title: Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype.
abstract: 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 alpha1,4 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: Galalpha1-4GalNAcbeta1-3Galalpha1-4GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-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 soybean agglutinin. This intermediate glycolipid (now designated NOR(int)) 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 NOR(int) was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids that are synthesized by sequential addition of Galalpha1,4 and GalNAcbeta1,3 to globoside.
authors.affiliation: Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Duk
authors.forename: Suddham
authors.initials: S
authors.lastname: Singh
authors.forename: Vernon N
authors.initials: VN
authors.lastname: Reinhold
authors.forename: Hubert
authors.initials: H
authors.lastname: Krotkiewski
authors.forename: Ewa
authors.initials: E
authors.lastname: Kurowska
authors.forename: Elwira
authors.initials: E
authors.lastname: Lisowska
mesh_terms: D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000276:immunology*; D005915:Globosides / Q000737:chemistry* / Q000276:immunology; D006384:Hemagglutination; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010504:Periodic Acid / Q000737:chemistry; D010641:Phenotype; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005915:Globosides; C519499:NOR2 glycolipid; D010504:Periodic Acid; C009288:metaperiodate