SERP-1, a Secreted Poxviral Serpin

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SERP-1, a Secreted Poxviral Serpin Grant McFadden1,* and Richard Moyer2 1

The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519)663-3184, fax: (519)663-3847, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03017.

SUMMARY More than half a dozen poxvirus-encoded members of the serpin superfamily of serine proteinase inhibitors have been discovered, but only one of these, SERP-1 of myxoma virus, functions as an extracellular immunomodulator. SERP-1 is expressed as a secreted glycoprotein that protects myxoma-infected cells from clearance by macrophages and other inflammatory cells. Biochemically, SERP-1 protein binds and inhibits a spectrum of serine proteinases, such as plasmin, urokinase and tissue plasminogen activator, but its precise mode of action in virusinfected lesions remains to be elucidated. The purified SERP-1 protein alone functions as a potent antiinflammatory reagent in various animal models of inflammation.

While SERP-1 is secreted, the orthopoxvirus serpin SPI-3 has a similar, if not identical, spectrum of inhibited proteinases (R. Moyer and P. Turner, unpublished data). SERP-1 was first discovered in 1990, as a single copy gene in a related poxvirus designated malignant rabbit fibroma virus, and as a repeated copy gene in the terminal inverted repeat sequences of myxoma virus (Upton et al., 1990). Expression studies indicated that the processed SERP-1 protein is secreted as a 55±60 kDa glycoprotein that forms inhibitory complexes with a variety of cellular serine proteinases (Lomas et al., 1993; Nash et al., 1998).

Alternative names SPI-4 (proposed by Turner et al., 1995).

BACKGROUND

Structure

Discovery

SERP-1 contains the consensus domain structural features of eukaryotic serpins, including the reactive center loop bearing an unusual P1±P10 pairing of Arg±Asn (see Figure 2).

At least eight poxvirus genes have been described that are members of the serpin superfamily of serine proteinase inhibitors (Turner et al., 1995; Messud-Petit et al., 1998). The only one of these that expresses a protein secreted from infected cells is SERP-1, which is encoded by the leporipoxvirus myxoma virus (Nash et al., 1997). Thus, SERP-1 is the only viral serpin that qualifies as a virokine, and is likely targeted to inhibit extracellular (or cell surface) proteinases.

Main activities and pathophysiological roles Like all serpins, SERP-1 forms 1:1 inhibitory complexes with serine proteinases. In the case of SERP-1,

1430 Grant McFadden and Richard Moyer the list of inhibitable proteinases includes tissue plasminogen activator, urokinase, and plasmin (Nash et al., 1997, 1998). These inhibitory studies were conducted in vitro with human proteinases, indicating that SERP-1 target proteinases are highly conserved in evolution. A reactive center loop mutant of SERP1 in which the P1±P10 site has been mutated to Ala± Ala (SAA: see Figure 2) does not bind or inhibit any of the normal proteinase substrates for SERP-1, indicating that the viral protein functions as a true secreted serpin and is classified as an enzymatically active virokine.

postinfection and remain at high levels throughout the viral replication cycle. Like all poxviruses, the transcriptional regulation is under virus control in cells that support the full replication cycle. Thus, SERP-1 expression is relatively independent of the type of infected cell.

PROTEIN

Accession numbers g332305

GENE AND GENE REGULATION

Sequence

Accession numbers

See Figure 1. M35233

Chromosome location

Description of protein

The SERP-1 gene is present as a single copy in the genome of malignant rabbit fibroma virus (which can be regarded as a myxoma virus substrain), and as two copies in the terminal inverted repeat sequences of myxoma virus, strain Lausanne (Upton et al., 1990).

SERP-1 is a classic eukaryotic serpin, and is expressed as a 55±60 kDa secreted glycoprotein. Although the protein has not been crystallized, alignment studies indicate an unusually extended C-terminus, and the absence of a predicted D-helix, when compared with other nonviral serpins (Lomas et al., 1993). Despite the potential absence of the D-helix, modeling studies predict that the ordering and orientation of the classic serpin protein domains remain similar to other serpins, such as ovalbumin and antichymotrypsin (Lomas et al., 1993).

Regulatory sites and corresponding transcription factors SERP-1 is expressed as a classic late poxvirus gene, and as such is transcribed after DNA replication has commenced and late transcription factors have been synthesized (Macen et al., 1993). The SERP-1 promoter sequence includes the consensus motifs of all late poxvirus genes (Moss, 1996). SERP-1 mRNA species are first detected at approximately 6 hours

Figure 1

Important homologies SERP-1 possesses sequence motifs common to all serpins, but the closest cellular homologs include anti-

Protein sequence of SERP-1.

MKYLVLVLCL TSCACRDIGL WTFRYVYNES DNVVFSPYGL TSALSVLRIA AGGNTKREID VPESVVEDSD AFLALRELFV DASVPLRPEF TAFFSSRFNT SVQRVTENSE NVKDVINSYV KDKTGGDVPR VLDASLDRDT KMLLLSSVRM KTSWRIVFDP SFTTDQPFYS GNVTYKVRMM NKIDTLKTET FTLRNVGYSV TELPYKRRQT AMLLVVPDDL GEIVRALDLS LVRFWIRNMR KDVCQVVMPK FSVESVLDLR DALQRLGVRD AFDPSRADFG QASPSNDLYV TKVLQTSKIE ADERGTTASS DTAITLIPRN ALTAIVANKP FMFLIYHKPT TTVLFMGTIT KGEKVIYDTE GRDDVVSSV

SERP-1, a Secreted Poxviral Serpin 1431

Figure 2 A schematic representation of the SERP-1 protein structure indicating the positions of the predicted  helices that form the serpin structural fold. Helix a (hA) is the signal sequence responsible for SERP-1 secretion and is not present in the mature protein. Helix D (hD), normally present in serpins, is missing in SERP-1, although this does not appear to be essential for inhibitory function. The reactive center loop (RCL) interacts with the active site of the proteinase and contains the critical P1±P10 residues. The stable inhibited serpin±enzyme complex is believed to be an acyl-enzyme linked covalently at the P1 residue. The RCL sequence of SERP-1 and the engineered noninhibitory mutant (SAA) are also shown.

trypsin, antichymotrypsin, and plasminogen activator inhibitor 1. The specificity of serpins is in part defined by the amino acid residues at the P1±P10 site, which in the case of SERP-1 is a unique pairing (Arg±Asn) not found in any other known serpin (Figure 2).

membrane-associated enzymes. This subject is still under active investigation.

IN VITRO ACTIVITIES

In vitro findings A variety of catalytic parameters, including apparent inhibition constraints, apparent second-order rate constant for inhibition, and the measured rate constant for dissociation of the inhibited complex, were determined for SERP-1 against a spectrum of host serine proteinases in vitro (Lomas et al., 1993; Nash et al., 1998). The most effectively inhibited substrates for SERP-1 in vitro were plasmin, tissue plasminogen activator, urokinase, and thrombin.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles

Polyclonal antisera against SERP-1 expressed from myxoma virus reveal a heterogeneous 55±60 kDa species which can be digested with N-glycosidase F to the predicted size of the amino acid sequence, indicating that the major posttranslational modification is N-linked glycosylation. N-Linked sugar residues are not believed to affect the serpin function of SERP-1 in terms of reaction parameters in vitro, but could affect protein half-life or tissue localization in vivo.

The role of SERP-1 in vivo has been investigated by targeted disruption of the gene, followed by analysis of the knockout virus infection in susceptible rabbits (Upton et al., 1990; Macen et al., 1993). Unlike the parental myxoma virus, which is uniformly fatal to the European rabbit (Oryctolagus cuniculus), the SERP-1-minus virus was severely attenuated in vivo despite the fact that it replicated normally in tissue culture (Macen et al., 1993). Histological analysis of infected lesions indicated that in the absence of SERP-1 expression a more effective inflammatory cell response resulted in a more rapid clearance of the infection (Macen et al., 1993). Thus, the biological role of SERP-1 can be operationally defined as antiinflammatory, even though the target serine proteinase(s) remains speculative.

RECEPTOR UTILIZATION

Species differences

The `true' biological serine proteinases that bind and mediate the anti-inflammatory properties of SERP-1 in infected tissues are still speculative, but candidate proteinases include a variety of soluble or

All in vitro inhibition assays of SERP-1 protein have been conducted with human serine proteinases, suggesting a broad species range for its inhibitory functions. However, a precise evaluation of the inhibition

Posttranslational modifications

1432 Grant McFadden and Richard Moyer constants with a single class of serine proteinase from multiple vertebrates has not been conducted.

Pharmacological effects SERP-1 protein expressed from myxoma, or vaccinia virus vectors that overexpress SERP-1, or CHOtransfected cells, has been purified and tested as an anti-inflammatory reagent in two models of inflammation: balloon-induced atherosclerosis and antigeninduced arthritis (Lucas et al., 1996; Maksymowych et al., 1996). In the coronary restenosis model, single infusions of purified SERP-1 protein in the picogram dose range at the time of balloon injury were able to block the influx of macrophages into the injured vasculature and thus reduce the resultant development of atherosclerotic plaque (Lucas et al., 1996). Similar anti-inflammatory properties of SERP-1 protein were noted after intra-articular injection in a rabbit model of collagen-induced arthritis (Maksymowych et al., 1996). In each case, the therapeutic dosage of SERP-1 protein was in the picogram to nanogram range, which is lower than other known antiinflammatory reagents but closer to the natural levels of secreted SERP-1 expression during virus infection.

References Lomas, D. A., Evans, D. L., Upton, C., McFadden, G., and Carrell, R. W. (1993). Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor. J. Biol. Chem. 268, 516±521.

Lucas, A., Liu, L.-Y., Macen, J., Nash, P., Dai, E., Stewart, M., Graham, K., Etches, W., Boshkov, L., Nation, P. N., Humen, D., Hobman, M. L., and McFadden, G. (1996). Virus-encoded serine proteinase inhibitor SERP-1 inhibits atherosclerotic plaque development after balloon angioplasty. Circulation 94, 2890±2900. Macen, J. L., Upton, C., Nation, N., and McFadden, G. (1993). SERP-1, a serine proteinase inhibitor encoded by myxoma virus, is a secreted glycoprotein that interferes with inflammation. Virology 195, 348±363. Maksymowych, W. P., Nation, N., Nash, P. D., Macen, J., Lucas, A., McFadden, G., and Russell, A. S. (1996). Amelioration of antigen-induced arthritis in rabbits treated with a secreted viral serine proteinase inhibitor. J. Rheumatol. 23, 878±882. Messud-Petit, F., Gelfi, J., Delverdier, M., Amardeilh, M.-F., Py, R., Sutter, G., and Bertagnoli, S. (1998). SERP-2, an inhibitor of the interleukin-1 -converting enzyme, is critical in the pathobiology of myxoma virus. J. Virol. 72, 7830±7839. Moss, B. (1996). In ``Virology'' (ed B. N. Fields, D. M. Knipe, and P. M. Howley), Replication of poxviruses, pp. 2637±2671. Lippincott Raven Press, New York. Nash, P., Lucas, A., and McFadden, G. (1997). In ``Chemistry and Biology of Serpins'' (ed F. C. Church, D. D. Cunningham, D. Ginsburg, M. Hoffman, S. R. Stone, and D. M. Tollefsen), SERP-1, a poxvirus-encoded serpin, is expressed as a secreted glycoprotein that inhibits the inflammatory response to myxoma virus infection, pp. 195±205. Oxford University Press, New York. Nash, P., Whitty, A., Handwerker, J., Macen, J., and McFadden, G. (1998). Inhibitory specificity of the anti-inflammatory myxoma virus serpin, Serp-1. J. Biol. Chem. 273, 20982±20991. Turner, P. C., Musy, P. W., and Moyer, R. W. (1995). In ``Viroceptors, Virokines and Related Immune Modulators Encoded By DNA Viruses'' (ed G. McFadden), Poxvirus serpins, pp. 67±88. R.G. Landes & Co, Austin, TX. Upton, C., Macen, J. L., Wishart, D. S., and McFadden, G. (1990). Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence. Virology 179, 618±631.