der p 1 was isolated from house dust mite fecal pellets ( allergon ) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti 
der p 1 mab ( clone 4c1 ; indoor biotechnologies ) , removal of contaminating serine proteases on immobilized soybean trypsin inhibitor ( sigma chemical co. ) , and finally fast protein liquid chromatography ( fplc ) to remove low - molecular - mass contaminants .
the purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer ( applied biosystems , inc . ) , sds - page analysis ( 15% gel ) , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 ( l - trans - epoxysuccinyl - leucylamido [ 4-guanidino]butane ) .
protein concentration was determined using a bicinchoninic acid ( bca ) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e ( 280 nm ) = 16.4 . before use ,
der p 1 was preactivated with 5 mm cysteine ( sigma chemical co. ) to regenerate its thiol group , which becomes oxidized during purification .
the catalytic activity of der p 1 was ascertained in a continuous rate ( kinetic ) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl ( boc)-gln - ala - arg7-amino-4-methyl - coumarin ( amc ; reference 6 ) . to block the proteolytic activity of cysteine - activated der p 1 , 1,000-fold molar excess of e-64 ( sigma chemical co. ) was used ; a similar molar ratio of the cysteine protease inhibitor iodoacetamide ( sigma chemical co. ) was used as another sulfhydryl reactive agent .
the cells ( 2  10 ) were suspended in rpmi ( gibco life technologies ) and stimulated for 3 d at 37c with con a ( 5 g / ml final concentration ) in the presence of il-2 ( 100 u / ml ) in a humidified atmosphere of 5% co2 .
cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 ( preactivated with 5 mm cysteine ) for 1 h at 37c in a total volume of 200 l serum - free aim v medium ( gibco life technologies ) .
the cells were then resuspended in rpmi containing 2% fcs , stained for 45 min at room temperature in the dark with fitc - labeled anti 
mouse cd25 mab ( clone amt-13 ; sigma chemical co. ) , and fixed with 5% formaldehyde .
the expression of other t cell surface markers , namely cd3 , cd4 , and cd8 , was monitored in the same way using appropriate pe- or fitc - labeled antibodies ( clone kt3 , beckman coulter ; and clones yts191.1 and kt15 [ serotec ltd .
five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1 , 10 g of e-64blocked der p 1 , 10 g of iodoacetamide - blocked der p 1 , 10 g of ova ( as proteolytically inactive antigen ; sigma chemical co. ) , or 10 g of ova with e-64 , respectively .
a tail bleed was obtained 1 wk before the start of immunization ( prebleed ) , and a total bleed was obtained by cardiac puncture 1 wk after the last injection ( final bleed ) .
the proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above .
serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass .
the optimal dilutions used here were 1/10 for detecting total ige , der p 1specific ige , and ova - specific ige and 1/20,000 , 1/40,000 , and 1/250 for detecting der p 1specific igg , igg1 , and igg2b , respectively .
mouse ige ( clone r35 - 72 ; pharmingen ) as capture antibody and a second biotinylated monoclonal anti 
der p 1specific ige , ova - specific ige ( measured using samples that have been depleted of igg on a protein g column [ pharmacia ] ) , and der p 1specific igg , igg1 , and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated ( for ige clone r35 - 118 and igg1 clone a85 - 1 [ pharmingen ] and for igg2b clone ab275 [ the binding site ] ) or alkaline phosphatase  conjugated isotype - specific antibodies ( for igg ; sigma chemical co. ) .
alkaline phosphatase  conjugated extravidine ( sigma chemical co. ) was used in conjunction with biotinylated antibodies .
unpaired student 's t test was used to compare levels of antibody responses between the different immunization groups ; p < 0.05 was considered significant .
der p 1 was isolated from house dust mite fecal pellets ( allergon ) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti 
der p 1 mab ( clone 4c1 ; indoor biotechnologies ) , removal of contaminating serine proteases on immobilized soybean trypsin inhibitor ( sigma chemical co. ) , and finally fast protein liquid chromatography ( fplc ) to remove low - molecular - mass contaminants .
the purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer ( applied biosystems , inc . ) , sds - page analysis ( 15% gel ) , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 ( l - trans - epoxysuccinyl - leucylamido [ 4-guanidino]butane ) .
protein concentration was determined using a bicinchoninic acid ( bca ) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e ( 280 nm ) = 16.4 . before use ,
der p 1 was preactivated with 5 mm cysteine ( sigma chemical co. ) to regenerate its thiol group , which becomes oxidized during purification .
the catalytic activity of der p 1 was ascertained in a continuous rate ( kinetic ) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl ( boc)-gln - ala - arg7-amino-4-methyl - coumarin ( amc ; reference 6 ) . to block the proteolytic activity of cysteine - activated der p 1 , 1,000-fold molar excess of e-64 ( sigma chemical co. ) was used ; a similar molar ratio of the cysteine protease inhibitor iodoacetamide ( sigma chemical co. ) was used as another sulfhydryl reactive agent .
spleen t cells were obtained from c57bl/6j mice using standard procedures . the cells ( 2  10 ) were suspended in rpmi ( gibco life technologies ) and stimulated for 3 d at 37c with con a ( 5 g / ml final concentration ) in the presence of il-2 ( 100 u / ml ) in a humidified atmosphere of 5% co2 .
cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 ( preactivated with 5 mm cysteine ) for 1 h at 37c in a total volume of 200 l serum - free aim v medium ( gibco life technologies ) .
the cells were then resuspended in rpmi containing 2% fcs , stained for 45 min at room temperature in the dark with fitc - labeled anti 
mouse cd25 mab ( clone amt-13 ; sigma chemical co. ) , and fixed with 5% formaldehyde .
the expression of other t cell surface markers , namely cd3 , cd4 , and cd8 , was monitored in the same way using appropriate pe- or fitc - labeled antibodies ( clone kt3 , beckman coulter ; and clones yts191.1 and kt15 [ serotec ltd .
five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1 , 10 g of e-64blocked der p 1 , 10 g of iodoacetamide - blocked der p 1 , 10 g of ova ( as proteolytically inactive antigen ; sigma chemical co. ) , or 10 g of ova with e-64 , respectively .
a tail bleed was obtained 1 wk before the start of immunization ( prebleed ) , and a total bleed was obtained by cardiac puncture 1 wk after the last injection ( final bleed ) .
the proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above .
serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass .
the optimal dilutions used here were 1/10 for detecting total ige , der p 1specific ige , and ova - specific ige and 1/20,000 , 1/40,000 , and 1/250 for detecting der p 1specific igg , igg1 , and igg2b , respectively .
mouse ige ( clone r35 - 72 ; pharmingen ) as capture antibody and a second biotinylated monoclonal anti 
der p 1specific ige , ova - specific ige ( measured using samples that have been depleted of igg on a protein g column [ pharmacia ] ) , and der p 1specific igg , igg1 , and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated ( for ige clone r35 - 118 and igg1 clone a85 - 1 [ pharmingen ] and for igg2b clone ab275 [ the binding site ] ) or alkaline phosphatase  conjugated isotype - specific antibodies ( for igg ; sigma chemical co. ) .
alkaline phosphatase  conjugated extravidine ( sigma chemical co. ) was used in conjunction with biotinylated antibodies .
unpaired student 's t test was used to compare levels of antibody responses between the different immunization groups ; p < 0.05 was considered significant .
der p 1 is a 25-kd cysteine protease whose structure has been modeled 7 on the crystal structure of papain , with which it shows considerable sequence similarities , most notably for residues involved in the enzyme active site 8 .
the proteolytic activity of der p 1 can be inhibited by e-64 , the class - specific inhibitor of microbial origin 9 .
this inhibition is brought about when cysteine within the der p 1 active site forms a thioether covalent bond with the epoxy group of e-64 .
this is an irreversible process that does not lead to significant structural changes , as evidenced by crystallographic studies of a papain  e-64 complex 10 .
we have purified der p 1 from fecal pellets using a multistep procedure and confirmed its purity by nh2-terminal sequencing , sds - page analysis , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and inhibited by e-64 and iodoacetamide ( fig .
1 ) . we have recently shown that der p 1 selectively cleaves human cd25 from the surfaces of peripheral blood t cells 4 . here
we demonstrate that der p 1 also selectively cleaves cd25 from cultured mouse spleen t cells ( fig .
2 ) , which is not surprising given the high degree of sequence homology that exists between human 11 and mouse 12 cd25 .
this observation has therefore provided the justification for using this animal species for testing our hypothesis , namely that the proteolytic activity of der p 1 is a major contributor to its allergenicity .
intraperitoneal immunization of groups of 10 cba / j mice with either proteolytically active or inactive ( e-64blocked ) der p 1 over a 6-wk period showed a statistically significant enhancement in total ige ( p < 0.01 ) and der p 1specific ige ( p < 0.02 ) responses in animals immunized with proteolytically active der p 1 .
this effect was ige specific , as der p 1specific igg , igg1 , and igg2b responses increased to the same extent with proteolytically active or inactive der p 1 ( fig .
we are not sure why the igg1 response did not follow that of ige , as these two isotypes are considered to be coregulated in the mouse .
however , the ige - restricted enhancement seen in response to immunization with proteolytically active der p 1 does suggest a mechanism that is unique to ige isotype switching / synthesis .
furthermore , our control experiments clearly show that the ige - specific effect observed here is not due to e-64 exerting a suppressive influence on ige production by a mechanism that is independent of its binding to the der p 1 enzyme active site ( fig .
, suppression of total ige ( p < 0.04 ) and der p 1specific ige ( p < 0.05 ) productions was also obtained when the proteolytic activity of der p 1 was blocked with iodoacetamide , another sulfhydryl reactive agent .
second , the ige antibody response to ova , a proteolytically inactive antigen , was not suppressed when the animals were immunized with ova plus e-64 .
our results are direct evidence that the cysteine protease activity of der p 1 induces a significant increase in ige responses .
such an effect is clearly consistent with the ability of der p 1 to proteolytically cleave mouse cd25 and induce a th2 response by modulating the balance between il-4 and ifn- 13 .
the recent demonstration in mice that leishmania mexicana cysteine proteinase  deficient mutants potentiate a th1 response , compared with the th2 response normally seen in response to infection with wild - type parasite 14 , is also of great relevance here .
allergic individuals could potentially be achieved by administering der p 1 in a catalytically inactive ( mutant ) form . on the other hand ,
exploring the potential th2 adjuvant property of the proteolytic activity of der p 1 would have important implications in defining principles for modulation of th1-mediated pathological conditions .
our demonstration that the cysteine protease activity of der p 1 enhances total ige production , apart from increasing der p 1-specific ige , suggests that this allergen may play a central role in destabilizing the microenvironment within target tissues to one that is proallergic and thus aids in the initiation and propagation of the allergic cascade .
in other words , the proteolytic activity of der p 1 may exert an ige - specific adjuvant effect .
the in vivo relevance of the proteolytic activity of der p 1 is further highlighted by reports demonstrating that it increases the permeability of the human respiratory epithelium to macromolecules 1516 .
such observations , together with our current findings showing a direct effect on the immune system , indicate that the proteolytic activity of der p 1 is a major contributor to its allergenicity .
 the house dust mite dermatophagoides pteronyssinus allergen der p 1 is the most immunodominant allergen involved in the expression of dust mite  specific immunoglobulin ( ig)e  mediated hypersensitivity . 
 the reason for this potent ige - eliciting property of der p 1 remains unknown , but there is mounting in vitro evidence linking the allergenicity of der p 1 to its cysteine protease activity . 
 here we demonstrate for the first time that immunization of mice with proteolytically active der p 1 results in a significant enhancement in total ige and der p 1specific ige synthesis compared with animals immunized with der p 1 that was irreversibly blocked with the cysteine protease inhibitor e-64 . 
 we conclude that the proteolytic activity of der p 1 is a major contributor to its allergenicity . 
Materials and Methods
Der p 1 Preparation.
CD25 Cleavage.
Immunization Protocol.
Antibody Detection.
Results and Discussion