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 ␣-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Gal␣133Gal-sequence and that anti-NOR agglutinins were common human anti-Gal␣133Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thinlayer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with ␣-galactosidase and ␤-N-acetylhexosaminidase was followed by highperformance thin-layer chromatography with product detection by lectins and the anti-Gb 4 monoclonal antibody. The results suggested that NOR1 was an ␣-galactosylated Gb 4 Cer with a ␤-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex134HexN133Hex134Hex134Hex linked to a ceramide composed of C 18 sphingosine and a C 24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Gal␣134GalNAc␤133Gal␣134Gal␤134 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Gal␣133Gal xenoantibodies.

Our results showed that two rare features of TS's RBCs, NOR polyagglutination and St(a) glycophorin, are inherited independently, and that NOR+ RBCs contain neutral glycolipids with an abnormal oligosaccharide structure, most likely terminated with alpha-galactosyl residues.

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.

A strong complement-fixing incomplete anti-Di^a was found in the serum of a Polish mother who gave birth to a newborn suffering from a severe haemolytic anaemia. The whole family was of Polish origin. 9,661 donor blood samples from different regions of Poland were tested against antiserum derived from that mother. Di^a antigen was present on red cells of 45 (0.46%) individuals. All of them were of Polish origin. In some cases, family studies were undertaken.

Human erythrocytes treated with purified human neutrophil elastase (HNE) or cathepsin G (CathG) were analysed by serological methods and by SDS-polyacrylamide gel electrophoresis followed by staining or immunoblotting with monoclonal antibodies. Both enzymes digested exhaustively glycophorins A, B and C, and HNE caused a partial digestion of band 3 protein. The degradation of other membrane proteins was not detectable by the methods used. Immunoblotting with the use of monoclonal antibodies against the defined epitopes of glycophorin A showed that HNE and CathG hydrolysed distinct peptide bonds in this antigen. The antibody PEP80, specific for the epitope in the cytoplasmic fragment of glycophorin A, gave patterns of bands which were characteristic for each of the two proteases. These bands could be distinctly identified in erythrocyte membrane samples containing only few percent of digested glycophorins. Therefore, the immunoblotting with this antibody may be useful as a sensitive assay for detecting the action of neutrophil proteases on red blood cells.