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Glycobiology Advance Access published online on November 21, 2006
Glycobiology, doi:10.1093/glycob/cwl071
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype

Maria Duk2, Suddham Singh3, Vernon N. Reinhold3, Hubert Krotkiewski2, Ewa Kurowska4 and Elwira Lisowska1,2

2 Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland
3 Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA
4 Institute of Hematology and Blood Transfusion, 00-957 Warsaw, Poland

1 To whom correspondence should be addressed: Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Rudolf Weigl St. 12, 53-114 Wroclaw, Tel. 48-71-337 1172; Fax: 48-71-337 1382; E-mail: lisowska{at}iitd.pan.wroc.pl

Received on July 11, 2006; revised on October 24, 2006; accepted on November 16, 2006

Rare polyagglutinable erythrocytes of NOR phenotype were found to contain two unique glycosphingolipids (designated NOR1 and NOR2). These components (not detected in normal erythrocytes) were reactive with Griffonia simplicifolia isolectin IB4 (GSL-IB4) and commonly present human anti-NOR antibodies. The NOR1 component has been reported to be a globoside containing a single galactose residue linked {alpha}1,4 to the terminal N-acetylgalactosamine. Here, we report the structural studies on a second glycolipid, NOR2, and a third novel component migrating in high-performance thin-layer chromatography (HPTLC) between NOR1 and NOR2. The structures were determined by a combination of ion trap sequential mass spectrometry (MALDI-QIT-TOF) and step-wise treatment with glycosidases, followed by identification of products on HPTLC plates with lectins and mouse monoclonal anti-NOR antibody. The NOR2 component was found to be a disaccharide extension of NOR1 with the following structure: Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4GalNAcß1-3Gal{alpha}1-4Galß1-4Glcß1-Cer. Treatment of NOR2 with {alpha}-galactosidase gave a glycolipid migrating between NOR1 and NOR2 which did not react with either GSL-IB4 or anti-NOR antibodies, but did react with GalNAc-specific soy bean agglutinin (SBA). This intermediate glycolipid (now designated NORint) was identified as a relatively abundant component of a neutral glycolipid fraction from NOR erythrocytes, suggesting its presence as a precursor to NOR2. The structure of NORint was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids which are synthesized by sequential addition of Gal{alpha}1,4 and GalNAcß1,3 to globoside.

Key words: antibodies / glycosphingolipids / lectins / sequential mass spectrometry / polyagglutination NOR


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