Kallikrein-like enzymes from Crotalus atrox venom.

The symptoms which immediately follow envenomation by many crotalid snakes include hypotension, hypovolemia, hemoconcentration, and shock. We have isolated and characterized two proteases (EI and EII) from the venom of Crotalus atrox which may be involved in the onset of these symptoms. EI and EII have molecular weights of 27,500 and 29,200 and isoelectric points of 4.7 and 4.3, respectively. Specific esterolytic activities of EI and EII on N alpha-p-tosyl-L-arginine methyl ester are 51.5 mumol min-1mg-1 and 48.1 mumol min-1 mg-1, respectively. Both enzymes are rather specific in their substrate requirements in that neither was demonstrated to have any proteolytic activity against either of the oxidized chains of insulin, or glucagon. Neither enzyme was shown to have plasmin or fibrinolytic activity. Both enzymes are able to cleave a kininogen analog to release bradykinin. This proteolytic activity is inhibited by aprotinin and phenylmethanesulfonyl fluoride but not by ethylenediaminetetraacetate. The enzymes are active upon the kallikrein substrates S2666 and S2302. The Km values of the enzymes with these substrates are similar to those reported for kallikrein. Structural similarity between the two enzymes was demonstrated by ultraviolet and circular dichroic spectroscopy, and amino acid analysis. Tryptic peptide mapping of the two native enzymes also suggested a large degree of structural similarity. Furthermore, sequence studies on the NH2-terminal regions of the enzymes indicate that they share a significant degree of sequence homology with porcine kallikrein and crotalase, a kallikrein-like enzyme from Crotalus adamanteus. The main physical difference between the two kallikreins reported here appears to be due to the carbohydrate moieties on the enzymes. At present the in vivo role of venom kallikreins in envenomation pathology is uncertain; however, it is possible that they play an important part in giving rise to the initial symptoms of hypotension and shock.

The symptoms which immediately follow envenomation by many crotalid snakes include hypotension, hypovolemia, hemoconcentration, and shock. We have isolated and characterized two proteases (E1 and EII) from the venom of Crotalus atrox which may be involved in the onset of these symptoms. E1 and E11 have molecular weights of 27,500 and 29,200 and isoelectric points of 4.7 and 4.3, respectively. Specific esterolytic activities of E1 and E11 on N"-p-tosyl-L-arginine methyl ester are 51.5 pmol min"mg" and 48.1 pmol min"mg", respectively. Both enzymes are rather specific in their substrate requirements in that neither was demonstrated to have any proteolytic activity against either of the oxidized chains of insulin, or glucagon. Neither enzyme was shown to have plasmin or fibrinolytic activity. Both enzymes are able to cleave a kininogen analog to release bradykinin. This proteolytic activity is inhibited by aprotinin and phenylmethanesulfonyl fluoride but not by ethylenediaminetetraacetate. The enzymes are active upon the kallikrein substrates 52666 and 52302. The K , values of the enzymes with these substrates are similar to those reported for kallikrein. Structural similarity between the two enzymes was demonstrated by ultraviolet and circular dichroic spectroscopy, and amino acid analysis. Tryptic peptide mapping of the two native enzymes also suggested a large degree of structural similarity. Furthermore, sequence studies on the NHz-terminal regions of the enzymes indicate that they share a significant degree of sequence homology with porcine kallikrein and crotalase, a kallikrein-like enzyme from Crotalus adamanteus. The main physical difference between the two kallikreins reported here appears to be due to the carbohydrate moieties on the enzymes. At present the in vivo role of venom kallikreins in envenomation pathology is uncertain; however, it is possible that they play an important part in giving rise to the initial symptoms of hypotension and shock.
The Western diamondback rattlesnake (Crotalus atrolc) is indigenous to a large area spanning the Southwestern United States. Upon envenomation, there are marked effects on the victim's cardiovascular system, respiratory system, somatic nerve system, and skeletal muscle (1). Since death is a relatively uncommon consequence of envenomation, much em-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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To whom correspondence should be addressed. phasis has been placed on research involving the dramatic local effects caused by envenomation such as hemorrhage, myonecrosis, inflammation, edema, and pain (2)(3)(4)(5)(6)(7). Another area of focus by investigators has been on the systemic effects of crotalid envenomation. These effects play an important role in giving rise to hemorrhage, hypotension, coagulation, hemolysis, and hemoconcentration. All of these factors serve to produce the overall symptom of crotalid poisoning sometimes called rattlesnake venom shock (8). It is the rapid onset of venom shock which probably plays a major role in prey immobilization and possibly death.
In the past, there have been reports of the hypotensive nature of certain crotalid venoms (9)(10)(11) and also of the isolation of kallikrein-like enzymes from viper and crotalid venoms (12)(13)(14). It has been proposed that these venom kallikreins along with other hypotensive factors in the venom serve in the production of venom shock (8). In this report, we discuss the isolation from the venom of C. atrox of two proteases with similar structural and functional properties as certain snake and mammalian kallikreins.

EXPERIMENTAL PROCEDURES AND RESULTS'
Amino Acid Composition and NHz-terminal Sequence Analysis- Table I contains the amino acid composition of E1 and EII. As can be seen, the compositions of each protein are nearly identical, suggesting homology between the two proteins. The total number of residues/molecule for E1 and E11 based upon the estimated protein fraction of the molecular weights are 216 and 219 residues, respectively. Fig. 1 shows the NH,-terminal sequences of E1 and E11 compared to another snake venom protease, crotalase (from Crotalus adamanteus venom), and porcine a chain kallikrein. In each case, the HPLC analysis of the first Edman degradation cycle on E1 and E11 showed the presence of only one PTH derivative (see Table VI in Miniprint). The NH,-terminal sequences of E1 and E11 are identical to each other up to residue number 21. A notable degree of sequence homology is also observed between EI, EII, crotalase, and kallikrein (15,16).
Portions of this paper (including "Experimental Procedures," part of "Results," Figs. 2-13 and Tables 11-VI) are presented in miniprint at the end of this paper. The abbreviations used are: PTH, phenylthiohydantoin; TAME, N"-p-tosyl-L-arginine methyl ester; PMSF, phenylmethanesulfonyl fluoride; HLPC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83 "133, cite the authors, and include a check or money order for $12.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Amino Terminal Sequence Comparison of E l and E l l W i t h S i m i l a r P r o t e a s e s
. I "

DISCUSSION
The overall mechanism of the toxic action of rattlesnake poisoning is somewhat unclear due to the complex nature of crotalid venoms. In general, rattlesnake venoms are not strongly neurotoxic with the exceptions being venoms from Crotalus durissus terrificus and Crotalus scutulatus. This is in sharp contrast to the venoms from Hydrophiidae and Elapidae snakes which are extremely neurotoxic. The primary, initial, overt action of crotalid envenomation seems to be onset of venom shock symptoms such as hypotension and hypovolemia, followed by rather considerable local tissue damage at the site of envenomation. To date, significant progress has been made in the understanding of the mechanism and factors involved in local tissue damage.
The initial transient hypotension that is common in rattlesnake envenomation is probably due, for the most part, to two types of factors present in the venom; one of the factors is the presence in some crotalid venoms of angiotensin-converting enzyme inhibitors. These interesting peptides act by inhibiting the conversion of angiotensin I to angiotensin I1 by the converting enzyme and thereby additionally serve to potentiate the pharmacological actions of bradykinin (31).
The other group of important factors involved in hypotension is the kallikrein-like enzymes. Bradykinin has been demonstrated to be released by the proteolytic action of venom kallikreins on bradykininogen in plasma, intestine, uterus, and smooth muscle (31, 32). Additionally, bradykininogen levels have been shown to be decreased following rattlesnake envenomation (9). Prior to this investigation, there have been reports of at least two well characterized snake venom proteases which were identified as having kallikrein-like enzymatic activity. One of these proteases was isolated from the venom of Vipera ammodytes ammodytes (12). This kallikrein was shown to be a glycoprotein of molecular weight 34,300. The protease had an isoelectric point of 7.2 and was six times as active as trypsin in releasing a kinin from plasma kininogen. As to whether the kallikrein released was lysyl-bradykinin or bradykinin was not discussed. Another kallikrein-like enzyme called crotalase, which was originally identified as a thrombin-like enzyme, has been isolated from the venom of C. adamanteus (13). Some of the distinguishing properties of this enzyme are molecular weight of 32,700, glycoprotein, serine esterase, and inhibition by specific chloromethylketone kallikrein inhibitors (13,33). This enzyme was shown to make up approximately 0.23% of the crude venom (13).
The two kallikreins (E1 and EII), as we now term the enzymes, isolated from C. atrox venom show some similarity to the enzymes mentioned above, particulariy crotalase. E1 and E11 are also very similar with regard to each other. The amino acid compositions and NH2-terminal amino acid sequences of both enzymes are similar. The conformations of E1 and E11 as examined by UV and CD spectroscopy also appear similar to a degree. However, the spectroscopic studies did demonstrate some differences in the fine structure of the two enzymes' conformations. These similarities of native conformations were further demonstrated by tryptic digestion studies on the two native enzymes. The HPLC elution profiles of the digestions were overall notably alike although at least one major difference was observed. As to whether this difference is a result of a slight conformation difference due to different primary structures or different carbohydrate moieties between the two enzymes is at present unclear. However, this study has shown that there are both qualitative and quantitative differences in the carbohydrates present in these enzymes. The effects these may have had on the mapping and spectroscopic studies is uncertain.
The two enzymes share like specificity for releasing the same peptides in identical order from the KS-1, KS-2, and KS-3 substrates. However, when their activities were examined with the chromogenic kallikrein substrates S2266 and S2302, some kinetic differences became evident. The relative reaction rates (normalized against trypsin) of E1 on both substrates were nearly identical whereas E11 demonstrated a higher rate with S2266. E1 had nearly identical K,,, values with both substrates; however, E11 had a larger K,,, with the glandular kallikrein substrate S2302 compared to the plasma kallikrein substrate S2266.
The C. atrox kallikreins seem to share some biochemical properties with the kallikreins from V. ammodytes ammodytes and C. adamanteus. The molecular weight of V. ammodytes kallikrein is 34,300 and crotalase is 33,000 compared to the molecular weights of 27,500 and 29,200 for E1 and EII, respectively. EI, EII, v. ammodytes kallikrein, and crotalase are all glycoproteins although they do not share exactly the same carbohydrate moieties. Both V. ammodytes kallikrein and crotalase are sialoglycoproteins whereas E1 does not contain sialic acid. EII, however, does contain sialic acid. With regards to the proteases' presence in crude venom, crotalase comprises approximately 0.23% by weight of the crude venoms compared

Kallikrein-like Enzymes from
Crotalus atrox Venom to 0.13% and 0.26% for E1 and EII, respectively. Finally, EI, EII, crotalase, and porcine kallikrein do possess homologous amino acid sequences in the NH2-terminal region, and, as more sequence data are reported on both C. utrox kallikreins and crotalase, it is likely further sequence homology will be uncovered. One very interesting difference between E1 and E11 and crotalase is the strong thrombin-like activity demonstrated by crotalase whereas no significant clotting ability was observed with either E1 or EII. In light of the many similar biochemical properties of EI, EII, and crotalase, far from insignificant is the observed amino acid sequence homology. The comparison of the complete primary structures of EI, EII, and crotalase may well lead to very interesting findings from the proteins' structures which shed light on the dual enzymatic activities of crotalase and the mechanism of action of E1 and EII. Additionally, it is noteworthy that these two crotalid snakes are closely related taxonomically, and therefore it is not surprising that the two snakes share similar toxins in their venoms. Consequently, in the future, it will also be interesting to examine what similarities and differences the other toxins in the respective venoms possess and their effects in the overall pathological nature of the venoms.
Finally, the isolation of kallikrein enzymes from crotalid venoms further emphasizes that one of the important aspects of crotalid poisoning is the hypotensive venom shock which occurs almost immediately following envenomation. It is this aspect of envenomation which probably plays an important, immediate role in debilitating the victim, which is then followed by the local effects at the site of envenomation. Further investigation of these and other kallikrein-like enzymes in crotalid venoms may lead to a more complete understanding of the nature of crotalid envenomation.

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The kininogen analog IS-1 Ya6 cleaved at two site8 by E 1 and E l l . The following is the description of El digestion Of Is-1.
At Oh msubation t m e the one malor peak at approximately 43 mi" vas identified by amino acld analysis as the intact KS-1 peptide (Figure 11 and

PTO-Cly-Ser-PrD-Phe-Arg-
resulting from the bond cleavage between -Arg13the first cleavage site l-Se~-V~l-Gln-V~1-Ser-NH21. In the 60 and 120 mi" Ser14-. Peak 2 was identified a6 the carboxyl terminal peptide fragment from digeBt10ns one can see that the peak 1 peptlde 1 8 being dlgested to yield two new fragments located at Peaks 3 and 4 . Ammo acid analyaie identlfied peak 4 as the C-terminal peptide ~-A T~-P~O -P~O -G~Y -P~~-S~~-P C O -P~~-~~T~) from the peak 1 peptide resulting from the bond cleavage at Lys4-Arg5. Peak 3 Y L~B identlfied ae the amino terminal fragment of the peak 1 peptide. NO further dzgestron was detected after 120 min.
Both enzymes appeared to Cleave the two peptlde bonds Of Is-1 in the same order and with nearly ldentlcal rates.  However, after extended periods of time 0 4 h ) very small. atyplcal Clots were sometimes Observed.
The rnechaniem of Productton of the atyplcal clots 16 unknown, therefore the thrombu-like actlvlty of E1 and Ell was considered to be negllglble.

Ifig. not presented)
The CD spectra for El and E11 do appear to have some r n~n o r dlffecences.
In the Peptide region ( figure   121. the El spectrum has mlnlma at 211 and 219nm wlth mean resldue weight ellptlcltes of -6205 and -6176 respectively.
In the case Of E11 a minimum is observed at 212nm and a broad mlnimurn at Both spectra appear LO represent protelna ulth predominantly O-helLcal structure 129, 301. HDVeVer, a8 apparent from their CD spectra, the conformatlans of both protelns do differ to some small extent.

WAVELENGTH (NM)
fig. 12. Circular dichroism spectra of E l and EII In the Peptide Ceqlon.
The CD spectra In the aromatic reglan of E1 and E11 IFlgUre 131 are to a large degree a180 similar. In both spectra of the arornatlc r e g~o n three rnlnma are observed.
In the El spectra the three malor n m m a a r e tound at 268, 214, and 280nn with molar elipticitles of -63,000, -60,500, and -57,300. The mnlrnurn at 268nm 1s likely due to an absorbance by phenylalanine: the broad, shallow, negatlve band centered around 305nm 1s llkely the ieSYlt of 274nm and 28On0i peaks are probably due to tryosine. tryptophan, 01 both. The dlsulfldea present LD the protelns. Overall, the appearance of the aromatic molar e l l p~~c l t~e s being very similar.
However, as I" the case of the pep-CD spectlum for E l l 16 qurte slmllar to that of EI, Ylth mlnlrna P051t10n and tlde r e g~o n spectra. some minor flne Structural dlfferences are apparent.
In both Instances the nature Of these differences are unknown. However the proteases, an observed vlth CD and W SpeCtrOBcopy, do appear to share