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The amino acid sequence and properties of an edema-inducing Lys-49 phospholipase A2 homolog from the venom of Trimeresurus mucrosquamatus

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' 1991 Else~.ier Science Puhlisher~. B.V. 0167-4838/91/$03.50

A D O N I S 016748389100166D

P ! { A P R ( ) 33x'~'~

The amino acid sequence and properties of an edema-inducing

Lys-49 phospholipase A~ homolog from the venom

of

Trimeresurus mucrosquamatus

C h e n - S h e n g Liu 1 J i n - M e i C h e n t, C h e n - H s i e n C h a n g i S h u n - W e n C h e n I C h e - M i n g T e n g 2 a n d l n n - H o T s a i i

/ ht~tttulc rS( Bt,/~gtla] ( ']tcnll~lfv, ,,lcad('~tia .~itrt~a. Itt.~tittll~" of ~loJlt't~ti~ ~11 ,~llz'~lc~..~r~lll¢)n(l[ mLll|t'tlPl ~ "Htl*l'r~ll~. "]~llpJ21 (]-~ti~aPt. ( hl~'~t/ iztld ) Ph~trnltl~o/ogtltd In~ttlutt'. ('o/leg(" of Ah,dt(tttc. N,ltnmal Tatwott lt~ll'cr~itl. Torpor. I ]~ltwa~t. ( fitntO

I Ret:ei'..ed 31 October 199~11

KeN *,~ord~,: Anlill~ acid .',equeflce: Pho~,pholipa~,.: A 2: Edema induction: Snake '..¢11om protein

Three phospholipase A z enzymes or homologs were purified from the venom of Trimeresurus mucrosquamatus (Taiv~an

habu). The most abundant one was found to be a phospholipase homolog without enzyme activity, and its complete amino acid sequence was determined using oligopeptide fragments derived from digestion by endopeptida.~es Glu-C, Asp-N, L~s-C and a-chymotrypsin, and by means o| gas-pha~ sequencing. The .,~quence revealed that the protein belonged to the Lys-49 family of snake venom phospholipase A z. This protein's function was characterized as edema-in4ueing. The Lys-49 protein has the potential to bind membrane phosphollpid and Ca z + (K d ~- 1 . 6 . 1 0 - 4 M) as shown by ultraviolet difference spectra; however, the catalytic site appeared to be inactive and the edematous respon~ was independent of the protein's hydrolytic activity.. Mast cells and platelets were shown to be subject Io activation by the Lys-49 protein.

Introduction

Snake venom is a rich source of both the structural and functional varieties of phospholipase A2 (PLA:. EC 3.1.1.41. For example, in the venom of king brown snake (Pseudechis auslrala'), there are more than thir- teen PLA2"s all differing in structures and enzyme capacities [1.2]. Besides a catalyst for the hydrolysis of phospholipids, PLA2 from snake venoms functions in other ways, such as playing the roles of neurotoxin [3], myotoxin [41, carditoxin [5], anticoagulant [61 and an edema-inducing principle [7.9]. Based on their primary structures, PL,~ were classified into two groups [8]. Group I comprises those from mammalian pancreatic juices and the venoms of snake families Elapidae and Itydrophidae while group II includes those froln the venoms of Crotalidae and Viperidae.

Tong et al. have isolated the most basic and abun- dant phospholipase A: homolog, termed FXXII-2 [9] or TMVPLA, 11 112,13], from the venom of Trimeresurus

Corre,~pt~ndence: I-H. Tsai, [nstitule of Biological Chemistry. Academia ~inica. p.o. Box 23-106, Taipei, Taiwan. China.

mucrosquamatus and shown it to be an edema-produc- ing principle [9-13]. In our work here, we have further purified this component by means of HPLC, and elucidated its primary structure, it turns out to be a member of Lys,-4q PLA, which was originally dis- covered in the venom of Agkistrodon p. pa'cit'orus by Maraganore and Heinrikson [14] and more recently in the venom of Trimeresuru~ flal'oviridis by Liu and Ohno el al. 115]. The abundant existence of the Lys-49 phos- pholipase A~ homologs in some Crotalidae and Viperidae venoms has been puzzling because their en- zyme activities are not easily detectable [20] and their biofunctions ambigt.ous 122]. This study may help to exphlin certain aspects of their structure and function.

Materials and Methtxis

Materials

Crude venom of T. mucrosqumnat,~ was supplied by Dr. M I. Liao of National Institute of Preventive medicine. Taipei, Taiwan. CM-Sephadex C-50 and Sep- hadex G-75 were obtained from Sigma. Chemcosorb ODS-H (Cis, 5 #) was obtained from Chemcosorb Scientific. Osaka. The enzymes used in the fragmenta-

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4 - t

___#4~

L

. . . 6 ' 1'~ ' F llll- 1

1

F Will 2'4 Time (re,n)

Fig, I. Purfflcalion of J M V - K 4 9 h~. rc',er,.c-pha:,,e IIPI.U. Ab,t~ut 1 or 2 mg o f FXXII-2 obltllned from ion-exchanger and gel-filtration chromatographi,'r, v.erc injo:ted on an HPI.C s~,stem with a ( ' h e i l f cosorb O D S - i l (CI,,) column i t 0 ~< 250 ram). Fhc clution ~a:, of footed with a linear gradient o f 27-40~ mobile pha~c ( o n 7 ~ I ' F A m acetonilri[e) o',er l h. Flow rate v.a~, 2 m l / m i n and the Jb~,orhance ',tale ;it 28(I nm wa,. 0 5. ~rro'*,, indicate the condition o f PI A : eIulcd

if FXXII-1 or W i l l I,~1 v, erc apphcd.

tion experinaents were lysyl endopeptidase (Wake, Japan), endoproteinar.e Glu-C (B~ehringer), endopro- teinase Asp-N (Boehringer) and a-chymotryosin (W~rthington). Other reagents, were of the highest qual- ity commercially available.

Isolation and purification

of

the protein

Fractions XVIIi, XXII-1 and XXII-2 were obtained from CM-Sephadex C-50 and G-75 chromatography of

T. mucrawuamatu~"

~,enom, as previously described 191.

The lyophilized sample was dissolved in 0.07~ trifluoro- acetic acid (TFA) and further purified by reverm-phase HPLC (Fig. I). The first emerging,, major component, denoted as TMV-K49, was used in the present :,tudy. The purity was assessed by SDS-PAGE and N-terminal sequencing.

En:o'me digestions and separatum of the pep/tales

After reduction and alkylation by iodoacetamide, the RCM protein (TMV-K49) was subjected to enzyme digestions as follows: (1) lysyl endopeptidase in 0.05 M Tris-HCI (pH 9.0) and 1 M guanidine HCI at 37°C for 3 h with substrate/enzyme ratio of 50/1 (w/w); (2) epdoproteinase Asp-N in 0.05 M sodium phosphate buffer (pH 7.5) and 1 M guanidine HCI at 37°C for overnight with substrate/enzyme ratio of 250/1 (w/w); (3) endoproteinase Glu-C in 0.05 M ammonium bi- carbonate (pH 8.0) and 1 M guanidine HCI at 37°C for 3 h with substrate/enzyme ratio of 40/1 (w/w): and

(4)

a-chymotrypsin in 0,05 M ammonium bicarbonate (pH adjusted to 6.5 by dilute acetic acid) at 25°C for I h with substrate/enzyme ratio of 110/1 (w/w).

[)igeslion was halted by addition of acetic acid and the aliquot ~as injected into a HPLC column. Sep- aration of the peptides was effccted by a linear gradient consisting of 0.07~7 TFA in water (solvent A) and O.07f7 TFA in acetonitri!e (solvent B). Amino acid composi- tions were determined bv HPLC separation of DABSYL-amino acids, after pre-column dabsylation of the amino acids generated by a 30-min gas-phase acid hydrolysis 116.17 I.

S('(~lll'#l{ U dUll'rFtlillOll#Jt

After IIPLC purificaiion, TMV-K49 and its oligo- peptide fragments were subjected to automatic amino acid sequencing. Edman degradation was carried out in a pulsed-liquid type sequenator (ml:del 477A, Appli~xl

Biosy~;ten'ts) accompanied with ;in on-line PTti anlino

acid analyzer. The progranl "normal-I' was used. .,l.,.~-;

q[pho.v~hohpuse

a(mv(,

l'hc activity wa.,, inea.,,ured using the pH-stat titration ineth~l [1.15]. Synthetic dipalmitoyl I-phosphatidyl- choline (Sigma) was emulsified at a concentration of 2.5 mM in a substrate solution containing 2.5 mM sotlium deoxycholate, 0.05 mM Na 2-EI)TA, and I(X} mM NaCI. After introducing 6 10 mM Ca('l> the PLA, sample (~< 50/tl) was added to 2.5 ml of the substratc and tht reaction proceeded at 37 °U, ;he antounts of fatty acid liberated being automaticalb, r tilrated at pH 7.5 with 4

mM N a O l l solution under purging N 2 gas on a pH-Ma[

apparatus (RTS 822, Radiometer, l,)cnmark). The rate of hydrolysis wa~ calculated from tile alkali consump- lion during the first 5 rain and was expressed as reel of fatty acid liberated/thin per mg protein.

UIIfavio/el diffi'rcnce specln)s('opy

Difference spectra were recorded on at double-bcant spectrophotometer (Hitachi I.I32(X0 using l-cm path- length cells. Protein concentr;~.tions in both cells were 50 p.M. Differertt amounts of ( a C I 2 (18 raM)sttv,.'k solu-

lion,,, wct~. added to the sanlpie cell and an equal viii. ,ff

buffer t~, the reference cell. Buffer solutions used ~n the titration.,, were 50 aim Tris-HCI and 0.I M NaCI (pH 8.0). Analyses of the spectral data on the interaction between phospholipase A, and ( a were carried out as previously described [18] for the Scatchard (~949) model of ligatid-protem interaction. For a single cla~s of binding .'.,ilcs the folk,wing equation holds,

( a .4/'..I .4,,,.,, )/[('a: * I = ' n ,,'~,i ) - ! 3.4/.l..t.,.,,/I( a )

where (JA/~A,u~,,) is the saturation degree, it the number of binding .sites, K,t the a~erage dissociation constant of the enzyme-Ca 2 ' complex,.s and [('a 2. ] the free calcium concentration. Thus a plot of

( A A /

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from which the value of n can he derived by extrapola- tion and the slope corresponds to - I/K,,.

Effect o n platelets and mast cells

Washed platelet suspension was oblaiiled from rab- bit blood ant(coagulated with EDTA (6 raM) by several centrifugations and the washing procedure as described previously [24 I. The final platelet pellets ~ere suspended in Tyrode's solution of the following composition (mM): NaCI (136.8), KCI (2.8). NaHCO~ (11.9), MgCI, (1.1), NaH:POa (0.33). CaCI: (I.0) and glucose (11.2). Plate- let aggregation was measured by the turbid(metric method of O'Brien [251 at 37°C using a Chrono-Log Lumiaggregometer. The platelet suspension was stirred at 1200 rpm just 1 rain b, efore the addition of venom protein. The percentage of aggregation was calculated assuming the absorbance of platelet suspension as 0q ~ aggregation and the ab~)rbance of Tyrode's solution as 1 ()O~'/aggregation.

The isolation of mast cells from a rat's peritoneal cavity, together" with the study of their release of hista- mine and ~8-glucuronidase, were as previously described 112.131.

Measurement of rat hind-paw c d c m . ~;t:# ¢,[]e('t on nert,e- must'le preparati, m

Wistar rats (180-220 g) were used. Hind-paw edema was induced by a single subplantar injection of 10/.tl of irritant in 0.05 M phosphate buffer saline (PBS, pH 7.4) and an equal voL of PBS into the right and left hind-paw, respectively. The vol. of both hind-paws of each rat were measured with a plethysmometer (Model 7150. tJgo Basil¢) at different times after the in.iection. Per-

cent hind-paw sv, elling was calcuated as following: hind-paw edema ("7)

right pa',, sol. left p;iv, ~.~,d. i mili~ll "~ol. imtlal ~,ol. / I(10

. ~;;gJlt piik-ifiiii;i[ ',tq. I c f l l:'l{i~;

f~iiT~Tft],l{,

I X

The data were also analyzed to compare the area under the time course curve. Neurotoxicity and myotoxicity were measured electrophysiologically as previously de.. ,~ribed [51.

Resulls

Determotation of amim, acid sequenc'e ~!f T M V.K49 In previous papers by Teng and the co-workers, three PLA,'s were isolated from the venom of T. mucro- squamat~ts and designated respectively as FX¥111, FXXII-I and FXXII-2 191 . FXVIll and FXXli-2 were named TMVPLA., I and II in the subsequent study o¢ their function [12,13]. By means of HPLC, we found that FXVlll (i.e., TMVPLA: II and FXXII-1 were rather pure (showing a single

peak),

but FXXII-2 were the combination of 75q~ TMV-K49 and 205 FXXII-1. As shown in Fig. !, HPLC effectively separated TMV- K49 (peak 4-1) from FXXII-I (peak 5t and another minor PLA.~ (peak 4 2). Homogeneity of the purified protein is confirmed by the results of automatic N- terminal amino acid sequencing and the single band with a molecular mass of 14 kDa in the SDS-PAGE pattern under non-reducing and reducing conditions ( not shown). Automatic a mi no acid sequencing of RCM protein established the N-terminal 47 amino acid ~ -

(a)

E-2 6

LE-8

(b)

LE-A LE-5 ~ m

2'o 3'o

s'0"

lb

2'o

3'0-4b

Time (rain) Time (rain)

Fig. 2. Preparative H P [ ( " peplide map,, tff t;ll endt+proteinase (+lu-C" dlge~,t and (hi IT~,yl endt+peplida+ dige~,l of TMV+K49. (a) Aliquot c~mluininll 0.1 mg digc~,t v, as applied In a Nu¢leosil (18 column (4.6 × 250 mini. ~'pl','ent A wa~, O,(t7q; TFA in waler, The gradient v.as Iinctar from IO-~Y~ ~)lvent B (0.07~ TFA in ac~tonimle) over ~0 rain and nZOnltored at 220 o111 with a tiny. rdI¢ of I ml/mm. Full absorbance was ,',,¢t at 0.0~. (b) Aliquot ¢ontainin 80.t ] mg dige,,t ~a~, chromatographed under tl~e same condition mentioned above except thai the elation wa~, carried out hy

(4)

. q .

(5)

(a)

r-

()

I0

2'0

3'0

&'0

5'0

6'0

7=0

Time (rain)

{b)

Ch-5

~b

~o

30

/.'0

5'0

60 7b

Time(min}

Fig 3. Peptide maps ~ff tal eod.~Froteinase Asp-N dig~.~t and [b} a-chymot~psin digest of TMV-K4q.

(a)

Aliquot containing 0.1 mg digest

~as

chromatographed a~ rnenti~med m the legend of Fig 2(a) excep! that the elution ga~ 5-60"; sokent B over 7(I rain and full ah~rbance ~as 0.1. (hi Aliquot comaining ILl 5 mg digest was chr~matographed under the same condition of (al e~cept that the elution v, as 5-45~/ ,okcnt B o',¢r 70 rain

and full ab,a3rhance was 0.2

quences. A lypical amino acid composition of RCM protein was obtained as follows: D l l . 9 (13) EIO.4 (9) CMC13.4 (14) $3.8 14) 1"4.5 (5) GIO.O 110) A5.6 (5) R5.2 (5) P5.8 (6) V7.9 (10) MI.8 (1) 13.2 (4) L7.0 (7) F3.0 (3) K13.1 (17) H2.1 (2) Y5.2 (6). Repeated experi- ments always showed the lower values for Lys and this can be attributed to incomplete hydrolysis of three Lys-Lys linkages in the molecule.

in Fig. 2a and b, the preparative HPLC of peptides from endoproteinase Glu-C and lysyl endopeptidase digests are displayed. Only those peptide peaks involved in the sequencing are numerated. Likewise. two other peptides maps arising from endoproteinase Asp-N and a-chymotrypsin digests are shown respectively in Fig. 3a and b. Amino acid compositions of the relevant peptides are shown in Table 1, as well as the location of each peptide. Since the peptide E-5 was the mixture of two peptides at about equal molarity, as evidenced from the PTH amino acids, the values of E-5-a and E-5-b were apportioned from the gross compositions of E-5. Likewise LE-8-a and LE-8-b received appropriate val- ues from that of LE-8. The two peptides. E-6 and E-7. were further purified by HPLC with less steep gradient to obtain the major peak, respectively, as E-6-3 and E-7-2 (not shown). Owing to the scarcity of material. peptides E-6-3 and E-7-2 were directly used for the sequencing. The former peptide produced the a!ignment of 31 amino acid residues beginning from Thr-13 to CMCys~3 whtle the latter, 15 amino acid residues from Thr-13 to Asn-27. Combining all these results of se- quencing the alignment of the whole sequence is shown in Fig. 4.

A primary structure comparison between the K49 homologs and some representative group !1 P L A , ' s is shown in Fig. 5. The common feature of Lys-49 PLAz's as opposed to the Asp-49 P L A , ' s is evident in the

replacement of Asp-49 by Lys-49 in the No. 1 to No. 4 PLA_,'s. Homology index calculation revealed their sim- ilarity: 79c/ for No. 2. 72e~ for No. 3. 5If/ for No. 5 and 55f/ for No. 6 as compared with No. 1.

Spectroscopit ~ titration of T M V-K49 with Ca: •

To study the calcium affinity of TMV-K49. we have monitored the ultraviolet spectral change of the protein Ul~m adding increasing amounts of Ca -'+ (Fig. 6). The absorbance increa~d at 264 nm while two troughs were observed at 290 nm and 240 nm (which were also observed in the Ca" ~ titration spectra of the K49 pro- lein from T. flarorl'iridis [15]). These two troughs are likely due to the respective perturbation of histidine and aromatic amino acid residues [18]. The dissociation constant ( K j I of Ca-" '-TMV-K49 complex was calcu- lated to be i . 6 - 1 0 4 M by a Scatchard plot analysis (Fig. 6 i n s t i l , where AA is the difference absorbance at 290 nm and AA,~, is that upon Ca -'~ maturation. Similar K,t values were obtained when the increases of absorbances at 264 nm corresponding to increasing [Ca-" 1 were analysed.

Hydrolytic actn'it.v of put._, T M V.K49

it was reported that FXXll-2 had moderate en- zymatic activity toward the mixed micelles of deoxy- cholate and dipalmitoyl phosphatidylcholine [9]. How- ever. the specific activity of FXXII-2 decreased when further purified by G-75 gel-filtration or by HPLC (Fig. 1 ). Its original hydrolytic activity appeared to be due to coeluted FXXII-1 [9] PLA, (corresponding to peak 5, Fig. ! ) plus another ve D' minor contaminating enzyme (peak 4-2. Fig. I ). The purified TMV-K49 after HPLC (peak 4-1, Fig. 1) had almost no enzymatic activity ( < 0.1)4 p m o l / m i n per rag) (Table 11). The absence of hydrolytic activity of TMV-K49 was not caused by

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1o 20 S-L-I-E-L-G-K-M-I-F-Q-E-T-G-F ;; r'',' ~ ~;~Y ( : - L - Y - L - - ROt1 pro~(,in - - E - I ~ g - S - b ' | E - 6 - 3 L E - 8 - a - - 30 40 5¢ C - N - C - G - V - G - N - R - G - F , - p - V - P - A - T - D R - C - C - ~ - V - H - ~ - C - C - - - R C M p r o t e i n - - E - 6 - 3 D N - 3 ~ C h - 4 - - 60 70 Y - K - K - V - T - G - C - D - P - I < - K- D - R - Y - S - Y - $ - W - E - ~ : - : ~ - A - I - V - C - Z - 2 ~ I L E - 2 - - 105 ~ D N - 3 ~ t Ch- 5 . . . ~ L E - ~ - b 8 0 96 G - E - K - N - P- P - C - L - I ( - Q - V - C - E - C - D - K - A - V - A - I - C - L - R - E - N - ~ E - 2 I E-3 I | L E - 4 - - - - L E - 1 0 ~ 11G ! 2 0 L - Q - T - Y - D - K - K - H - R ~ V - T - V - K - F - L - C - K - A - P - E - S - C E - 5 - ~ - - L E - 1 0 - - ~ L E - 6 - -- -- L E -- S ----

Fig. 4. Amino acid .cquence of TMV-K49 deduced from pept,de fragments. Ahbre~i,nmns are: E. GIu-C or Staph>kxa',ccal %'~, pro- teina,,e peptide,,: LE. b">| cndopeptida~ peptid~: DN cndoprc.-

teinase A~,p-N peptldes: Ch. chymol~pxm l",eptides.

inactivation during the H P L ( " since ~n the control ex- periment. ~hcrc partiall> purified f-XXII-2 v.~, in- cubated v.ith O.07g: TFA and 4(J~ CH~CNIv/,,) at 25°C for 6 h. the enzyme activity remained almost the same. Ti~erefore ,,vc ma> deduce that the enz~,me activ- it,, of pure TMV-K49 is bareh detec,able.

Phttrma~ ologt~ a l u( Itt'tttes o f T.~t I : - K 4 9

Results of previous analysis with the FXXII-2 (i.e., TMVPLA z II1 [10- 131 suggest thai this venom protein induces rat hind-paw edema in a dose-dependent manner, b e c a u ~ it stimulates serotonin and histamine release from PMN-leukocytes and mast cells. The edema-inducing activity of HPLC-purified TMV-K49 was confirmed in the pres,ent study Wig. 7). The purl- fled TMV-K49 (at Iov. dosage. 10/~g/ml) activated the platelet membrane by inducing shape change and caus- ing reversible aggregation, and it also activated mast cells to release histamine and #-glucuronidase (Table IlL Hov, cver. upon tenting. ,~e found that TMV-K49 had ve~' low m_,,otoxic and neurotoxic activit', at dosage as high as 100/~g/ml.

D i s c u s s i o n

Purification of the venom components from 7".

mtaro.~quamattt~ h) various chromatographies including

HPLC resolved at least three or four PLA. isoforms (Fig. 1). The pre~ent study has completed the amino acid sequence analysis of a nev. member of the Lys-49 family of PLA_. (i.e.. the K49 proteins) [14.21]. This and the pre,,iou:, reports [14.15] together suggest that this family of PLA:'.,, frequentl 3 occur as component.,, of the

Crotalidae snake venoms (especially t h o ~ of elgktstro-

don. Bothrop~ and Trtmere~'uru~) and that Nature has

preserved group !1 phonpholipase A. homolog,,, de~,oid

T A B L E I I

Enzlmatlt and hu~l,,y, nal properne~ r~f I~ho~ph~dipa~e~. 1: l~,laled (r,,m Trt.n re~uru~ mu< r~,~quam~.'u~ ~ on,,.:

Purified HPLC + PLA : acti', m. Edema + Platelet • +~ ret¢,~,,e ,++ m.~,t tell 'j

Protein elution. Fmol,+ rain per m@ ('~) a.~rcgatton 4~ I tb~taminc l~+~]ucuronldd~: B ,~'4'. en t

T M V - K 4 q 22 < 0 . 0 4 ~ 4 0 ~" 31.5 ~ , ~ ~ 2 i t

FXXII-I 32 123 _'14 '73.2 2" 5 t4

TMVPLA 2 I 37 160 30 PO.O

Conditions used as in Fig. I T M V - K 4 9 and F X X I b I V.er¢ obtained from HPLC of F X X I I -'~ and T M V P L ~ , I fw.m F X V I l l (qj.

b Percent hind-pa~ s~dling at 6 h after subplantar in}ccuon of ~em',m pro|era (5 ~g, pa~

• Percent a~rcgalion of washed rabbit piatclcts at 6 mm after the addmon of the ~en~nn protein I I0 pg m h a A t 15 rain after lhe addilion of the venom protein (10 pg/ml), mean values of three expcnrncnl~ arc ~ho~n

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( K 49 ) I T M V 2 T F V 3 A P P 4 B A V ( D 49 ) 5 T F V 6 A P P * w w w * * w 10 2 0 3 0 4 0 50 6 0 S L I E L G K M I F Q E T G K N - P V K N Y G L Y G C N C G V G N R G K P V D A T D R C C F V H K C C Y K K V T G C D P K S L V W L W K M I F Q E T G K E - A A K N Y G L Y G C N C G V G R R G K P K D A T D S C C Y V H K C C Y K K V T G C D P K S V L E L G K M I L Q E T G K N - A I T S Y G S Y G C N C G W G H R G q P K D A T D R C C F V H K C C Y K K L T D C N H K S L V E L G K M I L Q E T G K N - P L T S Y G V Y G C N C G V G S R H K P K D D T D R C C F V H K C C Y / G L W Q F E N M I I K V V K K - S G I L S Y S A Y G C Y C G W G G R G K P K D A T D R C C F V H D C C Y G K V T G C N P K N L F Q F E K L I K K M T G K - S G M L W Y S A Y G C Y C G W G G Q G R P K D A T D R C C F V H D C C Y G K V T G C N P K 70 80 90 100 110 120 I K D R Y S Y S W E - N K A I V C G E K N P P C L K Q V C E C D K A V A I C L R E N L G T Y D K - K H - R V T V K F L C K A - - P E S C 2 M D S Y S Y S W K - N K A I V C G E K N P P C L K W V C E C D K A V A I C L R E N L G T Y N K - K - Y T I Y P K F F C K K - - A D T C 3 T D R Y S Y S W K - N K A I I C E E K N - P C L K Q M C E C D K A V A I C L R E N L D T Y N K - K - Y K A Y F K L K C K K - - P D T C 5 L G K Y T Y S W Q G N - - I V C G G - D D P C D K E V C E C D R A A A I C F R D N L D T Y D R N K - Y W R Y P A S N C Q - E D S E P C 6 M D I Y T Y S V E - N G N I V C G G T N - P C K K Q I C E C D R A A A I C F R D N L L T Y D - S K T Y W K Y P K N - C T K E E S E P C

i-I~. 5 ( o m p a r i m ~ n between Ih¢ L~.,,-49 a n d Ihe A~,p-4q p h o s p h o h p a ' , e A: f a m d l e , f r o m s o m e ( r o t a h d a e ~.enomn. Abbre~i.Jtlons of senom,, are:

].~t%'. Ttuuere~urz~ mu~ro~quamulu.~ (the p r ~ e n t slud~,~: TF%. lrt.wre~uru~ [lat.ttrtdt~ l l S l : A P P . 4~zxtr,~lonp/~(tr.ru~ p ~ . ' o r . ~ [21L2t1: BA~,'.

Bothr,,p~ ~ttro.x 121]. An a,~leri~,k denote,, the .,pecial ,,ub,,tilulion m Ihe [.'..,-4q protein~ in conlr;a,,I to the other PLat ,~

of enzymatic activity [20]. which share a high degree of structural homolog 5 in Old and New World snakc~.

Being homologous to lhe group II ~enom PLA?s.

. . . .

Wave encj h n m

F,g. 6. [)ifference ~Ik'~tra of TMV-K4q reduced b~ ( a : " and the ~atchard phil (In~rll. The sample cu%elt¢ *:ontained purified TMV-

K 4 9 0.5 r n g / m l in 50 m M "[ri~4).l M NaCI (pH 8.0) at 2 5 ° ( in the

pre~ence of 0 . 0 5 . 0 . 1 . 0 . 3 . 0.7 or 1.4 m M [ C a : " I ~ - " . . . - - . . . . . . . . . r~pecti~elx . w h i l e the reference cu~.ett¢ c o n t a i n e d the s a m e c o n c e n t r a t l ( ~ of the protein a n d b u f f e r ~ ' a t c h a r d p l o h for o b t a i n i n g the dl~:aKiation c,~nslal~l or the .'nclal c o m p l e x v,a,,

ba,,ed on the ab~rbanc¢~ at 2 9 0 n m I [n~,erl)

Ihc K49 protein~ have ~ome ~tructural fealure,~ in c o m - mon (residue n u m b e r i n g as in Fig. 5): ~1) In their N - t e r m i n a l a - h e l i x . Glu-~,. Leu-5 is in contrasl to the h i g h b conserved G i n - 4 . Phe-5 in o t h e r P L A . ' s : (2) A~n-2S. (A~n. A r g . H i s o r Ser)-33 and Lys-49 in the

50- O---- O TIAV K49 @-- @ F3C(11- ! 40 = / O ~ - O ' ~ O w t . - ~ :,': / / / lill t/

i 2o~

/

K. c" O~ 0 1 2 3 4 5 6 Time

(hr)

Fig. 7, Time-course ~ff edema reduced b, three PLA: i,~lforms from

Y ,ltaro,Wamatu~ ~enom T~,|%'-K49. FXXII-I and TMVPI_A, I u~d "*ere put,bed b) ItPI.( + iFig I)+ ttmd pa~ edcmd v.a+ md,u:cd b, a ~ungl¢ ,ubplanlar mj¢aAnon of 5 #g protein in I0 pl buffer t~e

(8)

K49 proteins ,,ub.~,ituted the calcium binding ,,ite,~ "I;r- 28. (ily-33 and A~,p-49 im.arlabl~, found in ~ther PLA .',, [2Ui: (31 (;In-I I. L),,-53. (;lu-g#J. Val-95. I.eu-gq are c o n ~ r v e d in the K49 pr,~tcim, v, hereas in group II venom P L A . ' s l..vs-II. G1~-53. -,~0. Ala-95. Phe-99 are

con.',,¢rvcd (Fig. 5 and Ref. 20): (4) the K49 proteins are very basic (p# > 10). and contain m a n ) basic residue~ at region II0 122 (Fig. 5).

The K49 protein from A. p. plltlrorlll v.,ax sllo~n tl, have the capacil) to bind phospholipid [20,21] and in this study the activating e f f ~ t of T M V - K 4 9 on platelets

and mast cells (Table II) suggests that i1 binds to the biomembranes. Another basic and membrane-binding venom protein, cardioloxin, was al~* shown to cause edema in rat hind-paws [19]. The effective dosages for the edema induction b ) T M V - K 4 9 and cardiotoxin [19] are 2 5 y g and 5 15 y g per pay,. respectiv¢l 3. T h u , the edema-forming activit~ o f TMV-K49 ix more potent

than t h o ~ of the cardiotoxin [Iq]. cobra PLA: 113] and

also tbo~,e of the P L A . from the ~,ame ,,ChOre. FXXiI-I

and T M V P L A . I (Table il). The phospholipid-

bydrolysing activities are not parallel to the edema inducing activities of these proteins. The p-bromophen- ae~,l hmnllde-medified FXXII-2. a h o s e enzyme cata- lytic activities were completel} Iosl. ~,till induced pau edema and the effect ~,~as greatly suppressed b) heparin [131. which further supports the contention thai the basic charges, rather than the enzymatic h)drol)si,,, are resl~lnsible for the edema-inducing pmpert3 of TMV- K49. Furthermore. the P L A . inhibitor, ari.,,tolochic acid [23 I. did noi interfere with the effect of TMV-K49 ('not shov, n). but this edematous response ~ a s totall3 re- versed in the rats pretreated with aspirin in combination with anti-serotonin (methysergidel and anti-histamine Idiphenhydramine) 1131.

It was shown previomJy that the K4q protein from

A+ p. pi.wi~oru.~ venom had vet3 Io~ intra~,entricular

lethal potent',, low anticoagulant and hemol,,tic activi- ties and that it also had little effect on cardiac and neuromuscular tissues (70 and 35 p.g/ml of the nearl.~- purified K49 are required to be effective, respectilel) I I22]. Our results confirmed the low activity of "I-MV-K,Iq on the n e ~ e - m u ~ ' l e preparalion. Moreoter. thi~ and the previow, studies Ill 13] indicate that one of the major functions of the K49 famil) is direct binding to PMN leukoc)tes, platelets and ma~l cells Io cause relea,c ,,f serotonin, histamine and oth~.r inflamma jr,. mediatoP,. thur, causing vat,dilation and edema. ;t ~a_', found '.haz various proteins (PLA:'s. e s t e r a ~ s and proteina~esi in

T. mut'rosquamatu_~ venom had edema-inducing capabil-

itie~ 191 . T h u s TMV-K49 may be part of the s)nergistic sv.,,tem which contributes to the o,,erall edematou.', re- s p o n ~ c a u ~ d by the venom. Although the Trmu're~ur~L~

K49 proteins bind C a : " (Fig. 6 and Ref. 15). Ihe role of the pr¢~thetic Ca-"- remain to be investigated. The lack of enzyme activity of TMV-K49 ma,, be due to un-

(a~,orahl,2 c(~nformatli,i1 a! l h ¢ J c t l t e ~lle c<lu,,i.'d b ~, . u b M i l U t i O l l l~l" rl:,,iduc 4q and p(>~i~l'~ ~ l l l e r , u b M i l u - Iilln,~

1201.

R c c e n t l ' , , . t h e crs,~tallographic - t r u c l u r e o f t h e K 4 9

PLA:-homol'ag from .4ga~trod,,n ~ a , ,,ol',cd [26J. The

3-D m a p sho~ed an intacl pho,,phohpid binding ,.ire and , ,on,,cr~ed calal?,tic center tHi~-4g. T,,r-73 and A~,p-99) e~cepi 1_,,,,-49 di,,placing Asp-40 in the calcium-binding qte. H¢,',,.c~cr. the ,d~uclure-functlon relation,,hlp of the K49 protein remain, 1o be ,,ludied before il~, d~,namic ride tlt~ard sl'~cific cell t~pes leg.. ma,,I cell. PMNI can be u n d e r , , t ~ at the moh:cular le,.el.

Aeknowlt~l~enlenl,,

V(e extend our gratitude 1o Dr. ~,lin I. Liao for his gift of the ~,enom. to Dr. Jih-P?,ang Wang for carrlng

out edcmu experiment, and Mr. Pei-Jung Lu for techni-

cal a,,,,i,,iance. Thi,, x~,~rk i, ,upported hx a grant from Nalional ,~lence c +~uncil of l a l ~ a n . ( h i n a .

l l e f e r e ~ e ~

1 t,.ikj.aki. ( 'NlJtukl J arid l,im,~.J % (t'+~Jlh l~.tL,,~l 2!l. 1!9 32"

2 t.ik,.i.akt. ( . " l u t J m . | ~tmj kj,ixj-l~li~t t ,'l'4'q,l~ ]o~.l~t,li 2t4 3 2 9 - 3 3 9

3 T,al I H . LnJ H ( and (hang. I i 1 ' ~ ' l Bl.~.htm Bl~,h-~, ~¢.ta q16. 94 r~.

J Meh,. |). and %.lmcjinla. i i I~Jl'a~t To~l~,m Ix, 4.4~-.154 5 Lc~. ( ' l . the. ( . ( arid hk~.-r, l ) ~i*i--~ l-,m+.~m 151 ~55 ~5~ 6 B,,ffa G A. I~ffa. %.1( aod V, lrl=hcnm Jt ~t9"¢,= B~-h=rn

501-51 ~

'~ T e n g . ( M . ~ n ~ J P P c n g I t ( ar, d (Iw, ang ( ¢lql, q , l , ~ -

I~t (ILixJnl:. ( . .l~ 'll* . / a ~ ~l P ~nd 1(¢~= ( %,I tDISli T,ll.i~.,,o lq. l l 3 l ] t i

It ( h,u tt I ( h c n I I and Ic~7 t 'it t l(l~,tl t,,t~<on 2" 11:" 125 12 %llang J P and l o n g ( %1 ~l~;~l~, J Ph,ffm lJk~'ma~ 42. 13 Vlang I P and Tong. ( %1 ~lv'~q t.ur J Pl~arrrl~,~l t~). 347.3_'5.i 14 %|jragam~l+e. J ~ i l P,~.rman R A and Hemr!k~,n. R t . il9~'¢~t J

Proleirl ( h¢l'Ii e,. l ' 7 1 ~

1~ kri¢~h;. R ,Jn,,J ( heng. J,'i ~tv,"~ -ltrIJt ( hl~rri ~ . 2J75 2J TM I "~ ( l i j n g , ( ' S arsuJ | I U . ( N i P'IRY, I J ('bin [~h~'~'~l Y'~l¢, I'~. 12-19. l;~ llicll-'l~rl %1, ~t,. %'o|tie*k. JJ and ~ tl.ia.. ( ; H ~ IqI4! Bil~alem-

t - i n IJ. 14~lt-laa5

19 I,~ang. J P an<l l e n g ( M ¢1'.,'1~'1~ FJJr J Pllarm, l. o l 161. 0-1t4 20 %,,it l ~ n Bergh. ( I . S h ~ . ~ m . ~IJ 'l¢,tleij H M arid D< Haa.

C , H i I ~ " . h J ( t i t Bsl~.hcm 39. 37~--.~I~

(9)

2." Dhdl,m. [ ) ~ (,,ndrca. ! M.~ra~ar~orc. ! ~,1 Ilonr~ks, m R I , Ik:n]amm. N anJ R,~.nI:~rg P Ilq,xT) B~,~.hcm Pharma~,l II~ 172a 1 7 ~

2 ~ Ro~enlh.fl M I ) . Vi.hv.analh. B S and Fr.sn~m R ( ~lqxqi

24 leng. C M . ( h e n . '~ ~t K o ~,I, ( .m,l ()UTah ~ ( ~ t~)~'~ B,o- t'hlm Bloph~x ~¢1.~ q24. ~-~ 3t~2

2 ~ O'Br~cn. I R ~ l t ~ 2 ) J ( h n PJthol. 15.4.',2 4 ~

26 t l . l l a n d . I ) R . (lain." 3. k I . ~,lu~.hm~,r¢. S~,'. R'.dc, ! J . t:m~.- pahr. t I M . . Fmzcl B ( . llcmnk~)n. R ! Jnd ~;alcnpaush. K . I ) ~lO~)) .I B,~l ('hem 265. 1"7649 17656

數據

Fig,  I.  Purfflcalion  of  J M V - K 4 9   h~.  rc',er,.c-pha:,,e  IIPI.U.  Ab,t~ut  1  or  2  mg  o f   FXXII-2  obltllned  from  ion-exchanger  and  gel-filtration  chromatographi,'r,  v.erc  injo:ted  on  an  HPI.C  s~,stem  with  a  ( ' h e i l f   co
Fig.  2.  Preparative  H P [ ( &#34;   peplide  map,, tff t;ll endt+proteinase  (+lu-C&#34; dlge~,t and  (hi  IT~,yl endt+peplida+  dige~,l of TMV+K49
Fig  3. Peptide maps ~ff tal eod.~Froteinase Asp-N dig~.~t and [b} a-chymot~psin digest of TMV-K4q
Fig.  4.  Amino acid .cquence of  TMV-K49 deduced  from pept,de  fragments. Ahbre~i,nmns are:  E

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