Carbohydrate residues downstream of the terminal Galα(1,3)Gal epitope modulate the specificity of xenoreactive antibodies

Abstract

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Gala(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional a1,3-galactosyltransferase (a1,3GT), which transfers aGal to N-acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Gala(1,3)Gal is still expressed in a1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in a1,3GTÀ/À mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid-linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface-expressed Gala(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three-dimensional models suggest that the terminal a-linked Gal binds tightly into the antibody-binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.

The design and synthesis of an α-Gal trisaccharide epitope that provides a highly specific anti-Gal immune response

“…While this subtle change in a single functional group drastically alters antibody recognition, cases of antibody discrimination between these two functional groups has been documented and thus highlight the subtle importance of steric and electronic effects in determining antibody-carbohydrate epitope recognition. Similar discrimination has been reported in the isolation of anti-Gal monoclonal Abs, 35 but not in a polyclonal response. Our results demonstrate the ability of this glycoconjugate formulation to discriminate these epitopes almost entirely in the polyclonal response.…”

Humans Lack iGb3 Due to the Absence of Functional iGb3-Synthase: Implications for NKT Cell Development and Transplantation

“…The ability of this chimeric rat/human(exon5)-iGb3S molecule to synthesize Galα(1,3)Gal was determined by analysis of transfected CHOP cells. As expected, cells transfected with DNA encoding rat iGb3S displayed strong cell surface expression of the Galα(1,3)Gal epitope on glycolipid as determined by binding of the monoclonal antibody 15.101 [28] and human anti-Gal immunoglobulin (Ig) purified from normal human serum (Figure 3B). The 15.101 mAb has been shown to bind preferentially to Galα(1,3)Gal on iGb3 lipid [28].…”

“…As expected, cells transfected with DNA encoding rat iGb3S displayed strong cell surface expression of the Galα(1,3)Gal epitope on glycolipid as determined by binding of the monoclonal antibody 15.101 [28] and human anti-Gal immunoglobulin (Ig) purified from normal human serum (Figure 3B). The 15.101 mAb has been shown to bind preferentially to Galα(1,3)Gal on iGb3 lipid [28]. The chimeric molecule containing the majority of the catalytic domain of human iGb3S (rat/human(exon5)-iGb3S) was unable to synthesise the Galα(1,3)Gal epitope as staining was not observed with 15.101 or human anti-Gal Ig (Figure 3B).…”

Close-up of the Immunogenic α1,3-Galactose Epitope as Defined by a Monoclonal Chimeric Immunoglobulin E and Human Serum Using Saturation Transfer Difference (STD) NMR

“…Consequently, specificity for ␣-Gal units has been assessed in different settings using a variety of reagents, including lectins, polyclonal sera, and monoclonal antibodies (32). Despite these data and recent theoretical approaches (33)(34)(35), to the best of our knowledge, FIGURE 2. Immunoreactivity with allergens and Fc receptor molecules.…”