Expression of mutant H-2(d) proteins encoded by class I genes which alternatively process the 5' end of their transcripts

Mutation of the 3' splice sites bordering exon 2 of the H-2D(d) and H-2K(d) genes generated alternatively spliced transcripts when the constructs were transfected into L cells (J. Immunol. 143:1018). The H-2D(d) transcripts contained an additional 84 nucleotides derived from the first intervening sequence, whereas 60 extra bases were included in the H-2K(d) mRNA. Proteins derived from these transcripts were recognized by mAb. Moreover, both Ag served as recognition elements for CTL, and the mutant H-2K(d) molecule functioned as a restricting element for an Ag peptide. As a result of alternative splicing, the mutant proteins should have additional residues at their NH₂ termini to increase their lengths by 28 (D(d)) or 20 (K(d)) amino acids. Immunoprecipitation and analysis on SDS-PAGE demonstrated that the mutant H-2K(d) molecule was indeed larger than the normal H-2K(d) protein, but the mutant and wild-type H-2D(d) Ag were the same size. In addition, treatment of H-2D(d) mutant and normal Ag with N-glycanase produced molecules of equal size, demonstrating that the mutant protein was completely glycosylated. Limited amino acid sequencing of this Ag indicated that it was normal H-2D(d). Therefore, before its transfer to the cell surface, post-translational modifications remove the additional NH₂-terminal residues of the mutant D(d) but not K(d) protein.