Structure of a Neutral Glycosphingolipid Recognized by Human Antibodies in Polyagglutinable Erythrocytes from the Rare NOR Phenotype

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 ␣-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.

A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome

“…The dried plates were immersed in 0.05% polyisobutylmethacrylate (Aldrich, Steinheim, Germany) in hexane for 1 min, dried, sprayed with TBS (0.05 M Tris buffer, 0.15 M NaCl (pH 7.4)), and blocked in 5% BSA for 1 h. For antibody assays, the plates were successively overlain with 1) mouse monoclonal or rabbit polyclonal antibody diluted in TBS/1% BSA (TBS-BSA) for 1-1.5 h; 2) biotinylated goat anti-mouse Ig antibody or goat anti-rabbit Ig antibody conjugated with alkaline phosphatase (Dako, Glostrup, Denmark), diluted 1:5000 with TBS-BSA; 3) ExtrAvidin-alkaline phosphatase conjugate (Sigma) diluted 1:1000 with TBS/ BSA/0.2% Tween20 for 1 h; and 4) the substrate solution (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, Sigma). Other details were as described previously (3,4). Each HPTLC experiment was repeated three times.…”

“…Finally, washed cells were resuspended (5 ϫ 10 5 cells per 0.75 ml) and analyzed as described above. Extraction and Purification of Glycosphingolipids-The isolation and fractionation of glycolipids and the orcinol staining were performed using the standard procedures applied previously to erythrocytes (2,3). Briefly, glycolipids were extracted from 2102Ep cells with chloroform/methanol, and the neutral glycolipids were separated from the phospholipids and gangliosides, purified in peracetylated form, de-O-acetylated, and desalted.…”

“…To isolate individual glycolipids, the neutral glycolipids were fractionated on Kieselgel H type 60 columns (Merck, Darmstadt, Germany), and the fractions were analyzed by highperformance thin-layer chromatography (HPTLC) 2 using an anti-NOR antibody. Fractions containing the desired glycolipid were pooled, and the glycolipid was purified by preparative HPTLC followed by elution from the appropriate plate region, as described by (3).…”

“…6A. The monosodiated [B 2ϩ Na] ϩ ion at m/z 388.1 was chosen for further MS 3 analysis, and cross-ring fragmentation produced a distinct monosodiated 3,5 A 2 ion at m/z 259.0, which is diagnostic for 134 linked sugar residues (Fig. 6B).…”

“…The erythrocytes of NOR-positive individuals from the Polish T.S. family were found to contain unique neutral glycosphingolipids formed by the elongation of globoside (3,4): NOR1, Gal(␣1-4)GalNAc(␤1-3)Gal(␣1-4)Gal(␤1-4)GlcCer; NOR int , GalNAc(␤1-3)Gal(␣1-4)GalNAc(␤1-3)Gal-(␣1-4)Gal(␤1-4)GlcCer; and NOR2, Gal(␣1-4)GalNAc-(␤1-3)Gal(␣1-4)GalNAc(␤1-3)Gal(␣1-4)Gal(␤1-4)GlcCer. Identical NOR glycolipids were later identified in erythrocytes from a NOR-positive donor from the American family (5).…”