Heterogeneity of proteinase inhibitors in the water-soluble organic matrix from the oyster nacre.
We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.
Bédouet
Laurent
L
Département des Milieux et Peuplements Aquatiques, Muséum National d'Histoire Naturelle, UMR 5178, CNRS-MNHN Biologie des Organismes Marins et Ecosystèmes, ERT Valorisation de Molécules Bioactives d'Origine Marine, Paris, F-75231, France. bedouet@mnhn.fr
Duplat
Denis
D
Marie
Arul
A
Dubost
Lionel
L
Berland
Sophie
S
Rousseau
Marthe
M
Milet
Christian
C
Lopez
Evelyne
E
D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002853:Chromatography, Liquid / Q000662:veterinary; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D019286:Endopeptidase K / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors; D049911:Pinctada / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000662:veterinary; D014867:Water / Q000737:chemistry
D015853:Cysteine Proteinase Inhibitors; D011480:Protease Inhibitors; D014867:Water; D002403:Cathepsins; D019286:Endopeptidase K; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain