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• Dss2000 Cat 862 Board Dci Version 1

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AbstractExfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu 381–Gly 382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilizedstructure of Dsg1.

Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage ofDsg1 by ETA, ETB, and ETD demonstrated k cat/ K m values of 2–6 × 10 4 m –1 s –1, suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine proteaseinhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate.Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we showthat for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond.If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisitespecificity and efficiency of ETA may depend on the enzyme's binding upstream of the cleavage site with a very specific fit,like a key in a lock.

Staphylococcus aureus is a frequent bacterial pathogen in human disease. Skin infections are particularly prevalent, with impetigo caused by S.

Aureus one of the most common infectious diseases in children. Approximately 30% of impetigo patients develop bullous impetigo, which is caused by S. Aureus strains that produce exfoliative toxins (ETs) (, ). In bullous impetigo, blisters of the superficial epidermis occur at sites of infection. More extensive disease can occurin staphylococcal scalded skin syndrome (SSSS), in which ETs produced by a nidus of infection circulate systemically, usuallyin infants and young children, to produce more generalized blistering.

Although SSSS is much less common than bullous impetigo,outbreaks that occur in neonatal nurseries have important consequences for health care delivery (, ).In the 1970s Melish et al. (–) showed that ETs produced by S. Aureus injected into neonatal mice caused blister formation in skin similar to that seen in patients with bullous impetigo and SSSS.It remained unclear exactly how ETs cause this blister until it was discovered that they have structural and sequence homologiesto serine proteases, at which time it was suggested that they act as proteolytic enzymes that were predicted to cleave aftereither a glutamic or aspartic acid.

However, they could not be shown to cleave model polypeptide substrates. Their normal pathophysiologic substrate, if any,remained a mystery until it was realized that the blisters in bullous impetigo and SSSS show clinical and histological similarityto those of pemphigus foliaceus, an autoimmune disease in which pathogenic autoantibodies bind the desmosomal cell adhesionmolecule, desmoglein 1 (Dsg1). This observation led to the hypothesis that the molecular target of ETs is Dsg1. This hypothesis was validated by showingthat three different ET isotypes (ETA, ETB, and ETD) specifically cleave mouse and human Dsg1 (hDsg1) but not closely relatedmolecules such as Dsg3 or E-cadherin (–). Mutation of the predicted active serine in these ETs caused loss of their proteolytic activity, confirming that they areserine proteases.Dsg1 is in the desmosomal cadherin subfamily of the cadherin supergene family.

Desmosomal cadherins, similar to classic cadherins,contain four amino-terminal homologous domains (called extracellular (EC) domains 1–4), of about 100 amino acid each, thatare highly conserved among many cadherins. A fifth juxtamembrane domain (EC5) is not as well conserved within or among the cadherins. The structure of Dsg1, likeother cadherins, is maintained by calcium binding that occurs between EC domains (–). This calcium-stabilized structure is thought to be critical in maintaining the adhesive function of classical and, presumably,desmosomal cadherins (–).Recent studies show not only that ETs show a substrate specificity for Dsg1 but also that they hydrolyze only one peptidebond in human and mouse Dsg1 at exactly the same site, after glutamic acid 381 between EC3 and EC4.

Furthermore, cleavage of Dsg1 by ETs is dependent on the former's calcium-stabilized conformation ( i.e. Denatured or partially denatured Dsg1 cannot be hydrolyzed by ETs). Thus, unlike more typical serine proteases whose specificities are defined by simple amino acid sequences and which canhydrolyze many proteins and model polypeptides, ETs show exquisite specificity for one peptide bond in one correctly foldedsubstrate, Dsg1.In this study, we show that in addition to being exquisitely specific for a single peptide bond in Dsg1, ETs are efficientin hydrolyzing that peptide bond. We show that the catalytic site of ETA is protected from typical serine proteases inhibitorsand that domains in Dsg1 amino-terminal, but not carboxyl-terminal, to the cleavage site are necessary for its cleavage byETA. Finally, we show that although ETA binds to canine Dsg1 (cDsg1) it does not cleave it, but it will do so if 5 amino acidsthat are upstream of the cleavage site are mutated to hDsg1 residues. The results of these studies suggest that the exquisitespecificity and efficiency of ETA in cleavage of Dsg1 depend on the enzyme's binding upstream of the cleavage site with theproper fit, like a key in a lock, to properly align, and perhaps activate, its catalytic site. MATERIALS AND METHODSRecombinant ETs—DNA encoding the ETD mature sequence was amplified by PCR using pQE-ETD-His (provided by Dr.

Motoyuki Sugai, University ofHiroshima) as a template. PCR was also used to add nucleotides encoding the ETA signal peptide to the 5′-end of the ETD sequence and to add nucleotidesencoding the V5 and His tags to the 3′-end of the ETD sequence.

ETD-V5His was subcloned into the shuttle vector, pCE104. PCE104 with ETD-V5His was electroporated into RN4220 S. Aureus electrocompetent cells. We also used recombinant wild-type ETA, ETB, and ETAS195A (enzymatically active serine, at position 195 as defined by classicalchymotrypsin serine protease numbering, mutated to alanine) with V5 and His tags as previously described. Recombinant ETs were purified on Ni 2+-nitrilotriacetic acid columns (Qiagen, Valencia, CA) according to the manufacturer's protocol and then dialyzed against phosphate-bufferedsaline. Protein concentrations of ETs were estimated with a Protein Assay Kit (Bio-Rad) and on Simply Blue (a modified CoomassieG-250 solution; Invitrogen)-stained SDS-PAGE gels compared with standards and each other.Production and ET Cleavage of Human Desmoglein 1—The entire EC domain of human recombinant Dsg1 with a His and E tag (hDsg1E) on the carboxyl terminus was produced as a secretedprotein by baculovirus as previously described.

For kinetic analysis, hDsg1E was purified from insect cell culture supernatant on an anti-E tag column (Amersham Biosciences)and eluted using 0.1 mg/ml E tag peptide in 20 m m Tris, pH 7.4, 500 m m NaCl, 1 m m CaCl 2 (TBS plus Ca 2+). The eluant was dialyzed against TBS plus Ca 2+. 1 μ m purified hDsg1E (estimated with a protein assay kit (Bio-Rad)) was incubated with varying concentrations of ETA, ETB, andETD for various times in TBS plus Ca 2+ at 25 °C and then analyzed by SDS-PAGE stained with Simply Blue (Invitrogen). These gels were scanned for densitometric analysisby NIH Image software. The percentage of cleavage was calculated as a function of time, and resultant curves were analyzedby Igore Pro software (WaveMetrics, Inc., Lake Oswego, OR).1 μ m hDsg1E was chosen because of the previously estimated dissociation constant ( K d) of ∼10 μ m, determined for the interaction of an inactive (active serine at position 195 mutated to alanine) ETA with hDsg1E. This value suggests a lower limit for the K m, and at a substrate (hDsg1E) concentration ∼10-fold less than K m, the Michaelis-Menten equation predicts pseudo-first-order kinetics for any particular enzyme concentration, which was confirmedby the exponential decay of hDsg1E shown in.

1.Kinetics of cleavage of Dsg1 by ETs. A, purified ETA, ETB, and ETD were subjected to SDS-PAGE and then stained by Simply Blue, which showed one major band. B, purified hDsg1 (1 μ m) was incubated with 25 n m ETA and incubated for 0, 1, 5, 10, 20, 60, 120 min before boiling in SDS sample buffer.

Simply Blue staining of samples afterSDS-PAGE indicated hDsg1E at 80 kDa ( arrow) and the amino-terminal (50 kDa, arrowhead) and carboxyl-terminal (30 kDa, open arrow) cleavage products. C, time plots of the cleavage of hDsg1E by ETA, ETB, or ETD. Densitometry of stained SDS-PAGE indicates loss of hDsg1E ( squares) and production of cleaved products ( circles) with increasing time of digestion. D, plots of the natural log ( ln) of the density of the substrate, hDsg1E, against time show good linear fits for substrate concentrations down to ∼90% ofstarting concentrations, consistent with pseudo-first-order decay ( i.e.

Velocity of decay is directly proportional to the substrate concentration at a specific starting enzyme concentration). E, the exponential constant, k obs, was calculated for appearance of products ( filled circles) or disappearance of substrates ( open circles) for three concentrations of ETA (see “Results”).

For protease inhibitor studies, 1 μ m ETA or 1 μ m staphylococcus V8 protease (Roche Applied Science) was preincubated with 20 m m diiopropylfluorophosphate (DFP) (Calbiochem) or 2 m m 3,4-dichloroisocoumarin (DCI) (Sigma) or 0.1 units/μl α 2-macroglobulin (Roche Applied Science) at 25 °C for 30 min in TBS plus Ca 2+. Pretreated ETA or V8 was then incubated with 10 n m of hDsg1E in TBS plus Ca 2+ at 25 °C for 1 h and analyzed by Western blotting.Dsg1 Truncated Mutants and Chimeric Recombinants—PCR was used to construct cDNAs encoding various truncated hDsg1 molecules with E tags or E tag-glutathione S-transferase (GST) tags on their carboxyl termini. These recombinant constructs were subcloned into the baculovirus expressionvector pEVmod. Various domain-swapped chimeric molecules (Figs. And ) between the EC domains of hDsg1 and human Dsg3 (hDsg3) and between hDsg1 and mouse E-cadherin were produced by PCR and subcloned into the pEV-EHis cassette to add nucleotides encoding the E and His tags to their 3′-ends. CDsg1 cDNA was cloned by reverse transcriptase-PCR using RNA from the canine cell line MCA-B1 (from Dr. Susumu Tateyama; Universityof Miyazaki).

CDNA for the EC domains of cDsg1 was subcloned into baculovirus expression vector pEV-EHis as previously described. CDNAs encoding truncated cDsg1 and domain-swapped chimeric molecules between the EC domains of cDsg1 and hDsg1 were produced by PCR and subcloned into the pEV-E-GST cassette to add the E and GST tags to their carboxyl termini. Theentire EC domain of hDsg3 with a His and E tag (hDsg3E) on the carboxyl terminus was produced as previously described. Site-directed mutagenesis of cDsg1 was performed with the Genetailor mutagenesis kit (Invitrogen) following the manufacturer'sprotocol to make mutants so that selected amino acids were mutated into hDsg1 equivalents. Recombinant baculovirus expression vectors were co-transfected with BaculoGold DNA (BD Biosciences) to Sf9 cells, and baculovirusin culture supernatants was used to infect High Five cells as previously described. Approximately, 10 n m recombinant secreted protein in the High Five culture supernatant was incubated with 1 μ m ETA for cleavage analysis.

Degradation of hDsg1E was assayed by Western blotting with anti-E tag antibodies, as previouslydescribed. 3.Susceptibility of truncated hDsg1 to cleavage by ETA. A, comparison of truncated molecules of hDsg1. Black bars in the hDsg1E indicate presumptive calcium-binding amino acid residues. E381 indicates glutamic acid 381, after which ETs cleave. SP, signal peptide; Pro, propeptide sequence; E, E tag; GST, glutathione S-transferase tag. EGPYF PR and E PR are single letter amino acid codes (nonunderlined letters indicate normal hDsg1 sequence, and underlined letters indicatesequence of tag).

+, construct is cleaved by ETA; +/–, diminished cleavage; –, no cleavage. B, Western blotting with anti-E tag of truncated hDsg1E incubated with (+) or without (–) ETA. EC1–5 and EC1–4 show most efficientcleavage, but EC2–EC5 and EC2–4 also show some cleavage. EC3–EC5 was not cleaved by ETA. EC1–EC3 was no longer detected byanti-E tag after ETA incubation, suggesting that the E tag was cleaved but was not detected on the gel because of its smallsize. C, EC1–EC3 cleavage by ETA was confirmed with a longer tag consisting of both the E tag and GST tag on the carboxyl terminusof EC1–EC3. However, the deletion of 4 amino acids after the cleavage site ( EC1–3Δ) eliminates its susceptibility to ETA.

5.Susceptibility to cleavage by ETA of domain-swapped molecules between hDsg1, hDsg3, and E-cadherin. A, schematic diagram of domain-swapped molecules between hDsg1 ( white), hDsg3 ( gray), and mouse E-cadherin ( E-cad) ( dotted box). B, Western blotting with anti-E tag shows that hDsg3 with substitution of hDsg1 214–450 and hDsg1 214–398 (data not shown)were cleaved, whereas hDsg1 292–450 was not, suggesting that the junction of EC2-EC3 contains important sequences for susceptibilityto cleavage by ETA. However, E-cadherin containing hDsg1 214–450 was not cleaved, suggesting that E-cadherin does not providethe necessary overall conformation for cleavage. 6.ETA does not cleave cDsg1, but substitution of the EC2-EC3 junction from hDsg1 restores cDsg1 susceptibility to cleavage.

Dss2000 Cat 862 Board Dci Version 1

A, amino acid sequence alignment of hDsg1, mouse Dsg1, and cDsg1 around the cleavage site of exfoliative toxins. The arrow indicates the cleavage site by ETs. Note the very well conserved sequence in hDsg1, mouse Dsg1, and cDsg1 around the cleavagesite. B, amino acid sequence alignment of sequence upstream of the cleavage site.

The underlined amino acids in cDsg1 show nonidentical sequence. C, comparison of hDsg1E ( white), cDsg1E ( gray), truncated cDsg1EC1–3EGST, and cDsg1EC1–3EGST substituted with human sequences. D, anti-E tag Western blotting shows that cDsg1E, like hDsg3E, is not cleaved by ETA. E, Western blotting with anti-E tag shows that substitution of hDsg1 amino acid residues 214–334 in cDsg1 restored its susceptibilityto cleavage by ETA, but substitution of hDsg1 amino acid residues 214–257 in cDsg1 did not (data not shown). 7.Mutation of 5 amino acids in cDsg1 into human sequences restores its susceptibility to cleavage by ETA. A, amino acid sequences of point-mutated cDsg1 with residues mutated to human sequences underlined.

B, mutation of 5 amino acids in cDsg1, shown in construct c271–7, restores susceptibility for cleavage by ETA. C, mutated sequences (in black, thin arrows) in c271–7 superimposed on the c-cadherin crystal structure. Note that the mutated sequences are on a loop proximal to thecleavage site (in black, thick arrow). Immunoprecipitation—As in methods previously described , supernatants of High Five insect cells transduced with baculovirus encoding hDsg1E, cDsg1E, or hDsg3E were incubated withETAS195A and then used directly for immunoprecipitation. E tagged proteins were precipitated with anti-E tag-Sepharose (AmershamBiosciences) at 4 °C for 1 h. Immunoprecipitates were washed 10 times with 1% Triton X-100 TBS plus Ca 2+ and then eluted with Laemmli sample buffer at 100 °C.Western Blotting—Proteins in Laemmli sample buffer were separated by 4–20% SDS-PAGE (Bio-Rad) and then transferred to nitrocellulose sheets(Transblot; Bio-Rad). The sheets were incubated for 1 h in blocking buffer of 5% fat-free milk powder in phosphate-bufferedsaline.

The E tag antibody conjugated with horseradish peroxidase (Amersham Biosciences) or anti-ETA sheep polyclonal antibodyconjugated with horseradish peroxidase (Toxin Technology, Sarasota, FL), diluted in blocking buffer, was applied for 1 h atroom temperature. After four washes with 0.1% Tween 20 in phosphate-buffered saline, the signals were detected with chemiluminescence(ECL or ECL Plus; Amersham Biosciences). RESULTSKinetics of ET Cleavage of hDsg1—To investigate the efficiency of hydrolysis of Dsg1 by ETs, we measured hDsg1 cleavage by ETs as a function of time.Purified recombinant ETA, ETB, and ETD used in these experiments showed a single band by Simply Blue staining of SDS-PAGE.1 μ m hDsg1E was incubated with 12.5, 25, and 50 n m ETA or 25 and 50 n m of ETB and ETD. Both hDsg1E disappearance and product appearance were quantified over time from bands resolved by SDS-PAGE.SDS-PAGE of a representative cleavage time course with 25 n m ETA is shown in.

Reactions were stopped by the addition of SDS denaturing buffer and subsequent heating to 100 °C, resolved on a 4–20% gradientpolyacrylamide gel, and stained with Simply Blue. The substrate ( upper band, arrow) is cleaved with time to the products ( two lower bands, arrowhead and open arrow).Stained bands were quantified by densitometry, and the data were plotted as a function of time for analysis. Substrate consumption and product formation were fit to exponential curves defined by Equations and to obtain k obs, the observed rate constant for substrate consumption or product formation as follows, where S represents the substrate concentration (as determined by densitometry) and P represents concentration of theproducts.Observed rate constants obtained from disappearance and formation data were in good agreement, as expected. For a given ETconcentration, curves defined by first order kinetics ( i.e. Equations and ) fit the data points well.

This is best shown by the linear fit in, in which the natural log of S is plotted against time. Measurements were made at three (for ETA) or two (for ETB and ETD) ET concentrations. Values of k obs were directly proportional to ET concentrations, also consistent with pseudo-first-order reactions. The values for k cat/ K m, which is a measure of enzyme efficiency, were obtained from the slopes of plots of k obs versus ET concentrations, according to Equation, Values of 62,000, 26,000, and 19,000 m –1 s –1 were obtained for ETA, ETB, and ETD, respectively.

These values indicate efficient cleavage of a large protein substrate.The Broad Spectrum Serine Protease Inhibitors DFP, DCI, and α 2-Macroglobulin Do Not Effectively Inhibit the Enzymatic Activity of ETA—We tested the ability of small (DFP and DCI) and large (α 2-macroglobulin) broad spectrum serine protease inhibitors to inhibit the enzymatic activity of ETA. There was minimal, ifany, inhibition at the concentration tested, whereas both protease inhibitors did efficiently inhibit V8 protease at concentrationsequivalent to those of ETA , although an ∼10-fold larger excess of DCI did show inhibition (data not shown). These data suggest that the catalytic siteof ETA may be difficult to access in the proper alignment by some serine protease inhibitors or is relatively inactive inthe absence of its proper substrate. HDsg1 EC Domains That Are Upstream, but Not Downstream, of the Cleavage Site Are Critical for Its Hydrolysis by ETA—Previously, we have shown that the ability of ETs to cleave Dsg1 is not simply dependent on the amino acid sequence of thecleavage site but is dependent on the calcium-stabilized conformation of Dsg1. These results suggest that domains distant from the site of hydrolysis might influence the enzymatic efficiency of ETA.Because desmogleins have five well defined EC domains whose relationship to each other is stabilized by calcium, we firstdetermined whether all of these domains were necessary for cleavage by testing truncated molecules of hDsg1E for their susceptibilityto hydrolysis by ETA. These data indicated that EC5 is not necessary for cleavage. Loss of EC1 decreases the efficiency of cleavage, yet thetruncated molecule is still cleaved.

Loss of EC1-EC2 prevents cleavage. After incubation with ETA, the EC1–EC3 domain can no longer be detected with anti-E tag by Western blotting , presumably because of cleavage of the E tag, which is too small to detect on these gels. To confirm the cleavage of EC1–EC3,a GST tag was added after the E tag on the carboxyl-terminal end. Incubation of this new substrate, EC1–3EGST, with ETA clearlyshowed cleavage with the product now detectable. In this construct, the combination E and GST tag was added to hDsg1 only 4 amino acids after the cleavage site. Furtheranalysis indicates that if these 4 amino acids were truncated, the construct was no longer susceptible to cleavage.

Thesedata show that cleavage by ETA is not dependent on intact domains EC4 and EC5.Because truncations may result in a generally unstable overall structure, we used domain swapping between hDsg1E and hDsg3to further test which domains in Dsg1 are critical. These data suggest that sequences in hDsg1 between 213 and 450 are probably sufficient for cleavage. This conclusion wasconfirmed by insertion of just these sequences from Dsg1 into Dsg3.

In fact, finer analysis indicated that only amino acids 214–398 from the EC2-EC3 domain of Dsg1, inserted into Dsg3, weresufficient for cleavage (data not shown). Although sufficient in the overall structure of Dsg3, these amino acids must bein the proper overall conformation of a desmoglein, because when inserted into E-cadherin, they could no longer be hydrolyzedby ETA.5 Amino Acids 110 Residues Upstream of the Cleavage Site in Dsg1 Are Necessary for Cleavage—To further define which particular amino acids in the EC2-EC3 region of hDsg1 are critical for cleavage, we used cDsg1 becauseit is highly homologous to human and mouse Dsg1 and shares identical amino acids around the cleavage site yet is not hydrolyzed by ETA. Swapping sequences of hDsg1 into cDsg1 near the EC2-EC3 junction allowed its cleavage. We then determined which amino acids in this junction of cDsg1 were different from those in hDsg1 and mouse Dsg1, which,like hDsg1, is cleaved by ETs. Because cDsg1 is highly homologous to the human and mouse Dsg1, there were only a limited number of candidate amino acidsthat might be critical to allow cleavage of cDsg1.

Substitution of these amino acids from hDsg1 to cDsg1 allowed us to find5 amino acids in hDsg1 that are critical to allow hydrolysis of cDsg1 by ETA. These amino acids are about 110 residues upstream of the cleavage site.ETA Binding and Ability to Cleave Are Independent in Canine Dsg1—We speculated that the inability of ETA to hydrolyze cDsg1 could be due to its inability to bind that substrate, as with hDsg3, and substitution of amino acids from the hDsg1 sequence might restore binding and, therefore, cleavage. However, co-immunoprecipitationof an ETA with the active serine mutated to alanine with cDsg1 and hDsg1 showed that both could bind the enzyme. (A mutantmust be used to prevent hydrolysis of the hDsg1-positive control. These data suggest that cleavage of Dsg1 by ETA depends not only on binding but also on proper alignment and/or activationof the enzyme active site. DISCUSSIONExfoliative Toxins Are Efficient Enzymes—The calculated values of k cat/ K m of 2–6 × 10 4 m –1 s –1 show efficient enzymes.

These k cat/ K m values are similar to those for the best tetrapeptide synthetic substrates for the acidic amino acid-specific endopeptidasesS. Aureus V8 protease and glutamic acid-specific Streptomyces griseus proteinase, which are 6 × 10 3 and 9 × 10 5 m –1 s –1, respectively. These k cat/ K m values are also greater than those obtained for the hydrolysis of the esterolytic substrate ( N- t-Boc- l-Glu-α-phenyl ester) by ETA and ETB, which were reported to range from 3 to 6 × 10 3 m –1 s –1. It also should be considered that the values we calculated for k cat/ K m were for the hydrolysis of a large asymmetric protein, Dsg1. The interaction between two macromolecules is naturally goingto be less efficient than that of an enzyme with a small substrate due to fact that only a relatively small area of each largeprotein is interactive (–). For example, k cat/ K m values for enzyme-small model peptide substrate reactions can be as high as 10 8 to 10 9 m –1 s –1.

The basal rate constant for productive collisions ( i.e. Encounters with the enzyme and substrate in the proper orientation for hydrolysis) between two large proteins in the absenceof electrostatic effects is estimated at ∼10 5 m –1 s –1. The k cat/ K m values for ET hydrolysis of Dsg1 approach this value. From these data, it can be appreciated that ETs are efficient enzymes.Our data also suggest that ETA is more efficient than ETB and ETD, although this observation may not reflect significant biologicaldifferences, since all of the enzymes are extremely efficient and pathological.ETA Is Not Inhibited by Broad Spectrum Serine Protease Inhibitors—The broad spectrum serine protease inhibitors, DFP, DCI, and α 2-macroglobulin, do not efficiently inhibit ETA. These data suggest that somehow the active site of the enzyme is either inaccessibleor not in the proper conformation to interact with these inhibitors and is consistent with the idea that only by binding toits physiologic substrate does the catalytic site of ETA become properly activated and/or aligned with the peptide bond inDsg1 that it hydrolyzes.

Consistent with this idea, some crystal structure analyses of ETA and ETB have suggested that theiroxyanion hole is improperly formed and that the enzymes might require binding to the proper area to activate the catalyticsite (, ). However, one structural analysis of ETB suggests that the oxyanion hole is properly formed.Amino Acids Over 100 Residues Upstream of the Cleavage Site in hDsg1 Are Required for Susceptibility to Cleavage by ETA—Previous data indicate that ETs are not simply sequence-specific proteases, like typical serine proteases such as trypsinand chymotrypsin, but are dependent on the calcium-stabilized conformation of their substrate.

These observations may account for their exquisite specificity for Dsg1. Since the EC domain of Dsg1 contains well definedsubdomains, we determined which were necessary for susceptibility to cleavage by ETA. Interestingly, hDsg1 sequences in domainsEC2-EC3, amino-terminal to the scissile bond, are necessary for cleavage, but cleavage is more efficient if they are in theoverall structure of a desmoglein, and they are not sufficient for cleavage when inserted into the homologous region of E-cadherin.We also showed, using cDsg1, that within these domains there are 5 amino acids in hDsg1 that are critical to allow cleavage.Although highly homologous to hDsg1, cDsg1 is not cleaved by m ETA, consistent with the observation that ETA does not induce blister formation in dog skin (, ).

Substitution of these 5 amino acids from hDsg1 allows its cleavage. The location of these amino acids, as determined onthe homologous C-cadherin crystal structure , is on a loop proximal to the cleavage site , suggesting that this loop may be important in properly aligning the enzyme.Key in Lock Model for Specificity and Efficiency of ETA—Our present data, together with previous data showing the importance of calcium-stabilized conformation of Dsg1 for its cleavageby ETA , suggest that the enzyme binds the substrate upstream of the cleavage site, which may align the catalytic site with thescissile bond. Furthermore, our data on resistance of ETA to serine protease inhibitors is consistent with an inactive orinaccessible catalytic site that we hypothesize is activated and/or made accessible to the scissile bond when the proper bindingto the substrate occurs. Activation of ETA must require a very specific type of binding interaction, because although ETAbinds to cDsg1 it does not cleave it. However, changing 5 amino acids in a loop just upstream of the cleavage site in cDsg1allows its cleavage by ETA.

The structure of this loop might allow a very specific “fit” that leads to enzyme activation andcan, in this context, be thought of as similar to a key (ETA) in a lock (Dsg1).Finally, our kinetic data indicate that either an efficient enzyme once aligned properly or, alternatively, electrostaticinteractions between ETA and Dsg1 enhance the alignment beyond simple diffusion.The key in lock model that explains the specificity of ETs is not unique. For example, it has also has been applied to thespecificity of coagulation factors. One example is the specificity of thrombin for fibrinogen, another system involving theinteraction between two large proteins. Thrombin exhibits a number of unique structural features that provide specificity. The deep cleft bordering the activesite called the 60-insertion loop limits the accessibility of the active site. An exosite defined as a highly positively chargedregion away from the active site is complementary to a negatively charged region in fibrinogen. An aryl binding site requiresapolar residues to occupy certain positions of substrates.

Thus, the substrate must interact with all of these regions, likea key in a lock, to be hydrolyzed efficiently. Although our studies are not as detailed as those with thrombin, certain parallelscan be made.

Recognition of Dsg1 as a substrate is conformation-specific rather than sequence-specific and probably involvesa specific exosite interaction between ETA and a region of extracellular domain 3 of Dsg1. Although the value of k cat/ K m for hydrolysis of Dsg1 by ETA was not as high as that observed for thrombin hydrolysis of fibrinogen , Dsg1 is concentrated in vivo in desmosomes on cell surfaces. In these structures, hydrolytic rates may be much faster due to the effective concentrationof the substrate.In conclusion, ETs have evolved to efficiently and specifically hydrolyze one bond in one substrate through specific interactionswith that substrate, depending on both the substrate's amino acid sequence and conformation at and distal to the cleavagesite. The enzyme is thus able to efficiently and specifically target the exact molecule that allows S. Aureus to spread under the stratum corneum, the major barrier of the skin. Footnotes.1 The abbreviations used are: ET, exfoliative toxin; Dsg1 and -3, desmoglein 1 and 3, respectively; SSSS, staphylococcal scaldedskin syndrome; EC, extracellular; TBS, Tris-buffered saline; DFP, diiopropylfluorophosphate; DCI, 3,4-dichloroisocoumarin;GST, glutathione S-transferase; E tag, E peptide tag; hDsg1 and -3, human desmoglein 1 and 3, respectively; cDsg1, canine Dsg1. This work was supported by grants from the National Institutes of Health and a grant-in-aid for scientific research fromthe Ministry of Education, Science, and Culture of Japan.

The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be hereby marked “ advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ Recipient of the Japanese Society for Investigative Dermatology International Fellowship Shiseido Award for 2002. Received October 8, 2003.

Revision received November 7, 2003. The American Society for Biochemistry and Molecular Biology, Inc.