The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition.
The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.
Schick
C
C
Department of Pediatrics, Harvard Medical School, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
Brömme
D
D
Bartuski
A J
AJ
Uemura
Y
Y
Schechter
N M
NM
Silverman
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D000951:Antigens, Neoplasm; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C051072:squamous cell carcinoma-related antigen; D012697:Serine Endopeptidases