A deep mutational scan of the MHC class I-specific chaperone tapasin

Tapasin acts as the principal MHC-I-specific chaperone for facilitating folding and antigenic peptide loading of nascent MHC class I substrates in the cell. In cells where tapasin has been knocked out, the processing and surface trafficking of the human MHC-I allele HLA-A2 is substantially reduced. Over-expression of tapasin rescues HLA-A2 surface expression. Using this assay as the basis for a fluorescence-based selection, tapasin was deep mutationally scanned at 108 positions in the core, at the interface with MHC-I, and on the 'backside' distal from where MHC-I binds. Critical residues of tapasin for rescue of HLA-A2 processing map to sites that contact the underside of the MHC-I alpha-2 domain, to the surface contacting the MHC-1 beta2m and alpha-3 domains, and to the base of a protruding loop that rests above the peptide-binding groove (but not to the tip of the loop itself). Overall design: A synthetic sequence of tapasin was constructed. A library was generated of tapasin in which 108 positions were diversified with degenerate NNK codons. The library was transfected into tapasin-knock out Expi293F cells after 1500-fold dilution with a carrier plasmid; this ensures most cells express no more than a single sequence variant and there is a tight connection between genotype and phenotype. Illumina sequencing data to validate the introduction of indel mutations in exon 2 of the tapasin gene is in GEO Series Acc. No. GSE126206. Cells expressing the tapasin library were stained for surface HLA-A2 with R-phycoerythrin-conjugated anti-HLA-A2 clone BB7.2, and the 0.5% of cells with the highest fluorescence were collected by FACS. Following Illumina sequencing, the frequencies of mutations from transcripts in the sorted cells were compared to their respective frequencies in the naive plasmid library. The calculated enrichment ratios are a proxy for relative activity of the tapasin sequence variants.