The transmembrane domain and luminal C-terminal region independently support invariant chain trimerization and assembly with MHCII into nonamers

Abstract

Background: Invariant chain (CD74, Ii) is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations.

Results: Our results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules.

Conclusions: Altogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.

Keywords: Antigen presentation; CD74; MHCII; Nonamerization; RXR; Transmembrane domain; Trimerization domain.

Fig. 1

Formation of Ii trimers in absence of the TRIM domain. a Schematic representation of WT p35, p35_LIML and p35_LIMLTRIM. The glycosylation sites, the CLIP region, the TM and the RxR motif have been illustrated on the different p35 molecules. Leucine-based endosomal targeting motifs (di-leucine; open circles in p35) were mutated to alanines in p35_LIML and p35_LIMLTRIM. α-helices responsible for the C-terminal trimerization (TRIM domain) were deleted (Δ aa128 to 216 of p33) in the p35_LIMLTRIM mutant. Loss of the BU45 mAb epitope is coped with by addition of a myc/His-tag (detection 9e10 mAb). The Ii top view highlights how the trimerization domains oligomerize. The top view of the Ii_TRIM mutant shows the myc tag and the trimerization via the TM domains (circles) of Ii moieties. b HEK293T cells were transiently transfected with empty (mock) plasmid, p35, p35_LIML or p35_LIMLTRIM. Cells were lysed in presence of reducible cross-linker DSP (400 µg/ml). Proteins were separated by SDS-PAGE in non-reducing conditions. Samples were blotted using a rabbit anti-CLIP serum. Monomeric (Ii₁, TRIM₁), dimeric (Ii₂, TRIM₂) and trimeric (Ii_3, TRIM₃) forms are indicated on the right. Full-length blot is presented in Additional file 1: Figure 1A. c Histograms depicts the densitometry of Western blot bands (trimers and dimers) in relation to bands associated with monomers for each molecule. The densitometry was performed on blots from 3 independent experiments, as shown in (b). d HEK293T cells were transiently transfected with an empty plasmid (mock) or plasmids expressing p35, p35_LIMLTRIM or p35 and p35_LIMLTRIM. Cells were lysed and immunoprecipitated using 9e10. Immunoprecipitated material and cells lysates were analyzed by SDS-PAGE and immunoblotted for WT p35 using Pin.1. Ig identifies antibody heavy chain detected in lanes with beads and IP products. Full-length blot is presented in Additional file 1: Figure 1B

Fig. 2

Ii C-terminal TRIM is not necessary for binding to MHCIIs and to egress the ER. a Schematic representation of Δ20 and Δ20_TRIM. The lumenal glycosylation sites, CLIP region and TRIM are illustrated on the Ii molecule. Di-leucine endosomal targeting motifs (open circles) were removed following deletion of the first N-terminal cytoplasmic 20 aa (gray box) of p33 in Δ20 and Δ20_TRIM mutants. As for p35_LIMLTRIM, the TRIM domain was deleted (Δ aa128 to 216 of p33) in the Δ20_TRIM mutant and a myc/His-tag was added (detection 9e10 mAb). The Ii top view highlights how the trimerization domains oligomerize. The top view of Δ20_TRIM mutant shows the myc tag and trimerization via the TM (grey circles). b Ii Δ20, Δ20_TRIM, p35_LIML or p35_LIMLTRIM were transiently transfected in HEK293T cells. After 48 h, cells were stained to detect surface (black dotted line) and total (black line) Ii using mAbs BU45 or 9e10 (for Ii_TRIM mutants). c Cells were transfected as above together with DR and stained for surface and total Ii. d Cells from (c) and cells transfected as in (c) together with DM were stained for surface CLIP using CerCLIP.1 Ab. CLIP MFIs (mean fluorescence intensity) are expressed in a bar-chart. Error bars indicate the SD from at least five independent experiments. Student t-tests were performed; *p ≤ 0.001 and **p ≤ 0.05

Fig. 3

Trimerization through TRIM is sufficient for the formation of high-order complexes. a Schematic representation of WT DRα + DRβ/Ii and DRα + βSCD. Top view highlights αβ interaction with the CLIP domain. Top view of an Ii₃ with αβ dimers illustrates a nonamer (αβIi)₃. In βSCD, Ii and DRβ luminal domains are linked by a flexible gly₃/ser/gly₃ linker. Top views show association of DRα to βSCD and trimerization of the complex via TRIM. b HEK293T were mock-transfected or transfected with DRα + DRβ + Ii or with DRα + βSCD. Cell lysates, with or without EndoH, were analysed by WB and blotted for DRβ (XD5 mAb). Arrowhead represents EndoH resistant WT DRβ. Open arrowhead and arrowhead represent βSCD and a cleavage product of βSCD, respectively, both resistant to EndoH. c DRα + βSCD were transiently transfected in HEK293T. After 48 h, cells were stained to detect MHCII, Ii and CLIP (L243, BU45 and CerCLIP.1 mAbs, respectively) (left histogram). Cells were transfected as above as well as with DM and stained for surface CLIP or permeabilized to detect DM (Map.DM1 mAb) (right histogram). d Cells were either mock-transfected, transfected with DRα + βSCD alone or with p33 or p35. Cell lysates (right lanes) and material precipitated with the Ii-specific Pin.1 mAb (left lanes) were analyzed by WB. DRα was detected using the DA6.147 mAb. H chains of Ig used for IPs are indicated. e Samples from (d) were analyzed using the XD5.117 mAb specific for the DRβ1 extracellular domain. In e, d, Ig identifies antibody heavy chain detected in lanes with IP products. f–h Cells were transfected with DRα + βSCD alone or with p35_LIML or p35_LIMLTRIM. f Cell surface MFI for CLIP (CerCLIP.1 mAb) are represented in bar charts. g, h MFI ratios obtained for surface versus total expression of MHCII (using L243) and Ii (using BU45) are represented in bar charts. Error bars indicate the SD from five independent experiments. Student t-tests were performed; *p ≤ 0.001 and **p ≤ 0.05. Full-length blots from this figure are presented in Additional file 1: Figure 1C–E

Fig. 4

Lack of bidirectional TRIM interactions prevents formation pseudo-heptamers and/or pseudo-pentamers including βSCD. a Schematic representation of DRα and βSCD_TRIM. In βSCD_TRIM, the Ii_TRIM and DRβ luminal domains are linked by a flexible gly₃/ser/gly₃ linker. Top view on the right shows association of an α chain to the βSCD_TRIM. b DRα and βSCD_TRIM were transiently expressed in HEK293T cells (upper histogram). After 48 h, cells were stained to detect MHCII, Ii and CLIP using L243, 9e10 and CerCLIP.1 mAbs, respectively. Cells were transfected as above, as well as with DM and stained for surface CLIP. A fraction of the cells was permeabilized to detect DM using Map.DM1 mAb (lower histogram). c–e Cells were transfected with DRα and βSCD_TRIM alone or together with p35_LIML or p35_LIMLTRIM. After 48 h, cells were stained to detect surface CLIP (CerCLIP.1) (c). The surface over total expression ratio of MHCII (L243) (d) and Ii (BU45) (e) were calculated. Error bars indicate the SD from five independent experiments. Student t-tests were performed; *p ≤ 0.001

Fig. 5

The TM domain supports formation of nonameric complexes in the absence of TRIM. a Left; schematic representation of DRα, DRβ_myc and DRβ_KKAA. Right; illustration of the rational behind formation of pentamers and nonamers using the DRβ_KKAA. If pentameric (DRαβ)₁(Ii)₃ complexes can egress the ER, the ER-retained (DRα + DRβ_KKAA)₁(Ii_TRIM)₃ complex will not impact WT (DRα + DRβ)₁(Ii_TRIM)₃ complexes. In contrast, if formation of nonamers is the only outcome due to the expression of Iip35, the ER-retained DR_KKAA will trap a co-expressed WT DR that is incorporated in a same complex. b–f HEK293T cells were transiently transfected with DRα and DRβ_myc (DR) and/or DRα + DRβ_KKAA (DR_KKAA) together Δ20_TRIM or p35_LIMLTRIM. After 48 h, cells were analyzed by flow cytometry to evaluate surface to total expression ratio of MHCII using L243 (b). c Representative histograms showing surface expression of Ii and d bar graph shows surface to total expression ratio of Ii using BU45. e Representative histograms showing CLIP surface expression and f bar graph shows surface of CLIP using Cer-CLIP.1. Ctrl represent isotype control antibodies. b, d, e Error bars indicate the SD from at least three independent experiments. Student t-tests were performed; *p ≤ 0.001 and **p ≤ 0.05

Fig. 6

DR is retained by DR_KKAA upon formation of nonameric-like structures. a Schematic representation of αSCD and DRβ and illustration of the rationale behind the use of αSCD with DRβ_KKAA. Left; DRα and Ii luminal domains are linked by a flexible gly₃/ser/gly₃ linker (αSCD). Top view shows the association of αSCD with a β chain. Middle; when co-expressed with DRβ_KKAA, DRβ will still egress the ER if αSCD does not trimerize via the TRIM domain. Right; formation of a trimer through TRIM will force the incorporation of both DRβ and DRβ_KKAA in the same complex, which will be ER retained. b HEK293T cells were mock-transfected or transfected with αSCD or with αSCD and DRβ. Cell lysates were treated with or without EndoH and blotted for DRα (DA6.147). Open arrowhead and arrowhead represent EndoH resistant forms of αSCD with different types of complex sugars. Star represents the EndoH sensitive αSCD. Arrow represents cleavage products of αSCD. Full-length blot is showed in Additional file 1: Figure 1F. c–e HEK293T cells were transiently transfected with DRβ_myc (DR) and/or DRβ_KKAA together with αSCD. After 48 h, cells were analyzed by flow cytometry to evaluate CLIP surface expression, using CerCLIP.1 (c), MHCII (d) and Ii (e) surface over total expression ratio using L243 and BU45, respectively. Representative histograms of CLIP surface expression (c), MHCII surface and total expression (d) and Ii surface and total expression (e) are shown. Ctrl represent isotype control antibodies. Error bars indicate the SD from at least three independent experiments. Student t-tests were performed; *p ≤ 0.001 and **p ≤ 0.05