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. 2013 Dec 20;22(25):5262-75.
doi: 10.1093/hmg/ddt383. Epub 2013 Aug 15.

Armet/Manf and Creld2 Are Components of a Specialized ER Stress Response Provoked by Inappropriate Formation of Disulphide Bonds: Implications for Genetic Skeletal Diseases

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Free PMC article

Armet/Manf and Creld2 Are Components of a Specialized ER Stress Response Provoked by Inappropriate Formation of Disulphide Bonds: Implications for Genetic Skeletal Diseases

Claire L Hartley et al. Hum Mol Genet. .
Free PMC article

Abstract

Mutant matrilin-3 (V194D) forms non-native disulphide bonded aggregates in the rER of chondrocytes from cell and mouse models of multiple epiphyseal dysplasia (MED). Intracellular retention of mutant matrilin-3 causes endoplasmic reticulum (ER) stress and induces an unfolded protein response (UPR) including the upregulation of two genes recently implicated in ER stress: Armet and Creld2. Nothing is known about the role of Armet and Creld2 in human genetic diseases. In this study, we used a variety of cell and mouse models of chondrodysplasia to determine the genotype-specific expression profiles of Armet and Creld2. We also studied their interactions with various mutant proteins and investigated their potential roles as protein disulphide isomerases (PDIs). Armet and Creld2 were up-regulated in cell and/or mouse models of chondrodysplasias caused by mutations in Matn3 and Col10a1, but not Comp. Intriguingly, both Armet and Creld2 were also secreted into the ECM of these disease models following ER stress. Armet and Creld2 interacted with mutant matrilin-3, but not with COMP, thereby validating the genotype-specific expression. Substrate-trapping experiments confirmed Creld2 processed PDI-like activity, thus identifying a putative functional role. Finally, alanine substitution of the two terminal cysteine residues from the A-domain of V194D matrilin-3 prevented aggregation, promoted mutant protein secretion and reduced the levels of Armet and Creld2 in a cell culture model. We demonstrate that Armet and Creld2 are genotype-specific ER stress response proteins with substrate specificities, and that aggregation of mutant matrilin-3 is a key disease trigger in MED that could be exploited as a potential therapeutic target.

Figures

Figure 1.
Figure 1.
Armet and Creld2 are increased in V194D matrilin-3 mouse chondrocytes, cell culture models and growth plate cartilage. (A) Chondrocytes were isolated from the rib cartilage of 5-day-old Matn3 V194D [m/m] and wild-type (WT/WT) mice. Total protein from 1 × 105 cells was analysed by SDS–PAGE and western blotting using antibodies against Armet (∼18 kDa) and Creld2 (∼45 kDa). Equal protein loading was verified by Ponceau staining and three litters per genotype (∼5–10 mice pooled per litter) were analysed in three separate experiments. (B) Western blots were scanned and analysed by densitometry which demonstrated that there was a ∼4-fold increase in Armet and a ∼2-fold increase in Creld2 (independent t-test, **P < 0.01). (C) Cell lysate samples from CHO and HEK-293 cells expressing wild-type and V194D matrilin-3 were analysed by SDS–PAGE and western blotting and increased amounts of Armet and Creld2 were detectable in lysates from cells expressing the V194D mutation in both the full-length (FLM3) and single A-domain forms. Equal protein loading was verified by Ponceau staining. (D) Dual-labelling immunofluorescence microscopy confirmed that V194D matrilin-3 (green) and Armet (red) co-localized as an intracellular protein accretion (yellow/orange); DAPI was used to identify cell nuclei. (E) IHC using Armet and Creld2 antibodies on the tibia growth plates from 3-week-old wild-type (WT) and V194D mutant mice (mm). Chondrocytes in all zones of the mutant growth plate showed increased levels of intracellular staining for both Armet and Creld2. Interestingly, staining was also observed in the ECM of tibia growth plate cartilage. Scale bar is 100 µm; kDa = kilodaltons.
Figure 2.
Figure 2.
Creld2 and Armet are not up-regulated in mouse models of COMP-related PSACH-MED, but are increased in a model of MCDS. (A) IHC using Armet and Creld2 antibodies on tibia growth plates from 3-week-old Comp T585M (T585M), Comp D469del (D469) and matched wild-type (WT) mice. No increase in staining was observed for Armet or Creld2 in either mutant (mm) mouse model compared with wild-type (WT). (B) Chondrocytes were isolated from the cartilage of 5-day-old Comp T585M (T585M), Comp D469del (D469) and wild-type (WT) mice. Total protein from 1 × 105 cells was loaded per lane and analysed by SDS–PAGE and western blotting. No detectable differences in the levels of Creld2 and Armet were observed in cell extracts from mutant mice (T585M and D469) compared with wild-type (WT) controls. Equal protein loading was verified by Ponceau staining. (C) IHC on tibia growth plates from 3-week-old Col10a1 N617K (N617K) mice demonstrated both intracellular and ECM staining in the hypertrophic zone of growth plate cartilage. Scale bar is 100 µm; kDa = kilodaltons.
Figure 3.
Figure 3.
Armet and Creld2 interact in a complex with matrilin-3, but not with COMP. (A) Cell lysate proteins of HEK293 cells transfected with wild-type (WT) and V194D matrilin-3 expression constructs (FLAG-tagged full-length (FLM3) and the single A-domain) were immunoprecipitated with ANTI-FLAG affinity gel. SDS–PAGE and western blotting demonstrated that ERp72, Creld2 and Armet were co-precipitated with mutant matrilin-3 alone and that these interactions could be mediated by the A-domain (right panel). GAPDH confirmed equal loading and cell lysates from untransfected HEK 293 cells was used as a control. (B) Cell lysate proteins of HT1080 cells transfected with GFP alone or GFP-tagged wild-type COMP (WT COMP) and D469del mutant COMP (D469) expression constructs were immunoprecipitated with anti-GFP-sepharose beads. SDS–PAGE and western blotting did not identify any interactions between COMP and Armet or Creld2 (right panel). GAPDH confirmed equal loading and cell lysates from untransfected HT1080 cells was used as a control. Key: FLM3 = full-length matrilin-3; A-domain = A-domain alone comprising residues 77–263 of matrilin-3; kDa = kilodaltons.
Figure 4.
Figure 4.
Creld2 processes putative PDI-like activity, whereas Armet does not. Total cell lysate proteins from HT1080 cells stably transfected with Armet-V5, Creld2-V5 or ERp72-V5 substrate-trapping mutants were separated by SDS–PAGE and analysed by western blotting with an anti-V5 antibody. (A) There was no evidence of higher order mixed disulphides formed between putative substrate proteins and either wild-type Armet (WT) or the individual substrate-trapping mutants (N/C-CXXA, C-CXXA and N-CXXA). (B) In comparison, the control ERp72 substrate-trapping mutant (C-CXXA) demonstrated the formation of mixed disulphides with substrate proteins. (C) Both the N-CXXA and N/C-CXXA substrate-trapping mutants of Creld2 formed high-molecular weight mixed disulphides (left panel) that were resolved on reduction (right panel). In contrast, wild-type Creld2 (WT) and the C-CXXA trapping mutant did not form higher molecular weight aggregates with putative substrate proteins. (D) V5 co-immunoprecipitated proteins from the various Creld2 substrate-trapping cell lines were resolved by SDS–PAGE and viewed by silver staining or instant blue (insert). Total protein pools >50 kDa were excised from each lane of the instant blue gel for liquid chromatography-mass spectrometry/MS analysis. Key: N/C-CXXA = amino and carboxyl terminal double substrate-trapping mutant; C-CXXA = carboxyl terminal substrate-trapping mutant; N-CXXA = amino terminal substrate-trapping mutant; WT = wild- type Armet or Creld2; HT1080 = untransfected HT1080 cells; lysate = total protein lysate prior to V5 co-immunoprecipitation; R = reduced protein samples; NR = non-reduced protein samples; kDa = kilodaltons.
Figure 5.
Figure 5.
N-CXXA Creld2 substrate-trapping mutant shows specificity for mutant matrilin-3. Wild-type Armet-V5 and Creld2-V5 (WT) and the various substrate-trapping (N/C-CXXA, C-CXXA and N-CXXA) cell lines were co-transfected with wild-type (WT M3) or V194D mutant (V194D) matrilin-3 expression constructs. (A) Reducing SDS–PAGE and western blotting (anti-FLAG) on total cell lysate proteins confirmed the co-expression of WT and V194D matrilin-3 in all Armet cell lines (left panel: WT Armet and the various substrate-trapping lines). However, non-reducing SDS–PAGE and western blotting for Armet (anti-V5) did not detect any higher order mixed disulphides in all substrate-trapping and wild-type cell lines (right panel). (B) Co-immunoprecipitation with V5 followed by reducing SDS–PAGE and western blotting for Creld2 (anti-V5) and matrilin-3 (anti-FLAG) confirmed interactions between full-length V194D matrilin-3 and only the N/C-CXXA and N-CXXA substrate-trapping cell lines (left panel). When these samples were run under non-reducing conditions, the presence of matrilin-3 (anti-FLAG) containing higher order mixed disulphide complexes was demonstrated (right panel). Total cell lysate proteins from HT1080 cells (either untransfected or transfected with WT Creld2) were used controls. Key: WT M3 = wild-type matilin-3; V194D = V194D mutant matrilin-3; N/C-CXXA = amino and carboxyl terminal double substrate-trapping mutant; C-CXXA = carboxyl terminal substrate-trapping mutant; N-CXXA = amino terminal substrate-trapping mutant; WT = wild-type Armet or Creld2; HT1080 = untransfected HT1080 cells; kDa = kilodaltons.
Figure 6.
Figure 6.
Mutant matrilin-3 forms non-native disulphide bonded aggregates that can be resolved by deletion of the A-domain terminal cysteine residues. SDS–PAGE and western blot analysis confirmed the intracellular retention and aggregation over time (t = day 0 or day 3 following confluency after transfection) of V194D mutant matrilin-3 (V194D) in the cell lysate of (A) cellular models or (B) isolated mouse chondrocytes. (A; left panel) demonstrates that only wild-type (WT) matrilin-3 is secreted into the culture medium predominately as a tetramer, whereas as V194D mutant matrilin-3 (V194D) is retained initially as various oligomeric forms (t = 0), but these aggregate over time (t = 3) to form high-molecular weight aggregates. (A; right panel) confirms that aggregation is mediated through the A-domain by the presence of various oligomeric forms (1x to 5x). V194D and R121W are typical MATN3-MED mutations, whereas E252K is a known polymorphism and is comparable to wild type (WT). (C) In vitro substitution of the terminal cysteine residues of the A-domain (V194D_no cys) resolved mutant protein aggregation (V194D) and did not affect oligomerization of either the wild-type (WT_no cys) or mutant proteins when analysed by non-reducing SDS–PAGE and western blotting (anti-FLAG). (D) Substitution of the terminal cysteine residues with alanine promoted secretion of the mutant V194D protein (V194D-no cys) as visualized by reducing SDS–PAGE. Furthermore, (E) there was also selective reduction in the levels of ERp72, Creld2, and Armet, but not GRP78 or GRP94. Ponceau staining (not shown) and GAPDH were used as loading controls. Key: WT = wild-type matrilin-3; V194D = mutant matrilin-3; V194D_no cys & WT_no cys = matrilin-3 with cysteine residues 77 and 263 replaced with alanine (mutant and wild-type respectively); 293 = cell lysate from untransfected HEK293 cells; agg = non-native disulphide bonded aggregates of mutant matrilin-3; q = tetramer, t = trimer, d = dimer and m = monomers of matrilin-3; ns = non-specific band; kDa = kilodaltons. All gels show full-length matrilin-3 unless stated otherwise and the number of replicates in (E) is indicated.

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