Recruitment of MHC Class I Molecules by Tapasin into the Transporter Associated with Antigen Processing-Associated Complex Is Essential for Optimal Peptide Loading

Abstract

The ER protein tapasin (Tpn) forms a bridge between MHC class I H chain (HC)/β2-microglobulin and the TAP peptide transporter. The function of this TAP-associated complex was unclear because it was reported that soluble Tpn that has lost TAP interaction would be fully competent in terms of peptide loading and Ag presentation. We found, however, that only wild-type human Tpn (hTpn), but not three soluble hTpn variants, a transmembrane domain point mutant of hTpn (L410→F), wild-type mouse Tpn, nor a mouse-human Tpn hybrid, fully up-regulated peptide-dependent Bw4 epitopes when expressed in Tpn-deficient .220.B*4402 cells. Consistent with suboptimal peptide loading, the t1/2 of class I molecules was considerably reduced in the presence of soluble hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra and the class I-mediated inhibition of NK clones showed distinct differences to the hTpn transfectant. Only wild-type hTpn efficiently recruited HC and calreticulin (Crt) into complexes with TAP and endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in TAP association, but bound to class I molecules, Crt, and ERp57. Mouse Tpn associated with human TAP and ERp57 on the one hand, and with HC and Crt on the other, but failed to recruit normal amounts of HLA class I molecules into the TAP complex. We conclude that the loading with peptides conferring high stability requires the Tpn-mediated introduction of HC into the TAP complex, whereas the mere interaction with Tpn is not sufficient.

Intracellular peptide transporters in human – compartmentalization of the “peptidome”

“…First, it stabilizes TAP as TAP expression is drastically decreased in tapasin-deficient cells [5,25,51,75]. Second, by MHC class I binding and recruitment of ERp57 [19,71], tapasin coordinates and facilitates peptide loading of MHC class I molecules [50,62,83,92,102]. Tapasin-deficient mice show an impaired immune response and an altered peptide repertoire presented on MHC class I molecules [24].…”

Generation of a functional, soluble tapasin protein from an alternatively spliced mRNA

“…13 It also enhances the rate and extent of class I MHC loading with optimal peptides. [14][15][16][17] In tapasin-deficient cells, the repertoire of class I-bound peptide is thus skewed towards less stably binding ones, a phenomenon that is consistent with the phenotype of C57BL/6 mice that lack tapasin and B2705 expressed in 0.220. [18][19][20] Tapasin is a type I transmembrane glycoprotein with a dilysine ER-retention motif (KxKxx) in its cytoplasmic tail.…”

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function

“…Weak co-IP of TPSN Ex3 by ERp57 was albeit found, indicating that an indirect interaction via other PLC components might occur (discussed later). Similarly, calreticulin was found to co-precipitate TPSN Ex3 although to a lesser extent than tpn.Previous studies have shown that the TMSs confer the interaction between TAP and tpn [6,8,9]; therefore, as expected, binding to TAP was not affected by deletion of exon 3. Interestingly however, we detected a weak but reproducible interaction between TPSN Ex3 and MHC-I.…”

“…Proper peptide loading of MHC-I is controlled by several chaperones that form the peptide loading complex (PLC) in the ER [4]. In the PLC the MHC-I heterodimer associates with tapasin (tpn), calreticulin, ERp57, and indirectly with TAP; tpn and TAP interact via their transmembrane segments (TMSs), thereby stabilizing the structure and function of TAP [5][6][7][8][9]. It has been shown that both TAP1 and TAP2 subunits possess binding sites for tpn at their N-terminal TMSs [10][11][12].…”