Generation of a functional, soluble tapasin protein from an alternatively spliced mRNA.
The loading of newly synthesised MHC class I molecules (MHCI) with peptides requires the involvement of several endoplasmic reticulum (ER)-resident cofactors including calnexin, calreticulin, transporter associated with antigen processing, ERp57 and tapasin. In the absence of tapasin, MHC I complexes are loaded with suboptimal peptides and their recognition by cytotoxic T cells raised to high-affinity, immunodominant peptide epitopes is impaired. Here, we describe the cloning and functional assessment of an alternative spliced form of tapasin. From the EST database, we obtained a partially spliced tapasin cDNA that retained introns 4-6. When transfected into the tapasin-deficient cell line 0.220, the cDNA produced an alternatively spliced tapasin transcript that contained intron 5 (74 bp). This introduced a new stop codon that terminated translation immediately before the putative transmembrane domain and led to a tapasin molecule containing the lumenal domain plus 8 extra novel amino acids at its C-terminus. This molecule promoted peptide loading of HLA-B5 in 0.220 cell line, and restored normal HLA-B5 surface expression. However, the peptides loaded onto HLA-B5 were suboptimal compared to those loaded onto HLA-B5 in the presence of wild-type tapasin.
Gao
B
B
Rheumatology Unit, Institute of Child Health, University College London, UK.
Williams
A
A
Sewell
A
A
Elliott
T
T
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D000939:Epitopes / Q000378:metabolism; D020224:Expressed Sequence Tags; D005434:Flow Cytometry; D015151:Immunoblotting; D007136:Immunoglobulins / Q000235:genetics* / Q000378:metabolism*; D008285:Major Histocompatibility Complex / Q000502:physiology*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014162:Transfection
D017920:Antiporters; D017931:DNA Primers; D000939:Epitopes; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D010455:Peptides; D012333:RNA, Messenger; C107762:tapasin