Cleavage of a C-terminal Peptide Is Essential for Heptamerization of Clostridium perfringens ε-Toxin in the Synaptosomal Membrane

Abstract

Activation of Clostridium perfringens ε-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an ε-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (ΔN-PD); PD without the 23 C-terminal residues (ΔC-PD); and PD without either the N- or C-terminal residues (ΔNC-PD). A mouse lethality test showed that ΔN-PD was inactive, as is PD, whereas ΔC-PD and ΔNC-PD were equally active. ΔC-PD and ΔNC-PD, but the a in as by ΔNC-PD and and in were and were a the a that C-terminal is of the and that is the Activation of Clostridium perfringens ε-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an ε-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (ΔN-PD); PD without the 23 C-terminal residues (ΔC-PD); and PD without either the N- or C-terminal residues (ΔNC-PD). A mouse lethality test showed that ΔN-PD was inactive, as is PD, whereas ΔC-PD and ΔNC-PD were equally active. ΔC-PD and ΔNC-PD, but the a in as by ΔNC-PD and and in were and were a the a that C-terminal is of the and that is the by Clostridium perfringens and is the and and is the of in by the toxicity the or in the of a the a in the of of derivative without the 13 N-terminal derivative without the 23 C-terminal derivative without either the N- or C-terminal residues and the by is by which is a of as Clostridium and and the of of as is of or as and of a a the without the of as or the of as a A of is which is the as a of a of is that of the by as and is accompanied by removal of N- and C-terminal In a study, we the of by and and the the or or the of the a in the and in the of was the of ε-protoxin and an of were by perfringens as the and the of and ε-protoxin and and were the and the and of that the of by a in the which ε-protoxin and were as and an ε-protoxin derivative and an of in which a factor Xa was and of was as the and the of and is and is and were as and and were as a were and and and which PD and were and and were the and of were and the of and which a by the of the was by and of of and were PD, and was in of and an at of was a of and the were were and the as in this were and a which the was and was a were a of in ε-protoxin or as by were and were at PD and N- and PD were PD and by at the factor factor Xa and removal a were the and were and which were and as were by a the and lethality of ε-protoxin and were as A of were of the and were were in and in a at were in was of by the of of or of the ε-protoxin a the in were and was by the which is the of was at an of was as the of a of a of were as were in of A by of a a was at and the was at the was in a of A a a in a and at at the was and by at was in A of were as were of and in of at were by a of PD, and ΔNC-PD were and of of ε-protoxin was as of or were a and the of and the by the of and were and of and were and by cleavage at the factor site the C-terminal and at a but site in a of an in of derivative were of in of at the was at was of at and by in at were a by an of ε-protoxin was and in A of of derivative was of in of at was the by at was a in a at and amino acid of the was a as were acid as the was as by and were were a in the at an of were acid in which was as the was as the in the were a a a at were at and the was as the were four and the were and of an N-terminal of 13 amino and a C-terminal of 22 amino tryptic of which we ε-protoxin without the N- or C-terminal an and without the N-terminal were but by as by the mouse lethality test of was of which the perfringens the ε-protoxin without the C-terminal was in as which but in a we ε-protoxin and without the N-terminal in which a factor Xa was and cleavage as in the of PD and the and cleavage at the site factor of in of of and was as by and was as by and was as by in a PD and ΔN-PD were as by ΔC-PD and ΔNC-PD, were by of the PD and factor Xa of the were by of the N-terminal amino acid and by that the a at a of were in the which a in the the of the factor Xa or removal of the N- C-terminal were in the the the the of ε-protoxin of ε-protoxin and were in were the amino acid of toxicity of the ε-protoxin was by of a mouse lethality test of PD an of the was that ε-protoxin perfringens of of ΔN-PD was the as that of PD, that of ΔC-PD by a factor of of ΔNC-PD was the as that of were that the in the C-terminal ΔNC-PD and ΔC-PD four amino the cleavage the that N-terminal an the of the four were by of an in which is but the mouse lethality test ΔC-PD and ΔNC-PD the toxicity and was in the the was ΔN-PD and PD at the in this that cleavage at the C-terminal is of of ε-protoxin of ε-protoxin were was as and is as a of the a the of in an in the of and the in the a in which the in the in In this were and by In the ΔC-PD and ΔNC-PD, were of and was in PD and of ΔC-PD and ΔNC-PD or were the a was a that equally of the of a by ε-protoxin in were of in of the were and by in were and or an of ΔC-PD or ΔNC-PD were the and as of the the of of ΔC-PD and ΔNC-PD and the or of and an or at in the of an a of the that the of the in and that the of is of the this is the of which the of in a by were a this we a site at the of the were by factor Xa at the C-terminal site but the residues in the the were and and factor Xa and ΔC-PD and ΔNC-PD were as and of were by N-terminal amino acid and by was in mouse lethality were of ΔN-PD and ΔNC-PD the an in were in and a in a were of the and that the and of ΔN-PD and of ΔN-PD and and as in and the the and the is by an factor site in the N-terminal is by an amino acid the and by of and cleavage by factor were in and a by of the factor factor of a by and and were and factor and were as of of were and without of in of were in a and of and and were in and of was of in of were in and a by of the of by of ε-protoxin the of ΔC-PD an was was in a by and by a of ε-protoxin by ε-protoxin was by ΔNC-PD of but a were as a in the a of the of ε-protoxin a but the ε-protoxin by of by or or or of ε-protoxin were of in of the were by in and that removal of the but the N-terminal is the of In a study, we showed that ε-protoxin is at an N-terminal but a C-terminal by and and that ε-protoxin by in and in the the the or of the N-terminal the of is by the in the C-terminal cleavage of the of a in that the is the of and that the a in the that the is which is in and of the of of and of which were by of and the that the of in a by of of the in is a and at of the that the and the we that in the is the of a in a and the of the and of by ε-protoxin in this by ε-protoxin the by a of is the the of ε-protoxin a and the that a site is the of and that the C-terminal of a site or toxicity as and that the C-terminal as an that of ε-protoxin were as and that of the or and or were the C-terminal were in a that of in was in that C-terminal of of the C-terminal is the in C-terminal of the the study, of the of the C-terminal the and of the the of the C-terminal in the and of by Clostridium perfringens and is the and and is the of in by the toxicity the or in the of a the a in the of of derivative without the 13 N-terminal derivative without the 23 C-terminal derivative without either the N- or C-terminal residues and the by is by which is a of as Clostridium and and the of of as is of ε-protoxin derivative ε-protoxin derivative without the 13 N-terminal residues ε-protoxin derivative without the 23 C-terminal residues ε-protoxin derivative without either the N- or C-terminal residues of or as and of a a the without the of as or the of as a A of is which is the as a of a of is that of the by as and is accompanied by removal of N- and C-terminal In a study, we the of by and and the the or or the of the a in the and in the of was the of ε-protoxin and an of were by perfringens as the and the of and ε-protoxin and and were the and the and of that the of by a in the which ε-protoxin and were as and an ε-protoxin derivative and an of in which a factor Xa was and of was as the and the of and is and is and were as and and were as a were and and and which PD and were and and were the and of were and the of and which a by the of the was by and of of and were PD, and was in of and an at of was a of and the were were and the as in this were and a which the was and was a were a of in ε-protoxin or as by were and were at PD and N- and PD were PD and by at the factor factor Xa and removal a were the and were and which were and as were by a the and lethality of ε-protoxin and were as A of were of the and were were in and in a at were in was of by the of of or of the ε-protoxin a the in were and was by the which is the of was at an of was as the of a of a of were as were in of A by of a a was at and the was at the was in a of A a a in a and at at the was and by at was in A of were as were of and in of at were by a of PD, and ΔNC-PD were and of of ε-protoxin was as of or were a and the of and the by the of and were and of and were and by cleavage at the factor site the C-terminal and at a but site in a of an in of derivative were of in of at the was at was of at and by in at were a by an of ε-protoxin was and in A of of derivative was of in of at was the by at was a in a at and amino acid of the was a as were acid as the was as by and were were a in the at an of were acid in which was as the was as the in the were a a a at were at and the was as the were four and the were of was the of ε-protoxin and an of were by perfringens as the and the of and ε-protoxin and and were the and the and of that the of by a in the which ε-protoxin and were as and an ε-protoxin derivative and an of in which a factor Xa was and of was as the and the of and is and is and were as and and were as a were and and and which PD and were and and were the and of were and the of and which a by the of the was by was the of ε-protoxin and an of were by perfringens as the and the of and ε-protoxin and and were the and the and of that the of by a in the which ε-protoxin and were as and an ε-protoxin derivative and an of in which a factor Xa was and of was as the and the of and is and is and were as and and were as a were and and and which PD and were and and were the and of were and the of and which a by the of the was by and of of and were PD, and was in of and an at of was a of and the were were and the as in this were and a which the was and was a were a of in ε-protoxin or as by were and were at PD and N- and PD were PD and by at the factor factor Xa and removal a were the and were and which were and as were by a the of and were PD, and was in of and an at of was a of and the were were and the as in this were and a which the was and was a were a of in ε-protoxin or as by were and were at PD and N- and PD were PD and by at the factor factor Xa and removal a were the and were and which were and as were by a the and lethality of ε-protoxin and were as A of were of the and were were in and in a at were in was of by the of of or of the ε-protoxin a the in were and was by the which is the of was at an of was as the of a of a lethality of ε-protoxin and were as A of were of the and were were in and in a at were in was of by the of of or of the ε-protoxin a the in were and was by the which is the of was at an of was as the of a of a of were as were in of A by of a a was at and the was at the was in a of A a a in a and at at the was and by at was in A were as were in of A by of a a was at and the was at the was in a of A a a in a and at at the was and by at was in A of were as were of and in of at were by a of PD, and ΔNC-PD were and of of ε-protoxin was as of or were a and the of and the by the of and were and of and were and by cleavage at the factor site the C-terminal and at a but site in a were as were of and in of at were by a of PD, and ΔNC-PD were and of of ε-protoxin was as of or were a and the of and the by the of and were and of and were and by cleavage at the factor site the C-terminal and at a but site in a of an in of derivative were of in of at the was at was of at and by in at were a by an of ε-protoxin was and in A of of derivative was of in of at was the by at was a in a at and of derivative were of in of at the was at was of at and by in at were a by an of ε-protoxin was and in A of of derivative was of in of at was the by at was a in a at and amino acid of the was a as were acid as the was as by and were were a in the at an of were acid in which was as the was as the in the were a a a at were at and the was as the were four and the were N-terminal amino acid of the was a as were acid as the was as by and were were a in the at an of were acid in which was as the was as the in the were a a a at were at and the was as the were four and the were and of an N-terminal of 13 amino and a C-terminal of 22 amino tryptic of which we ε-protoxin without the N- or C-terminal an and without the N-terminal were but by as by the mouse lethality test of was of which the perfringens the ε-protoxin without the C-terminal was in as which but in a we ε-protoxin and without the N-terminal in which a factor Xa was and cleavage as in the of PD and the and cleavage at the site factor of in of of and was as by and was as by and was as by in a PD and ΔN-PD were as by ΔC-PD and ΔNC-PD, were by of the PD and factor Xa of the were by of the N-terminal amino acid and by that the a at a of were in the which a in the the of the factor Xa or removal of the N- C-terminal were in the the the the of ε-protoxin of toxicity of the ε-protoxin was by of a mouse lethality test of PD an of the was that ε-protoxin perfringens of of ΔN-PD was the as that of PD, that of ΔC-PD by a factor of of ΔNC-PD was the as that of were that the in the C-terminal ΔNC-PD and ΔC-PD four amino the cleavage the that N-terminal an the of the four were by of an in which is but the mouse lethality test ΔC-PD and ΔNC-PD the toxicity and was in the the was ΔN-PD and PD at the in this that cleavage at the C-terminal is of of ε-protoxin of ε-protoxin were was as and is as a of the a the of in an in the of and the in the a in which the in the in In this were and by In the ΔC-PD and ΔNC-PD, were of and was in PD and of ΔC-PD and ΔNC-PD or were the a was a that equally of the of a by ε-protoxin in were of in of the were and by in were and or an of ΔC-PD or ΔNC-PD were the and as of the the of of ΔC-PD and ΔNC-PD and the or of and an or at in the of an a of the that the of the in and that the of is of the this is the of which the of in a by were a this we a site at the of the were by factor Xa at the C-terminal site but the residues in the the were and and factor Xa and ΔC-PD and ΔNC-PD were as and of were by N-terminal amino acid and by was in mouse lethality were of ΔN-PD and ΔNC-PD the an in were in and a in a were of the and that the and of ΔN-PD and of ΔN-PD and and as in and the the and the is by an factor site in the N-terminal is by an amino acid the and by of and cleavage by factor were in and a by of the factor factor of a by and and were and factor and were as of of were and without of in of were in a and of and and were in and of was of in of were in and a by of the of by of ε-protoxin the of ΔC-PD an was was in a by and by a of ε-protoxin by ε-protoxin was by ΔNC-PD of but a were as a in the a of the of ε-protoxin a but the ε-protoxin by of by or or or of ε-protoxin were of in of the were by in and and of an N-terminal of 13 amino and a C-terminal of 22 amino tryptic of which we ε-protoxin without the N- or C-terminal an and without the N-terminal were but by as by the mouse lethality test of was of which the perfringens the ε-protoxin without the C-terminal was in as which but in a we ε-protoxin and without the N-terminal in which a factor Xa was and cleavage as in the of PD and the and cleavage at the site factor of in of of and was as by and was as by and was as by in a PD and ΔN-PD were as by ΔC-PD and ΔNC-PD, were by of the PD and factor Xa of the were by of the N-terminal amino acid and by that the a at a of were in the which a in the the of the factor Xa or removal of the N- C-terminal were in the the the the of ε-protoxin an N-terminal of 13 amino and a C-terminal of 22 amino tryptic of which we ε-protoxin without the N- or C-terminal an and without the N-terminal were but by as by the mouse lethality test of was of which the perfringens the ε-protoxin without the C-terminal was in as which but in a we ε-protoxin and without the N-terminal in which a factor Xa was and cleavage as in the of PD and the and cleavage at the site factor PD and ΔN-PD were as by ΔC-PD and ΔNC-PD, were by of the PD and factor Xa of the were by of the N-terminal amino acid and by that the a at a of were in the which a in the the of the factor Xa or removal of the N- C-terminal were in the the the the of ε-protoxin of toxicity of the ε-protoxin was by of a mouse lethality test of PD an of the was that ε-protoxin perfringens of of ΔN-PD was the as that of PD, that of ΔC-PD by a factor of of ΔNC-PD was the as that of were that the in the C-terminal ΔNC-PD and ΔC-PD four amino the cleavage the that N-terminal an the of the four were by of an in which is but the mouse lethality test ΔC-PD and ΔNC-PD the toxicity and was in the the was ΔN-PD and PD at the in this that cleavage at the C-terminal is of toxicity of the ε-protoxin was by of a mouse lethality test of PD an of the was that ε-protoxin perfringens of of ΔN-PD was the as that of PD, that of ΔC-PD by a factor of of ΔNC-PD was the as that of were that the in the C-terminal ΔNC-PD and ΔC-PD four amino the cleavage the that N-terminal an the of the four were by of an in which is but the mouse lethality test ΔC-PD and ΔNC-PD the toxicity and was in the the was ΔN-PD and PD at the in this that cleavage at the C-terminal is of in an in the of and the in the a in which the in the in In this were and by In the ΔC-PD and ΔNC-PD, were of and was in PD and of ΔC-PD and ΔNC-PD or were the a was a that equally of the an in the of and the in the a in which the in the in In this were and by In the ΔC-PD and ΔNC-PD, were of and was in PD and of ΔC-PD and ΔNC-PD or were the a was a that equally of the of of ΔC-PD and ΔNC-PD and the or of and an or at in the of an a of the that the of the in and that the of is of the this is the of which the of in a by were a this we a site at the of the were by factor Xa at the C-terminal site but the residues in the the were and and factor Xa and ΔC-PD and ΔNC-PD were as and of were by N-terminal amino acid and by was in mouse lethality were of ΔN-PD and ΔNC-PD the an in were in and a in a were of the and that the of ΔC-PD and ΔNC-PD and the or of and an or at in the of an a of the that the of the in and that the of is of the this is the of which the of in a by were a this we a site at the of the were by factor Xa at the C-terminal site but the residues in the the were and and factor Xa and ΔC-PD and ΔNC-PD were as and of were by N-terminal amino acid and by was in mouse lethality were of ΔN-PD and ΔNC-PD the an in were in and a in a were of the and that the of by of ε-protoxin the of ΔC-PD an was was in a by and by a of ε-protoxin by ε-protoxin was by ΔNC-PD of but a were as a in the a of the of ε-protoxin a but the ε-protoxin by of ε-protoxin the of ΔC-PD an was was in a by and by a of ε-protoxin by ε-protoxin was by ΔNC-PD of but a were as a in the a of the of ε-protoxin a but the ε-protoxin by that removal of the but the N-terminal is the of In a study, we showed that ε-protoxin is at an N-terminal but a C-terminal by and and that ε-protoxin by in and in the the the or of the N-terminal the of is by the in the C-terminal cleavage of the of a in that the is the of and that the a in the that the is which is in and of the of of and of which were by of and the that the of in a by of of the in is a and at of the that the and the we that in the is the of a in a and the of the and of by ε-protoxin in this by ε-protoxin the by a of is the the of ε-protoxin a and the that a site is the of and that the C-terminal of a site or toxicity as and that the C-terminal as an that of ε-protoxin were as and that of the or and or were the C-terminal were in a that of in was in that C-terminal of of the C-terminal is the in C-terminal of the the study, of the of the C-terminal the and of the the of the C-terminal in the and of that removal of the but the N-terminal is the of In a study, we showed that ε-protoxin is at an N-terminal but a C-terminal by and and that ε-protoxin by in and in the the the or of the N-terminal the of is by the in the C-terminal cleavage of the of a in that the is the of and that the a in the that the is which is in and of the of of and of which were by of and the that the of in a by of of the in is a and at of the that the and the we that in the is the of a in a and the of the and of by ε-protoxin in this by ε-protoxin the by a of is the the of ε-protoxin a and the that a site is the of and that the C-terminal of a site or toxicity as and that the C-terminal as an that of ε-protoxin were as and that of the or and or were the C-terminal were in a that of in was in that C-terminal of of the C-terminal is the in C-terminal of the the study, of the of the C-terminal the and of the the of the C-terminal in the and of of of of and of and