The ER folding sensor UGGT1 acts on TAPBPR-chaperoned peptide-free MHC I
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eLife assessment
This valuable study reports a complete in vitro system where different steps and direct interactions between different components of MHCI maturation can be monitored, hence leading to a better mechanistic understanding of MHC I maturation. The evidence supporting the findings is currently incomplete and would benefit from clarification of some key issues. This work will be of interest to immunologists and biochemists.
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Abstract
Adaptive immune responses are triggered by antigenic peptides presented on major histocompatibility complex class I (MHC I) at the surface of pathogen-infected or cancerous cells. Formation of stable peptide-MHC I complexes is facilitated by tapasin and TAPBPR, two related MHC I-specific chaperones that catalyze selective loading of suitable peptides onto MHC I in a process called peptide editing or proofreading. On their journey to the cell surface, MHC I complexes must pass a quality control step performed by UGGT1, which senses the folding status of the transiting N-linked glycoproteins in the endoplasmic reticulum (ER). UGGT1 reglucosylates non-native glycoproteins and thereby allows them to revisit the ER folding machinery. Here, we describe a reconstituted in-vitro system of purified human proteins that enabled us to delineate the function of TAPBPR during the UGGT1-catalyzed quality control and reglucosylation of MHC I. By combining glycoengineering with liquid chromatography-mass spectrometry, we show that TAPBPR promotes reglucosylation of peptide-free MHC I by UGGT1. Thus, UGGT1 cooperates with TAPBPR in fulfilling a crucial function in the quality control mechanisms of antigen processing and presentation.
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eLife assessment
This valuable study reports a complete in vitro system where different steps and direct interactions between different components of MHCI maturation can be monitored, hence leading to a better mechanistic understanding of MHC I maturation. The evidence supporting the findings is currently incomplete and would benefit from clarification of some key issues. This work will be of interest to immunologists and biochemists.
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Reviewer #1 (Public Review):
In this manuscript, the authors use purified human proteins to assess the factors required for the reglucosylation of MHC-I and describe an elegant, mass-spectrometry-based assay to assess reglucosylation. This process is an essential quality-control step for peptide-MHC-I complexes before they are trafficked to the cell surface. Earlier studies have established TAPBPR as a tapasin-like peptide editor of MHC-I outside the peptide loading complex. The ER chaperone UGGT1 has also been shown to interact with MHC-I loaded with a low-affinity peptide, reglucosylating it to allow re-interaction with the peptide loading complex via calreticulin. That TAPBPR facilitates the interaction of UGGT1 with MHC-I was described by Boyle and co-workers in 2017. In that study, a free cysteine on TAPBPR was shown to be …
Reviewer #1 (Public Review):
In this manuscript, the authors use purified human proteins to assess the factors required for the reglucosylation of MHC-I and describe an elegant, mass-spectrometry-based assay to assess reglucosylation. This process is an essential quality-control step for peptide-MHC-I complexes before they are trafficked to the cell surface. Earlier studies have established TAPBPR as a tapasin-like peptide editor of MHC-I outside the peptide loading complex. The ER chaperone UGGT1 has also been shown to interact with MHC-I loaded with a low-affinity peptide, reglucosylating it to allow re-interaction with the peptide loading complex via calreticulin. That TAPBPR facilitates the interaction of UGGT1 with MHC-I was described by Boyle and co-workers in 2017. In that study, a free cysteine on TAPBPR was shown to be essential for the interaction between TAPBPR and UGGT1, although there was no inter-molecular disulfide linkage formed. The data in the current in vitro study suggests that while TAPBPR is an essential facilitator of reglucosylation of the HLA-A*68:02 allele, the free Cys on TAPBPR is not required to bridge the interaction between MHC-I and UGGT1.
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Reviewer #2 (Public Review):
In this manuscript, authors had to circumvent some challenges in protein design that included the generation of peptide-receptive MHCI and a defined Man9GlcNAc2 glycan tree on the MHC I recognizable by UGGT1. Production of peptide-receptive MHCI was achieved by forming a fos/jun dimerized single-chain MHC1-fos with TAPBPR-jun in the presence of the α-mannosidase I inhibitor kifunensine. Glucozylation of MHCI by UGGT1 was monitored on protease-cleaved MHCI/TAPBPR, and liquid chromatography-mass spectrometry was used to monitor reglucosylation. Authors have provided convincing evidence that TAPBPR is sufficient and necessary for glucosylation of MHC 1, hence TAPBPR in addition to serving as an accessory protein in regulating peptide selection has a second function in quality control and fitness of newly …
Reviewer #2 (Public Review):
In this manuscript, authors had to circumvent some challenges in protein design that included the generation of peptide-receptive MHCI and a defined Man9GlcNAc2 glycan tree on the MHC I recognizable by UGGT1. Production of peptide-receptive MHCI was achieved by forming a fos/jun dimerized single-chain MHC1-fos with TAPBPR-jun in the presence of the α-mannosidase I inhibitor kifunensine. Glucozylation of MHCI by UGGT1 was monitored on protease-cleaved MHCI/TAPBPR, and liquid chromatography-mass spectrometry was used to monitor reglucosylation. Authors have provided convincing evidence that TAPBPR is sufficient and necessary for glucosylation of MHC 1, hence TAPBPR in addition to serving as an accessory protein in regulating peptide selection has a second function in quality control and fitness of newly synthesized MHC I during maturation.
The strength of the study lies in the generation of a complete in vitro system where different steps and direct interactions between different components of MHCI maturation can be monitored, hence leading to a better mechanistic understanding of MHC I maturation. However, some potential weakness might be that the major finding of the manuscript describing the critical role of TAPBPR as a chaperon in optimizing peptide selection and regulation of MHC I glucosylation and reglucosylation has been previously reported. Nonetheless, the current study further establishes and better defines some prior findings, thus quite valuable.
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