SERP-1, a Secreted Poxviral Serpin

4 Pages·2000·0.105 MB·English

by  McFadden G., Moyer R.

SERP-1, a Secreted
Poxviral Serpin
Grant McFadden1,* and Richard Moyer2
1The John P. Robarts Research Institute and Department of Microbiology and Immunology,
The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada
2Department of Molecular Genetics and Microbiology, University of Florida College of Medicine,
PO Box 100266, Gainesville, FL 32610-0266, USA
*corresponding author tel: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2000.03017.
SUMMARY While SERP-1 is secreted, the orthopoxvirus serpin
SPI-3hasasimilar,ifnotidentical,spectrumofinhib-
ited proteinases (R. Moyer and P. Turner, unpub-
More than half a dozen poxvirus-encoded members
lisheddata).SERP-1wasfirstdiscoveredin1990,asa
of the serpin superfamily of serine proteinase inhib-
single copy gene in a related poxvirus designated
itors have been discovered, but only one of these,
malignant rabbit fibroma virus, and as a repeated
SERP-1 of myxoma virus, functions as an extracell-
copy gene in the terminal inverted repeat sequences
ular immunomodulator. SERP-1 is expressed as a
of myxoma virus (Upton et al., 1990). Expression
secreted glycoprotein that protects myxoma-infected
studies indicated that the processed SERP-1 protein
cells from clearance by macrophages and other
is secreted as a 55–60kDa glycoprotein that forms
inflammatory cells. Biochemically, SERP-1 protein
inhibitory complexes with a variety of cellular serine
binds and inhibits a spectrum of serine proteinases,
proteinases (Lomas et al., 1993; Nash et al., 1998).
such as plasmin, urokinase and tissue plasminogen
activator, but its precise mode of action in virus-
infectedlesionsremainstobeelucidated.Thepurified
Alternative names
SERP-1 protein alone functions as a potent antiin-
flammatory reagent in various animal models of
SPI-4 (proposed by Turner et al., 1995).
inflammation.
Structure
BACKGROUND
Discovery SERP-1 contains the consensus domain structural
features of eukaryotic serpins, including the reactive
center loop bearing an unusual P1–P10 pairing of
Atleasteightpoxvirusgeneshavebeendescribedthat
Arg–Asn (see Figure 2).
are members of the serpin superfamily of serine pro-
teinase inhibitors (Turner et al., 1995; Messud-Petit
et al., 1998). The only one of these that expresses a Main activities and
protein secreted from infected cells is SERP-1, which
pathophysiological roles
isencodedbytheleporipoxvirusmyxomavirus(Nash
et al., 1997). Thus, SERP-1 is the only viral serpin
that qualifies as a virokine, and is likely targeted to Like all serpins, SERP-1 forms 1:1 inhibitory com-
inhibit extracellular (or cell surface) proteinases. plexeswith serineproteinases.In thecaseofSERP-1,
1430 Grant McFadden and Richard Moyer
the list of inhibitable proteinases includes tissue postinfection and remain at high levels throughout
plasminogenactivator,urokinase,andplasmin(Nash the viral replication cycle. Like all poxviruses, the
et al., 1997, 1998). These inhibitory studies were con- transcriptional regulation is under virus control in
ducted in vitro with human proteinases, indicating cells that support the full replication cycle. Thus,
that SERP-1 target proteinases are highly conserved SERP-1 expression is relatively independent of the
inevolution.AreactivecenterloopmutantofSERP- type of infected cell.
1 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,
PROTEIN
indicating that the viral protein functions as a true
secreted serpin and is classified as an enzymatically
Accession numbers
active virokine.
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 SERP-1isaclassiceukaryoticserpin,andisexpressed
genomeofmalignantrabbitfibromavirus(whichcan as a 55–60kDa secreted glycoprotein. Although the
protein has not been crystallized, alignment studies
beregardedasamyxomavirussubstrain),andastwo
indicate an unusually extended C-terminus, and the
copies in the terminal inverted repeat sequences of
absence of a predicted D-helix, when compared with
myxoma virus, strain Lausanne (Upton et al., 1990).
other nonviral serpins (Lomas et al., 1993). Despite
thepotentialabsenceoftheD-helix,modelingstudies
Regulatory sites and corresponding predictthattheorderingandorientationoftheclassic
serpin protein domains remain similar to other
transcription factors
serpins, such as ovalbumin and antichymotrypsin
(Lomas et al., 1993).
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
Important homologies
synthesized (Macen et al., 1993). The SERP-1 pro-
moter sequence includes the consensus motifs of all
late poxvirus genes (Moss, 1996). SERP-1 mRNA SERP-1 possesses sequence motifs common to all
species are first detected at approximately 6 hours serpins,buttheclosestcellularhomologsincludeanti-
Figure 1 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 membrane-associated enzymes. This subject is still
protein structure indicating the positions of the under active investigation.
predicted (cid:11) helices that form the serpin structural
fold.Helixa(hA)isthesignalsequenceresponsiblefor
SERP-1 secretion and is not present in the mature
protein. Helix D (hD), normally present in serpins, is
IN VITRO ACTIVITIES
missing in SERP-1, although this does not appear to
beessentialforinhibitoryfunction.Thereactivecenter
loop (RCL) interacts with the active site of the In vitro findings
proteinase and contains the critical P1–P10 residues.
The stable inhibited serpin–enzyme complex is be-
A variety of catalytic parameters, including apparent
lieved to be an acyl-enzyme linked covalently at the
P1 residue. The RCL sequence of SERP-1 and the inhibition constraints, apparent second-order rate
engineered noninhibitory mutant (SAA) are also constant for inhibition, and the measured rate con-
shown. stant 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
trypsin,antichymotrypsin,andplasminogenactivator
inhibitor1.Thespecificityofserpinsisinpartdefined Normal physiological roles
bytheaminoacidresiduesattheP1–P10 site,whichin
thecaseofSERP-1isauniquepairing(Arg–Asn)not
The role of SERP-1 in vivo has been investigated by
found in any other known serpin (Figure 2).
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
Posttranslational modifications
parental myxoma virus, which is uniformly fatal to
the European rabbit (Oryctolagus cuniculus), the
Polyclonal antisera against SERP-1 expressed from SERP-1-minus virus was severely attenuated in vivo
myxoma virus reveal a heterogeneous 55–60kDa despite the fact that it replicated normally in tissue
specieswhichcanbedigestedwithN-glycosidaseFto culture (Macen et al., 1993). Histological analysis of
the predicted size of the amino acid sequence, indi- infected lesions indicated that in the absence of
cating that the major posttranslational modification SERP-1expressionamoreeffectiveinflammatorycell
is N-linked glycosylation. N-Linked sugar residues response resulted in a more rapid clearance of the
are not believed to affect the serpin function of infection (Macen et al., 1993). Thus, the biological
SERP-1 in terms of reaction parameters in vitro, but role of SERP-1 can be operationally defined as anti-
could affect protein half-life or tissue localization inflammatory, even though the target serine protei-
in vivo. nase(s) remains speculative.
RECEPTOR UTILIZATION Species differences
The ‘true’ biological serine proteinases that bind and All in vitro inhibition assays of SERP-1 protein have
mediate the anti-inflammatory properties of SERP-1 been conducted with human serine proteinases, sug-
in infected tissues are still speculative, but candi- gesting a broad species range for its inhibitory func-
date proteinases include a variety of soluble or tions. However, a precise evaluation of the inhibition
1432 Grant McFadden and Richard Moyer
constantswithasingleclassofserineproteinasefrom Lucas,A.,Liu,L.-Y.,Macen,J.,Nash,P.,Dai,E.,Stewart,M.,
multiple vertebrates has not been conducted. 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.
Pharmacological effects
Circulation94,2890–2900.
Macen,J. L.,Upton,C., Nation, N., andMcFadden,G.(1993).
SERP-1 protein expressed from myxoma, or vaccinia SERP-1, a serine proteinase inhibitor encoded by myxoma
virus, is a secreted glycoprotein that interferes with inflamma-
virus vectors that overexpress SERP-1, or CHO-
tion.Virology195,348–363.
transfected cells, has been purified and tested as an
Maksymowych, W. P., Nation, N., Nash, P. D., Macen, J.,
anti-inflammatory reagent in two models of inflam- Lucas, A., McFadden, G., and Russell, A. S. (1996).
mation: balloon-induced atherosclerosis and antigen- Amelioration of antigen-induced arthritis in rabbits treated
induced arthritis (Lucas et al., 1996; Maksymowych with a secreted viral serine proteinase inhibitor. J. Rheumatol.
23,878–882.
et al., 1996). In the coronary restenosis model, single
Messud-Petit, F., Gelfi, J., Delverdier, M., Amardeilh, M.-F.,
infusions of purified SERP-1 protein in the picogram
Py,R.,Sutter,G.,andBertagnoli,S.(1998).SERP-2,aninhi-
dose range at the time of balloon injury were able to
bitorofthe interleukin-1(cid:12)-converting enzyme, is critical inthe
block the influx of macrophages into the injured vas- pathobiologyofmyxomavirus.J.Virol.72,7830–7839.
culature and thus reduce the resultant development Moss,B.(1996).In‘‘Virology’’(edB.N.Fields,D.M.Knipe,and
of atherosclerotic plaque (Lucas et al., 1996). Similar P. M. Howley), Replication of poxviruses, pp. 2637–2671.
LippincottRavenPress,NewYork.
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Nash,P.,Lucas,A.,andMcFadden,G.(1997).In‘‘Chemistryand
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