Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-alphaGal antibody.
Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6' OH of Galbeta(1 --> 4)Glcbeta-OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). UDP-[6-(3)H]Gal studies indicated that alpha1,3-galactosyltransferase recognized the C-6' modified Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and alpha1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15-22. Galalpha(1 --> 3)Galbeta(1 --> 4)Glcbeta-OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6" modified derivatives (23-30). An ELISA bioassay was performed utilizing human anti-alphaGal antibodies to study the binding affinity of the derivatized epitopes (6, 15-30). Modifications made at the C-6' position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6" position resulted in significant or complete abrogation of recognition. The results indicate that the C-6' OH of the alphaGal trisaccharide epitope is not mandatory for antibody recognition.
Wayne State University, Detroit, MI 48202, USA.
Peter R
PR
Andreana
Przemyslaw
P
Kowal
Adam J
AJ
Janczuk
Peng George
PG
Wang
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D005692:Galactose Oxidase / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000378:metabolism; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D007797:Laminin; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; D005692:Galactose Oxidase; D005700:Galactosyltransferases