Date of Award
Open Access Dissertation
Doctor of Philosophy (PhD)
Recognition of MHC class I/peptide complexes is required for the generation of CD8+ T cell responses. Peptide loading onto MHC class I/beta2m dimers occurs in the ER and involves both specific proteins and cellular chaperones. Tapasin is essential for peptide loading onto most MHC class I alleles, and it forms a mixed disulfide with the glycoprotein specific oxidoreductase ERp57. I have characterized the biochemical requirements for tapasin/ERp57 conjugate formation and addressed potential functions for ERp57 in peptide loading. Tapasin specifically recruits ERp57 into a mixed disulfide at the expense of free ERp57 in the ER. Other components of the MHC class I peptide loading complex are not required for conjugate formation, and, in contrast to models of glycoprotein folding, conjugate formation does not require the generation of monoglucosylated glycans.Once associated, tapasin has evolved to inhibit the reductase activity of the ERp57 a domain leading to the retention of ERp57 in the loading complex over the course of normal peptide loading. In contrast, calreticulin undergoes cycles of binding and release, and its presence in the loading complex is dependent upon MHC class I. ERp57 is a core structural component of the MHC class I loading complex, and it is permanently sequestered there by tapasin.Finally, I have further characterized cells expressing a tapasin mutant unable to form the conjugate. Loading complex assembly is impaired in these cells, and peptide loading is inefficiently catalyzed by this mutant. Additionally, ERp57 binding stabilizes the structural integrity of tapasin. When endogenous ERp57 expression was suppressed using RNAi, all residual ERp57 was bound to tapasin, and both protein disulfide isomerase and ERp72, other ER resident oxidoreductases, formed mixed disulfides with tapasin. When redox mutant ERp57 proteins were over-expressed in these cells, only slight changes were seen in MHC class I trafficking. My data indicate that ERp57 is a key structural component of the MHC class I loading complex and likely does not exhibit any redox activity in this context. The key features of ERp57 responsible for its function remain unknown.
Peaper, David Ryan, "Biochemical and functional characterization of the tapasin/ERp57 conjugate" (2007). Yale Medicine Thesis Digital Library. 2230.