8d49ae6fa63c1189913cd33f5682377a pmc_31470614.pdf 0f56278c6dcb5399afda1c83e3fc67a07f587d80 pmc_31470614.pdf ed80457cdab6ab4fd4f57b1a958acffa3f6c7a514af83caa760d1a3f8c0f1967 pmc_31470614.pdf Title: Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor Subject: A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses. Keywords: Nerita versicolor; proteinaceous inhibitor; recombinant production; carboxypeptidase; picomolar inhibition; biotechnological and biomedical applications Author: Giovanni Covaleda-Cortés, Martha Hernández, Sebastián Alejandro Trejo, Manuel Mansur, Sergi Rodríguez-Calado, Javier García-Pardo, Julia Lorenzo, Josep Vendrell, María Ángeles Chávez, Maday Alonso-del-Rivero and Francesc Xavier Avilés Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Thu Aug 29 12:22:15 2019 CEST ModDate: Thu Aug 29 12:22:15 2019 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4299275 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SMEUFM+URWPalladioL-Ital Type 1 Custom yes yes no 114 0 BTTJQU+URWPalladioL-Bold Type 1 Custom yes yes no 115 0 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BAKLGD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 349 0 BAKMEB+TimesNewRoman CID TrueType Identity-H yes yes yes 350 0 BAKPLO+Symbol Type 1C Custom yes yes yes 394 0 BAKKOB+PalatinoLinotype CID TrueType Identity-H yes yes yes 395 0 BAKKPC+PalatinoLinotype TrueType WinAnsi yes yes no 396 0 BAKLFB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 397 0 BAKLGD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 398 0 BAKMEB+TimesNewRoman CID TrueType Identity-H yes yes yes 399 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 441 0 BAKKPC+PalatinoLinotype TrueType WinAnsi yes yes no 460 0 BAKLFB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 461 0 BAKLGD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 462 0 BAKMEB+TimesNewRoman CID TrueType Identity-H yes yes yes 463 0 BAKKPC+PalatinoLinotype TrueType WinAnsi yes yes no 489 0 BAKLFB+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 490 0 BAKLGD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 491 0 BAKMEB+TimesNewRoman CID TrueType Identity-H yes yes yes 492 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2020-11-19 06:10:24 CET RepresentationInformation: pmc_31470614.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2020-08-03 03:40:02 CEST Size: 4299275 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 FreeObjects: 7 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: ぉのぴひはつふっぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸲ Item: Title: げづびふぬぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮ㄩ‾㸊⡜㌷㙜㌷㝜〰ぉびはぬちぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぐふひどてどっちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㈩‾㸊⡜㌷㙜㌷㝜〰ぐひどねちひべぜ〴ぜ〰こづぱふづのっづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぃとちひちっぴづひどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぴとづぜ〴ぜ〰げづっはねぢどのちのぴぜ〴ぜ〰うはひねぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴ぜ〰ちのつぜ〴ぜ〰こづぬづっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰ぉのぜ〴ぜ〰ざどぶはぜ〴ぜ〰がはっちぬどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Children: Item: Title: ぉびはぬちぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぐふひどてどっちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㈩‾㸊⡜㌷㙜㌷㝜〰ぐひどねちひべぜ〴ぜ〰こづぱふづのっづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぃとちひちっぴづひどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぴとづぜ〴ぜ〰げづっはねぢどのちのぴぜ〴ぜ〰うはひねぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴ぜ〰ちのつぜ〴ぜ〰こづぬづっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰ぉのぜ〴ぜ〰ざどぶはぜ〴ぜ〰がはっちぬどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: ぐひどねちひべぜ〴ぜ〰こづぱふづのっづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㌩‾㸊⡜㌷㙜㌷㝜〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぃとちひちっぴづひどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぴとづぜ〴ぜ〰げづっはねぢどのちのぴぜ〴ぜ〰うはひねぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴ぜ〰ちのつぜ〴ぜ〰こづぬづっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰ぉのぜ〴ぜ〰ざどぶはぜ〴ぜ〰がはっちぬどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぃとちひちっぴづひどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぴとづぜ〴ぜ〰げづっはねぢどのちのぴぜ〴ぜ〰うはひねぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㐩‾㸊⡜㌷㙜㌷㝜〰ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴ぜ〰ちのつぜ〴ぜ〰こづぬづっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰ぉのぜ〴ぜ〰ざどぶはぜ〴ぜ〰がはっちぬどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴ぜ〰ちのつぜ〴ぜ〰こづぬづっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㔩‾㸊⡜㌷㙜㌷㝜〰ぉのぜ〴ぜ〰ざどぶはぜ〴ぜ〰がはっちぬどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: ぉのぜ〴ぜ〰ざどぶはぜ〴ぜ〰がはっちぬどぺちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㈮㘩‾㸊⡜㌷㙜㌷㝜〰ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: ぁびびちべびぜ〴ぜ〰はのぜ〴ぜ〰ぃづぬぬぜ〴ぜ〰ぃふぬぴふひづぜ〴ぜ〰ちのつぜ〴ぜ〰あどはぴづっとのはぬはでどっちぬぜ〴ぜ〰ぐはぴづのぴどちぬぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸳ Item: Title: いどびっふびびどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳散瑩潮⸴ Item: Title: きちぴづひどちぬびぜ〴ぜ〰ちのつぜ〴ぜ〰きづぴとはつびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮ㄩ‾㸊⡜㌷㙜㌷㝜〰ぐふひどてどっちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎちぴふひちぬぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㈩‾㸊⡜㌷㙜㌷㝜〰ぐひどねちひべぜ〴ぜ〰こぴひふっぴふひづぜ〴ぜ〰いづぴづひねどのちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㌩‾㸊⡜㌷㙜㌷㝜〰げづっはねぢどのちのぴぜ〴ぜ〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰あどはばとべびどっちぬぜ〴ぜ〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰ひぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸴⸳⸱ Children: Item: Title: ぐふひどてどっちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎちぴふひちぬぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㈩‾㸊⡜㌷㙜㌷㝜〰ぐひどねちひべぜ〴ぜ〰こぴひふっぴふひづぜ〴ぜ〰いづぴづひねどのちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㌩‾㸊⡜㌷㙜㌷㝜〰げづっはねぢどのちのぴぜ〴ぜ〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰あどはばとべびどっちぬぜ〴ぜ〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰ひぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸴⸳⸱ Item: Title: ぐひどねちひべぜ〴ぜ〰こぴひふっぴふひづぜ〴ぜ〰いづぴづひねどのちぴどはのぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㌩‾㸊⡜㌷㙜㌷㝜〰げづっはねぢどのちのぴぜ〴ぜ〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰あどはばとべびどっちぬぜ〴ぜ〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰ひぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸴⸳⸱ Item: Title: げづっはねぢどのちのぴぜ〴ぜ〰ぐひはつふっぴどはのぜ〴ぜ〰ちのつぜ〴ぜ〰あどはばとべびどっちぬぜ〴ぜ〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰ひぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸴⸳⸱ Children: Item: Title: ぃぬはのどので〬ぜ〴ぜ〰ぅへばひづびびどはのぜ〴ぜ〰ちのつぜ〴ぜ〰ぐふひどてどっちぴどはのぜ〴ぜ〰どのぜ〴ぜ〰ぐどっとどちぜ〴ぜ〰ぐちびぴはひどびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獵扳散瑩潮⸴⸳⸲ Item: Title: あどはばとべびどっちぬぜ〴ぜ〰ぁのちぬべびづびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㐩‾㸊⡜㌷㙜㌷㝜〰ぅのぺべねちぴどっぜ〴ぜ〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰ぴとづぜ〴ぜ〰ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㔩‾㸊⡜㌷㙜㌷㝜〰えどびぴはぬはでべぜ〴ぜ〰ちのつぜ〴ぜ〰ぉねねふのはてぬふはひづびっづのっづぜ〴ぜ〰ぅへばづひどねづのぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㘩‾㸊⡜㌷㙜㌷㝜〰ぃべぴはぴはへどっどぴべぜ〴ぜ〰ぅぶちぬふちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㜩‾㸊⡜㌷㙜㌷㝜〰ぃづぬぬふぬちひぜ〴ぜ〰さばぴちにづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰きはぬづっふぬちひぜ〴ぜ〰ちのつぜ〴ぜ〰うふのっぴどはのちぬぜ〴ぜ〰こぴちぢどぬどぴべぜ〴ぜ〰はてぜ〴ぜ〰ぐぃぉぜ〴ぜ〰どのぜ〴ぜ〰あぬははつぜ〴ぜ〰ぐぬちびねちぜ〴ぜ〰ちのつぜ〴ぜ〰こづひふねぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: ぅのぺべねちぴどっぜ〴ぜ〰ぁのちぬべびどびぜ〴ぜ〰はてぜ〴ぜ〰ぴとづぜ〴ぜ〰ぉのとどぢどぴはひべぜ〴ぜ〰ぁっぴどぶどぴべぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㔩‾㸊⡜㌷㙜㌷㝜〰えどびぴはぬはでべぜ〴ぜ〰ちのつぜ〴ぜ〰ぉねねふのはてぬふはひづびっづのっづぜ〴ぜ〰ぅへばづひどねづのぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㘩‾㸊⡜㌷㙜㌷㝜〰ぃべぴはぴはへどっどぴべぜ〴ぜ〰ぅぶちぬふちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㜩‾㸊⡜㌷㙜㌷㝜〰ぃづぬぬふぬちひぜ〴ぜ〰さばぴちにづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰きはぬづっふぬちひぜ〴ぜ〰ちのつぜ〴ぜ〰うふのっぴどはのちぬぜ〴ぜ〰こぴちぢどぬどぴべぜ〴ぜ〰はてぜ〴ぜ〰ぐぃぉぜ〴ぜ〰どのぜ〴ぜ〰あぬははつぜ〴ぜ〰ぐぬちびねちぜ〴ぜ〰ちのつぜ〴ぜ〰こづひふねぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: えどびぴはぬはでべぜ〴ぜ〰ちのつぜ〴ぜ〰ぉねねふのはてぬふはひづびっづのっづぜ〴ぜ〰ぅへばづひどねづのぴびぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㘩‾㸊⡜㌷㙜㌷㝜〰ぃべぴはぴはへどっどぴべぜ〴ぜ〰ぅぶちぬふちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㜩‾㸊⡜㌷㙜㌷㝜〰ぃづぬぬふぬちひぜ〴ぜ〰さばぴちにづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰きはぬづっふぬちひぜ〴ぜ〰ちのつぜ〴ぜ〰うふのっぴどはのちぬぜ〴ぜ〰こぴちぢどぬどぴべぜ〴ぜ〰はてぜ〴ぜ〰ぐぃぉぜ〴ぜ〰どのぜ〴ぜ〰あぬははつぜ〴ぜ〰ぐぬちびねちぜ〴ぜ〰ちのつぜ〴ぜ〰こづひふねぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: ぃべぴはぴはへどっどぴべぜ〴ぜ〰ぅぶちぬふちぴどはのぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㜩‾㸊⡜㌷㙜㌷㝜〰ぃづぬぬふぬちひぜ〴ぜ〰さばぴちにづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰きはぬづっふぬちひぜ〴ぜ〰ちのつぜ〴ぜ〰うふのっぴどはのちぬぜ〴ぜ〰こぴちぢどぬどぴべぜ〴ぜ〰はてぜ〴ぜ〰ぐぃぉぜ〴ぜ〰どのぜ〴ぜ〰あぬははつぜ〴ぜ〰ぐぬちびねちぜ〴ぜ〰ちのつぜ〴ぜ〰こづひふねぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: ぃづぬぬふぬちひぜ〴ぜ〰さばぴちにづぜ〴ぜ〰はてぜ〴ぜ〰ぎぶぃぉぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰きはぬづっふぬちひぜ〴ぜ〰ちのつぜ〴ぜ〰うふのっぴどはのちぬぜ〴ぜ〰こぴちぢどぬどぴべぜ〴ぜ〰はてぜ〴ぜ〰ぐぃぉぜ〴ぜ〰どのぜ〴ぜ〰あぬははつぜ〴ぜ〰ぐぬちびねちぜ〴ぜ〰ちのつぜ〴ぜ〰こづひふねぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: きはぬづっふぬちひぜ〴ぜ〰ちのつぜ〴ぜ〰うふのっぴどはのちぬぜ〴ぜ〰こぴちぢどぬどぴべぜ〴ぜ〰はてぜ〴ぜ〰ぐぃぉぜ〴ぜ〰どのぜ〴ぜ〰あぬははつぜ〴ぜ〰ぐぬちびねちぜ〴ぜ〰ちのつぜ〴ぜ〰こづひふねぜ〴〩਼㰠⽓ 䝯呯 䐠⡳畢獥捴楯渮㐮㠩‾㸊⡜㌷㙜㌷㝜〰げづてづひづのっづび Item: Title: げづてづひづのっづび 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