ABH blood group antigens in O-glycans of human glycophorin A
Section snippets
Isolation of O-linked oligosaccharides
GPA was isolated and purified as we described previously [15], [17]. O-linked oligosaccharides from 20 mg samples of GPA were released by reductive β-elimination by a protocol adapted from Likhosherstov et al. [18]. The reaction was stopped by the addition of 50% acetic acid, and borate salts were removed by gel filtration on a BioGel P-2 (Bio-Rad Laboratories, Richmond, VA, USA) column eluted with water. The major fraction, containing reduced O-glycans and short N-linked glycopeptides, was
Reductive β-elimination and composition analysis of O-linked glycans
The O-glycans were released from purified GPA-A, GPA-B, and GPA-O using reductive β-elimination, and separated from the short N-linked glycopeptides. Standard GC/MS sugar analysis of the reduced O-glycans revealed that the major components were galactose and N-acetylgalactosamine; fucose and N-acetylglucosamine were also detected, but not quantified due to their low amounts (data not shown). The presence of sialic acid was confirmed by a colorimetric method. A part of each preparation of
Discussion
The ABH and Lewis blood group antigens are present not only on erythrocytes, but are widely distributed in other tissues and for this reason are called histo-blood group antigens [1]. The ABH antigens are synthesized by addition of fucose (H antigen-specific) and then N-acetylgalactosamine (A antigen-specific) or galactose (B antigen-specific) to three major precursor structures attached to proteins or lipids:
Type 1 chain: Galβ1-3GlcNAcβ1-R.
Type 2 chain: Galβ1-4GlcNAcβ1-R.
Type 3 chain:
Acknowledgments
We thank Dr. S. Bochenek (Regional Centre of Blood Transfusion, Wrocław, Poland) for the samples of human erythrocytes. We are also indebted to K. Izydorczyk for assistance in preparing the graphics. This work was supported by the State Committee for Scientific Research (KBN, Warsaw) Grant No. 6P04A 052 20.
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Both authors contributed equally to this work.