Anti-Gal is a natural antibody, which constitutes as much as 1% of circulating IgG in humans and displays a distinct specificity for the structure Galal-*3Gal. This

In 1985, we reported that a naturally occurring human antibody (anti-Gal), produced as the most abundant antibody (1% of immunoglobulins) throughout the life of all individuals, recognizes a carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (the α-gal epitope). Since that time, an extensive literature has developed on discoveries related to the α-gal epitope and the anti-Gal antibody, including the barrier they form in xenotransplantation and their reciprocity in mammalian evolution. This review covers these topics and new avenues of clinical importance related to this (α-gal epitope/ anti-Gal) unique antigen/antibody system in improving the efficacy of viral vaccines and in immunotherapy against cancer.

A natural IgG antibody (anti-Gal) with alpha-galactosyl binding specificity has been found in large amounts (0.5 - 1.0% of serum IgG) in all individuals tested. It has been purified by affinity chromatography on a column of melibiose-Sepharose. In addition to its affinity for normal and pathological senescent human red cells, the antibody readily interacts with rabbit red blood cell (RRBC) glycolipids with alpha-galactosyl terminal residues. Two types (glycosidic linkages of 1----3 vs. 1----4) of rabbit red cells glycolipids with terminal alpha-galactosyl residues were tested for antibody binding. The antibody specifically bound to glycolipids with Gal alpha 1----3 terminal residues, and treatment of these glycolipids with alpha-galactosidase abolished binding. Hemagglutination inhibition studies with oligosaccharides of known structure also showed that the antibody binds specifically to glycoconjugates with an alpha 1----3 terminal galactose residue. Anti-Gal did not bind to a human B-active glycolipid, indicating that fucose-linked alpha 1----2 to the penultimate galactose prevents anti-Gal binding. The anti-Gal specificity for RRBC glycolipids also paralleled that of the alpha-galactosyl-specific Bandeiraea simplicifolia lectin. The possible reasons for the occurrence of this unique antibody in human serum are discussed.

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.

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A well-defined antigen/antibody system was used to evaluate the effect of immune tolerance on the spectrum of specificities of natural antibodies. The antibody used in this study, anti-Gal, is a naturally occurring, polyclonal IgG that constitutes 1% of the circulating IgG in humans. We have previously shown that anti-Gal, purified from AB sera, specifically interacts with glycosphingolipids bearing a Gal alpha 1----3Gal epitope, but not with the closely related B antigen in which the penultimate galactose of the Gal alpha 1----3Gal epitope is fucosylated Gal alpha 1----3(Fuc alpha 1----2)Gal. This narrow specificity was assumed to be the result of an effective immune tolerance mechanism that prevents the expression of antibody clones that can recognize both the Gal alpha 1----3Gal and the self B epitopes. If the assumption that immune tolerance determines the range of anti-Gal specificity is correct, then anti-Gal from individuals lacking the B antigen (A and O blood types) would be expected to interact with both Gal alpha 1----3Gal and Gal alpha 1----3(Fuc alpha 1----2)Gal epitopes. In this study, anti-Gal from the serum of individuals of various blood types was purified by affinity chromatography on Gal alpha 1----3Gal adsorbent and tested for its reaction with the B antigen. Whereas anti-Gal from AB and B individuals only reacted with Gal alpha 1----3Gal epitopes, anti-Gal from A and O individuals reacted with both Gal alpha 1----3Gal and B epitopes. Furthermore, it was determined that the majority of anti-B reactivity in A and O individuals is in fact anti-Gal antibodies capable of recognizing both Gal alpha 1----3Gal and B epitopes. It can be concluded from these results that immune tolerance accurately controls the spectrum of natural antibody specificities by preventing the production of antibody clones that can interact with self antigens.

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometrybased analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycopro-

Alpha3-fucosyltransferases (alpha3-FucTs) catalyze the final step in the synthesis of a range of important glycoconjugates that function in cell adhesion and lymphocyte recirculation. Six members of this family of enzymes have been cloned from the human genome, and their expression pattern has been shown to be highly regulated. Each enzyme has a unique acceptor substrate binding pattern, and each generates a unique range of fucosylated products. Results from a range of studies have provided information on amino acids in the FucT sequence that contribute to the differential acceptor specificity for the FucTs, and to the binding of the nucleotide sugar donor GDP-fucose. These results, in conjunction with results obtained from the analysis of the disulfide bond pattern, have provided useful clues about the spatial distribution of amino acids that influence or directly contribute to substrate binding. This information is reviewed here, and a molecular fold prediction is presented which has been constructed based on the available information and current modeling methodology.