Comparison of Acid Hydrolytic Conditions for Asn-Linked Oligosaccharides

Fan, Jintu; Namiki, Yoshihiro; Matsuoka, Koji; Lee, Y. C.

doi:10.1006/abio.1994.1280

Cited by 46 publications

(22 citation statements)

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“…The pellet was resuspended in 10 ml MilliQ and treated with RNase (10 μg ml −1 ) and DNase (10 μg ml −1 , 37°C, 2 h) and afterward with pronase E (100 μg ml −1 , 37°C for 24 h). Cell wall carbohydrates were extensively washed with SuperQ (35 000× g for 30 min), treated with trypsin (100 μg ml −1 , 37°C, 2 h), washed with SuperQ and lyophilized before the sample was subjected to hydrolysis with TFA according to published procedures (Fan et al ., 1994). Carbohydrate analysis was performed on a Dionex ICS‐3000 Ion Chromatography System (Dionex / Thermo Scientific, 1228 Titan Way, P.O.…”

Section: Methodsmentioning

confidence: 99%

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Beek

Breton

Ferenbach

et al. 2015

Molecular Microbiology

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SummaryThe sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac A in a S. mutans rml D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.

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Section: Methodsmentioning

confidence: 99%

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Beek

Breton

Ferenbach

et al. 2015

Molecular Microbiology

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“…This result indicated that TFA hydrolysis of glycoproteins at 100°C was not enough to release all sugars from glycoproteins. Fan et al (1994) also described incomplete digestion of glycoproteins with TFA. When fetuin was hydrolyzed with 6 M HCl at 100°C, amino sugars were completely released after 2 h incubation, but neutral sugars were decomposed very quickly ( Figure 1C), as expected in sugar stability test.…”

Section: New Hydrolysis Methodsmentioning

confidence: 99%

“…At present, the most widely used method is the acid hydrolysis followed by quantitative analysis with high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Townsend et al, 1997). Glycoproteins have been digested with two different acids separately to determine amino and neutral sugars, since each kind of monosaccharides had different hydrolyzing yields and stabilities against acids (Fan et al, 1994). The amounts of amino sugars are often determined with strong acid hydrolysis using HCl or sulfuric acid, while neutral sugars are underestimated because of their unstability.…”

Section: Introductionmentioning

confidence: 99%

An improved method for quantitative sugar analysis of glycoproteins

Kim

Kim²,

Ha³

et al. 2000

Exp Mol Med

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Although there are numerous methods available to hydrolyze glycans utilizing strong acids, it all requires lengthy steps to obtain quantitative yield. We have developed a new simple one-step method for analysis of amino and neutral monosaccharides of glycoproteins quantitatively. Free monosaccharides were found to be stable during hydrolysis of glycans with 6 N HCl at 80 o C up to 2 h. Using this condition, analysis of free monosaccharides hydrolyzed from the bovine fetuin showed sugar composition of Gal : Man : GlcN : GalN = 13.2 : 11.0 : 15.5 : 2.6, which is closely matched with the reported value of 12.4 : 9.6 : 17.2 : 2.7 (Townsend et al., ABRF News 8: 14, 1997). This method was shown to be applicable to varieties of well-characterized glycoproteins, erythropoietin, fibrinogen and soybean agglutinin. The amounts of sugars released under the condition were very close to the experimental values by other procedures or to the theoretical ones. This condition was found to be suitable for direct sugar analysis of fetuin, which have been immobilized onto polyvinylidene difluoride membrane. Based on these results, it support that the 6 N HCl/80 o C/2 h is the simplest method for quantitative analysis of monosaccharide composition of glycoproteins.

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“…The saccharide composition of the plasma membrane was determined by high-performance anion exchange chromatography with pulsed amperometric detector (HPAEC-PAD) analysis of monosaccharides released under conditions of acid hydrolysis [27,28]. HPAEC conditions: isocratic elution with 12 mM NaOH, 0.5 mL/min, 25°C, column Carbopac PA20 (Dionex), quadruple potential waveform (Dionex, www.dionex.com, Technical Note 21 (1998), Application update 141 (2000)), and with the usual six monosaccharides as standards (L-fucose (Fuc), D-galactose (Gal), D-glukose (Glc), D-mannose, D-galactosamine (GalN), and D-glucosamine (GlcN)).…”

Section: In Vitro Proliferation Assaymentioning

confidence: 99%

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells

et al. 2010

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To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.

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Comparison of Acid Hydrolytic Conditions for Asn-Linked Oligosaccharides

Cited by 46 publications

References 0 publications

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

An improved method for quantitative sugar analysis of glycoproteins

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells

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