Use of large-scale hydrazinolysis in the preparation of N-linked oligosaccharide libraries: application to brain tissue.
In this report, we describe the preparation of a library of N-linked glycans from whole murine brain obtained by the large-scale hydrazinolysis of an acetone powder of the tissue followed by chromatographic procedures. 84% of the characterized oligosaccharides were found to be anionic, the remainder neutral. The anionic species were successively neutralized by neuraminidase (29%), aq. hydrofluoric acid (30%), and methanolysis (26%), indicating that approximately equal portions were sensitive to desialylation, dephosphorylation and desulfation, respectively. The presence of the sulfated fraction was confirmed by direct 35SO4 metabolic labelling. A residual partially characterized fraction was found to be anionic through possession of carboxylic acid groups, unrelated to sialic acid. The purified oligosaccharides, in the absence of their original protein conjugates, were shown to retain those immunological characteristics essential for recognition by a specific monoclonal antibody, LS (412), that is known to recognize a carbohydrate epitope present on a number of neural adhesion molecules and functional in neural cell adhesion. These properties confirm the viability of scaling up the size of the hydrazinolysis procedure and adapting it to whole tissue for the production of glycan libraries and for the probing of structures of interest.
Wing
D R
DR
Glycobiology Institute, Department of Biochemistry, Oxford, UK.
Rademacher
T W
TW
Field
M C
MC
Dwek
R A
RA
Schmitz
B
B
Thor
G
G
Schachner
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism*; D002236:Carbohydrate Conformation; D005591:Chemical Fractionation; D005260:Female; D006834:Hydrazines / Q000378:metabolism*; D008297:Male; D051379:Mice; D009844:Oligosaccharides / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000302:isolation & purification*; D013462:Sulfur Radioisotopes
D000911:Antibodies, Monoclonal; D006834:Hydrazines; D009844:Oligosaccharides; D011134:Polysaccharides; D013462:Sulfur Radioisotopes