Quail cystatin: Isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B

FEBS Letters

Mentele, Reinhard²  Auerswald, Ennes A²  Eckerskorn, Christoph⁴  Machleidt, Werner¹  Wittmann, Josef⁵  Kolb, Helmut J³  Fritz, Hans²  Gerhartz, Bernd²  Engh, Richard A⁶  Torquato, Richardo² 

[1]Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, LMU München, Goethestr. 33, D-80336 Munich, Germany

[2]Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt, LMU München, Nußbaumstr. 20, D-80336 Munich, Germany

[3]Klinisch-Chemisches Institut am Städtischen Krankenhaus Harlaching, Sanatoriumsplatz 1, D-81545 Munich, Germany

[4]Max-Planck-Institut für Biochemie, Proteinanalytik, D-82152 Martinsried, Germany

[5]Institut für Physiologische Chemie, Physiolog. Chemie und Ernährungsphysiologie, LMU München, Veterinärstr. 13, D-80539 Munich, Germany

[6]Max-Planck-Institut für Biochemie, Strukturforschung, D-82152 Martinsried, Germany

关键词: Cystatin;    Cysteine proteinase inhibitor;    Cathepsin B;    Inhibition kinetics;    Mass spectrometry;    Molecular interaction;    catB;    cathepsin B;    cc;    chicken cystatin;    CNBr;    cyanogen bromide;    E-64;    l-3-carboxy-2;    3-trans-epoxy-propionyl-leucyl-amido-(4-guanidino)butane;    NH-Mec;    7-(4-methyl) coumaryl-amide;    Z;    benzyloxycarbonyl;    qc;    quail cystatin. Enzymes: papain (EC 3.4.22.2);    cathepsin;    (EC 3.4.22.1).;   

DOI  :  10.1016/S0014-5793(97)00806-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Quail cystatin, a new cysteine proteinase inhibitor protein of the cystatin superfamily, was purified from egg albumen of Japanese quail Coturnix coturnix japonica. Amino acid sequencing and mass spectrometry revealed the complete 116 amino acid residue primary structure of a phosphorylated form (13 173 Da). The inhibitor has a 90% sequence identity with chicken cystatin. Its interaction with papain is rapid and tight (K _i=4.4 pM; k _on=1.8×10⁷ M⁻¹ s⁻¹; k _off=0.8×10⁻⁴ s⁻¹) and very similar to that of chicken cystatin. Surprisingly, however, cathepsin B was inhibited 15-fold more strongly by quail cystatin (K _i=47 pM; k _on=19×10⁷ M⁻¹ s⁻¹; k _off=9×10⁻⁴ s⁻¹) than by chicken cystatin (K _i=784 pM; k _on=2.9×10⁷ M⁻¹ s⁻¹; k _off=24×10⁻⁴ s⁻¹). Intuitive comparative conformational inspection of related inhibitors and of cognate enzymes suggest that: (i) the 3D structure of quail cystatin is nearly identical to that of chicken cystatin, (ii) quail cystatin can interact with cathepsin B analogous to the stefin B–papain interaction, if the `occluding loop' of cathepsin B possesses an `open' conformation, (iii) the greater inhibition of cathepsin B by quail cystatin compared to chicken cystatins probably arises from two additional ionic interactions between residues Arg¹⁵ and Lys¹¹² of the inhibitor and Glu¹⁹⁴ and Asp¹²⁴ of the enzyme, respectively. The two potential salt bridges are located outside of the known contact regions between cystatins and peptidases of the papain family.

【 授权许可】

Unknown