1999

Asiatic Herpetological Research

Vol.8, pp. 107-110

Immuno-chemical Study of TSV-PA, a Specific Plasminogen Activator from theVenom of Trimeresurus stejnegeri
Yun Zhang Wen-hui Lee, Yu-liang Xiong, and Wan-yu Wang
1.

Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China
Abstract.- Rabbit antibodies were prepared against purified TSV-PA, a specific plasminogen activator from thevenom of Trimeresurus stejnegeri. They strongly cross-reacted with Crotalinae snake venoms like Trimeresurus
Trimeresurus

mucrosquamatus, Agkistrodon halys and Agkistrodon acutus. In contrast,
immunological cross-reactions with Elapidae snake venoms, Ophiophagus Hannah, Naja naja atra wererelatively lower and no cross-reactions with Bungarus fasciatus, Bungarus multicinctus venoms. On the otherstejnegeri,

hand, enzymatic assays only revealed the existence of plasminogen activation activity in the venom of
Trimeresurus stejnegeri. Except trypsin, anti-TSV-PA sera and antibodies did not cross-react with other serine
proteases, such as physiological urokinase-type plasminogen activator (u-PA)
activator (t-PA).

and tissue-type plasminogenAnti-TSV-PA antibodies inhibited both the amidolytic activity and plasminogen activation

activity of TSV-PA, but they did not inhibit the plasminogen activation activity of u-PA and t-PA.

Key words.- Snake venoms, antibodies, serine proteases'To whom correspondence should be addressed: Dr. Yun Zhang, Kunming Institute of Zoology, Chinese Acad-emy of Sciences, Kunming 650223, Yunnan, China. Tel: (86-871 5194279; Fax: (86-871 5191823.
))

Introductions.c.

TSV-PA, a specific plasminogen activator from thevenom of Trimeresurus stejnegeri, is a serine proteasewhich specifically cuts the Arg560- Val56 bond of1

plasminogen and forms plasmin (Zhang et al., 1995).

Sequence analysis indicates that TSV-PA is a typicalsnake venom serine protease which show great

homology with other snake venom serine proteases,
such as thrombin-like enzymes (Itoh et al., 1987;
Shieh et al., 1988), protein C activator (McMullen et1989), and factor V activator (Tokunaga et al.,1988). Serine proteases are widely distributed inal.,

at2weekintervals.

Boosts, which were per-formed when the serum titers were decreasing, wereachieved by injecting s.c. the same components, in thepresence of incomplete Freund adjuvant. The immunization protocol was performed by the dose ofadministered antigen 100 (ig per rabbit.
Microtitration plates (96 wells) were coated inphosphate-buffered saline (PBS) by over night incubation of antigen (5 (ig/ml) and saturation was carriedout with 3% BSA in PBS. Plates were washed with

PBS containing 0.1% Tween-20. The solutions to be
PBS containing 3%tested (100 |il/well), diluted insnake venoms especially in Crotalinae snake venoms

BSA, were incubated(Stocker, 1990). TSV-PA is the first specific plasmino-Peroxidase-labelled goat antibodies anti-rabbit immu-

gen activator found in snake venoms. In this investigation, we reported the immuno-chemical study of TSV-

noglobulins (Biosys, Compiegne) were addedPA.

washed. Substrate for peroxidase (O-phenylenedi-

Material and Methods

pH 7.3, 0.01% H 2 G*2) were added and the absorbance
was recorded at 405 nm with a Dynatech microplate

1:2000 dilutions,1hour at 37 °C, then washed.

incubatedamine dihydrochloride in 10

Snake Venoms were from the stock of Kunming Insti-1houratata37 °C andmM sodium phosphate,tute of Zoology, Chinese Academy of Sciences. TSV-reader.

PA from Trimeresurus stejnegeri venom is purified as

Antibodies (IgG) were purified from the sera by
double ammonium sulfate precipitations (35% final

described previously (Zhang et al., 1995). TSV-PA
was denatured by adding (3-mecaptoethanol to 1%

concentration), and then dialyzed against PBS. Inhibi-concentration and boiled at 100 °C for 5 min.

tion of enzymatic activity was carried out by preincu-

Native TSV-PA and denatured TSV-PA were emulsi-

bating for 30 min at 37 °C a fixed concentration of theenzyme (5 Ug/ml) with variable amounts of antibodies

final

fied with

50% Freund adjuvant and were administered

Vol. 8, p. 108

Asiatic Herpetological Research

Table 1The titers of1999

the antibodies against TSV-PA (aspecific plasminogen activator from the venom of Trim.

eresurus stejnegen)>oCDAntigencE<i>

CEIgG concentration(mg/ml)

Figure 1. Inhibition of plasminogen activation by IgGagainst native and denatured TSV-PA. Fixed concentrations of TSV-PA, human two chains lower-molecularweight urokinase (u-PA) or human two chains tissue
type plasminogen activator (t-PA) were incubated withvarious concentrations of antibodies against TSV-PA
for 30 min in 37 °C. Then theremaining plasminogenactivation activity was determined and expressed as

the percentage of the original values. (O) TSV-PA test,(•) TSV-PA control, (A) TSV-PA test with anti-denatured TSV-PA antibodies,
u-PA test, (<3>) u-PA con-()trol,() t-PA test, () t-PA control.

and by testing the residual enzymatic activity. Plasmi-

nogen activation activity and amidolytic activity ofthe enzymes were assayed as describedal.

by Zhang et(1995).

Results

We immunized rabbits with purified TSV-PA, both inits

native and denatured forms. Table

1

shows that theantiserum directed against native TSV-PA has a muchhigher ELISA titer than that directed against denatured TSV-PA, showing that native TSV-PA is much

more antigenic than denatured TSV-PA. On the otherhand, antiserum directed against native TSV-PA has amuch lower ELISA titer when coating the plate withdenatured TSV-PA, indicating that large parts of theantibodies raised against nativeTSV-PA are against

comformational epitopes of the protein.
In the first series of experiments, we examined theimmunological cross-reactions by ELISA. The anti-TSV-PA antibodies (from here, if not specially noted,anti-TSV-PA serum and antibodies means those raised
against nativeTSV-PA) cross-reacted with TSV-PAand Trimeresurus stejnegen venom. In the mean time,they cross-reacted with several other Crotalinae snakevenoms in which we did not find the activity of plasminogen activation. From the ELISA titers, we cansee, first, the immunological cross-reaction with ven-oms from Crotalinae snakes are much higher thanwith those from Elapidae snake venoms. Second,Asiatic Herpetological Research

1999

Vol. 8, p.

40

20

1

TSV-PAbatroxobin
trypsin I
t-PA
u-PA

VFGGDECNINEHRSLWLFN
VIGGDECDINEHPFLAFMYY

TSV-PAbatroxobin
trypsin I
t-PA
u-PADSNNFQLLFGVHSKKILNEDEQTRDPKEKFFCPNRKKDDEV DKDIMLIKLDSNRRFMRIHLGKHAGSVANYDEWRYPKEKFICPNKKKNVIT DKDIMLIRLDRYKSRIQVRLGEHNINVLEGDEQF-INAAKIIKHPNYSSWTL--NNDIMLIKLSSP-HHLTVILG-RTYRWPGEEEQKFEVEKYIVHKEFDDDT--YDNDIALLQLKSKKEDYIVYLG-RSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSSNG--FLCGGTLINQDWWTAAHCSPR YFCGMTLINQEWVLTAAHCNSGYHFCGGSLINDQWWSAAHCIVGGYTCPEHSVPYQVSLIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPIIGGEFTTIENQPWFAAIYRRHR-GGSVTYVCGGSLISPCWVISATHCFID-YP—8060——140120100

TSV-PAbatroxobin
trypsin It-PA
u-PA

1()9SVSNSEH
IAPLSLPSSPPSVGSVCRIMGWGKTIPTKEIYPDVPHCANPVKNSEH
IAPLSLPSNPPSVGSVCRIMGWGAITTSEDTYPDVPHCANPVKLNAR
VAPVALPSAC
APAGTQCLISGWGNTLSNGVNNPDLLQCVDDSSRCAQESSWRTVCLPPADLQLPDW-TECELSGYGKHEALSPFYSERLKEAHKEGRCAQPSRTIQTICLPSM-YNDPQFGTSCEITGFGKENSTDYLYPEQLKMTV
180160

TSV-PAbatroxobin
trypsin It-PA
u-PA

INILDHAVCRTA-YSWRQVANTTLCAGILQGGINLFNNTVCREA-Y--NGLPAKTLCAGVLQGGKDSCQGDSGGPWCNGAPVLSQADCEAA-YP-GEITSSMICVGFLEGGVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDSCQGDSGGPLVCSLVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTTSV-PAbatroxobintrypsin It-PAu-PA

FQGIVSWGGHPCGQPGEPGVYTKVFDYLDWIKSIIAGNKDATCPPFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKTATCPLQGIVSWG-YGCALPDNPGVYTKVCNFVGWIQDTIAANDGRMTLVGIISWG-LGCGQKDVPGVYTKVTNYLDWIRDNMRPQGRMTLTGIVSWG-RGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL

RDTCHFDSGGPLICNG
IDTCGGDSGGPLICNG

234

220200I

Q
Q

Figure 2. Amino acid sequence comparison of TSV-PA with batroxobin, rat trypsin, human t-PA and u-PA.
The sequence comparison was performed with a Clustal V software package in a computer. Sequences were fromthe following sources: TSV-PA, Zhang et al. 1995); batroxobin, Itoh et al. 1987); rat trypsin, MacDonald et al.
1982); human t-PA and u-PA protease domains, Pennica et al. (1983) and Steffens et al. (1982).
(((In second series of experiments,

we tested thefigure

1and the inhibition of amidolytic activity ofinhibitory effect of antibodies directed against TSV-TSV-PA, we could find that anti-native-TSV-PA anti-

PA on the enzymatic activities of TSV-PA, u-PA, t-PA
and other proteases. Figure 1 shows the plasminogenbodies inhibited both amidolytic and plasminogenactivation activities of TSV-PA, on the other hand,activation inhibition by antibodies against both native

anti-denatured-TSV-PA

and denatured TSV-PA. In agreement with the results

amidolytic activity of TSV-PA, but inhibited plasmi-

above, anti-TSV-PA antibodies did not inhibit thenogen activation by TSV-PA. These results indicate

plasminogen activation activity of u-PA and t-PA. On
the contrast, both anti-native-TSV-PA and anti-dena-that the inhibition of plasminogen activation by antidenatured-TSV-PA antibodies is not caused by thetured-TSV-PA antibodies inhibited plasminogen acti-

block of catalytic centre but by the block of the sub-TSV-PA. Further experiments expressedstrate binding site. Probably, this is caused by a pieceof peptide in the enzyme which involves in the bind-vation bythat only anti-native-TSV-PA antibodies inhibited theamidolytic activity of TSV-PA, but anti-denatured-

TSV-PA antibodies did not. Combining the results ofantibodies

didnotinhibit

ing of the substrate, especially for large protein substrate binding, and which appears as a sequential

Asiatic Herpetological ResearchVol. 8, p. 110

1999

epitope. Both anti-native and anti-denatured TSV-PA
antibodies did not inhibit the thrombin-like activity ofItoh, N.,

Trimeresunts stejnegeri and Agkistrodon acutus ven-gene for batroxobin, a thrombin-like snake venom
enzyme. Journal of Biological Chemistry 263: 7628-oms.

N. Tanaka, I. Funakoshi, T. Kawasaki, S.
Mihashi and I. Yamashina. 1988. Organization of theFigure 2 shoes the sequence comparison among
TSV-PA, batroxobin, rat trypsin, human t-PA and uPA. TSV-PA shares 63% sequence identity with7632.

batroxobin (a thrombin-like enzyme), 42% with rat
trypsin, but only 23% with human u-PA and 21% withenzyme from the venom of Crotalus viridis viridis

human t-PA. The sequence comparison results are in

967:92-102.

agreement with the immuno-chemical studies above.

MacDonald, R. J., S. J. Stary and G. H. Swift. 1982.
The primary sequence of rat trypsin I, as deduced

Discussion(Prairie rattlesnake).

Biochimica et Biophysica Acta

from its cDNA. Journal of Biological Chemistry 257:

Snake venoms contain numerous different proteaseson blood cascade (Stocker, 1990). Biochemically, two main classes of these protease arerecognized: serine proteases and mealloproteinases.
which

Komori, Y, T. Nikai and H. Sugihara. 1988. Biochemical and physiological studies on a kallikrein-likeact

For one group of venom serine proteases, they share a9724-9732.

McMullen, B. A., K. Fujikawa and W. Kisiel. 1989.
Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom. Biochemistry28: 674-679.

trypsin homologous catalytic domain and the molecu-Pennica, D., W. E. Holmes, W. J. Kohr, R. N. Harkins,

kDa-35 kDa

G. A. Vehar, C. A. Ward, W. F. Bennett, E. Yelverton,depending on the carbohydrate content of the enzyme.
Even the sequence similarity among them are around

Goeddel and D.
P. H. Seeburg, H. L. Heyneker, D.
Collen. 1983. Cloning and expression of human tis-65-70%, for example thrombin-like enzymes (Itoh etsue-type plasminogen
Nature 301: 214-221.

larweights

areusuallyaround25

protein C activator(McMullen et al., 1989), factor V activator (Tokunagaal..

1987; Shieh etet al.,al.,

1988),

1988), kallikrein-likeenzyme (Komori et al.,1988) and newly determined plasminogen activator(Zhang et al., 1995), this group of venom serine proteaseisspecificity.

their highly

homology primary sequence verse theirhighly divergent substrate specificity (for protein substrate) and the determination of their substrate binding

extremely significant in our understanding ofprotein structure-function relationship and further thesite isprotein reconstruction for medical purpose in future.

activator

cDNA in E.

coli.

Shieh, T. G., S. I. Kawabata, H. Kihara. M. Ohno andS.

Iwanaga. 1988. Amino acid sequence of a coagu-lant enzyme, flavoxobin, from Trimeresurus flavoviri-dius venom. Journal of Biochemistry 103: 596-605.

characterized by their highly divergent sub-Unlike their trypsin homologous,
they are usually highly specific. The explanation ofstrate

VSteffens,

G. J., W. A. Gunler, F. Otting, E. Frankus

1982. The complete amino acidsequence of low molecular mass urokinase fromandL.

Flohe.

human urine. Hoppe-Seyler Zeitschrift fur Physiologische Chemie 363: 1043-1058.
Stocker, K.

1990. Snakevenom

proteins affecting

hemostasis and fibrinolysis. Pp97-130. In K. Stocker(ed),Medical Use of Snake Venom Proteins.

CRCPress, Inc., New York.

Acknowledgments
The study was supported in part by the national 863
plan, the Director fund of Chinese Academy of Sci-ences, and in part by the fund of Yunnan Science andTechnology Commission C0085M.

N. Tanaka, S. Mihashi andZhang, Y, A. Wisner, Y L. Xiong and C. Bon. 1995. A
novel plasminogen activator from Trimeresurus stejI.

Yamashina.

1987. Molecular cloning and sequence analysis ofcDNA for batroxobin, a thrombin-like snake venomenzyme. Journal of Biological Chemistry 262: 31323135.

enzyme (RVV-V) from Russell's viper venom. Identification of isoproteins RVV-Va, -Vb and Vr and theircomplete amino acid sequence. Journal of BiologicalChemistry 263: 17471-17481.

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