TY - JOUR
T1 - Simple modifications of the serpin reactive site loop convert SCCA2 into a cysteine proteinase inhibitor
T2 - A critical role for the P3' proline in facilitating RSL cleavage
AU - Luke, Cliff
AU - Schick, Charles
AU - Tsu, Christopher
AU - Whisstock, James C.
AU - Irving, James A.
AU - Brömme, Dieter
AU - Juliano, Luiz
AU - Shi, Guo Ping
AU - Chapman, Harold A.
AU - Silverman, Gary A.
PY - 2000/6/20
Y1 - 2000/6/20
N2 - The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of cats by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in cats inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild- type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of cats by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.
AB - The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of cats by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in cats inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild- type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of cats by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.
UR - http://www.scopus.com/inward/record.url?scp=0034691279&partnerID=8YFLogxK
U2 - 10.1021/bi000050g
DO - 10.1021/bi000050g
M3 - Article
C2 - 10852705
AN - SCOPUS:0034691279
VL - 39
SP - 7081
EP - 7091
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 24
ER -