Anti-α-galactosyl antibodies recognizing epitopes terminating with α1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

Abstract

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

“…1). The content of NOR2 glycolipid in erythrocytes is much lower than that of NOR1, but the former is easily detectable because it reacts more strongly with antibodies, most likely because of its longer oligosaccharide chain and/or double Gal(␣1-4)GalNAc moiety (7). In contrast to erythrocytes, 2102Ep teratocarcinoma cells have Gb3Cer as their most abun- dant glycolipid and show a much lower Gb4Cer content, suggesting that these cells have relatively low ␤3GalNAc-transferase activity.…”

“…It is highly specific for Gal(␣1-4)GalNAc and reacts exclusively with NOR glycolipids in human erythrocytes (7). For some experiments, the anti-NOR antibody was purified and biotinylated as described by Duk et al (18).…”

“…The NOR glycolipids contain a unique Gal(␣1-4)GalNAc-moiety that has not been described in any other mammal. The glycolipids that terminate with Gal(␣1-4)GalNAc (NOR1 and NOR2) are recognized by anti-NOR antibodies (6,7), which are commonly present in human sera. The NOR-positive individuals apparently do not show any morbid symptoms, but their erythrocytes are agglutinated by most human sera, creating a risk of complications during transfusion of NOR erythrocytes.…”

Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

“…The NOR-related oligosaccharides, Galα1-4GalNAc (NORdi), Galα1-4GalNAcβ1-3Gal (NOR-tri), and Galα1-4Galβ1-3Galβ1-4Glc-sp [(Gal) 3 Glc], and conjugates of NOR-tri with polyacrylamide (NOR-tri-PAA) and human serum albumin (NOR-tri-HSA) were obtained by chemical syntheses [9,10]. The disaccharides Galα1-4Gal and Galα1-3Gal were purchased from Glycorex (Lund, Sweden).…”

“…These glycolipids are globoside extension products terminating with the unique sequence Galα1-4GalNAcβ1-3Gal [6,7]. Two types of human anti-NOR antibodies were identified that are specific either to Galα1-4GalNAc/Galβ1-3Gal or to Galα1-4GalNAc [8,9]. The Galα1-4GalNAc sequence has not been found so far in mammals, but anti-NOR antibodies seem to be widespread in animal sera.…”