Single Peptide Bond Research Articles

The Temperature Dependence of Hydrogen Bonds Is More Uniform in Stable Proteins: An Analysis of NMR h3JNC' Couplings in Four Different Protein Structures.

Long-range HNCO NMR spectra for proteins show crosspeaks due to 1JNC', 2JNC', 3JNCγ, and h3JNC' couplings. The h3JNC' couplings are transmitted through hydrogen bonds and their sizes are correlated to hydrogen bond lengths. We collected long-range HNCO data at a series of temperatures for four protein structures. P22i and CUS-3i are six-stranded beta-barrel I-domains from phages P22 and CUS-3 that share less than 40% sequence identity. The cis and trans states of the C-terminal domain from pore-forming toxin hemolysin ΙΙ (HlyIIC) arise from the isomerization of a single G404-P405 peptide bond. For P22i and CUS-3i, hydrogen bonds detected by NMR agree with those observed in the corresponding domains from cryoEM structures of the two phages. Hydrogen bond lengths derived from the h3JNC' couplings, however, are poorly conserved between the distantly related CUS-3i and P22i domains and show differences even between the closely related cis and trans state structures of HlyIIC. This is consistent with hydrogen bond lengths being determined by local differences in structure rather than the overall folding topology. With increasing temperature, hydrogen bonds typically show an apparent increase in length that has been attributed to protein thermal expansion. Some hydrogen bonds are invariant with temperature, however, while others show apparent decreases in length, suggesting they become stabilized with increasing temperature. Considering the data for the three proteins in this study and previously published data for ubiquitin and GB3, lowered protein folding stability and cooperativity corresponds with a larger range of temperature responses for hydrogen bonds. This suggests a partial uncoupling of hydrogen bond energetics from global unfolding cooperativity as protein stability decreases.

Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): We acknowledge funding from Ministerio de Ciencia e Innovación (Madrid, Spain) through grant FJC2021 047055-I and European Research Council (ERC) through grant H2020-ERC-2020-COG (ProtMechanics-Live). Background/Introduction Stresses exerted during myocardial contraction and relaxation cycles are mainly braced by mechanical proteins located in the extracellular matrix (ECM) and cardiomyocytes (CMs). Titin (TTN) is a gigantic protein that scaffolds the sarcomere (i.e. the basic unit of contraction). Since most of the CM cytoskeleton is occupied by sarcomeres, titin is an essential structural hub for the maintenance of a homeostatic level of mechanical tension. Purpose It has been shown that mechanical disruption of the ECM affects the equilibrium forces between CMs and the ECM, leading to morphological changes in sarcomeres. However, potential alteration of tensional homeostasis resulting from defective mechanics of CM proteins remains unknown due to the lack of tools for in vivo studies. Therefore, the aim of this study is to explore the in vivo response of the myocardium to the abrupt cessation of the mechanical properties of titin. Methods For this purpose, we have experimentally challenged CMs to a tensional unloading by means of a titin cleavage model (TEVs-TTN) [1], both in vitro, using neonatal CM (NMCM), and in vivo. In this model, a cassette with a tobacco etch virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (myoAAV), we were able to cleave titin in living CMs, ceasing only its mechanical properties. Samples were analyzed using an array of techniques such as histology, immunofluorescence and bulk and single-nucleus RNAseq. Results Our results show that while in NMCM we obtain a complete titin cleavage, in vivo we observe mosaic expression of TEVp and no more than 30% cleavage of the protein. In both cases, TEVp expression in TEVs-TTN does not affect cell viability. However, mild myocardial titin cleavage leads to a fibrotic response in less than six days, characterized by an increase in interstitial collagen and an expansion of cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this fibrotic response occurs without activation of the canonical TGFβ pathway. Conclusion Taken together, our results suggest that the loss of titin mechanical-structural function in cardiomyocytes derived from the cleavage of a single peptide bond elicits a fast fibrotic compensatory response in the myocardium through intercellular communication with fibroblasts.

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.

Albumin-Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells.

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

Semisynthesis of Human Ribonuclease-S.

Since its conception, the ribonuclease S complex (RNase S) has led to historic discoveries in protein chemistry, enzymology, and related fields. Derived by the proteolytic cleavage of a single peptide bond in bovine pancreatic ribonuclease (RNase A), RNase S serves as a convenient and reliable model system for incorporating unlimited functionality into an enzyme. Applications of the RNase S system in biomedicine and biotechnology have, however, been hindered by two shortcomings: (1) the bovine-derived enzyme could elicit an immune response in humans, and (2) the complex is susceptible to dissociation. Here, we have addressed both limitations in the first semisynthesis of an RNase S conjugate derived from human pancreatic ribonuclease and stabilized by a covalent interfragment cross-link. We anticipate that this strategy will enable unprecedented applications of the "RNase-S" system.

Peptide Bond Ultraviolet Absorption Enables Vibrational Cold-Ion Spectroscopy of Nonaromatic Peptides.

Peptide-bond VUV absorption is inherent to all proteins and peptides. Although widely exploited in top-down proteomics for photodissociation, this absorption has never been spectroscopically characterized in the gas phase. We have measured VUV/UV photofragmentation spectrum of a single peptide bond in a cryogenically cold protonated dipeptide. Although the spectrum appears to be very broadband and structureless, vibrational pre-excitation of this and even larger cold peptides significantly increases the UV dissociation yield for some of their photofragments. We use this effect to extend the technique of IR-UV photofragmentation vibrational spectroscopy, developed for aromatic peptides, to nonaromatic ones and demonstrate measurements of conformation-specific and nonspecific IR spectra for di- to hexa-peptides.

A fragment of SPARC reflecting increased collagen affinity shows pathological relevance in lung cancer – implications of a new collagen chaperone function of SPARC

ABSTRACTThe matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

Peptide bond detection via graphene nanogaps: a proof of principle study.

Solid-state nanopores and nanogaps are emerging as promising tools for single molecule analysis. 2D materials, such as graphene, can potentially reach the spatial resolution needed for nucleic acid and protein sequencing. In the context of the density functional theory, atomistic modeling and non-equilibrium Green's function calculation, we show that glycine based polypeptide chains translocating across a nano-gap between two semi-infinite graphene nano-ribbons leave a specific transverse current signature for each peptide bond. The projected density of states and bond current analyses reveal a complex scenario with a role played by the adjacent α-carbons and side chains and by the orbitals of the partially resonant double bond involving C, N and O atoms of the peptide bond. In this context, specific fingerprints of the atoms involved in the peptide bonds are found. The same scenario is evidenced also for peptides involving alanine residues. The signal measured can be considered as a specific fingerprint of peptide bonds between small and neutral amino acids with no polar/charge effects. On this basis, a newly conceived nano-device made of a graphene based array of nano-gap is proposed as a possible route to approach peptide sequencing with atomic resolution.

What lurks beneath (the membrane): a mechanistic exploration of rhomboid proteolysis

At the turn of the millennium an extraordinary class of enzymes that cut proteins inside the cell membrane was discovered. Rhomboid proteases are the largest family of these ancient membrane‐immersed enzymes. Although deceptively simple, the act of hydrolyzing a single peptide bond inside the water‐excluding environment of the cell membrane has evolved to trigger cell signaling events during bacterial growth and animal embryogenesis, as well as mitochondrial quality control throughout the life of a cell. The membrane is a challenging environment for catalysis. We have been pioneering new quantitative methods to study rhomboid catalysis directly inside the membrane and combining them with modern X‐ray crystallography approaches. I will discuss an integrated but unexpected model for how rhomboid proteases function, and the promising new opportunities that it raises for combating devastating parasitic diseases that have plagued mankind for millennia, including malaria.Support or Funding InformationThis work was supported by NIH grants 2R01AI066025 and R01AI110925, the Howard Hughes Medical Institute, and the David &amp; Lucile Packard Foundation.

N-Methylated α-Amino Acids And Peptides: Synthesis And Biological Activity.

The simplest and minimal modification of a single amino acid or peptide bonds is represented by N-methylation. This can improve the pharmacokinetic properties of biologically active peptides as well as resulting in analogues that show specific biological activity such as enzyme inhibitors, receptor antagonists and agonists, building blocks in combinatorial chemistry for the screening of new potential drugs. Further, structural and conformational studies performed with N-methylated analogues of natural amino acids and peptides enabled to (i) produce stable foldamers with different topology with respect to the helix of natural and endogenous peptides, (ii) confer to modified peptides high stability against proteases and (iii) enhance lipophilicity and bioavailability for pharmacological purposes. Consequentially, it is crucial to provide optically pure N-methyl-amino acids and N-methylated peptides with a large supply. The present report will focus on the results obtained in the last decade in the field of chemical synthetic methodologies for the N-methylation of amino acids.

Dysfunctional Antibodies in the Tumor Microenvironment Associate with Impaired Anticancer Immunity.

Studies have demonstrated that cancer-associated matrix metalloproteinases (MMP) can generate single peptide bond cleavages in the hinge region of immunoglobulin G1 (IgG1). This study investigated the cleavage of endogenous IgGs by MMPs in the tumor microenvironment and the consequences of the IgG hinge cleavage for humoral immunity. We investigated the occurrence of single peptide bond cleaved IgGs (scIgG) in tumor tissues and plasma samples collected from a cohort of breast cancer patients (n = 60). Samples from healthy people (n = 20) were used as the control. Antibody hinge cleavage was detected by multiple assays, including IHC, ELISA, and flow cytometry. A correlation analysis was conducted between scIgG levels and patient clinical parameters. Levels of scIgGs in tumors were significantly higher than in normal tissues. In addition, scIgG levels in tumors were enriched compared with that in the plasma of the same patients. The appearance of scIgGs in tumor tissues was associated with altered host IgG content and decreased IgG1. Increased tumor scIgGs were found to be positively correlated with adverse clinical factors, such as elevated tumor-associated macrophages, increased expression of MMP9 and other MMPs, and local metastasis to axillary lymph nodes. The study contributes to mounting evidence for the presence of hinge-cleaved antibodies with reduced Fc immune effector function in the tumor microenvironment. The results highlight a link between tumor scIgGs and poor patient outcomes, and reveal a component of compromised humoral immunity within tumors that could point to new immunotherapeutic strategies to rescue host immunity.

A novel therapeutic strategy to rescue the immune effector function of proteolytically inactivated cancer therapeutic antibodies.

Primary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for therapeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derangement of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading proteinase (IdeS). Similar to cancer-related matrix metalloproteinases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost antibody-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-suppressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzumab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity.

Electrostatically Enhanced Association of a Pim Kinase Substrate Revealed by Stochastic Detection

A wide variety of analytes may be detected stochastically by observing the modulation of ionic current flowing through a single engineered protein pore. Rate and equilibrium constants for the interaction of an analyte with a binding site are obtained without a need for labels, such as fluorescent tags. We engineered alpha-hemolysin pores containing peptide sensor elements within a single trans-side beta-barrel loop. Site-specific proteolysis produced pores bearing peptides tethered by a single N-terminal peptide bond. These pores were used for the single-molecule detection and study of kinase-peptide interactions. Kinetics and affinities of the Pim kinases (Pim-1, Pim-2, Pim-3) for their consensus substrate Pimtide, and of cAMP-dependent protein kinase for an inhibitory peptide, were found to be in good agreement with previously reported values. Distinct current noise behaviors were observed between kinases while bound, which form an additional basis for analyte discrimination. Pim kinases exhibited unusually high association rate constants for their consensus substrate (∼107-108 M−1 s−1), extrapolated to zero applied membrane potential. We found this to be due to electrostatic enhancement, which in vivo may constitute an additional layer of control between cell signaling pathways.

Investigation of peptide splicing using two‐peptide‐chain analogs of trypsin inhibitor SFTI‐1

This study examines peptide splicing catalyzed by serine proteinases. A series of two-peptide-chain analogs of trypsin inhibitor SFTI-1 were designed and synthesized via the solid-phase method. All consisted of two peptide chains (also called N- and C-terminal fragments) joined together by one disulfide bridge. The analogs were incubated with bovine β-trypsin or bovine α-chymotrypsin. Analysis of MS data analysis showed that, after enzyme-catalyzed degradation of the single peptide bond between the Lys and Ser residues located at the C-terminus of the C-terminal peptide chain, a new peptide bond was formed. This bond brought together the separated peptide chains, and, as a result, monocyclic SFTI-1 was recovered. This proteolytic route of peptide rearrangement appears to be similar to peptide splicing catalyzed by proteasomes. However, the proteasome is much more complex than 'classical' serine proteinases.

PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF) multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1) repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein) domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606) in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs). In this review, we a) discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b) address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c) indicate the direction of future investigations.

The activation of factor VII by a variety of potential activators

Factor VII (VII) is an inactive zymogen, which upon proteolysis of a single peptide bond, is activated to the serine‐protease form factor VIIa (VIIa). It is known that in vitro factor Xa (Xa), factor IXa (IXa), thrombin, factor XIIa (XIIa), VIIa, and plasmin can activate VII to VIIa.In the present study, the activation of VII by potential in vivo activators was evaluated under identical conditions, in the absence and presence of cofactors. In order to avoid the influence of VIIa autoactivation to the analysis of VII activation by other activators, VII mutant (Sc195A) was generated, in which the active site serine residue has been mutated to alanine. Experiments were performed on phospholipid vesicles either in the presence of relipidated membrane‐TF, soluble TF, or none.The results show that among Xa, IXa, VIIa‐TF, meizothrombin, thrombin and XIIa, Xa and IXa are the most potent activators of VII. TF facilitates VII activation by 3 to 9‐fold, various from different activators. The cofactors for protease Xa and IXa do not influence the activation of VII. Preliminary data also shows that IXaα is one of the potent activators of VII. This study shed light on how VII get activated in physiological conditions.

Thrombotic Thrombocytopenic Purpura and Anti-Thrombotic Therapy Targeted to Von Willebrand Factor

Resting endothelial cells lining the inner surface of blood vessels have anti-thrombotic and anti-inflammatory actions, critical for maintaining normal vascular homeostasis. Upon localized or systemic stimulation, endothelial cells are activated to secrete bioactive molecules; among them is von Willebrand factor (VWF). Freshly secreted VWF is enriched in ultra-large (UL) forms that are anchored to endothelial cells to form long string-like structures, to which platelets tether and aggregate. This prothrombotic event is normally prevented by proteolytic cleavage of ULVWF multimers by ADAMTS-13 at a single peptide bond of Tyr1605-Met1606 in the A2 domain. The cleavage reduces the size and adhesion activity of (UL)VWF multimers. Lacking this cleaving activity due to mutations in the ADAMTS13 gene or autoantibodies against the metalloprotease is associated with systemic microvascular thrombosis found in patients with thrombotic thrombocytopenic purpura (TTP). Recombinant ADAMTS-13 has the potential to be a therapeutic agent to reduce prothrombotic activity of ULVWF multimers. Alternatively, blocking an interaction between ULVWF and its platelet receptor could achieve the same therapeutic goal. This review discusses potentials of using recombinant ADAMTS-13 and VWF-blocking agents as therapeutics for TTP and other acquired ADAMTS-13 deficiencies.

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin

The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77-G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.

Activation of the Zymogen to Urokinase-Type Plasminogen Activator Is Associated with Increased Interdomain Flexibility

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab ( fragment antigen- binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed.

Removal of amino-terminal extracellular domains of desmoglein 1 by staphylococcal exfoliative toxin is sufficient to initiate epidermal blister formation

In both bullous impetigo and staphylococcal scalded-skin syndrome (SSSS), exfoliative toxins (ETs) produced by Staphylococcus aureus cause superficial intraepidermal blisters. ETs are known to cleave specifically a single peptide bond in the extracellular domains 3 and 4 of desmoglein (Dsg) 1. However, the precise mechanisms underlying ET-induced epidermal blister formation remain poorly understood. To determine whether cleavage of Dsg1 by an ET is sufficient to induce blister formation in vivo or if the subsequent internalization of cleaved Dsg1 or other desmosomal components is required. Skin samples obtained from neonatal mice injected with ETA were analyzed by time-lapse immunofluorescence and transmission electron microscopy for desmosomal components. Epidermal blister formation was observed as early as 60 min after ETA treatment. At this time, the amino-terminal extracellular domains of Dsg1 disappeared from the surface of keratinocytes, while the cleaved carboxy-terminal domain of Dsg1 (Dsg1-C) as well as the extracellular domains of desmocollin 1 (Dsc1-N) remained on the cell surface. Half-split desmosomes with intracytoplasmic dense plaques and attached tonofilaments were recognized ultrastructurally on the split surface of keratinocytes at 60 min. Subsequent to this, Dsg1-C and Dsc1-N gradually disappeared from the surface layer of keratinocytes. Our findings suggest that the removal of amino-terminal extracellular domains of Dsg1 by ETs is sufficient to initiate epidermal blister formation in bullous impetigo and SSSS.