Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases.
The type 1 domain of thyroglobulin is a protein module (Thyr-1) that occurs in a variety of secreted and membrane proteins. Several examples of Thyr-1 modules have been previously identified as inhibitors of the papain family of cysteine proteinases. Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog of transferrin with a pair of such Thyr-1 modules located in the N-lobe. Saxiphilin is now characterized as a potent inhibitor of three cysteine proteinases as follows: papain, human cathepsin B, and cathepsin L. The stoichiometry of enzyme inhibition reveals that both Thyr-1 domains of saxiphilin inhibit papain (apparent K(i) = 1. 72 nm), but only one of these domains inhibits cathepsin B (K(i) = 1. 67 nm) and cathepsin L (K(i) = 0.02 nm). Physical association of saxiphilin and papain blocked from turnover at the active-site cysteine residue can be detected by cross-linking with glutaraldehyde. The rate of association of saxiphilin and cathepsin B is strongly pH-dependent with an optimum at pH 5.2, reflecting control by at least two H(+)-titratable groups. These results further demonstrate that various Thyr-1 domains are selective inhibitors of cysteine proteinases with utility in the study of protein interactions and degradation.
Lenarcic
B
B
Departments of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. brigita.lenarcic@ijs.si
Krishnan
G
G
Borukhovich
R
R
Ruck
B
B
Turk
V
V
Moczydlowski
E
E
D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000494:pharmacology*; D002401:Cathepsin B / Q000037:antagonists & inhibitors*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010450:Endopeptidases; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors*; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013954:Thyroglobulin / Q000737:chemistry*
D029845:Amphibian Proteins; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; C027525:saxitoxin-binding protein, Rana catesbeiana; D013954:Thyroglobulin; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain