Calreticulin and Calnexin Interact with Different Protein and Glycan Determinants During the Assembly of MHC Class I

Harris, Michael; Yu, Yanbo; Kindle, Cathy S.; Hansen, Ted H.; Solheim, Joyce C.

doi:10.4049/jimmunol.160.11.5404

Cited by 108 publications

(2 citation statements)

References 61 publications

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“…Calreticulin, or CRT, functions as an "eat me" signal and is an ER lectin chaperone family member. It is crucial in triggering phagocytosis and subsequent immune responses [ 41 , 99 , 100 ]. The translocation of Calreticulin to the cell surface is prompted by cellular stress and DNA damage.…”

Section: Regulation Of Phagocytic Signaling Pathways In Macrophagesmentioning

confidence: 99%

Don't eat me/eat me signals as a novel strategy in cancer immunotherapy

Khalaji,

Yancheshmeh,

Farham

et al. 2023

Heliyon

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Section: Regulation Of Phagocytic Signaling Pathways In Macrophagesmentioning

confidence: 99%

Don't eat me/eat me signals as a novel strategy in cancer immunotherapy

Khalaji,

Yancheshmeh,

Farham

et al. 2023

Heliyon

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“…However, the role of tapasin is not limited to a structural function but it is also a major facilitator of peptide exchange on MHC I and thereby directly involved in the quality control of MHC I loading [54][55][56] . ERp57 is a member of the protein disulfate isomerase (PDI) family 57,58 . These proteins can form, break, or rearrange intra-and inter-molecular disulfide bonds.…”

Section: Tapasin and Erp57mentioning

confidence: 99%

Mechanistic and structural studies of the MHC I peptide loading complex

Domnik¹

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The peptide loading complex (PLC) is a central machinery in adaptive immunity ensuring antigen presentation by major histocompatibility complex class I (MHC I) molecules to immune cells. If nucleated cells present foreign antigenic peptides from various origins (e.g., viral infected or cancer cells) on their cell surface they are targeted and eliminated by effector cells of the immune system to protect the organism against the hazard. The antigen presentation process starts with proteasomal degradation. Peptide loading and quality control of most, if not all, MHC I is performed by the PLC. Despite the main components, architecture, and general functions of this labile and multi-subunit assembly have been described, knowledge about the inner mechanics of MHC I loading and quality control in the PLC is limited. Detailed structural insights into the interactions and functions of key elements are lacking. In this PhD thesis, structural and functional aspects of the PLC in peptide loading and quality control of MHC I are unraveled, and the PLC was analyzed from an evolutionary perspective. First, composition and architecture of native PLC isolated from different mammalian species was analyzed. Comparison of detergent-solubilized PLC from cow and sheep spleens with PLC isolated from human source showed a compositional conservation in mammals, with the central components TAP, ERp57, tapasin, calreticulin, and the MHC I heterodimer were conserved in these species. Negative-stain electron microscopy (EM) analyses revealed an identical overall architecture of PLCs from human, sheep, and cow with two major densities at opposing sides of the plane of the detergent micelle corresponding to endoplasmic reticulum (ER) luminal and cytosolic domains. Interestingly, the glucose-regulated protein 78 (GRP78) was associated only with the PLC from sheep and cow as revealed by mass spectrometry. This ER chaperone is involved in initial folding steps of MHC I but was not co-purified with human PLC, rendering it an interesting target for future functional and in-depth structural studies. The human PLC was stabilized by reconstitution in membrane mimicking systems that replace the detergent, which is necessary to solubilize the complex. This stabilization allowed detailed structural analysis by single-particle cryogenic electron microscopy (cryo-EM). The structure of the MHC I editing module in the PLC, composed of tapasin, ERp57, calreticulin, MHC I, and β-2-microglobulin (β2m), was solved at an overall resolution of 3.7 Å. Within the structure, two important features were visualized: (i) the editing loop of tapasin, which is directly involved in peptide proofreading of MHC I; (ii) the A-branch of the Asn86 tethered N-linked glycan on MHC I. Both features are crucial elements in the quality control and peptide editing process on MHC I. The editing loop interacts with the peptide binding groove in MHC I. It disturbs the interaction between a cargo peptide C terminus and the F-pocket in the binding groove by displacing Tyr84 and the helices α1 and α2. The helix displacement widens the F-pocket which allows a faster peptide exchange on MHC I. The glycan is bound in its monoglucosylated form (Glc1Man9GlcNAc2) by the lectin domain of calreticulin. The A-branch of this glycan is stretched between MHC I Asn86 and the lectin domain, leading to the hypothesis that the glycan will be released from calreticulin once MHC I is loaded with a favored peptide (pMHC I). For investigation of the glycan status of MHC I, intact protein liquid chromatography coupled mass spectrometry (LC-MS) was performed under denaturating conditions. An allosteric coupling between peptide loading and removal of the terminal glucose by α-Glucosidase II (GluII) was discovered. In addition, the PLC remained fully intact after peptide loading, which demonstrated GluII action on the PLC once MHC I is loaded. With establishing GluII as transient interaction partner, this work deepens the knowledge of the molecular sociology of the PLC and how the PLC is involved in the endoplasmic reticulum quality control (ERQC). Further investigation of the ER aminopeptidases ERAP1 and ERAP2 showed that these enzymes neither alone nor together stably interact with the PLC. In contrast, both work independent from the PLC on free peptides in the ER. LC-MS analysis of the PLC components revealed a very unusual glycosylation pattern of tapasin. Tapasin was observed with N-linked glycans ranging from the full glycan (Man9GlcNAc2) to heavily trimmed glycans, where only a single GlcNAc remained attached to Asn233. In the PLC, tapasin is probably shielded from degradation by ERQC and can remain functional and intact without a full N-linked glycan.

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HLA-B polymorphism affects interactions with multiple endoplasmic reticulum proteins

et al. 2000

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To explore the nature of amino acid substitutions that influence association with TAP, we compared a site‐directed mutant of HLA‐B*0702 (Y116D) to unmutated HLA‐B7 in regard to TAP interaction. We found that the mutant had stronger association with TAP, and, in addition, with tapasin and calreticulin. These data confirm the importance of position 116 for TAP association, and indicate that (1) an aspartic acid at the 116 position can facilitate the interaction, and (2) association with tapasin and calreticulin is affected along with TAP. Furthermore, we tested three natural subtypes of HLA‐B15, and found that a B15 subtype with a tyrosine at position 116 (B*1510) was strongly associated not only with TAP, but also with tapasin and calreticulin. In contrast, two B15 subtypes with a serine at position 116 (B*1518 and B*1501) exhibited very little or no association with any of these proteins. Thus, very closely related HLA‐B subtypes can differ in regard to interaction with the entire assembly complex. Interestingly, when their surface expression was tested by flow cytometry, the HLA‐B15 subtypes with little to no detectable intracellular assembly complex association had a slightly, yet consistently, higher level of the open heavy chain form than did the B15 subtype with intracellular assembly complex association. These data suggest that the relatively low strength or short length of interaction between endoplasmic reticulum proteins and natural HLA class I molecules can decrease their surface stability.

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Calreticulin and Calnexin Interact with Different Protein and Glycan Determinants During the Assembly of MHC Class I

Cited by 108 publications

References 61 publications

Don't eat me/eat me signals as a novel strategy in cancer immunotherapy

Don't eat me/eat me signals as a novel strategy in cancer immunotherapy

Mechanistic and structural studies of the MHC I peptide loading complex

HLA-B polymorphism affects interactions with multiple endoplasmic reticulum proteins

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