Transepithelially Transported Pro-phenoloxidase in the Cuticle of the Silkworm, Bombyx mori

Pro-phenoloxidase (proPO) in insects is activated through the action of a protease cascade triggered by minute amounts of microbial cell wall components. It is an important molecule for the defense against invading microorganisms and for the repair of wounds. In the accompanying paper (Asano, T., and Ashida, M. (2001)J. Biol. Chem. 276, 11100–11112), a proPO isoform, proPO-HS, in the hemolymph of the silkworm, Bombyx mori, is reported to be transported to the cuticle. The transported proPO isoform was recovered from the cuticle and named proPO-CS. The elution profiles of proPO-CS and proPO-HS in reversed-phase high performance liquid chromatography (HPLC) were found to be different, giving a basis to the inference that proPO-CS is a modified form of proPO-HS. In the present study, we investigated the nature of the modifications occurring in proPO-CS, in which proteolytically and chemically cleaved fragments originating from the subunits of proPO-CS and proPO-HS were analyzed by reversed-phase HPLC, amino acid sequencing, and mass spectrometry. A subunit of the heterodimeric proPO-CS was found to contain five or six methionine sulfoxides, and another subunit was found to contain one methionine residue oxidized to the sulfoxide. All of the oxidized methionyl residues were identified. Other than oxidation of the methionyl residues, no additional modification of proPO-CS was found. In the model structure of each subunit of proPO-CS constructed by protein modeling with the known structures of the horseshoe crab, Limulus polyphemus, hemocyanin type II subunit as templates, the methionine residues identified as methionine sulfoxide had high degrees of accessibility to the solvent. The implication of the oxidation at the methionine residues is discussed in relation to the mechanism of transepithelial transport of proPO from the hemolymph to the cuticle. Pro-phenoloxidase (proPO) in insects is activated through the action of a protease cascade triggered by minute amounts of microbial cell wall components. It is an important molecule for the defense against invading microorganisms and for the repair of wounds. In the accompanying paper (Asano, T., and Ashida, M. (2001)J. Biol. Chem. 276, 11100–11112), a proPO isoform, proPO-HS, in the hemolymph of the silkworm, Bombyx mori, is reported to be transported to the cuticle. The transported proPO isoform was recovered from the cuticle and named proPO-CS. The elution profiles of proPO-CS and proPO-HS in reversed-phase high performance liquid chromatography (HPLC) were found to be different, giving a basis to the inference that proPO-CS is a modified form of proPO-HS. In the present study, we investigated the nature of the modifications occurring in proPO-CS, in which proteolytically and chemically cleaved fragments originating from the subunits of proPO-CS and proPO-HS were analyzed by reversed-phase HPLC, amino acid sequencing, and mass spectrometry. A subunit of the heterodimeric proPO-CS was found to contain five or six methionine sulfoxides, and another subunit was found to contain one methionine residue oxidized to the sulfoxide. All of the oxidized methionyl residues were identified. Other than oxidation of the methionyl residues, no additional modification of proPO-CS was found. In the model structure of each subunit of proPO-CS constructed by protein modeling with the known structures of the horseshoe crab, Limulus polyphemus, hemocyanin type II subunit as templates, the methionine residues identified as methionine sulfoxide had high degrees of accessibility to the solvent. The implication of the oxidation at the methionine residues is discussed in relation to the mechanism of transepithelial transport of proPO from the hemolymph to the cuticle. pro-phenoloxidase S-type cuticular proPO F-type cuticular proPO S-type hemolymph proPO F-type hemolymph proPO methionine sulfoxide pro-phenoloxidase-activating enzyme lysylendopeptidase polyacrylamide gel electrophoresis reversed-phase high performance liquid chromatography octadecyl matrix-assisted laser desorption ionization mass spectrometry post-source decay The chitinous exoskeleton of the insect is a nonliving of and protein from a of that the of the the and of the and The of and The cuticle is a structure the of the insect and Biol. a the and the a of the that the insect to and and a for the defense against by the by the of microorganisms to and by the to the M. of in The and by form the that a by the important in cuticular of the in the chitinous exoskeleton of the insect to be by to be of cuticular to be in and the insect to the and to be transported to the cuticle the of the hemolymph to be transported from the hemolymph to the cuticle and in insects M. M. M. the for the transepithelial transport of in in to transepithelial and protein transport in in which the of of as A and investigated in we the mechanism of pro-phenoloxidase of M. of in that the cuticle proPO and the protease cascade for the the cuticle were found to contain the of the proPO that cuticular proPO is transported from the hemolymph M. the silkworm, Bombyx mori, proPO from the hemolymph and cuticle M. Biol. Chem. The from the hemolymph named and proPO-HS, and the from the cuticle named and proPO-CS. The and the in polyacrylamide gel electrophoresis and to as F-type proPO-HS and proPO-CS to the in and to as S-type F-type than S-type in from the hemolymph and cuticle to be heterodimeric M. Biol. Chem. of the subunits of proPO-HS and is the subunits different, and five amino acid M. Biol. Chem. the accompanying paper M. Biol. Chem. proPO-HS is reported to be transported from the hemolymph to the cuticle and to be recovered as proPO-CS from the cuticle. is to be transported the of The proPO-CS and proPO-HS in as by gel or in by a elution profiles in reversed-phase high performance liquid chromatography an octadecyl were that modifications in cuticular that proPO-CS from was transported from the hemolymph to the cuticle was the of the M. Biol. Chem. It that the to modification an important in the transport of hemolymph proPO to the cuticle. of the nature of the modification to be the in of the mechanism of transepithelial protein transport in we that the modification in proPO-CS is oxidation of methionine All of the oxidized methionine residues identified. is the the of transported protein in the that is to the of The is to the of through in the M. The of the proPO subunit to be in M. proPO in the of and M. Ashida, proPO to be transported from the hemolymph to the cuticle M. Biol. Chem. that hemolymph proPO is transported from the hemolymph to the of transepithelial or of as A and in of the of the the as It is that the by at the transported to the and in the mechanism by which transported is that a protein is transported by through insect the proPO to be a molecule that a to the mechanism of transepithelial protein transport in the cuticular proPO had the as of hemolymph proPO were activated by a pro-phenoloxidase-activating enzyme M. Biol. Chem. subunits of cuticular proPO were at the as of hemolymph proPO subunits in that cuticular proPO is a modified form of hemolymph that hemolymph transported from hemolymph to the cuticle M. M. M. no of the transported of the nature of the modification of transported be the to the of transepithelial transport of from hemolymph to the cuticle in the hemolymph of the silkworm, mori, proPO to as proPO-HS and proPO-HS to be transported to the cuticle and proPO-CS, is that is transported as proPO-HS M. Biol. Chem. In the present study, modification occurring in proPO-CS was In proPO-CS was in The in the were named and The and were to be modified of subunit of and the was to six methionine sulfoxides, at and and the was to five at and The was to be a modified form of subunit of and to a at mass for the subunits of proPO-CS, of mass of the in protease of the amino acid of the in the and the of of methionine by mass spectrometry were with the that modification of the subunits of proPO-CS oxidation of the methionine residues in the present In the of F-type and that and the amino acid and that the modifications as in proPO-CS and M. Ashida, of methionyl residues in by or reported It was that the oxidized methionyl residues in the cuticular proPO were the in the present the or hemolymph proPO be for of elution profiles in that the oxidation of methionyl residues in cuticular proPO In another an with hemolymph proPO was to cuticular and of in the was the a subunit of hemolymph proPO was recovered in a high that the oxidation of methionine residues of proPO the of cuticular cuticular proPO to in the oxidized form in mass of proPO-CS was to be by gel and the was for the mass of proPO-HS. proPO-HS to be of the mass to that proPO-CS is a heterodimeric the proPO-CS was to be a of proPO-CS one of which is of and and the of which is of and in the by proPO is a protein to the of of which the structures Biol. Biol. hemocyanin is to the proPO in than is hemocyanin structures hemocyanin type II subunits of were from protein in and the one was from protein in the of Biol. to the structures Biol. modeling of proPO subunits was with structures as The model structures were to of the the model of methionine residues were residues identified to be in cuticular proPO subunits were to high in the with the and the and the residues of The accessibility of of and of which were identified as methionine in proPO-CS, be in the modeling the were in the as in the present protein and of were to high and in the were as oxidized methionine residues in to be is that the methionyl residues at the subunits of proPO and to the the proPO residues in and of methionine is to be to the or residues the the high of the methionine residues as methionine in proPO-CS that the model structures in the present protein modeling the structures of proPO that of oxidation of methionine residues of and of In the of the to M. Biol. Chem. that from the cuticle hemolymph proPO and cuticular proPO to of which the were and that the by to be by the oxidation of methionyl The of oxidation was the transport of proPO from hemolymph to the cuticular proPO-CS the was transported to the the proPO-HS was transported to the cuticle M. Biol. Chem. The model for proPO transport be transport as in M. and It is that the for proPO to the oxidized proPO from residues of to in Biol. Chem. M. M. M. Biol. Chem. Biol. Chem. Biol. Chem. of methionyl residues to a of the Biol. Chem. The at the oxidized methionyl residues of cuticular proPO the of to the proPO of the cuticular proPO for the that oxidation of the methionine residues to the of which is to be transported from the hemolymph to the cuticle. It is that the a in the cuticular proPO from from the cuticle the of no insect that to be transported from the hemolymph to the cuticle. is known methionyl residues of transported the of insect in M. M. M. the of proPO in A of of insects was constructed from the reported were in The subunits and to each subunits to the in which is to as type proPO and to the in which is to as type II proPO In the of oxidized proPO the methionine residue to of was found in type subunits for a proPO to is the type proPO subunits with the of the subunits of proPO to of of the type II proPO subunits of insects were found to a methionine residue at the to to the that the methionine residue to or of a in the transport of from hemolymph to the cuticle in The proPO which a methionine residue to or of of the at protein to be of than no of a of the proPO subunit to form a proPO and is known proPO the subunit is transported to the cuticle. In a as as six for proPO subunits known to be no of the transported to the cuticle. is to in insects transported from hemolymph to the cuticle and the transported proPO methionine at the to or of of the of of in the transport of proPO subunits be proPO at the methionine residues were we in the of the oxidized methionyl residues in cuticular proPO to the of and of the of the the oxidation to an of the of the transepithelial transport of hemolymph for cuticle and to an to hemolymph proPO to cuticular proPO by with identified as in cuticular The of an and an of the methionine residues All of the methionyl residues to the of proPO to be to be a mechanism by which methionyl residues It is that hemolymph proPO is to an the from hemolymph to the the of is the of a high of methionine sulfoxide in cuticular be methionyl residues oxidized to sulfoxide as in Chem. the of the methionine residues oxidized to in cuticular to be the implication of oxidation in the transepithelial transport of hemolymph proPO to the cuticle is of The chitinous exoskeleton of the insect is a nonliving of and protein from a of that the of the the and of the and The of and The cuticle is a structure the of the insect and Biol. a the and the a of the that the insect to and and a for the defense against by the by the of microorganisms to and by the to the M. of in The and by form the that a by the important in cuticular of the in the chitinous exoskeleton of the insect to be by to be of cuticular to be in and the insect to the and to be transported to the cuticle the of the hemolymph to be transported from the hemolymph to the cuticle and in insects M. M. M. the for the transepithelial transport of in in to transepithelial and protein transport in in which the of of as A and investigated in In we the mechanism of pro-phenoloxidase of M. of in that the cuticle proPO and the protease cascade for the the cuticle were found to contain the of the proPO that cuticular proPO is transported from the hemolymph M. In the silkworm, Bombyx mori, proPO from the hemolymph and cuticle M. Biol. Chem. The from the hemolymph named and proPO-HS, and the from the cuticle named and proPO-CS. The and the in polyacrylamide gel electrophoresis and to as F-type proPO-HS and proPO-CS to the in and to as S-type F-type than S-type in from the hemolymph and cuticle to be heterodimeric M. Biol. Chem. of the subunits of proPO-HS and is the subunits different, and five amino acid M. Biol. Chem. In the accompanying paper M. Biol. Chem. proPO-HS is reported to be transported from the hemolymph to the cuticle and to be recovered as proPO-CS from the cuticle. is to be transported the of The proPO-CS and proPO-HS in as by gel or in by a elution profiles in reversed-phase high performance liquid chromatography an octadecyl were that modifications in cuticular that proPO-CS from was transported from the hemolymph to the cuticle was the of the M. Biol. Chem. It that the to modification an important in the transport of hemolymph proPO to the cuticle. of the nature of the modification to be the in of the mechanism of transepithelial protein transport in we that the modification in proPO-CS is oxidation of methionine All of the oxidized methionine residues identified. is the the of transported protein in the that is to the of The is to the of through in the M. The of the proPO subunit to be in M. proPO in the of and M. Ashida, proPO to be transported from the hemolymph to the cuticle M. Biol. Chem. that hemolymph proPO is transported from the hemolymph to the of transepithelial or of as A and in of the of the the as It is that the by at the transported to the and in the mechanism by which transported is that a protein is transported by through insect the proPO to be a molecule that a to the mechanism of transepithelial protein transport in the cuticular proPO had the as of hemolymph proPO were activated by a pro-phenoloxidase-activating enzyme M. Biol. Chem. subunits of cuticular proPO were at the as of hemolymph proPO subunits in that cuticular proPO is a modified form of hemolymph that hemolymph transported from hemolymph to the cuticle M. M. M. no of the transported of the nature of the modification of transported be the to the of transepithelial transport of from hemolymph to the cuticle in the hemolymph of the silkworm, mori, proPO to as proPO-HS and proPO-HS to be transported to the cuticle and proPO-CS, is that is transported as proPO-HS M. Biol. Chem. In the present study, modification occurring in proPO-CS was In proPO-CS was in The in the were named and The and were to be modified of subunit of and the was to six methionine sulfoxides, at and and the was to five at and The was to be a modified form of subunit of and to a at mass for the subunits of proPO-CS, of mass of the in protease of the amino acid of the in the and the of of methionine by mass spectrometry were with the that modification of the subunits of proPO-CS oxidation of the methionine residues in the present In the of F-type and that and the amino acid and that the modifications as in proPO-CS and M. Ashida, of methionyl residues in by or reported It was that the oxidized methionyl residues in the cuticular proPO were the in the present the or hemolymph proPO be for of elution profiles in that the oxidation of methionyl residues in cuticular proPO In another an with hemolymph proPO was to cuticular and of in the was the a subunit of hemolymph proPO was recovered in a high that the oxidation of methionine residues of proPO the of cuticular cuticular proPO to in the oxidized form in mass of proPO-CS was to be by gel and the was for the mass of proPO-HS. proPO-HS to be of the mass to that proPO-CS is a heterodimeric the proPO-CS was to be a of proPO-CS one of which is of and and the of which is of and in the by proPO is a protein to the of of which the structures Biol. Biol. hemocyanin is to the proPO in than is hemocyanin structures hemocyanin type II subunits of were from protein in and the one was from protein in the of Biol. to the structures Biol. modeling of proPO subunits was with structures as The model structures were to of the the model of methionine residues were residues identified to be in cuticular proPO subunits were to high in the with the and the and the residues of The accessibility of of and of which were identified as methionine in proPO-CS, be in the modeling the were in the as in the present protein and of were to high and in the were as oxidized methionine residues in to be is that the methionyl residues at the subunits of proPO and to the the proPO residues in and of methionine is to be to the or residues the the high of the methionine residues as methionine in proPO-CS that the model structures in the present protein modeling the structures of proPO that of oxidation of methionine residues of and of In the of the to M. Biol. Chem. that from the cuticle hemolymph proPO and cuticular proPO to of which the were and that the by to be by the oxidation of methionyl The of oxidation was the transport of proPO from hemolymph to the cuticular proPO-CS the was transported to the the proPO-HS was transported to the cuticle M. Biol. Chem. The model for proPO transport be transport as in M. and It is that the for proPO to the oxidized proPO from residues of to in Biol. Chem. M. M. M. Biol. Chem. Biol. Chem. Biol. Chem. of methionyl residues to a of the Biol. Chem. The at the oxidized methionyl residues of cuticular proPO the of to the proPO of the cuticular proPO for the that oxidation of the methionine residues to the of which is to be transported from the hemolymph to the cuticle. It is that the a in the cuticular proPO from from the cuticle the of no insect that to be transported from the hemolymph to the cuticle. is known methionyl residues of transported the of insect in M. M. M. the of proPO in A of of insects was constructed from the reported were in The subunits and to each subunits to the in which is to as type proPO and to the in which is to as type II proPO In the of oxidized proPO the methionine residue to of was found in type subunits for a proPO to is the type proPO subunits with the of the subunits of proPO to of of the type II proPO subunits of insects were found to a methionine residue at the to to the that the methionine residue to or of a in the transport of from hemolymph to the cuticle in The proPO which a methionine residue to or of of the at protein to be of than no of a of the proPO subunit to form a proPO and is known proPO the subunit is transported to the cuticle. In a as as six for proPO subunits known to be no of the transported to the cuticle. is to in insects transported from hemolymph to the cuticle and the transported proPO methionine at the to or of of the of of in the transport of proPO subunits be proPO at the methionine residues were we in the of the oxidized methionyl residues in cuticular proPO to the of and of the of the the oxidation to an of the of the transepithelial transport of hemolymph for cuticle and to an to hemolymph proPO to cuticular proPO by with identified as in cuticular The of an and an of the methionine residues All of the methionyl residues to the of proPO to be to be a mechanism by which methionyl residues It is that hemolymph proPO is to an the from hemolymph to the the of is the of a high of methionine sulfoxide in cuticular be methionyl residues oxidized to sulfoxide as in Chem. the of the methionine residues oxidized to in cuticular to be the implication of oxidation in the transepithelial transport of hemolymph proPO to the cuticle is of the that is to the of The is to the of through in the M. The of the proPO subunit to be in M. proPO in the of and M. Ashida, proPO to be transported from the hemolymph to the cuticle M. Biol. Chem. that hemolymph proPO is transported from the hemolymph to the cuticle. The of transepithelial or of as A and in of the of the the as It is that the by at the transported to the and in the mechanism by which transported is that a protein is transported by through insect the proPO to be a molecule that a to the mechanism of transepithelial protein transport in the cuticular proPO had the as of hemolymph proPO were activated by a pro-phenoloxidase-activating enzyme M. Biol. Chem. subunits of cuticular proPO were at the as of hemolymph proPO subunits in that cuticular proPO is a modified form of hemolymph that hemolymph transported from hemolymph to the cuticle M. M. M. no of the transported of the nature of the modification of transported be the to the of transepithelial transport of from hemolymph to the cuticle in In the hemolymph of the silkworm, mori, proPO to as proPO-HS and proPO-HS to be transported to the cuticle and proPO-CS, is that is transported as proPO-HS M. Biol. Chem. In the present study, modification occurring in proPO-CS was In proPO-CS was in The in the were named and The and were to be modified of subunit of and the was to six methionine sulfoxides, at and and the was to five at and The was to be a modified form of subunit of and to a at mass for the subunits of proPO-CS, of mass of the in protease of the amino acid of the in the and the of of methionine by mass spectrometry were with the that modification of the subunits of proPO-CS oxidation of the methionine residues in the present In the of F-type and that and the amino acid and that the modifications as in proPO-CS and M. Ashida, of methionyl residues in by or reported It was that the oxidized methionyl residues in the cuticular proPO were the in the present the or hemolymph proPO be for of elution profiles in that the oxidation of methionyl residues in cuticular proPO In another an with hemolymph proPO was to cuticular and of in the was the a subunit of hemolymph proPO was recovered in a high that the oxidation of methionine residues of proPO the of cuticular cuticular proPO to in the oxidized form in The mass of proPO-CS was to be by gel and the was for the mass of proPO-HS. proPO-HS to be of the mass to that proPO-CS is a heterodimeric the proPO-CS was to be a of proPO-CS one of which is of and and the of which is of and in the by The proPO is a protein to the of of which the structures Biol. Biol. hemocyanin is to the proPO in than is hemocyanin structures hemocyanin type II subunits of were from protein in and the one was from protein in the of Biol. to the structures Biol. modeling of proPO subunits was with structures as The model structures were to of the the model of methionine residues were residues identified to be in cuticular proPO subunits were to high in the with the and the and the residues of The accessibility of of and of which were identified as methionine in proPO-CS, be in the modeling the were in the as in the present protein and of were to high and in the were as oxidized methionine residues in to be is that the methionyl residues at the subunits of proPO and to the the proPO residues in and of methionine is to be to the or residues the the high of the methionine residues as methionine in proPO-CS that the model structures in the present protein modeling the structures of proPO that of The oxidation of methionine residues of and of In the of the to M. Biol. Chem. that from the cuticle hemolymph proPO and cuticular proPO to of which the were and that the by to be by the oxidation of methionyl The of oxidation was the transport of proPO from hemolymph to the cuticular proPO-CS the was transported to the the proPO-HS was transported to the cuticle M. Biol. Chem. The model for proPO transport be transport as in M. and It is that the for proPO to the oxidized proPO from residues of to in Biol. Chem. M. M. M. Biol. Chem. Biol. Chem. Biol. Chem. of methionyl residues to a of the Biol. Chem. The at the oxidized methionyl residues of cuticular proPO the of to the proPO of the cuticular proPO for the that oxidation of the methionine residues to the of which is to be transported from the hemolymph to the cuticle. It is that the a in the cuticular proPO from from the cuticle the of no insect that to be transported from the hemolymph to the cuticle. is known methionyl residues of transported the of insect in M. M. M. the of proPO in A of of insects was constructed from the reported were in The subunits and to each subunits to the in which is to as type proPO and to the in which is to as type II proPO In the of oxidized proPO the methionine residue to of was found in type subunits for a proPO to is the type proPO subunits with the of the subunits of proPO to of of the type II proPO subunits of insects were found to a methionine residue at the to to the that the methionine residue to or of a in the transport of from hemolymph to the cuticle in The proPO which a methionine residue to or of of the at protein to be of than no of a of the proPO subunit to form a proPO and is known proPO the subunit is transported to the cuticle. In a as as six for proPO subunits known to be no of the transported to the cuticle. is to in insects transported from hemolymph to the cuticle and the transported proPO methionine at the to or of of the of of in the transport of proPO subunits be proPO at the methionine residues were we in the of the oxidized methionyl residues in cuticular proPO to the of and of the of the the oxidation to an of the of the transepithelial transport of hemolymph for cuticle and to an to hemolymph proPO to cuticular proPO by with identified as in cuticular The of an and an of the methionine residues All of the methionyl residues to the of proPO to be to be a mechanism by which methionyl residues It is that hemolymph proPO is to an the from hemolymph to the cuticle. the of is the of a high of methionine sulfoxide in cuticular be methionyl residues oxidized to sulfoxide as in Chem. the of the methionine residues oxidized to in cuticular to be the implication of oxidation in the transepithelial transport of hemolymph proPO to the cuticle is of to for of the amino acid of cuticular proPO at the of the present and to for of of the by ionization mass