Synthesis of UDN-N-[1-14C]acetyl d-glycosamine and UDP-N-[1-14C]acetyl-d-galactosamine from [1-14C]acetate

Rao, Aniruddha; Mendicino, Joseph

doi:10.1016/0003-2697(78)90535-3

Cited by 16 publications

(2 citation statements)

References 12 publications

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“…Other enzymatic methods for synthesizing UDP-GlcNAc that start from GlcNAc or GlcN have been described. − However, isotope-labeled GlcNAc and GlcN can be cost prohibitive and are available with only limited labeling patterns, unlike the scheme presented here that can be used to produce a wide array of custom labeling patterns starting with inexpensive starting materials, including glucose and glutamine. Numerous chemical methods are also available and can be adapted for the synthesis of GlcNAc analogues; however, these methods are less efficient than the one-pot enzymatic method presented here (not limited to refs − ).…”

Section: Resultsmentioning

confidence: 99%

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Barb

2014

Biochemistry

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Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

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Section: Resultsmentioning

confidence: 99%

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Barb

2014

Biochemistry

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“…For incorporation into hyaluronate the substrates [3HIUDP-GlcNAc and [3H]UDP-GlcA were synthesized from [3HIUTP and GlcNAc-P and Glc-P, respectively, by modified procedures of Rao & Mendicino (1978) and Strominger et al (1957).…”

Section: Incorporation Ofuridine Into Hyaluronate In Vitromentioning

confidence: 99%

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth

Prehm¹

1983

Biochemical Journal

153

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Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5'-nucleotidase are suggested as modulators of hyaluronate synthesis.

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UDP-GlcNAc: Gal?3GalNAc-Mucin: (GlcNAc ? GalNAc) ?6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Gal?3(GlcNAc?6) GalNAc-Mucin (GlcNAc ? Gal)?3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

1991

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Two specific beta-N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a beta 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal beta 3GalNAc-mucin to yield Gal beta 3(GlcNAc beta 6)GalNAc-Mucin and a beta 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal beta 3(GlcNAc beta 6)GalNAc-mucin to yield GlcNAc beta 3Gal beta 3 (GlcNAc beta 6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The beta 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal beta 1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal beta 1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the beta 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal beta 1,3GalNAc chains was 0.53 microM; for UDP-N-acetylglucosamine, 12 microM; and for Gal beta 1,3GalNAc alpha NO2 phi, 4 mM. The activity of the beta 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal beta 3GalNAc chains in Cowper's gland mucin glycoprotein. The best substrate for the partially purified beta 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal beta 1,3(GlcNAc beta 6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal beta 1,3GalNAc side chains. The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the beta 6- and beta 3-glucosaminyltransferases were shown to be Gal beta 3(GlcNAc beta 6) GalNAc and GlcNAc beta 3 Gal beta 3(GlcNAc beta 6)GalNAc respectively. Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a beta 6-glucosaminyltransferase converts Gal beta 3GalNAc chains in mucin glycoproteins to Gal beta 3(GlcNAc beta 6)GalNAc chains.(ABSTRACT TRUNCATED AT 400 WORDS)

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Synthesis of UDN-N-[1-14C]acetyl d-glycosamine and UDP-N-[1-14C]acetyl-d-galactosamine from [1-14C]acetate

Cited by 16 publications

References 12 publications

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth

UDP-GlcNAc: Gal?3GalNAc-Mucin: (GlcNAc ? GalNAc) ?6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Gal?3(GlcNAc?6) GalNAc-Mucin (GlcNAc ? Gal)?3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

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Synthesis of UDN-N-[1-14C]acetyl d-glycosamine and UDP-N-[1-14C]acetyl-d-galactosamine from [1-14C]acetate

Cited by 16 publications

References 12 publications

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth

UDP-GlcNAc: Gal?3GalNAc-Mucin: (GlcNAc ? GalNAc) ?6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Gal?3(GlcNAc?6) GalNAc-Mucin (GlcNAc ? Gal)?3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

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Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1