Abstract
Tapasin plays an important role in the quality control of major histocompatibility complex (MHC) class I assembly, but its precise function in this process remains controversial. Whether tapasin participates in the assembly of HLA-G has not been studied. HLA-G, an MHC class Ib molecule that binds a more restricted set of peptides than class Ia molecules, is a particularly interesting molecule, because during assembly, it recycles between the endoplasmic reticulum (ER) and the cis-Golgi until it is loaded with a high affinity peptide. We have taken advantage of this unusual trafficking property of HLA-G and its requirement for high affinity peptides to demonstrate that a critical function of tapasin is to transform class I molecules into a high affinity, peptide-receptive form. In the absence of tapasin, HLA-G molecules cannot bind high affinity peptides, and an abundant supply of peptides cannot overcome the tapasin requirement for high affinity peptide loading. The addition of tapasin renders HLA-G molecules capable of loading high affinity peptides and of transporting to the surface, suggesting that tapasin is a prerequisite for the binding of high-affinity ligands. Interestingly, the "tapasin-dependent" HLA-G molecules are not empty in the absence of tapasin but are in fact associated with suboptimal peptides and continue to recycle between the ER and the cis-Golgi. Together with the finding that empty HLA-G heterodimers are strictly retained in the ER and degraded, our data suggest that MHC class I molecules bind any available peptides to avoid ER-mediated degradation and that the peptides are in turn replaced by higher affinity peptides with the aid of tapasin.
Highlights
The HLA-G/E114Q mutant, in which residue 114 was replaced by the neutrally charged glutamine, fell between HLA-G wild type and HLA-G/E114H in the spectrum of tapasin dependence. These results indicate that, like HLA class Ia molecules, the tapasin dependence of HLA-G is influenced by the nature of the amino acid at position 114
We addressed the question of whether tapasin is involved in the assembly and trafficking of HLA-G
Our results demonstrate that tapasin is not required for suboptimal peptide binding but is critical for loading of high affinity peptides onto HLA-G
Summary
HLA-G, an MHC class Ib molecule, is expressed primarily in trophoblast cells and has limited polymorphism [2]. HLA-G is a interesting molecule in protein trafficking, because it recycles between the ER and the cis-Golgi until it is loaded with a high affinity peptide [19]. This feature makes it possible to estimate whether HLA-G molecules are loaded with high affinity or low affinity peptides. Because HLA-G binds a restricted set of peptides [23, 24] and recycles between the ER and cis-Golgi, it is an attractive model for investigating the roles of individual components of the ER peptide-loading complex in the assembly of functional MHC class I molecules. We propose that a critical function of tapasin is to transform the peptide-binding groove of HLA-G into a high affinity, peptidereceptive form, which could promote the replacement of low affinity peptides with high affinity peptides
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