Relationship between Immunogenicity and Protein Structure at Amino Acid Substitution Sites of Blood Group Antigens

• Immunogenicity of RBC antigens is related to protein secondary and tertiary structure at AA substitution sites that create the antigens.

• The most immunogenic AA substitutions are located in rigid, ordered regions with reduced accessibility.

Polypeptide blood group antigens, many of which are created by single exofacial amino acid (AA) substitutions, have varying immunogenicities. Why some AA substitutions are more immunogenic than others is little understood. Using AlphaFold2, an artificial intelligence system that predicts three-dimensional protein structure along with multiple other structure analysis programs, we investigated protein structure at sites of AA substitutions that create nine clinically-significant blood group antigens. Based on structure predictions, the AA substitutions that create the four most immunogenic of the nine antigens (K, Jka, Lua, E) were typically buried or partially buried in rigid, ordered protein regions, usually helices and β-strands. This was reflected by their lower mean relative solvent accessibility (RSA) than the five less immunogenic antigens (c, M, Fya, C, S; 0.13 versus 0.81; P=.003), and higher mean AlphaFold2 confidence score (92.5 versus 48.3; P=.001; scores <50 predict protein disorder). Substitutions creating the five least immunogenic antigens (c, Fya, M, C, S) were all predicted to be in flexible regions with high accessibility, either in surface-accessible loops (C, c) or disordered coils (Fya, M, S). Scatter plots revealed a positive linear correlation of immunogenicity with confidence score (R2=0.826; P=.0007) and percent helix/β-strand in 15-mers centered around the substitution sites (R2=0.763; P=.0021), and a negative linear correlation with RSA (R2=0.688; P=.0057). Therefore, based on an informatics analysis, the protein secondary and tertiary structure at AA substitution sites that create blood group antigens are significant correlates and potential determinants of immunogenicity.