Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.
Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.
Nakajima
Kazuki
K
Disease Glycomics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.
Kitazume
Shinobu
S
Angata
Takashi
T
Fujinawa
Reiko
R
Ohtsubo
Kazuaki
K
Miyoshi
Eiji
E
Taniguchi
Naoyuki
N
D002241:Carbohydrates / Q000737:chemistry*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002241:Carbohydrates; D008562:Membrane Glycoproteins; D009711:Nucleotides