Quail cystatin: isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B.
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 (Ki = 4.4 pM; k(on) = 1.8x10(7) M(-1) s(-1); k(off) = 0.8x10(-4) s(-1)) and very similar to that of chicken cystatin. Surprisingly, however, cathepsin B was inhibited 15-fold more strongly by quail cystatin (Ki = 47 pM; k(on) = 19x10(7) M(-1) s(-1); k(off) = 9x10(-4) s(-1)) than by chicken cystatin (Ki = 784 pM; k(on) = 2.9x10(7) M(-1) s(-1); k(off) = 24x10(-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.
Gerhartz
B
B
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgischen Klinik und Poliklinik, LMU München, Germany.
Engh
R A
RA
Mentele
R
R
Eckerskorn
C
C
Torquato
R
R
Wittmann
J
J
Kolb
H J
HJ
Machleidt
W
W
Fritz
H
H
Auerswald
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002645:Chickens; D003370:Coturnix; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment
D015891:Cystatins; D002401:Cathepsin B