Artigo Acesso aberto Revisado por pares

A pH ‐ and ionic strength‐dependent conformational change in the neck region regulates DNGR ‐1 function in dendritic cells

2016; Springer Nature; Volume: 35; Issue: 22 Linguagem: Inglês

10.15252/embj.201694695

ISSN

1460-2075

Autores

Pavel Hanč, Oliver Schulz, Hanna Fischbach, Stephen R. Martin, Svend Kjær, Caetano Reis e Sousa,

Tópico(s)

Immune Response and Inflammation

Resumo

Article17 October 2016Open Access Source DataTransparent process A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells Pavel Hanč Pavel Hanč Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Oliver Schulz Oliver Schulz Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Hanna Fischbach Hanna Fischbach Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Stephen R Martin Stephen R Martin Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK Search for more papers by this author Svend Kjær Svend Kjær Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK Search for more papers by this author Caetano Reis e Sousa Corresponding Author Caetano Reis e Sousa [email protected] orcid.org/0000-0001-7392-2119 Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Pavel Hanč Pavel Hanč Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Oliver Schulz Oliver Schulz Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Hanna Fischbach Hanna Fischbach Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Stephen R Martin Stephen R Martin Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK Search for more papers by this author Svend Kjær Svend Kjær Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK Search for more papers by this author Caetano Reis e Sousa Corresponding Author Caetano Reis e Sousa [email protected] orcid.org/0000-0001-7392-2119 Immunobiology Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Author Information Pavel Hanč1, Oliver Schulz1, Hanna Fischbach1, Stephen R Martin2, Svend Kjær2 and Caetano Reis e Sousa *,1 1Immunobiology Laboratory, The Francis Crick Institute, London, UK 2Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK *Corresponding author. Tel: +44 20 3796 1310; E-mail: [email protected] The EMBO Journal (2016)35:2484-2497https://doi.org/10.15252/embj.201694695 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNGR-1 is receptor expressed by certain dendritic cell (DC) subsets and by DC precursors in mouse. It possesses a C-type lectin-like domain (CTLD) followed by a poorly characterized neck region coupled to a transmembrane region and short intracellular tail. The CTLD of DNGR-1 binds F-actin exposed by dead cell corpses and causes the receptor to signal and potentiate cross-presentation of dead cell-associated antigens by DCs. Here, we describe a conformational change that occurs in the neck region of DNGR-1 in a pH- and ionic strength-dependent manner and that controls cross-presentation of dead cell-associated antigens. We identify residues in the neck region that, when mutated, lock DNGR-1 in one of the two conformational states to potentiate cross-presentation. In contrast, we show that chimeric proteins in which the neck region of DNGR-1 is replaced by that of unrelated C-type lectin receptors fail to promote cross-presentation. Our results suggest that the neck region of DNGR-1 is an integral receptor component that senses receptor progression through the endocytic pathway and has evolved to maximize extraction of antigens from cell corpses, coupling DNGR-1 function to its cellular localization. Synopsis The neck region of DNGR-1, a dendritic cell receptor that mediates cross-presentation of dead cell-associated antigens, can undergo a pH- and ionic strength-dependent conformational change. This allows internalized receptors to sense their relocalization from the cell surface to low pH endosomes and promotes endosomal extraction of antigens from engulfed cell corpses for cross-presentation. The neck region of DNGR-1 undergoes a conformational change in a pH- and ionic strength-dependent manner. The conformational change happens predominantly at the level of tertiary structure. The integrity of the neck region is essential for the ability of DNGR-1 to promote cross-presentation. The conformational state impacts DNGR-1 function. Introduction Recognition of damage-associated molecular patterns (DAMPs), molecules released or exposed by cells upon injury, is essential for maintenance of homeostasis and tissue repair (Zelenay & Reis e Sousa, 2013). In vertebrates, it can also lead to adaptive immune responses against proteins present within dead cells, including cancer neoantigens (Zelenay & Reis e Sousa, 2013). DNGR-1 (also known as CLEC9A) is a vertebrate DAMP receptor expressed by certain types of dendritic cells (DCs), leukocytes that couple innate and adaptive immunity. DNGR-1 specifically binds to a DAMP exposed by cells that have lost their plasma membrane integrity (Sancho et al, 2009). We and others have previously identified the DNGR-1 ligand as the filamentous form of actin (F-actin) (Ahrens et al, 2012; Zhang et al, 2012) and determined the structure of the ligand–receptor complex (Hanč et al, 2015). DNGR-1 can act as an endocytic receptor, yet it is dispensable for uptake of dead cell debris by DCs (Huysamen et al, 2008; Sancho et al, 2008, 2009). Rather, DNGR-1 recognition of F-actin favors the cross-presentation of dead cell-associated antigens by DCs that internalized dead cell debris (Sancho et al, 2009; Iborra et al, 2012; Zelenay et al, 2012). The exact mechanism by which DNGR-1 exerts this function remains elusive but is thought to involve regulation of the maturation of endosomes containing dead cell material (Sancho et al, 2009; Iborra et al, 2012; Zelenay et al, 2012). DNGR-1 is a type II transmembrane protein and a member of the C-type lectin superfamily. In mouse, multiple DNGR-1 isoforms have been found, of which only two retain the entire ligand-binding domain and the transmembrane region (termed “long” and “short”) (Huysamen et al, 2008; Sancho et al, 2008). In human, a single isoform exists that corresponds to the mouse “short” receptor and behaves as a glycosylated homodimer (Huysamen et al, 2008). In contrast, the mouse receptor has been described as non-glycosylated and monomeric in one report (Huysamen et al, 2008) or as a glycosylated dimer in another (Sancho et al, 2008). The extracellular domain of DNGR-1 consists of a single C-type lectin-like domain (CTLD) and a membrane-proximal neck region. The neck of the long isoform of murine DNGR-1 differs from the short and the human isoforms by the presence of an extra exon coding for additional 26 amino acids (Huysamen et al, 2008; Sancho et al, 2008). The intracellular portion of DNGR-1 contains a short domain termed hemITAM, which resembles that found in the related C-type lectin dectin-1 (Rogers et al, 2005) and which permits signaling via Syk (Huysamen et al, 2008; Sancho et al, 2009). The CTLD of human DNGR-1 has been crystalized (Zhang et al, 2012) and shown to be solely responsible for the ability of DNGR-1 to bind to F-actin (Ahrens et al, 2012; Zhang et al, 2012; Hanč et al, 2015). On the other hand, the structure and properties of the neck region and their relevance to the biology of the receptor have not been explored. Although originally seen primarily as serving a scaffold function, the neck region is emerging as an important determinant of the properties of various C-type lectin receptors (Back et al, 2009; Tabarani et al, 2009; Manzo et al, 2012). Here, we report that the neck region of mouse and human DNGR-1 allows the receptor to exist in two distinct conformations that interconvert in response to changes in pH and ionic strength. The ability of DNGR-1 to adopt different conformations as a function of pH and ionic strength suggests that this receptor may be intrinsically altered as it travels through the endocytic pathway and we present data indicating that this contributes to its function in cross-presentation of dead cell-associated antigens. Results DNGR-1 is a glycosylated disulfide-bonded homodimer To assess their properties, we expressed the entire extracellular domains (ECDs) of mouse DNGR-1 long and short isoforms, as well as the single human isoform, as soluble recombinant FLAG-tagged proteins (Ahrens et al, 2012; Hanč et al, 2015). By non-reducing SDS–PAGE followed by Western blotting for FLAG, all ECDs ran as dimers, with a minor fraction in the form of higher order oligomers (Fig 1A). The dimers and oligomers could be reduced to their constituent monomers using dithiothreitol (DTT) or β-mercaptoethanol (β-ME) (Fig 1A). Mutating the conserved cysteine in the neck region to serine (C94S in the long mouse isoform ECD) had a strong adverse effect on expression efficiency (data not shown) but resulted in a protein that ran as a monomer under both reducing and non-reducing conditions (Fig 1B). Finally, all ECDs were glycosylated as their electrophoretic mobility could be augmented, at least in part, by treatment with the glycosidase PNGase F (Fig 1C). The deglycosylation treatment did not change the two-band pattern of DNGR-1 ECDs mobility, consistent with a previous report (Huysamen et al, 2008). Importantly, PNGase F treatment only removes N-bound glycans which do not contain an α(1-3)-fucose bound to the core N-acetyl glucosamine. It is therefore possible that the two bands correspond to glycoforms that differ in O-glycosylation or α(1-3)-fucosylated N-glycans. In summary, the ECDs of both human and mouse DNGR-1 behave as glycosylated dimers. Figure 1. DNGR-1 is a glycosylated disulfide-bonded dimer Western blot analysis of supernatants containing long and short mouse and human DNGR-1 ECD proteins under reducing and non-reducing conditions. Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR-1 ECD proteins under reducing and non-reducing conditions. C94S mutant-containing supernatant was 200× concentrated before analysis. Western blot analysis of supernatants containing the indicated DNGR-1 ECD proteins after treatment with PNGase F overnight. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Source data are available online for this figure. Source Data for Figure 1 [embj201694695-sup-0001-SDataFig1.pdf] Download figure Download PowerPoint Low pH and ionic strength can induce formation of reduction-resistant dimers of DNGR-1 Surprisingly, we noticed that under certain conditions, the dimers of DNGR-1 could maintain their dimeric status even in the presence of reducing agents (Fig 2A). Buffers of lower pH and ionic strength were most effective in inducing the reduction-insensitive state (Fig 2A). We confirmed this by testing 36 buffers of different ionic strengths (range 15–250 mM) and pH (range pH 6.5–8.05) in which buffering capacity was kept constant (Table 1). After analysis by SDS–PAGE in the presence of 100 mM DTT, we observed a clear trend toward higher abundance of reduction-insensitive dimers as a function of a decrease in pH and ionic strength (Fig 2B). Figure 2. DNGR-1 can form reduction-resistant dimers 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1–250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS–PAGE and Western blot. 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS–PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH. 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS–PAGE and Western blot. 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat denaturation in Laemmli buffer was tested by reducing SDS–PAGE and Western blot. Data information: HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +. Source data are available online for this figure. Source Data for Figure 2 [embj201694695-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint Table 1. Buffers used to assess the influence of pH and ionic strength on the conformational state of DNGR-1 Buffer pH Ionic strength (mM) 15 mM Bis-Tris 6.5 15 50 100 150 200 250 15 mM BES 7.1 15 50 100 150 200 250 15 mM MOPS 7.2 15 50 100 150 200 250 15 mM HEPES 7.5 15 50 100 150 200 250 15 mM Tricine 8.05 15 50 100 150 200 250 15 mM Trizma base 8.06 15 50 100 150 200 250 A reversible conformational change is responsible for the formation of reduction-insensitive dimers As the neck region is responsible for dimerization of DNGR-1, we hypothesized that a pH- and ionic strength-dependent conformational change in this portion of the protein could be responsible for the reduction insensitivity of DNGR-1 dimers. To test this possibility, we subjected the reduction-insensitive form of long mouse DNGR-1 ECD to mildly denaturing (Laemmli buffer) or strongly denaturing (8 M urea) conditions in the presence of DTT and analyzed the samples by SDS–PAGE and Western blot. As predicted, strongly denaturing conditions almost completely abolished the ability of DNGR-1 to resist reduction, while under weakly denaturing conditions the reduction-insensitive dimers could be observed. In the absence of reducing agents, the protein maintained its dimeric status regardless of the denaturing conditions (Fig 2C). To the same end, we gradually increased the stringency of the heat denaturation step by increasing the time of boiling in Laemmli buffer. When analyzed by SDS–PAGE and Western blot, DNGR-1 ECD appeared exclusively as a monomer after 5 min of boiling under neutral conditions, while the protein kept under mildly acidic conditions (MES pH 6.1) was not completely reduced even after 15 min of the same treatment (Fig 2D). To evaluate the reversibility of the conformational change, we subjected long mouse DNGR-1 ECD to mildly acidic conditions (MES pH 6.1) to induce the reduction-resistant state, dialyzed the protein back into PBS, and tested its reduction sensitivity. As expected, we observed reduction-resistant dimers under mildly acidic conditions (Fig 3A). However, the protein that had been MES-treated and transferred back into PBS showed no sign of increased resistance to DTT treatment (Fig 3A), suggesting that the conformational change is indeed reversible. Figure 3. Formation of reduction-resistant dimers is reversible and conserved between DNGR-1 isoforms 1 μg of purified long mouse DNGR-1 ECD was transferred into 10 mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS–PAGE and Western blot. Supernatants after production of indicated murine proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio. 1 μg of purified human DNGR-1 ECD was diluted into the same buffers and all samples were analyzed using reducing SDS-PAGE and Western blot. Data information: HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +. Source data are available online for this figure. Source Data for Figure 3 [embj201694695-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint Finally, to confirm that the ability of the ECD to undergo a conformational change is not an isoform-specific property, we tested the reduction sensitivity of the two mouse isoforms and the human isoform. Under neutral conditions (PBS), all the proteins were readily reducible while, when subjected to weakly acidic conditions (pH 6.1), they showed increased resistance to reduction (Fig 3B). These data indicate that the ability to undergo a pH-dependent conformational change is an evolutionarily conserved property of DNGR-1. DNGR-1 neck region is necessary and sufficient for the formation of reduction-resistant dimers We expressed a chimeric ECD, in which the neck region of DNGR-1 was fused to the CTLD of another C-type lectin receptor, dectin-1 (Fig 4A). Dectin-1 is a monomer but the chimeric ECD behaved as a disulfide-bonded homodimer (Fig 4B), consistent with the fact that the neck region of DNGR-1 bears the requisite cysteine for receptor dimerization (see above). Importantly, the chimeric protein was refractory to DTT-mediated reduction under the same conditions that induced the reduction-insensitive state in DNGR-1 ECD (Fig 4C). Dectin-1 alone showed no such phenotype (Fig 4C). We conclude that the neck region of DNGR-1 is necessary and sufficient for the formation of reduction-resistant dimers. Figure 4. The neck of DNGR-1 is sufficient for formation of reduction-resistant dimers Schematic representation of DNGR-1, dectin-1, and the chimeric protein consisting of the neck region of DNGR-1 and CTLD of dectin-1. Supernatants containing ECD of long mouse DNGR-1, dectin-1, and the chimeric receptor were prepared for SDS–PAGE and Western blot under non-reducing conditions. Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS–PAGE and Western blot under reducing conditions. Data information: HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +. Source data are available online for this figure. Source Data for Figure 4 [embj201694695-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint The conformational change happens at the level of tertiary structure In order to further characterize the conformational change, we made use of circular dichroism (CD). Far-UV CD spectra reflect the secondary structure content of the protein while near-UV CD signals derive from the three aromatic residues (Phe, Tyr, and Trp) and, in some cases, from disulfide bonds, and reflect protein tertiary structure (Martin & Schilstra, 2008). We first measured far-UV CD spectra for both long mouse and human DNGR-1 isoforms under neutral (PBS) or mildly acidic (MES pH 6.1) conditions, and observed almost perfect overlap between the two (Fig 5A). Detailed analysis of both mouse and human isoforms revealed only a minor increase in the content of alpha helical structure under low pH conditions, corresponding at most to four residues (Table 2). Interestingly, a different content of disordered structure was observed in the mouse versus human isoform (44% versus 37%) corresponding to ~26 residues. This is precisely the length of the stretch of amino acids encoded in the extra exon present in the long mouse isoform, suggesting that this part of the mouse protein is unstructured. Figure 5. A change in the tertiary structure is responsible for the formation of reduction-resistant dimers Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition). Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two). Schematic representation of the suggested conformational states of DNGR-1. Download figure Download PowerPoint Table 2. Content of the elements of secondary structure in long mouse and human DNGR-1 ECD α-helix β-sheet β-turn Random Long mouse ECD PBS 16.1% 22.5% 17.1% 44.3% MES 17.8% 21.8% 17.0% 43.5% Human ECD PBS 22.3% 21.3% 19.5% 37.2% MES 24.2% 20.7% 20.8% 34.2% With no major change in the secondary structure, we envisaged that mutual repositioning of the two neck regions within the dimer might be at the root of the observed reduction resistance. To test this hypothesis, we measured near-UV CD spectra of long mouse DNGR-1 under neutral and mildly acidic (pH 6.1) conditions (Fig 5B). The sample in PBS showed significant near-UV CD intensity extending to long wavelengths (> 350 nm), characteristic of a contribution from a disulfide bond. This long wavelength intensity was not observed in the low pH sample, suggesting that the disulfide dihedral angle is different at the two pHs. Although the difference spectrum (PBS–MES) is characteristic of a disulfide contribution it is also possible that the changes observed around 290 nm indicate that there is a change in the environment of at least one tryptophan residue. Thus, taken together, our near-UV and far-UV CD data suggest that a change in the tertiary but not secondary structure happens when DNGR-1 undergoes the transition to the reduction-insensitive form. Our data are consistent with a model in which repositioning of the neck regions within the dimer results in protection of the disulfide bond or in making the disulfide bond dispensable for maintaining the dimeric status of DNGR-1. To make a clear distinction between the two conformational states, we henceforth refer to the reduction-sensitive form as “type-1 dimer” and the reduction-insensitive form as “type-2 dimer” (Fig 5C). Mutations in the neck region affect type-2 dimer formation and dimerization of DNGR-1 We observed type-2 dimer formation in both mouse and human DNGR-1 (see above), suggesting that the part of the neck region involved in the process is conserved between the two. In order to pinpoint its location, we genetically removed overlapping blocks of 10–11 amino acids from the conserved part of the neck [K57–L66 (Δ1), L64–I73 (Δ2), L72–L82 (Δ3), N81–T90 (Δ4), R87–A96 (Δ5), and Q95–S104 (Δ6) (Fig 6A)], expressed the resulting constructs as soluble ECD proteins, and tested their ability to form type-2 dimers. The mutants devoid of the first two blocks (Δ1 and Δ2) were expressed comparably to the wild-type (WT) ECD and showed no phenotype with regard to type-2 dimer formation. The Δ3 mutant also displayed no obvious phenotype but expressed very poorly, while the Δ6 mutant failed to express at all (Fig 6B). Interestingly, the Δ4 mutant expressed as efficiently as the WT, but showed enhanced type-2 dimer formation even under neutral conditions (Fig 6B). The Δ5 mutant lacks the dimerization cysteine (C94) and consequently expressed as a monomer (Fig 6B). Unlike the C94S mutant, however, the Δ5 mutant expressed at levels comparable to the WT (Fig 6C). Figure 6. Distinct parts of the neck region contribute to regulation of type-2 dimer formation A. Sequences of the neck regions of human and long and short isoforms of mouse DNGR-1 aligned in Clustal X software. Parts of the neck corresponding to the block deletions are depicted as green bars. B, C. Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS–PAGE and Western blot under reducing or non-reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. D. Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10 mM MES pH 6.1 buffers and the lysates were analyzed by SDS–PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Data information: Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +. Source data are available online for this figure. Source Data for Figure 6 [embj201694695-sup-0005-SDataFig6.pdf] Download figure Download PowerPoint To assess whether the effects observed with the deletion mutants were a result of the loss of specific amino acids or a non-specific consequence of segments of the neck region getting “out of sync” due to part of the helices being removed, we substituted all the residues within blocks 3, 4, and 6 with strings of alanines. Replacing all the residues in block 4 with alanine (Δ4A) resulted in an ECD protein that behaved like the Δ4 mutant in that it formed type-2 dimers in neutral conditions (Fig 6C). Interestingly, and unlike the Δ3 and Δ6 deletion mutants, the mutants with alanines in blocks 3 and 6 (Δ3A and Δ6A) were able to be expressed, although, in the case of Δ6A, to a lower extent than the WT ECD. Notably, while the Δ3A mutant showed no phenotype with respect to type-2 dimer formation, the Δ6A mutant failed to form type-2 dimers under conditions which were effective in inducing their formation in the WT protein (Fig 6C). Under non-reducing conditions, all mutants except the Δ5 ran as dimers (Fig 6C), confirming that the inability of the Δ6A ECD to form type-2 dimers is not because it lost the ability to make the disulfide bond. We noticed a putative N-glycosylation site (NxT sequence) (Gavel & von Heijne, 1990) at the boundary of blocks 3 and 4, which is disrupted in both Δ3 and Δ4 mutants (Fig 6A). Consistent with this site being glycosylated in mouse DNGR-1, both Δ3A and Δ4A mutants showed slightly increased electrophoretic mobility compared to the WT ECD or the Δ6A mutant (Fig 6C). As Δ3A and Δ4A mutants do not exhibit concordant phenotypes, however, the glycosylation appears not to be involved in type-2 dimer formation. Finally, to confirm that the phenotypes we observed for the ECD mutant proteins were applicable to the full-length transmembrane receptor, we expressed full-length WT, Δ4, and Δ6A DNGR-1 in cells. We lysed the cells in either 10 mM MES pH 6.1 or PBS-based buffers and analyzed the samples by reducing SDS–PAGE and Western blot with anti-DNGR-1 mAb. The WT receptor appeared exclusively as a type-1 dimer in the PBS sample, while lysis in the MES buffer induced type-2 dimer formation (Fig 6D). On the other hand, the Δ4 mutant appeared in the form of type-2 dimers in both buffers, while the Δ6A mutant exhibited no type-2 dimer formation in either buffer (Fig 6D). Thus, the data with full-length receptor recapitulate those obtained with ECD proteins. Interestingly, in the case of the type-2 dimer containing samples (WT in MES buffer and Δ4 mutant in both conditions), we reproducibly observed multiple bands, seemingly corresponding to reduction-resistant oligomers (Fig 6D), indicating that, in the context of the full-length receptor, complexes of higher order than dimers are possible. Type-2 dimer formation does not affect the ability of DNGR-1 to bind F-actin, signal to NFAT, or undergo internalization A pH-induced conformational change in another C-type lectin receptor, DEC205, has recently been suggested to allow the protein to recognize a ligand in apoptotic and necrotic cells (Cao et al, 2015). Consequently, we set out to determine whether the conformational change in DNGR-1 affects its ability to bind its ligand. To this end, we utilized a dot blot assay (Ahrens et al, 2012; Hanč et al, 2015) in which decreasing amounts of in vitro polymerized actin were spotted onto a membrane, which was then incubated with equal amounts of DNGR-1 WT, Δ4, or Δ6A mutant ECDs in PBS or MES pH 6.1 buffer. Binding was revealed with anti-FLAG antibody and strength of signal quantified by densitometry. Using this setup, we could not observe any differences in the binding ability of WT, Δ4, or Δ6A DNGR-1 ECDs in either buffer (Fig 7A), indicating that type-2 dimer switching is not involved in the regulation of DNGR-1 ligand binding. A slight trend toward lower binding in the MES buffer could be seen in some experiments, likely attributable to a small pH-induced conformational change in the actin filaments themselves (Oda et al, 2001). Figure 7. Type-2 dimer formation does not affect DNGR-1 ligand binding, coupling to Syk or receptor internalization A. Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and qu

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