demonstrate a strong correlation of H₂O₂ production and the presence of the spxB gene in dental plaque. Using Real-Time RT PCR we show that spxB is expressed in freshly isolated human plaque samples from several donors and that the expression is relative constant when followed over time in one individual. This is the first demonstration of an oral community encoded gene expressed in vivo suggesting a functional role of spxB in oral biofilm physiology. This also demonstrates a possible strategy to connect the microbial potential of the metagenome to its functionality in future studies by identifying similar highly conserved genes in the oral microbial community.     

Effect of Aglycon Structure on Saccharide Elongation by Cells

Alkyl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc primers) with different aglycon moieties were synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation. Dodecyl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐C12), tridecan‐7‐yl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐2C6), and pentacosan‐13‐yl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐2C12) primers were synthesized by glycosylation of dodecan‐1‐ol, tridecan‐7‐ol, and pentacosan‐13‐ol, respectively, with peracetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids. After 48 h incubation, results showed that GlcNAc‐C12 was elongated to give NeuAc‐Gal‐GlcNAc‐C12. GlcNAc‐2C6 was also elongated to afford Gal‐GlcNAc‐2C6 and NeuAc‐Gal‐GlcNAc‐2C6. On the other hand, GlcNAc‐2C12 primer was not elongated. Significantly, the results demonstrated that the amount of glycosylated product increased 1.5‐times by modifying the aglycon structure of GlcNAc from C₁₂ to 2 C₆ despite having almost the same number of C‐units.