One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors
Snakes contain three types of phospholipase A<sub>2</sub> (PLA<sub>2</sub>)-inhibitory proteins in their blood, PLIα, β, and γ, which protect them from their own venom, PLA<sub>2</sub>. PLIβ is the snake ortholog of leucine-rich α<sub>2</sub> glycoprot...
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MDPI AG
2024-03-01
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| author | Ryoichi Shirai Kana Shibata Shinobu Fujii Rikiro Fukunaga Seiji Inoue |
| author_facet | Ryoichi Shirai Kana Shibata Shinobu Fujii Rikiro Fukunaga Seiji Inoue |
| author_sort | Ryoichi Shirai |
| collection | DOAJ |
| description | Snakes contain three types of phospholipase A<sub>2</sub> (PLA<sub>2</sub>)-inhibitory proteins in their blood, PLIα, β, and γ, which protect them from their own venom, PLA<sub>2</sub>. PLIβ is the snake ortholog of leucine-rich α<sub>2</sub> glycoprotein (LRG). Since autologous cytochrome <i>c</i> (Cyt <i>c</i>) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt <i>c</i> affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. <i>Laticauda semifasciata</i> and <i>Naja kaouthia</i> LRGs showed no inhibitory activity against <i>L. semifasciata</i> PLA<sub>2</sub> and weak inhibitory activity against <i>Gloydius brevicauda</i> basic PLA<sub>2</sub>. <i>Elaphe climacophora</i> PLIβ had weaker inhibitory activity against <i>G. brevicauda</i> basic PLA<sub>2</sub> than <i>G. brevicauda</i> and <i>Elaphe quadrivirgata</i> PLIs, which are abundant in blood and known to neutralize <i>G. brevicauda</i> basic PLA<sub>2</sub>. <i>Protobothrops flavoviridis</i> LRG showed no inhibitory activity against basic venom PLA<sub>2</sub>, PL-X, or <i>G. brevicauda</i> basic PLA<sub>2</sub>. Binding analysis of <i>P. flavoviridis</i> LRG using surface plasmon resonance showed very strong binding to snake Cyt <i>c</i>, followed by that to horse Cyt <i>c</i>, weak binding to yeast Cyt <i>c</i>, and no binding to <i>P. flavoviridis</i> PL-X or BPI/II. We also deduced the amino acid sequences of <i>L. semifasciata</i> and <i>P. flavoviridis</i> LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA<sub>2</sub>, but, whether it actually functions as a PLA<sub>2</sub>-inhibitory protein, PLIβ, depends on the snake. |
| first_indexed | 2024-04-24T17:47:31Z |
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| spelling | doaj.art-a82b4decf8a74291912961ce0e6dded22024-03-27T14:06:21ZengMDPI AGToxins2072-66512024-03-0116312610.3390/toxins16030126One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> InhibitorsRyoichi Shirai0Kana Shibata1Shinobu Fujii2Rikiro Fukunaga3Seiji Inoue4Department of Biochemistry, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki 569-1094, Osaka, JapanDepartment of Biochemistry, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki 569-1094, Osaka, JapanDepartment of Biochemistry, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki 569-1094, Osaka, JapanDepartment of Biochemistry, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki 569-1094, Osaka, JapanDepartment of Biochemistry, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki 569-1094, Osaka, JapanSnakes contain three types of phospholipase A<sub>2</sub> (PLA<sub>2</sub>)-inhibitory proteins in their blood, PLIα, β, and γ, which protect them from their own venom, PLA<sub>2</sub>. PLIβ is the snake ortholog of leucine-rich α<sub>2</sub> glycoprotein (LRG). Since autologous cytochrome <i>c</i> (Cyt <i>c</i>) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt <i>c</i> affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. <i>Laticauda semifasciata</i> and <i>Naja kaouthia</i> LRGs showed no inhibitory activity against <i>L. semifasciata</i> PLA<sub>2</sub> and weak inhibitory activity against <i>Gloydius brevicauda</i> basic PLA<sub>2</sub>. <i>Elaphe climacophora</i> PLIβ had weaker inhibitory activity against <i>G. brevicauda</i> basic PLA<sub>2</sub> than <i>G. brevicauda</i> and <i>Elaphe quadrivirgata</i> PLIs, which are abundant in blood and known to neutralize <i>G. brevicauda</i> basic PLA<sub>2</sub>. <i>Protobothrops flavoviridis</i> LRG showed no inhibitory activity against basic venom PLA<sub>2</sub>, PL-X, or <i>G. brevicauda</i> basic PLA<sub>2</sub>. Binding analysis of <i>P. flavoviridis</i> LRG using surface plasmon resonance showed very strong binding to snake Cyt <i>c</i>, followed by that to horse Cyt <i>c</i>, weak binding to yeast Cyt <i>c</i>, and no binding to <i>P. flavoviridis</i> PL-X or BPI/II. We also deduced the amino acid sequences of <i>L. semifasciata</i> and <i>P. flavoviridis</i> LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA<sub>2</sub>, but, whether it actually functions as a PLA<sub>2</sub>-inhibitory protein, PLIβ, depends on the snake.https://www.mdpi.com/2072-6651/16/3/126phospholipase A<sub>2</sub> inhibitorphospholipase A<sub>2</sub>leucine-rich α<sub>2</sub>-glycoproteincytochrome <i>c</i>ViperidaeElapidae |
| spellingShingle | Ryoichi Shirai Kana Shibata Shinobu Fujii Rikiro Fukunaga Seiji Inoue One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors Toxins phospholipase A<sub>2</sub> inhibitor phospholipase A<sub>2</sub> leucine-rich α<sub>2</sub>-glycoprotein cytochrome <i>c</i> Viperidae Elapidae |
| title | One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors |
| title_full | One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors |
| title_fullStr | One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors |
| title_full_unstemmed | One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors |
| title_short | One-Step Affinity Purification of Leucine-Rich α<sub>2</sub>-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A<sub>2</sub>-Inhibitory Activities as β-Type Phospholipase A<sub>2</sub> Inhibitors |
| title_sort | one step affinity purification of leucine rich α sub 2 sub glycoproteins from snake sera and characterization of their phospholipase a sub 2 sub inhibitory activities as β type phospholipase a sub 2 sub inhibitors |
| topic | phospholipase A<sub>2</sub> inhibitor phospholipase A<sub>2</sub> leucine-rich α<sub>2</sub>-glycoprotein cytochrome <i>c</i> Viperidae Elapidae |
| url | https://www.mdpi.com/2072-6651/16/3/126 |
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