Abstract
NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.
Highlights
NOR is a rare inheritable polyagglutination phenomenon that has been described in two families
Preparation of NOR Glycosphingolipid Samples—The total neutral glycosphingolipid fraction isolated from NOR erythrocytes was fractionated on the silica gel column, and the fractions containing NOR1 were pooled and further purified by preparative High-performance thin-layer chromatography (HPTLC)
The NOR1 glycolipid could be distinguished by specific reactions with lectins or antibodies, and ion trap analysis ensured it to be free of contaminating material by stepwise isolation and molecular disassembly (MSn)
Summary
Erythrocytes—The blood of a healthy NOR-positive person (T.S.) (100 ml) was donated, and the red blood cells were serologically typed as A2, MNss, P1. The samples eluted from the TLC plate were passed through the Sephadex G-25 superfine column in chloroform/methanol/water (60:30:4.5, v/v) and dried. The samples were dissolved in a chloroform/methanol/water (30:60:8, v/v) solution and applied to the DEAESephadex A-25 column prepared and packed using the same solvent. The erythrocytes were centrifuged, exhaustively washed with PBS, mixed with 2 ml of human serum (blood group AB), and incubated for 90 min at room temperature. HPTLC Lectin and Antibody Assays—The glycolipid samples, solubilized in chloroform/methanol (2:1, v/v), were applied to HPTLC plates (Kieselgel 60, Merck) and developed with chloroform/methanol/water (55:45:10, v/v). The immunostaining with antibodies was done but instead of biotinylated lectins, the plates were overlaid with mouse or human antibody solutions and with biotinylated goat antibodies against mouse Ig (Dako, Glostrup, Denmark) or human Ig (Sigma) diluted with TBS containing 1% bovine serum albumin. The plates were incubated with the primary antibody for 2.5 h at room temperature, except for mAb
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