Inhibitory Specificity of the Anti-inflammatory Myxoma Virus Serpin, SERP-1

Abstract

SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissuetype plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg 319 -Asn 320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr 315 -Leu 316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from k inh ‫؍‬ 3 ؋ 10 5 M ؊1 s ؊1 with thrombin to ϳ600 M ؊1 s ؊1with C1s, while steady-state inhibition constants ranged from K I ‫؍‬ 10 pM with thrombin to ϳ100 nM with C1s. Stoichiometries of inhibition varied between SI ‫؍‬ 1.4 ؎ 0.1 for uPA to SI ‫؍‬ 13 ؎ 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with K I (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by k inh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.

Functional Analysis of Toxoplasma gondii Protease Inhibitor 1

“…TgPI-1 43 and TgPI-1 41 Secretion into ESA Is Unaffected by BAPTA-AM-TgPI-1 possesses a hydrophobic N-terminal sequence that has the properties of a signal sequence, suggesting that it might be a secretory protein. In an effort to determine whether TgPI-1 was secreted, two-dimensional gels of T. gondii ESA were transferred to nitrocellulose and probed with R␣rTgPI-1 (Fig.…”

“…Rabbit antibodies generated against rTgPI-1 (R␣rTgPI-1) recognize two polypeptides in extracellular tachyzoites, bradyzoites, and sporozoites, TgPI-1 43 and TgPI-1 41 , named according to their apparent molecular weight as determined by SDS-PAGE (Fig. 2B, lanes 1-5).…”

Characterization and functional analysis of Serp3: a novel myxoma virus-encoded serpin involved in virulence

“…SERP-1 is a secreted factor that can inhibit plasmin, urokinase-type plasminogen activator, tissue-type plasminogen activator, thrombin and J.-L. Guerin and others J.-L. Guerin and others factor Xa (Lomas et al, 1993 ;Macen et al, 1993 ;Maksymowych et al, 1996 ;Nash et al, 1997Nash et al, , 1998. Serp2 remains associated within the cells and has both an antiinflammatory and an anti-apoptotic activity (Messud-Petit et al, 1998 ;Petit et al, 1996).…”

“…Two copies of serp-1 (Upton et al, 1990) are located in the ITR of MV and encode a secreted factor that is implicated in modulation of host inflammatory responses (Macen et al, 1993 ;Nash et al, 1997Nash et al, , 1998. The serp2 gene has been identified near the right ITR (Petit et al, 1996) and is closely related to CPV-encoded CrmA.…”

“…Serpins (serine proteinase inhibitors ;Carrell & Travis, 1985) have been described in poxviruses, such as cowpox virus (CPV) (Pickup et al, 1986), rabbitpox virus (Brooks et al, 1995 ;Macen et al, 1998) and vaccinia virus (VV) (Boursnell et al, 1988 ;Kotwal & Moss, 1988 ;Smith et al, 1989 ;Turner & Moyer, 1992). These serpins are named SPI-1, SPI-2 (or CrmA in CPV) and SPI-3 .…”