Inhibition of the cysteine proteinases cathepsins K and L by the serpin headpin (SERPINB13): a kinetic analysis

• Published in: Archives of biochemistry and biophysics 2003-01, Vol.409 (2), p.367-374
• Main Authors: Jayakumar, Arumugam, Kang, Ya’an, Frederick, Mitchell J, Pak, Stephen C, Henderson, Ying, Holton, Paula R, Mitsudo, Kenji, Silverman, Gary A, EL-Naggar, Adel K, Brömme, Dieter, Clayman, Gary L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Baculoviridae - genetics; Cathepsin K; Cathepsin L; Cathepsins - antagonists & inhibitors; Cathepsins - drug effects; Cathepsins - metabolism; Cell Line, Transformed; Cysteine Endopeptidases - drug effects; Cysteine Endopeptidases - metabolism; Cysteine proteinases; Headpin; Humans; Insect cells; Keratinocytes - cytology; Kinetics; Lysosomes - enzymology; Molecular Sequence Data; Molecular Weight; Protein Structure, Tertiary; Proteinase inhibition; Recombinant Fusion Proteins - pharmacology; RNA, Messenger - metabolism; Serine proteinase inhibitor; Serine Proteinase Inhibitors - pharmacology; Serpins - chemistry; Serpins - isolation & purification; Serpins - pharmacology; Sodium Dodecyl Sulfate - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spodoptera - cytology; Spodoptera - genetics; Spodoptera - virology
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/S0003-9861(02)00635-5

Headpin (SERPINB13) is a novel member of the serine proteinase inhibitor (Serpin) gene family that was originally cloned from a keratinocyte cDNA library. Western blot analysis using a headpin-specific antiserum recognized a protein with the predicted M r of 44 kDa in lysates derived from a transformed keratinocyte cell line known to express headpin mRNA. Similarity of the reactive-site loop (RSL) domain of headpin, notably at the P1–P1 ′ residues, with other serpins that inhibit cysteine and serine proteinases suggests that headpin may inhibit similar proteinases. This study demonstrates that recombinant headpin indeed inhibits cathepsins K and L, but not chymotrypsin, elastase, trypsin, subtilisin A, urokinase-type plasminogen activator, plasmin, or thrombin. The second-order rate constants ( k a) for the inhibitory reactions of rHeadpin with cathepsins K and L were 5.1±0.6×10 4 and 4.1±0.8×10 4 M −1 s −1 , respectively. Headpin formed SDS-stable complexes with cathepsins K and L, a characteristic property of inhibitory serpins. Interactions of the RSL domain of headpin with cathepsins K and L were indicated by cleavage of headpin near the predicted P1–P1 ′ residues by these proteinases. These results demonstrate that the serpin headpin possesses specificity for inhibiting lysosomal cysteine proteinases.