d42ea16f51de611ceb56b5c6b6e77160 pmc_29495576.pdf 59eb5279c458ef27065c1254be45c133008bc193 pmc_29495576.pdf 92300ed88da442eaaeb37ceeb26bf6f2f7c3a9730ca5fd124085895e560fb1f5 pmc_29495576.pdf Title: Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña Subject: Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs. Keywords: cystine-knot miniproteins; carboxypeptidase inhibitor; plant inhibitor; Solanum tuberosum; protease; Andean potatoes Author: Juliana Cotabarren, Mariana Edith Tellechea, Sebastián Martín Tanco, Julia Lorenzo, Javier Garcia-Pardo, Francesc Xavier Avilés and Walter David Obregón Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.15; modified using iText® 7.0.1 ©2000-2016 iText Group NV (AGPL-version) CreationDate: Wed Feb 28 09:42:37 2018 CET ModDate: Wed Feb 28 09:45:49 2018 CET 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: 3365508 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 127 0 OXBKYU+TeXGyreAdventor-Bold Type 1 Custom yes yes no 128 0 MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 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Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-01-19 07:01:47 CET RepresentationInformation: pmc_29495576.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2018-04-16 03:49:29 CEST Size: 3365508 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 FreeObjects: 1 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〠 Item: Title: げづびふぬぴび〠ちのつ〠いどびっふびびどはの〠 Children: Item: Title: ぉつづのぴどてどっちぴどはの〠ちのつ〠ぉのどぴどちぬ〠ぃとちひちっぴづひどぺちぴどはの〠はて〠ち〠ぎちぴどぶづ〠きづぴちぬぬはっちひぢはへべばづばぴどつちびづ〠ぉのとどぢどぴはひ〠てひはね〠こ〮〠ぴふぢづひはびふね〠びふぢびば〮〠ちのつどでづのふね〠っぶ〮〠ぃとふひぱふづヱち〠 Item: Title: ぐふひどてどっちぴどはの〠ちのつ〠きぁがいぉ〭ごくう〠きちびび〠こばづっぴひはねづぴひどっ〠ぁのちぬべびどび〠はて〠ぴとづ〠ぎちぴどぶづ〠ぃちひぢはへべばづばぴどつちびづ〠ぉのとどぢどぴはひ〠 Item: Title: ぃぬはのどので〠ちのつ〠こづぱふづのっづ〠ぁのちぬべびどび〠はて〠ぴとづ〠ぎはぶづぬ〠ぃべびぴどのづ〭かのはぴ〠きづぴちぬぬはっちひぢはへべばづばぴどつちびづ〠ぉのとどぢどぴはひ〠〨っとふぐぃぉ〩〠 Item: Title: ぅへばひづびびどはの〬〠ぐふひどてどっちぴどはの〬〠ちのつ〠きぁがいぉ〭ごくう〠きちびび〠こばづっぴひはねづぴひべ〠ぁのちぬべびどび〠はて〠ひっとふぐぃぉ〠 Item: Title: いづぴづひねどのちぴどはの〠はて〠ぴとづ〠ぉのとどぢどぴどはの〠かどのづぴどっ〠ぃはのびぴちのぴび〠〨かど〩〠ちでちどのびぴ〠ぁ〯あ〭ごべばづ〠きぃぐび〠 Item: Title: ぉつづのぴどてどっちぴどはの〠ちのつ〠いづ〠ぎはぶは〠こづぱふづのっどので〠はて〠ぴとづ〠ぎちぴどぶづ〠っとふぐぃぉ〠 Item: Title: きちぴづひどちぬび〠ちのつ〠きづぴとはつび〠 Children: Item: Title: きちぴづひどちぬび〠ちのつ〠げづちでづのぴび〠 Item: Title: ぃひふつづ〠ぅへぴひちっぴ〠ぐひづばちひちぴどはの〠 Item: Title: ごはぴちぬ〠ぐひはぴづどの〠けふちのぴどぴちぴどはの〠 Item: Title: ぃちひぢはへべばづばぴどつちびづ〠ぁ〠ぉのとどぢどぴどはの〠きづちびふひづねづのぴび〠ちのつ〠ぉぃ〵〰〠いづぴづひねどのちぴどはの〠 Item: Title: ごとづひねちぬ〠こぴちぢどぬどぴべ〠ぅへばづひどねづのぴび〠ちのつ〠のっとふぐぃぉ〠ぐふひどてどっちぴどはの〠ぢべ〠ぁててどのどぴべ〠ぃとひはねちぴはでひちばとべ〠 Children: Item: Title: ごとづひねちぬ〠こぴちぢどぬどぴべ〠ぅへばづひどねづのぴび〠 Item: Title: ぁててどのどぴべ〠ぃとひはねちぴはでひちばとべ〠ぐふひどてどっちぴどはの〠 Item: Title: ぃぬはのどので〠ちのつ〠ぅへばひづびびどはの〠はて〠ひっとふぐぃぉ〠 Children: Item: Title: ぃぬはのどので〠ちのつ〠こづぱふづのっづ〠ぁのちぬべびどび〠はて〠っとふぐぃぉ〠 Item: Title: ぅへばひづびびどはの〠ちのつ〠ぐふひどてどっちぴどはの〠はて〠ひっとふぐぃぉ〠 Item: Title: ぇづのづひちぬ〠ぃとちひちっぴづひどぺちぴどはの〠はて〠きづぴとはつび〠 Children: Item: Title: ごひどび〭ごひどっどのづ〭こいこ〭ぐぁぇぅ〠 Item: Title: ぉびはづぬづっぴひはてはっふびどので〠〨ぉぅう〩〠 Item: Title: きぁがいぉ〭ごくう〠きちびび〠こばづっぴひはねづぴひどっ〠ぁのちぬべびどび〠 Item: Title: ぃはのっぬふびどはのび〠 Item: Title: げづてづひづのっづび Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OXBKYU+TeXGyreAdventor-Bold FontSubset: true FirstChar: 68 LastChar: 73 FontDescriptor: FontName: OXBKYU+TeXGyreAdventor-Bold Flags: Symbolic FontBBox: -679, -304, 1661, 1236 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ALMJEC+EURM10 FontSubset: true FirstChar: 11 LastChar: 33 FontDescriptor: FontName: ALMJEC+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: CZNUER+CMEX10 FontSubset: true FirstChar: 113 LastChar: 113 FontDescriptor: FontName: CZNUER+CMEX10 Flags: Symbolic FontBBox: -24, -2960, 1454, 772 FontFile: true Font: BaseFont: 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Annotations: Annotation: Subtype: Link Rect: 437, 727, 474, 738 Annotation: Subtype: Link Rect: 481, 727, 516, 738 Annotation: Subtype: Link Rect: 100, 676, 137, 686 Annotation: Subtype: Link Rect: 143, 676, 179, 686 Annotation: Subtype: Link Rect: 356, 637, 393, 648 Annotation: Subtype: Link Rect: 399, 637, 435, 648 Annotation: Subtype: Link Rect: 378, 611, 416, 622 Annotation: Subtype: Link Rect: 422, 611, 458, 622 Annotation: Subtype: Link Rect: 136, 573, 173, 583 Annotation: Subtype: Link Rect: 180, 573, 216, 583 Annotation: Subtype: Link Rect: 374, 533, 411, 544 Annotation: Subtype: Link Rect: 417, 533, 453, 544 Annotation: Subtype: Link Rect: 122, 496, 160, 506 Annotation: Subtype: Link Rect: 100, 405, 137, 415 Annotation: Subtype: Link Rect: 307, 379, 344, 390 Annotation: Subtype: Link Rect: 336, 340, 373, 351 Annotation: Subtype: Link Rect: 379, 340, 415, 351 Annotation: Subtype: Link Rect: 100, 302, 137, 312 Annotation: Subtype: Link Rect: 143, 302, 179, 312 Annotation: Subtype: Link Rect: 380, 276, 418, 286 Annotation: Subtype: Link Rect: 424, 276, 460, 286 Annotation: Subtype: Link Rect: 343, 250, 381, 260 Annotation: Subtype: Link Rect: 387, 250, 423, 260 Annotation: Subtype: Link Rect: 402, 211, 438, 222 Annotation: Subtype: Link Rect: 444, 211, 479, 222 Annotation: Subtype: Link Rect: 100, 173, 137, 183 Annotation: Subtype: Link Rect: 143, 173, 179, 183 Annotation: Subtype: Link Rect: 100, 134, 137, 144 Annotation: Subtype: Link Rect: 143, 134, 179, 144 Annotation: Subtype: Link Rect: 261, 94, 298, 105 Annotation: Subtype: Link Rect: 305, 94, 341, 105 Annotation: Subtype: Link Rect: 414, 69, 451, 80 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 377, 727, 414, 738 Annotation: Subtype: Link Rect: 421, 727, 457, 738 Annotation: Subtype: Link Rect: 245, 702, 282, 712 Annotation: Subtype: Link Rect: 194, 663, 232, 674 Annotation: Subtype: Link Rect: 238, 663, 274, 674 Annotation: Subtype: Link Rect: 324, 611, 361, 622 Annotation: Subtype: Link Rect: 368, 611, 404, 622 Annotation: Subtype: Link Rect: 76, 569, 166, 600 Annotation: Subtype: Link Rect: 234, 563, 424, 574 Checksum: 5b6d5d7d Type: CRC32 Checksum: d42ea16f51de611ceb56b5c6b6e77160 Type: MD5 Checksum: 59eb5279c458ef27065c1254be45c133008bc193 Type: SHA-1 Checksum: 92300ed88da442eaaeb37ceeb26bf6f2f7c3a9730ca5fd124085895e560fb1f5 Type: SHA-256