Pyridylamination as a means of analyzing complex sugar chains.
Herein, I describe pyridylamination for versatile analysis of sugar chains. The reducing ends of the sugar chains are tagged with 2-aminopyridine and the resultant chemically stable fluorescent derivatives are used for structural/functional analysis. Pyridylamination is an effective "operating system" for increasing sensitivity and simplifying the analytical procedures including mass spectrometry and NMR. Excellent separation of isomers is achieved by reversed-phase HPLC. However, separation is further improved by two-dimensional HPLC, which involves a combination of reversed-phase HPLC and size-fractionation HPLC. Moreover, a two-dimensional HPLC map is also useful for structural analysis. I describe a simple procedure for preparing homogeneous pyridylamino sugar chains that is less laborious than existing techniques and can be used for functional analysis (e.g., sugar-protein interaction). This novel approach was applied and some of the results are described: i) a glucosyl-serine type sugar chain found in blood coagulation factors; ii) discovery of endo-beta-mannosidase (EC 3.2.1.152) and a new type plant alpha1,2-L-fucosidase; and iii) novel substrate specificity of a cytosolic alpha-mannosidase. Moreover, using homogeneous sugar chains of a size similar to in vivo substrates we were able to analyze interactions between sugar chains and proteins such as enzymes and lectins in detail. Interestingly, our studies reveal that some enzymes recognize a wider region of the substrate than anticipated.
Hase
Sumihiro
S
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, Japan.
D000586:Amination; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D006801:Humans; D008361:Mannosidases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology; D011725:Pyridines / Q000737:chemistry*; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000378:metabolism
D002241:Carbohydrates; D011725:Pyridines; D008361:Mannosidases; D005644:alpha-L-Fucosidase