Mechanisms of blister formation by staphylococcal toxins

Many children suffer from the bacterial skin diseases bullous impetigo and staphylococcal scalded skin syndrome (SSSS). Staphylococcus aureus, which produces exfoliative toxins (ETs), causes these diseases. Recently, it was proven that ETs cleave the cell adhesion molecule desmoglein (Dsg) 1, which plays an important role in maintaining the proper structure and barrier function of the epidermis. Surprisingly, Dsg1 is also the antibody target in the autoimmune disease pemphigus foliaceus. Skin biopsies from pemphigus foliaceus patients show the same pathology as those from bullous impetigo and SSSS patients. The crystal structure of ET suggests that it is a serine protease with an inactive catalytic site, which may become activated when ET binds a specific receptor. This receptor binding is thought to cause a change in conformation that exposes the catalytic site. It has recently been shown that Dsg1 specifically binds and activates ET, which in turn cleaves the bound Dsg1 at only one peptide bond. This process is absolutely dependent on the calcium-dependent conformation of Dsg1. These data suggest that ETs have a very high specificity for human Dsg1, and that S. aureus uses ETs to disrupt the barrier of the human epidermis in order to survive and proliferate on the human body.     

Pseudomonas aeruginosa exotoxin A induces human mast cell apoptosis by a caspase-8 and -3-dependent mechanism

Mast cells play an important role in both allergy and innate immunity. Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines. Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P. aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium. ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process. Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis. ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate. Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not. Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis. Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors. Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.     

Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.

Desmoglein (Dsg) is a cadherin-type adhesion molecule found in desmosomes. Dsg1 and Dsg3 are the target Ags in the autoimmune blistering diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. To map conformational epitopes of Dsg1 and Dsg3 in PF and PV, we generated Dsg1- and Dsg3-domain-swapped molecules and point-mutated Dsg3 molecules with Dsg1-specific residues by baculovirus expression. The swapped domains were portions of the N-terminal extracellular domains of Dsg1 (1-496) and Dsg3 (1-566), which have similar structures but distinct epitopes. The binding of autoantibodies to the mutant molecules was assessed by competition ELISAs. Domain-swapped molecules containing the N-terminal 161 residues of Dsg1 and Dsg3 yielded >50% competition in 30/43 (69.8%) PF sera and 31/40 (77.5%) PV sera, respectively. Furthermore, removal of Abs against the 161 N-terminal residues of Dsg1 by immunoadsorption eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. Within these N-terminal regions, most of the epitopes were mapped to residues 26-87 of Dsg1 and 25-88 of Dsg3. Furthermore, a point-mutated Dsg3 molecule containing Dsg1-specific amino acid substitutions (His(25), Cys(28), Ala(29)) reacted with anti-Dsg1 IgG, thus defining one of the epitopes of Dsg1. Using the predicted three-dimensional structure of classic cadherins as a model, these findings suggest that the dominant autoimmune epitopes in both PF and PV are found in the N-terminal adhesive surfaces of Dsgs.     

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.     

Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms

The four isoforms of desmosomal cadherin desmogleins (Dsg1-4) are expressed in epithelial tissues in a differentiation-specific manner. Extensive sequencing of the human genome has revealed only one copy of the Dsg1 gene. However, we recently cloned two novel additional mouse Dsg1 genes, Dsg1-beta and -gamma, which flank the original Dsg1-alpha on chromosome 18. Sequence conservation between the Dsg1 isoforms diverged significantly at exon 11, particularly in the region that encodes for the extracellular anchoring (EA) domains. Computational analysis revealed very low hydrophilic potential of the Dsg1-gamma EA compared with the corresponding sequences of Dsg1-alpha and -beta, suggesting that the Dsg1-gamma EA domain may have a stronger affinity to the cell membrane. We generated antibodies using synthetic peptides or recombinant proteins localized within the EA domains. These antibodies were tested for their specificity and were then used to demonstrate expression of Dsg1 isoforms in various tissues. In the epidermis, all Dsg1 isoforms were differentially expressed in the differentiating cell layers. In the hair follicle, all Dsg1 isoforms were present throughout the entire process of its development and cycling but the expression of Dsg1 isoforms is subject to significant hair cycle-dependent changes. Dsg1-beta and -gamma, but not Dsg1-alpha, were detected in the sebaceous gland epithelium and the stratified epithelium of the stomach. Finally, Dsg1-alpha and Dsg1-beta, but not Dsg1-gamma, are proteolytically cleaved by exfoliative toxin A. These results suggest that the developmental complexity of mouse tissues, including skin and hair, may play a significant role in the evolutionary driving force to maintain multiple Dsg1 genes in mouse.     

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.     

Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.     

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.     

Conformational selection and collagenolysis in Type III collagen

Matrix metalloproteases (MMPs) cleave native collagen at a single site despite the fact that collagen contains more than one scissile bond that can, in principle, be cleaved. For peptide bond hydrolysis to occur at one specific site, MMPs must (1) localize to a region near the unique scissile bond, (2) bind residues at the catalytic site that form the scissile bond, and (3) hydrolyze the corresponding peptide bond. Prior studies suggest that for some types of collagen, binding of noncatalytic MMP domains to amino acid sequences in the vicinity of the true cleavage site facilitates the localization of collagenases. In the present study, our goal was to determine whether binding to the catalytic site also plays a role in determining MMP specificity. To investigate this, we computed the conformational free energy landscape of Type III collagen at each potential cleavage site. The free energy profiles suggest that although all potential cleavage sites sample unfolded states at relatively low temperatures, the true cleavage site samples structures that are complementary to the catalytic site. By contrast, potential cleavage sites that are not cleaved sample states that are relatively incompatible with the MMP active site. Furthermore, our findings point to a specific role for arginine residues in modulating the structural stability of collagen near the collagenase cleavage site. These data imply that locally unfolded potential cleavage sites in Type III collagen sample distinct unfolded ensembles, and that the region about the true collagenase cleavage site samples states that are most complementary to the MMP active site. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1.

Plasmin is shown to specifically cleave vitronectin at the Arg361-Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-1 inhibitor of plasminogen activator (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI-1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.     

Decreasing the distance between the two conserved sequence elements of histone pre-messenger RNA interferes with 3' processing in vitro.

Histone mRNA 3' end formation requires the presence of two cis-acting conserved sequence elements: a stem-loop structure upstream from the site of cleavage and a purine-rich region downstream from the site of cleavage called the histone downstream element (HDE). Possible interactions between these two elements and their respective binding factors were investigated by a series of deletions (1-7 nt) in the region between the two. The efficiency of processing decreased as the stem-loop and the HDE were moved closer together. In contrast with the documented ability of the U7 snRNP to direct cleavage at a fixed distance from the HDE in insertion mutants (Scharl & Steitz, 1994), all deletion substrates for which processing was observed were cleaved at or 1-nt upstream from the wild-type site. The reason for the inability of the system to cleave closer to the stem-loop remains unclear, but the removal of stem-loop binding protein(s) (SLBP) did not activate upstream cleavage events. Thus, although the processing machinery measures the distance between the cleavage site and the HDE of mammalian histone pre-mRNAs, there is a barrier limiting how far upstream cleavage can occur. These data allow a reevaluation of the sites of 3' end processing in known histone pre-mRNAs.     

Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition

The structure-specific endonuclease activity of the human XPF-ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF-ERCC1 cleaves DNA structures such as stem-loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem-loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF-ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3'-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix-hairpin-helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF-HLD shows a preference for binding stem-loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF-ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.     

Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage

RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.     

Modeling the bound conformation of Pemphigus Vulgaris-associated peptides to MHC Class II DR and DQ Alleles

Background

Pemphigus vulgaris (PV) is a severe autoimmune blistering disorder characterized by the presence of pathogenic autoantibodies directed against desmoglein-3 (Dsg3), involving specific DR4 and DR6 alleles in Caucasians and DQ5 allele in Asians. The development of sequence-based predictive algorithms to identify potential Dsg3 epitopes has encountered limited success due to the paucity of PV-associated allele-specific peptides as training data.

Results

In this work we constructed atomic models of ten PV associated, non-associated and protective alleles. Nine previously identified stimulatory Dsg3 peptides, Dsg3 96–112, Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977, were docked into the binding groove of each model to analyze the structural aspects of allele-specific binding.

Conclusion

Our docking simulations are entirely consistent with functional data obtained from in vitro competitive binding assays and T cell proliferation studies in DR4 and DR6 PV patients. Our findings ascertain that DRB1*0402 plays a crucial role in the selection of specific self-peptides in DR4 PV. DRB1*0402 and DQB1*0503 do not necessarily share the same core residues, indicating that both alleles may have different binding specificities. In addition, our results lend credence to the hypothesis that the alleles DQB1*0201 and *0202 play a protective role by binding Dsg3 peptides with greater affinity than the susceptible alleles, allowing for efficient deletion of autoreactive T cells.     

Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.     

Different stability of ligand-receptor complex formed with two endothelin receptor species, ETA and ETB.

There are at least two types of endothelin receptors, ETA and ETB, present in various tissues. We found that although biotinylated ET-1 could bind to both ETA and ETB receptors, the stability of the formed ligand-receptor complexes was different. When the preformed complexes of receptor (solubilized from canine brain and lung membranes) and biotinylated ET-1 were subjected to avidin agarose column chromatography, most of the ETA activity was recovered in the pass-through fraction and the remainder was recovered in the 0.5 M KSCN eluate as ligand-free forms. On the other hand, the ETB activity bound firmly to the avidin agarose column was eluted with 1.5 M KSCN. The detection of the complex of 125I-ET-1 and its receptor by SDS-PAGE run at a low temperature was only possible with the ETB fractions and the complex of 125I-ET-1 and ETA was unstable during the separation. These results suggest that the conformation of the ligand binding sites of canine ETA and ETB as well as the stability of their ligand-receptor complexes to SDS are significantly different. Similar observations were also obtained for human ETA and ETB receptors.