The defense against the invasion of viruses and tumors relies on the presentation of viral and tumor-derived peptides to CTL by cell surface MHC class I molecules. Previously, we showed that the ubiquitously expressed protein amyloid precursor-like protein 2 (APLP2) associates with the folded form of the MHC class I molecule Kd. In the current study, APLP2 was found to associate with folded Kd molecules following their endocytosis and to increase the amount of endocytosed Kd. In addition, increased expression of APLP2 was shown to decrease Kd surface expression and thermostability. Correspondingly, Kd thermostability and surface expression were increased by down-regulation of APLP2 expression. Overall, these data suggest that APLP2 modulates the stability and endocytosis of Kd molecules.
Tapasin influences the quantity and quality of MHC/peptide complexes at the cell surface; however, little is understood about the structural features that underlie its effects. Because tapasin, MHC class I, and TAP are transmembrane proteins, the tapasin transmembrane/cytoplasmic region has the potential to affect interactions at the endoplasmic reticulum membrane. In this study, we have assessed the influence of a conserved lysine at position 408, which lies in the tapasin transmembrane/cytoplasmic domain. We found that substitutions at position K408 in tapasin affected the expression of MHC class I molecules at the cell surface, and down-regulated tapasin stabilization of TAP. In addition to affecting TAP interaction with tapasin, the substitution of alanine, but not tryptophan, for the lysine at tapasin position 408 increased the amount of tapasin found in association with the open, peptide-free form of the HLA-B8 H chain. Tapasin K408A was also associated with more folded, β2-microglobulin-assembled HLA-B8 molecules than wild-type tapasin. Consistent with our observation of a large pool of tapasin K408A-associated HLA-B8 molecules, the rate at which HLA-B8 migrated from the endoplasmic reticulum was slower in tapasin K408A-expressing cells than in wild-type tapasin-expressing cells. Thus, the alanine substitution at position 408 in tapasin may interfere with the stable acquisition by MHC class I molecules of peptides that are sufficiently optimal to allow MHC class I release from tapasin.
Presentation of antigenic peptides to T lymphocytes by MHC class I molecules is regulated by events involving multiple endoplasmic reticulum proteins, including tapasin. By studying the effects of substitutions in the tapasin Ig-like domain, we demonstrated that H-2Ld/tapasin association can be segregated from reconstitution of folded Ld surface expression. This finding suggests that peptide acquisition by Ld is influenced by tapasin functions that are independent of Ld binding. We also found that the presence of a nine-amino acid region in the Ig-like domain of mouse or human tapasin is required for association with Ld, and certain point substitutions in this sequence abrogate human, but not mouse, tapasin association with Ld. These data are consistent with a higher overall affinity between Ld and mouse tapasin compared with human tapasin. In addition, we found that other point mutations in the same region of the tapasin Ig-like domain affect MHC class I surface expression and Ag presentation. Finally, we showed that the cysteine residues in the Ig-like domain of tapasin influence tapasin’s stability, its interaction with the MHC class I H chain, and its stabilization of TAP. Mutagenesis of these cysteines decreases tapasin’s electrophoretic mobility, suggesting that these residues form an intramolecular disulfide bond. Taken together, these results reveal a critical role for the tapasin Ig-like domain in tapasin function.
Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-Ld cells. One mutant has a deletion of nine amino acid residues (tapasin Δ334–342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with Ld and their effects on Ld surface expression. We found that tapasin Δ334–342 was unable to bind to the Ld H chain, and yet it facilitated Ld assembly and expression. Tapasin Δ334–342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of Ld. Tapasin mutant H334F/H335Y, unlike tapasin Δ334–342, bound to Ld. Expression of tapasin H334F/H335Y in 721.220-Ld reduced the proportion of cell surface open forms of Ld and retarded the migration of Ld from the endoplasmic reticulum. In total, our results indicate that the 334–342 region of tapasin influences Ld assembly and transport.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.