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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: J Invest Dermatol. 2021 Aug 2;142(2):323–332.e8. doi: 10.1016/j.jid.2021.07.154

Differential Pathomechanisms of Desmoglein 1 Transmembrane Domain Mutations in Skin Disease

Stephanie E Zimmer 1,2, Takuya Takeichi 3, Daniel E Conway 4, Akiharu Kubo 5, Yasushi Suga 6, Masashi Akiyama 3, Andrew P Kowalczyk 1,7
PMCID: PMC9109890  NIHMSID: NIHMS1799481  PMID: 34352264

Abstract

Dominant and recessive mutations in the desmosomal cadherin, desmoglein (DSG) 1, cause the skin diseases palmoplantar keratoderma (PPK) and severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, respectively. In this study, we compare two dominant missense mutations in the DSG1 transmembrane domain (TMD), G557R and G562R, causing PPK (DSG1PPK-TMD) and SAM syndrome (DSG1SAM-TMD), respectively, to determine the differing pathomechanisms of these mutants. Expressing the DSG1TMD mutants in a DSG-null background, we use cellular and biochemical assays to reveal the differences in the mechanistic behavior of each mutant. Super-resolution microscopy and functional assays showed a failure by both mutants to assemble desmosomes due to reduced membrane trafficking and lipid raft targeting. DSG1SAM-TMD maintained normal expression levels and turnover relative to wildtype DSG1, but DSG1PPK-TMD lacked stability, leading to increased turnover through lysosomal and proteasomal pathways and reduced expression levels. These results differentiate the underlying pathomechanisms of these disorders, suggesting that DSG1SAM-TMD acts dominant negatively, whereas DSG1PPK-TMD is a loss-of-function mutation causing the milder PPK disease phenotype. These mutants portray the importance of the DSG TMD in desmosome function and suggest that a greater understanding of the desmosomal cadherin TMDs will further our understanding of the role that desmosomes play in epidermal pathophysiology.

INTRODUCTION

Epidermal integrity relies on intercellular junctions such as desmosomes to protect against abrasion, infection, and water loss. Desmosomes mediate strong cell–cell adhesion by mechanically coupling adjacent cells through a series of protein interactions involving desmosomal cadherins and intracellular adaptor proteins that anchor intermediate filaments to the plasma membrane (Broussard et al., 2020; Zimmer and Kowalczyk, 2020). Desmosomes are targeted in numerous skin fragility diseases caused by autoantibodies, bacterially produced toxins, and gene mutations (Brooke et al., 2012; Lee and McGrath, 2021; Nitoiu et al., 2014; Samuelov and Sprecher, 2015; Stahley and Kowalczyk, 2015). Among genetically inherited diseases, haploinsufficiency of the desmosomal cadherin, desmoglein (DSG) 1, causes a relatively mild disorder called palmoplantar keratoderma (PPK) in which epidermal thickening occurs along the palms and soles (Hennies et al., 1995). In contrast, severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome has broader effects arising from epidermal thickening, fragility, and barrier defects typically caused by recessively inherited DSG1 loss of function mutations (Samuelov et al., 2013).

DSG1 is critical in the epidermis where it promotes differentiation and becomes the main DSG mediator of desmosomal adhesion in the upper epidermal layers (Getsios et al., 2009; Harmon et al., 2013). The differences in severity between PPK and SAM syndrome may be due to gene dosage; PPK arises from DSG1 haploinsufficiency, whereas the complete loss of functional DSG1 in SAM syndrome produces a more severe clinical presentation (Has et al., 2015). Recently, we reported an unusual case of SAM syndrome (referred to as DSG1SAM-TMD) in which a dominantly inherited missense mutation in DSG1 caused an arginine substitution at residue G562 in the DSG1 transmembrane domain (TMD) (Lewis et al., 2019). In this study and in a related study (Takeuchi et al., unpublished data), we report a second instance of a missense mutation in the DSG1TMD. This mutation also causes an arginine substitution, occurring at position G557 within the DSG1TMD, but the patient presents with PPK (referred to as DSG1ppk-TMD). Despite the genetic similarities between these cases, the phenotypic disparity suggests differing pathomechanisms underlying an important role for the DSGTMD in desmosomal processes.

In this study, we sought to determine the disease mechanisms of DSG1PPK-TMD versus DSG1SAM-TMD mutations and to establish a role for the DSGTMD in desmosomal processes. By expressing these mutants in a DSG-null background, we identified defects in membrane trafficking and lipid raft targeting. Both DSG1PPK-TMD and DSG1SAM-TMD failed to support normal desmosomal processes such as assembly and/or maintenance and were deficient in cell–cell adhesion strength. Mechanistically, DSG1PPK-TMD is highly unstable, rapidly undergoing lysosomal and proteasomal degradation. In contrast, DSG1SAM-TMD is expressed at levels similar to those of wild-type (WT) DSG1 and maintains normal cell surface stability. These findings reveal that different mutations within the DSG1TMD cause different types of inherited human skin disease by distinct mechanisms and suggest that different therapeutic strategies should be explored for treating these related disorders.

RESULTS

DSG1PPK-TMD and DSG1SAM-TMD support the formation of fewer, smaller, and weaker desmosomes

We previously reported a heterozygous missense mutation in DSG1TMD (G562R) that disrupts DSG1 association with lipid rafts, causing SAM syndrome (Lewis et al., 2019). In this study and in related work (Takeuchi et al., unpublished data), we report a second heterozygous DSG1TMD missense mutation G1669A resulting in a different glycine-to-arginine substitution in the DSG1TMD (G557R) (Supplementary Figure S1), which causes PPK rather than SAM syndrome. To understand how these two DSG1TMD mutations cause different skin diseases, we stably expressed mouse DSG (Dsg1a) open reading frame with the corresponding disease-causing TMD mutations (DSG1WT, DSG1PPK-TMD, DSG1SAM-TMD) in DSG-null A431 cells. Dsg1a shares 78% amino acid sequence identity with DSG1; most differences are in the extracellular anchor. A431 cells are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies (Bornslaeger et al., 1996; Roberts et al., 2016; Setzer et al., 2004). Our analysis of DSG isoform expression in these cells verified that DSG2 is the predominant DSG expressed (Schäfer et al., 1994) (Figure 1a). Therefore, we used CRISPR to knockout DSG2 and verified its loss by immunofluorescence and western blot (Figure 1b and c). The DSG2-knockout cells exhibit desmosomal protein mislocalization without changes in the expression levels and loss of adhesion strength as assessed using a dispase-based monolayer fragmentation assay (Figure 1b–e and Supplementary Figures S2 and S3). These data indicate that loss of DSG2 expression in A431 cells reduces desmosome adhesive function.

Figure 1. DSG-null cells exhibit mislocalized DP and reduced desmosome function.

Figure 1.

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(a) Widefield immunofluorescence images showing DSG2 but not DSG1 or DSG3 are expressed in A431 cells. Cells known to express each DSG species were used for positive controls: DSG1-GFP–transfected A431 cells; HaCaTs express DSG2 and DSG3 endogenously. (b) Western blot showing DSG2 knockout in A431 cells with no change in DP levels. β-Actin was used as the loading control. (c) Widefield immunofluorescence images showing a lack of DSG2 staining in DSG-null cells and DP mislocalization. (d) Images show the monolayer fragmentation of A431 parentals versus that of DSG-null cells. (e) Quantification of d. Bar = 10 μm. Error bars show the mean ± SEM (n = 7). ****P < 0.0001. DSG, desmoglein; DP, desmoplakin.

The DSG-null A431 cells were used as a model system to assess the function of DSG1SAM-TMD and DSG1PPK-TMD. DSG-null A431 cells stably expressing DSG1WT-GFP, DSG1PPK-TMD-GFP, or DSG1SAM-TMD-GFP (Figure 2a) were established using a lentiviral system and bulk sorted to create populations with roughly equal expression patterns (Supplementary Table S1). Immunofluorescence (Figure 2b) and western blot analysis (see additional analysis in Figure 6) revealed reduced levels of DSG1PPK-TMD-GFP relative to DSG1WT-GFP or DSG1SAM-TMD-GFP. Therefore, the brightness and contrast for DSG1PPK-TMD-GFP images were adjusted to assess localization differences between the DSG1 variants (Supplementary Figure S4 shows the unadjusted images). Total desmoplakin (DP), desmocollin 2, plakoglobin (PG), and E-cadherin levels were unaffected (Supplementary Figure S5). We found that both DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP could be detected with DP in cell border–localized puncta. However, whereas DP is properly localized at borders in cells expressing DSG1WT-GFP, it remained mislocalized in cells expressing DSG1PPK-TMD-GFP or DSG1SAM-TMD-GFP. Quantifying the number of puncta positive for DSG1-GFP and DP revealed fewer puncta per border in the mutant-expressing cells than in those expressing DSG1WT-GFP (Figure 2c). These findings suggest that G:R substitutions in the DSG1TMD compromise the ability of DSG1 to support normal desmosome formation.

Figure 2. DSG1PPK-TMD and DSG1SAM-TMD support the formation of fewer, weaker desmosomes.

Figure 2.

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(a) Amino acid sequences of DSG1WT, DSG1PPK, and DSG1SAM TMDs (blue = TMD residues, gray = non-TMD residues). (b) Widefield immunofluorescence images show the localization of DSG1WT-GFP, DSG1PPK-TMD-GFP, and DSG1SAM-TMD-GFP with DP. (c) Quantification of border puncta positive for DP and DSG1-GFP. Error bars show the mean ± SEM (n = 3). (d) Images show the monolayer fragmentation of DSG-null cells versus that of DSG-null cells expressing DSG1WT-GFP, DSG1PPK-TMD-GFP, or DSG1SAM-TMD-GFP. (e) Quantification of fragments in d. Bar = 10 μm. Error bars show the mean ± SEM (n = 7). **P < 0.01, ****P < 0.0001. DSG, desmoglein; DP, desmoplakin; PPK, palmoplantar keratoderma; SAM, severe dermatitis, multiple allergies, and metabolic wasting; TMD, transmembrane domain; WT, wild-type.

Figure 6. Inhibiting protein degradation rescues DSG1PPK-TMD levels.

Figure 6.

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(a) Western blot of whole-cell lysates shows reduced DSG1PPK-TMD-GFP levels. DP levels are unchanged. β-Actin shows equal loading. Two lanes per sample. (b) Quantification of DSG1-GFP blot in a. (c) Western blots depict the DSG1-GFP levels after 0, 1, 3, or 6 hours of MG132 treatment. β-Actin shows equal loading. (d) Quantification of blots in c. (e) Western blots show DSG1-GFP levels after 0, 1, 3, or 6 hours of primaquine treatment. β-Actin shows equal loading. (f) Quantification of blots in e. Error bars show the mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ****P < 0.0001. DP, desmoplakin; DSG, desmoglein; ns, nonsignificant; PPK, palmoplantar keratoderma; TMD, transmembrane domain.

To assess the adhesive potential of the various cell lines, we utilized a dispase-based monolayer fragmentation assay in which increased monolayer fragmentation correlates with decreased cell–cell adhesion strength (Huen et al., 2002). DSG1WT-GFP expression restored adhesion in the DSG-null A431 cells, whereas DSG1SAM-TMD-GFP failed to rescue desmosome function. DSG1PPK-TMD-GFP exhibited an intermediate adhesive phenotype (Figure 2d and e). These results suggest that the DSG1SAM-TMD mutation compromises desmosomal adhesion more than the DSG1PPK-TMD mutation.

Desmosomes are characterized by the formation of mirror-image cytoplasmic plaque structures that appear as railroad track staining patterns when resolved by super-resolution optical imaging (Stahley et al., 2016a, 2016b). To determine the cause of the reduced function in desmosomes formed from DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP, we used structured illumination microscopy to examine desmosome organization (Figure 3a). We localized the DSG1-GFP variants with DP, a mature desmosome marker, and quantified images by measuring desmosome length and desmosomes per border. Cells expressing DSG1WT-GFP formed more, longer desmosomes than cells expressing DSG1PPK-TMD-GFP or DSG1SAM-TMD-GFP (Figure 3b and c). Together, these findings suggest that DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP support the formation of desmosomes that are fewer, smaller, and weaker than desmosomes formed from DSG1WT-GFP.

Figure 3. DSG1PPK-TMD and DSG1SAM-TMD desmosomes are smaller than DSG1WT desmosomes.

Figure 3.

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(a) SIM images stained for GFP and DP depict cell borders from DSG-null cells expressing DSG1WT-GFP, DSG1PPK-TMD-GFP or DSG1SAM-TMD-GFP. (b) Quantification of the number of desmosomes per border. (c) Quantification of desmosome length. Desmosomes are defined as regions of the membrane where DP railroad tracks sandwich DSG1-GFP fluorescence signal; an example of desmosome versus nondesmosome is shown in white boxes in a. Bar = 1 μm. Error bars show the mean ± SEM (n = 3). **P < 0.01, ****P < 0.0001. DSG, desmoglein; DP, desmoplakin; PPK, palmoplantar keratoderma; SAM, severe dermatitis, multiple allergies, and metabolic wasting; SIM, structured illumination microscopy; TMD, transmembrane domain; WT, wild-type.

DSG1PPK-TMD and DSG1SAM-TMD impact desmosome assembly by disrupting raft association

Triton X-100 insolubility of desmosomal proteins is an indication of cytoskeletal attachment and overall desmosome assembly status (Lewis et al., 2019; Pasdar and Nelson, 1989; Stahley et al., 2014). Using a Triton X-100 solubility assay, we found that DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP exhibited increased solubility compared with DSG1WT-GFP (Supplementary Figure S6a). E-cadherin solubility remained unchanged across cell lines (Supplementary Figure S6b). This finding suggests that DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP do not readily assemble cytoskeleton-attached desmosomes.

Desmosomes are intermediately sized lipid rafts (Lewis et al., 2019), highly ordered, cholesterol- and sphingolipid-enriched regions of the cell membrane important in various cell processes (Sezgin et al., 2017). Certain TMD properties target single-pass transmembrane proteins, such DSGs, to lipid rafts (Lorent et al., 2017). To determine how the arginine residues in DSG1PPK-TMD and DSG1SaM-TMD could alter the TMD properties that drive raft association of membrane proteins, we modeled each TMD α-helix using the Robetta server (Kim et al., 2004) (Figure 4a). Whereas the DSG1WT TMD is predicted to neatly traverse the membrane, the DSG1PPK-TMD and DSG1SAM-TMD models predict that arginine breaks the α-helix, effectively shortening the TMD from the normal 24 residues to 11 residues for DSG1PPK-TMD and 16 residues for DSG1SAM-TMD. Length is one of several TMD properties used to predict TMD raft affinity by calculating the energy needed for raft association (Lorent et al., 2017). We used this raft affinity model to predict the energy necessary for DSG1WT, DSG1PPK-TMD, or DSG1SAM-TMD to associate with lipid rafts and found that DSG1PPK-TMD and DSG1SAM-TMD would require more energy for raft association than DSG1WT.

Figure 4. DSG1TMD G:R substitutions reduce raft association.

Figure 4.

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(a) Robetta server modeling of DSG1wt, DSG1PPK-TMD and DSG1SAM-TMD. Blues = TMD residues; gray = non-TMD residues; red = arginine. Raft affinities (ΔGraft are displayed beneath the models. DSG1SAM-TMD adapted from Lewis et al. (2019). (b) Western blots show DRM isolation by sucrose gradient fractionation. Calnexin = non-DRM fraction marker; flotillin-2 = DRM fraction marker. The remaining blots depict DSG1-GFP. (c) Quantification of DSG1-GFP blots in b. (d) Western blots show DRM isolation by sucrose gradient fractionation of DSC2. (e) Quantification of blots in d. (f) Western blots show DRM isolation by sucrose gradient fractionation of PG. (g) Quantification of blots in f. Error bars show the mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ****P < 0.0001. DRM, detergent-resistant raft membrane; DSG, desmoglein; PG, plakoglobin; PPK, palmoplantar keratoderma; SAM, severe dermatitis, multiple allergies, and metabolic wasting; TMD, transmembrane domain; WT, wild-type.

To verify this predicted loss of DSG1 raft association in the DSG1TMD mutants, we used sucrose gradient fractionations to isolate detergent-resistant raft membranes (DRMs) from nondetergent, nonraft membranes (non-DRMs). Raft association was verified by blotting for the DRM marker, flotillin-2, and the non-DRM marker, calnexin (Figure 4b). Significantly more DSG1WT-GFP fractionated with DRMs, suggesting defective raft targeting of both DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP (Figure 4b and c). Assessing the raft association of other desmosomal proteins, we found that desmocollin 2 raft association was unaffected (Figure 4d and e), whereas less PG associated with rafts in cells expressing DSG1PPK-TMD-GFP or DSG1SAM-TMD-GFP compared with those expressing DSG1WT-GFP (Figure 4f and g), suggesting that desmocollin 2 but not PG associates with rafts independently of DSG. Together, these findings suggest that the reduced number, size, and strength of desmosomes formed by DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP may be due to defective raft targeting and the sequestration of PG away from desmosomal raft domains.

DSG1PPK-TMD and DSG1SAM-TMD exhibit distinct trafficking defects

The difference in TMD length predicted by modeling would also be expected to affect DSG1 subcellular localization (Sharpe et al., 2010). At a steady state, we found increased colocalization of DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP with the endoplasmic reticulum (ER) marker, VAPB, relative to that of DSG1WT-GFP (Supplementary Figure S7a and b). We had previously observed that DSG1SAM-TMD-GFP was retained in the Golgi after a calcium switch (Lewis et al., 2019). In this study, we observed increased colocalization of both DSG1SAM-TMD-GFP and DSG1PPK-TMD-GFP with the Golgi marker, GM130, at a steady state (Supplementary Figure S7c and d). These observations suggest that DSG1PPK-TMD and DSG1SAM-TMD exhibit delayed trafficking through the biosynthetic pathway to the cell surface.

Despite apparent trafficking defects, immunofluorescence images show plasma membrane localization of DSG1PPK-TMD-GFP and DSG1SAM-TMD-GFP (Figure 2b). Our previous work showed that steady-state surface levels of DSG1SAM-TMD-GFP are equal to that of DSG1WT-GFP (Lewis et al., 2019). Using surface biotinylation, we found reduced steady-state surface levels of DSG1PPK-TMD-GFP (Figure 5a and b). To determine whether this was due to differences in surface stability, we performed a pulse chase using an antibody against the DSG1 ectodomain. Cells were fixed but not permeabilized 0, 1, 3, or 6 hours after antibody treatment and imaged to compare the remaining cell surface levels between DSG1WT-GFP, DSG1PPK-TMD-GFP, and DSG1SAM-TMD-GFP (Figure 5c and d and Supplementary Figure S8). More DSG1PPK-TMD-GFP was lost after the first hour than DSG1WT-GFP or DSG1SAM-TMD-GFP; this trend continued up to 6 hours, suggesting that DSG1PPK-TMD-GFP is less stable at the cell surface.

Figure 5. Reduced DSG1PPK-TMD surface levels are due to decreased surface stability.

Figure 5.

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(a) Western blot depicting the surface biotinylation levels of DSG1WT-GFP and mutants. − depicts unbiotinylated condition; + depicts biotinylated condition. (b) Quantification of the blot in a. (c) Widefield immunofluorescence images of cells stained with an antibody targeting DSG1 ectodomain and fixed 0, 1, 3, or 6 hours after staining. (d) Quantification of c shows the loss of DSG1 surface levels over time. Bar = 10 μm. Error bars show the mean ± SEM (n = 4). *P < 0.05, ***P < 0.001. DSG, desmoglein; ns, nonsignificant; PPK, palmoplantar keratoderma; TMD, transmembrane domain; WT, wild-type.

Low DSG1PPK-TMD expression levels are caused by increased protein turnover rates

To determine whether retention in the ER and increased surface turnover of DSG1PPK-TMD-GFP resulted in reduced steady-state protein levels, quantitative real-time reverse transcriptase–PCR and western blot analysis were conducted for whole-cell lysates of cells expressing DSG1WT-GFP, DSG1PPK-TMD-GFP, or DSC1SAM-TMD-GFP mutants. DSG1PPK-TMD-GFP protein levels were reduced compared with those of DSG1WT-GFP and DSG1SAM-TMD-GFP, but mRNA levels were consistent across cell lines (Figure 6a and b and Supplementary Figure S9). In addition, cells were treated with MG132 or primaquine to inhibit proteasomal or lysosomal degradation, respectively (Figure 6c–f). MG132 and primaquine treatments increased steady-state levels of each DSG1 variant over a 6-hour time course; the effects were most dramatic for DSG1PPK-TMD-GFP, restoring DSG1PPK-TMD-GFP to levels similar to those seen for DSG1WT-GFP and DSG1SAM-TMD-GFP. Cycloheximide treatment resulted in more rapid loss of DSG1PPK-TMD-GFP levels than of DSG1WT-GFP, with DSG1SAM-GFP being intermediary (Supplementary Figure S10). These data indicate that the low levels of DSG1PPK-TMD-GFP are caused by increased protein degradation rates relative to those of DSG1WT-GFP and DSG1SAM-TMD-GFP.

DISCUSSION

The DSG1 mutations reported in this paper are unique among >30 known DSG1 mutations causing PPK or SAM syndrome (Akbar et al., 2019). The DSG1 ectodomain-encoding region harbors the majority of currently known mutations, most of which cause premature translation termination and transcript degradation by nonsense-mediated mRNA decay (Abi Zamer et al., 2019). The DSG1PPK-TMD and DSG1SAM-TMD mutations studied in this work both occur in the TMD but cause disease by different mechanisms. Using a DSG-null background, we find that neither mutant supports normal desmosomal processes. However, whereas DSG1SAM-TMD is expressed at the cell surface and accumulates at steady-state levels similar to those of DSG1WT, DSG1PPK-TMD exhibits lower expression levels owing to rapid turnover through lysosomal and proteasomal pathways. Our findings suggest that DSG1PPK-TMD undergoes primarily proteasomal degradation that likely occurs in association with ER compartments where the mutant accumulates after biosynthesis. DSG1PPK-TMD that escapes ER retention or degradation mechanisms traffics to the cell surface and assembles with other desmosomal proteins. However, the cell surface pool of DSG1PPK-TMD pool is unstable, presumably owing to increased endocytic rates and subsequent lysosomal degradation. In contrast, DSG1SAM-TMD largely escapes proteasomal degradation and traffics slowly to the cell membrane where its stability at the surface permits a dominant-negative activity that may occur through competition with WT desmosomal cadherins for the binding of intracellular adaptor proteins such as PG. These findings explain in part the mechanisms by which these two unique DSG mutations cause different types of skin disease.

TMDs are stretches of hydrophobic residues allowing proteins to traverse lipid bilayers in an energetically favorable manner. Uncommon within TMDs, arginines mostly occur at the cytoplasmic-facing end of the α-helix where they interact with phospholipid headgroups, terminate TMDs, and determine membrane protein topology (Elazar et al., 2016; Parks and Lamb, 1993; Reddy et al., 2014; Sharpe et al., 2010). An inappropriately placed arginine can disrupt normal TMD behavior (Partridge et al., 2004) from synthesis to trafficking and stability. We observed retention in intracellular organelles (Supplementary Figure S7), reduced protein and surface stability (Supplementary Figures S5 and S6), and reduced raft association (Supplementary Figure S4). DSG1PPK-TMD and DSG1SAM-TMD models predict that the arginine residues present in the mutants snorkel at the membrane:cytoplasmic interface (Öjemalm et al., 2016; Schow et al., 2011) (Figure 4a) where they may stabilize the mutant TMDs in a trade-off that reduces the energetic costs of arginine but disrupts lipid packing and protein behavior. This snorkeling is predicted to create kinks in the α-helix to maintain the remaining hydrophobic residues within the hydrophobic region of the lipid bilayer, effectively shortening the 24-residue DSG1TMD to just 11 residues for DSG1PPK-TMD and 16 residues for DSG1SAM-TMD. TMD length guides both subcellular localization and raft association of single-pass transmembrane proteins; such proteins with short TMDs localize to ER and Golgi, whereas those with longer TMDs localize to the cell membrane and potentially associate with rafts (Diaz-Rohrer et al., 2014; Sharpe et al., 2010). The arginine in DSG1PPK-TMD and DSG1SAM-TMD likely disrupts DSG1 behavior beginning at synthesis. ER membranes can conform to TMDs with just 10 residues (Jaud et al., 2009), but arginine decreases the efficiency of translocon-mediated TMD insertion with the greatest effect occurring in the middle (Dorairaj and Allen, 2007; Hessa et al., 2007, 2005). The increased proteasomal degradation of DSG1PPK-TMD (Figure 6) along with ER and Golgi retention (Supplementary Figure S7) that we observed may be the combined result of inefficient ER insertion and disruptive hydrophobic mismatch. The DSG1SAM-TMD arginine is sufficiently distant from the TMD center that its impact on insertion efficiency would be substantially less while still causing some ER and Golgi retention. Ultimately, the effect of arginine on TMDs is position dependent, likely forming the basis for the differing pathomechanisms of DSG1PPK-TMD versus that of DSG1SAM-TMD.

Even with these handicaps, each mutant protein traffics to the surface and forms desmosomes, albeit fewer, smaller, and weaker than those formed in cells expressing DSG1WT. The DSG1TMD is important in desmosomal processes, including in function, through its ability to drive raft association (Lewis et al., 2019; Resnik et al., 2019, 2011; Stahley et al., 2014). The loss in raft association we observed is likely a major factor in the reduced desmosome size and number, along with reduced cell–cell adhesion strength, observed in cells expressing DSG1sam-TMD or DSG1PPK-TMD.

Many of our experiments showed apparently similar behavior between DSG1PPK and DSG1SAM, so how might one G:R substitution cause a mild disorder, whereas the other causes a severe disorder? In the context of patient skin, both DSG1PPK and DSG1SAM are expressed alongside WT DSG1. Desmosomal protein levels are elevated in the palm and sole skin relative to the skin in other parts of the body, allowing for larger desmosomes to form (Wan et al., 2003). The key behavioral differences we identified between DSG1PPK and DSG1SAM involved stability. Increased protein turnover and decreased cell surface stability of DSG1PPK would ultimately lead to a haploinsufficient disease mechanism in which DSG1WT protein levels are sufficient for normal desmosome assembly throughout most of the body but insufficient for larger desmosomes needed to maintain epidermal integrity in the palms and soles. Furthermore, DSG1 expression is not only critical for desmosome adhesive strength in the upper epidermis but also for initiating epidermal differentiation by decreasing RAS/MAPK pathway activity (Getsios et al., 2009; Harmon et al., 2013). In the presence of DSG1PPK, DSG1WT protein levels may be insufficient to drive appropriate epidermal differentiation in the palms and soles. Indeed, elevated RAS/MAPK signaling has been observed in the skin of patients with PPK (Harmon et al., 2013), and PPK symptoms have been observed in individuals diagnosed with RASopathies (Hammers and Stanley, 2013). How DSG1SAM might affect the RAS/MAPK pathway is unclear, although patient skin samples showed epidermal thickening consistent with differentiation defects (Lewis et al., 2019). In contrast to DSG1PPK, DSG1SAM maintains normal turnover rates and cell surface stability, allowing this mutant to interfere with normal desmosomal processes regardless of skin location, resulting in the more severe phenotype. Therefore, this mutant is well-poised to act dominant negatively, although the exact mechanism requires further experiments to parse out.

In summary, our findings show a key role for the DSG1TMD in desmosome processes, including in function as well as in the differential effects of G:R mutations based on TMD residue position. Additional work with the DSG1TMD will further our understanding of desmosome mechanisms such as assembly, maintenance, and disassembly and their contribution to epidermal differentiation and disease.

MATERIALS AND METHODS

For full materials and methods, please refer to the Supplementary Materials and Methods.

Microscopy

Widefield images acquired on a Nikon Ti-E inverted microscope (×100/1.49 NA oil immersion objective) equipped with a Hamamatsu C11440-22CU camera were deconvolved using Microvolution (Bruce and Butte, 2013). Three-dimensional structured illumination microscopy done on a Nikon N–SIM Super Resolution system on an Eclipse Ti-E microscope (×100/1.49 NA oil immersion objective, 488- and 561-nm solid-state lasers) equipped with an EMCCD (DU-897; Andor Technology, Belfast, United Kingdom) was reconstructed with the N-SIM Analysis module in NIS-Elements (version 5.02; Nikon, Tokyo, Japan). Ten z-stacks per condition per replicate were acquired.

Structural predictions

TMD sequences were analyzed using the Robetta structure prediction server (Kim et al., 2004).

Biochemical Assays

Triton solubility, surface biotinylation, and DRM isolation assays were previously described (Lewis et al., 2019; Lingwood and Simons, 2007). Protein turnover was determined by treating confluent cells grown in 12-well plates with 10 μM MG132 (474790, MilliporeSigma, Burlington, MA) or 200 μM primaquine bisphosphate (160393, Sigma-Aldrich, St. Louis, MO) for 0, 1, 3, or 6 hours at 37 °C. On ice, cells were washed with cold PBS+, lysed in RIPA (PBS+, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 10 mM Tris-hydrogen chloride, 140 mM sodium chloride, 1 mM EDTA, 0.5 mM EGTA, protease inhibitor [11836153001, Roche, Basel, Switzerland]), scraped, transferred to an Eppendorf tube, and mixed 1:1 with 2X Laemmli buffer containing 5% β-mercaptoethanol. Samples were heated to 95 °C for 10 minutes with vortexing before running SDS-PAGE and immunoblotting.

Dispase-based fragmentation assay

Confluent cells cultured in 24-well plates were treated with 1 U/ml dispase (Corning, Corning, NY) for 15 minutes at 37 °C. PBS+-rinsed released monolayers were transferred to 1.5 ml Eppendorf tubes and subjected to mechanical stress on an orbital shaker at 350 r.p.m. for 45 seconds. Fragments transferred to a 24-well plate were fixed and stained with 1% paraformaldehyde and methylene blue. Fragments imaged on an Elispot scanner (Cellular Technology Limited, Shaker Heights, OH) were counted with Fiji (Schindelin et al., 2012).

Statistics

Error bars represent SEM. Significance was determined by one-way ANOVA and Dunnett’s posthoc. Statistical analysis of immunofluorescence, immunoblotting, and dispase assay experiments was conducted on results from three, four, and seven independent replicates, respectively.

Supplementary Material

1

ACKNOWLEDGMENTS

The authors thank Ryan Hobbs and Natella Maglakelidze for their help in running quantitative real-time reverse transcriptase–PCR. The Kowalczyk laboratory is supported by the National Institutes of Health (Bethesda, MD) grants R01AR050501 and R01AR048266. SEZ was supported by the National Institutes of Health training grant T32GM008367. DEC was supported by the National Institutes of Health grants R03AR068096 and R35GM119617. MA was supported by Advanced Research and Development Programs for Medical Innovation funding (19gm0910002h0105) from the Japan Agency for Medical Research and Development (Tokya, Japan); by a Grant-in-Aid for Scientific Research (B) (18H02832) from the Japan Society for the Promotion of Science (Tokyo, Japan); and by Health and Labor Sciences Research Grants, Research on Intractable Diseases (20FC1052) from the Ministry of Health, Labor and Welfare of Japan. This research project was also supported by the Emory University Integrated Cellular Imaging Microscopy Core, the Emory Flow Cytometry Core, and the Emory Integrated Genomics Core (Atlanta, GA).

Abbreviations:

DRM

detergent-resistant raft membrane

DSG

desmoglein

DP

desmoplakin

ER

endoplasmic reticulum

PG

plakoglobin

PPK

palmoplantar keratoderma

SAM

severe dermatitis, multiple allergies, and metabolic wasting

TMD

transmembrane domain

WT

wild-type

Footnotes

CONFLICT OF INTEREST

The authors state no conflicts of interest.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2021.07.154.

Data availability statement

No datasets were generated or analyzed.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1799481-supplement-1.pdf (2.3MB, pdf)

Data Availability Statement

No datasets were generated or analyzed.

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