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Serine protease inhibitors of North American leeches
Comp. Biochem. Physiol. Vol. 84B, No. 1, pp. 117-124, 1986 Printed in Great Britain
0305-0491/86 $3.00+ 0.00 © 1986 Pergamon Press Ltd
SERINE PROTEASE INHIBITORS OF NORTH AMERICAN LEECHES ALLAN M. GOLDSTEIN, ERIK H. MURER and GEORGE WEINBAUM Research Division, Department of Medicine, The Graduate Hospital, Philadelphia, PA 19146, USA (Tel.: 215-893-7628)
(Received 19 August 1985)
Abstract--1. Serine protease inhibitors in extracts from three North American leeches, Nephelopsis obscura, Erpobdella punctata and Hemopis marmorata have been separated by anion exchange chromatography and the activity pattern against human granulocyte elastase and porcine chymotrypsin and trypsin determined. 2. All three leech species contained a major peak with anti-trypsin activity, but Hemopis was unique in that the trypsin inhibitor was equally active against chymotrypsin. 3. Nephelopsis was rich in anti-elastase activity of two types, one which was also active against chymotrypsin, and one which was a specific elastase inhibitor. 4. Erpobdella contained inhibitors against elastase and chymotrypsin but with major activity against the latter.
INTRODUCTION
of protease inhibitors of granulocyte elastase. The interest in granulocyte elastase inhibitors is related to the possible utilization of such inhibitors therapeutically for the arrest or prevention of emphysema (Stone, 1983).
Since the demonstration that the anticoagulant of Hirudo medicinalis was a specific thrombin inhibitor (Markwardt, 1970) the serine protease inhibitors of this leech have been the subject of extensive studies (Fritz and Krejci, 1976; Seemuller et al., 1977; Jochum et al., 1983). Four different types of protease inhibitors have been characterized, (a) hirudin (Markwardt, 1970) with specificity against thrombin and trypsin, (b) bdellin, a low-molecular-weight protein with inhibitory activity against trypsin and plasmin (Fritz and Krejci, 1976), (c) a highmolecular-weight protein with inhibitory activity against trypsin (Seemuller et al., 1977), and (d) eglins, two low-molecular-weight polypeptides with inhibitory activity against chymotrypsin, granulocyte elastase, subtilisin and cathepsin G (Seemuller et al., 1977). The interest in the protease inhibitors of H, medicinalis has not caused analysis of protease inhibitors from other types of leeches. One exception is the South American bloodsucker Haementeria ghilianii, which was shown by Budzynski et al. (1981a, b) to contain an enzyme in its salivary glands that degraded fibrin and fibrinogen, thus preventing coagulation of the host's blood. This leech did not contain a hirudin-type protease inhibitor. Murer et al. (1984) showed that salivary gland extracts from H. ghilianff and the related species H. officinalis contained inhibitors of trypsin, chymotrypsin, plasmin and granulocyte elastase. One result of the interest in the protease inhibitors of Hirudo medicinalis is that it is now difficult to obtain this leech in large quantities. We have therefore undertaken a study of protease inhibitors from leeches which are available in North America, one of the most available being the "bait leech" Nephelopsis obscura. Our primary aim has been to determine whether any of these leeches may be a good source
MATERIALS AND METHODS
Source of inhibitors Leeches were obtained from bait stores in Minnesota. Nephelopsis obscura (the ribbon leech) is the primary species sold at bait stores. It is distinguished from other leeches by behavior (it will stay in a bucket half full with water, while the horse leech Hemopis will climb out.) Nephelopsis prefers shallow, stagnant water and is bred commercially in ponds (DNR Report 1979). Erpobdella punctata (the dog leech) was found occasionally among the purchased Nephelopsis leeches (I0 Erpobdella leeches in 1 lb Nephelopsis leeches). Hemopis marmorata (the horse leech) is discarded by the bait salesmen, but we were able to obtain four big specimens, which were classified by family and used for protease inhibitor preparation. The leeches were identified by Dr D. J. Klemm of the Environmental Protection Agency, Cincinnati, OH (Klemm, 1982). Preparation of leech material (a) Leeches were prepared by the method used by Markwardt for Hirudo medicinalis (Markwardt, 1970). The leeches were fixed twice for 24 hr each in 95% ethanol, then cut into 0.5-1 cm wide slices. The sliced tissue was extracted twice for 30 min each with 10 volumes of 40% aqueous acetone, followed by centrifugation at 500 g for 5 min. The extract was diluted with 1/2 volume 80% acetone (pH 4.3-4.5) and the formed precipitate removed by centrifugation. This precipitate was inactive as a protease inhibitor. The pH of the supernatant was readjusted to 6 with dilute ammonia, and the acetone removed under vacuum. The extract volume was reduced 90% by freeze drying and acidified to pH 2-2.5 with 20% trichloroacetic acid. Nine volumes of pure acetone was added, and the formed precipitate collected by centrifugation. In many preparations heavy drops of active material appeared below the acetone layer, dark brown of color. These were collected with a 117
118
ALLAN M. GOLDSTEIN et al.
Pasteur pipette. The active material was dialysed against water using a membrane with a mol. wt cut-off of 1000 daltons (crude extract). The crude extract was dialyzed against the equilibrium buffer for application to chromatography columns. Proteases
Chymotrypsin a (3 x chrystallized) and porcine trypsin were purchased from Sigma Chemical Company, St. Louis, MO. Porcine trypsin type lI from Sigma was used to prepare purified pancreatic elastase by the method of Shotton (1976). The preparation was free from trypsin activity and had less than 1% chymotrypsin. Granulocyte elastase was prepared from human leukocytes. Human peripheral blood was collected by venipuncture, anticoagulated with EDTA and separated by dextran sedimentation and 30 sec lysis of red blood cells with distilled water (Sloan et al., 1981). The isolated neutrophils ( > 80% pure) were frozen and thawed 3 times in 0.l% Triton X100 in water or saline. The pellets were sedimented by centrifugation at 18,000g in an Eppendorf centrifuge and extracted with 0.96 M NaCI buffered to pH 8.3 with 0.1 M Tris HCI. The pellet from this high salt extraction was discarded, while the supernatant was used directly as soluble granulocyte elastase. Protease substrates. The substrate for chymotrypsin was Meo-Suc-Arg-Pro-Tyr-paranitroanilide (pNA) (Kabi substrate S-2586), Helena Laboratories, Beaumont, TX and for granulocyte elastase L-pyroglutamyl-Pro-Val-pNA (Kabi substrate S-2484), also from Helena Laboratories, Beaumont, TX. For determination of the binding constant was used Bzoxy-Suc-Ala-Ala-Pro-Vat-pNA (MAPVA) (Sigma Chemical Co., St. Louis, MO). Substrate for pancreatic elastase was Suc-Ala-Ala-Ala-pNA (SAPNA) (Peptide Research Institute, Osaka, Japan) and for trypsin benzoylarginyl-pNA (BAPNA) (Sigma Chemical Co). Protease assays
Reaction mixtures for measurement of protease activity were: (a) chymotrypsin, 425/d 0.1 M Tris-HC1, pH 8.3, containing 0.96 M NaC1 + 100/~1 water or protease inhibitor + 50 #1 chymotrypsin (0.8/~g/ml) + 25 #1 S-2586 (2.4mM in water); (b) pancreatic elastase, 600/d 0.2M Tris-HC1, pH 8.0 + 100 #1 water or protease inhibitor+2.5#1 elastase (0.4mg/ml)+ 100#1 SAPNA (2mg/ml in 0.2M Tris-HC1, pH 8.0); (c) trypsin, 680#1 0.1 M Tris-HCl, pH 8.0, containing 10 mM CaC12 + 100 #1 water or protease inhibitor+ 10#1 trypsin (0.1 mg/ml)+ 10 #I BAPNA (0.1 M in DMSO); (d) granulocyte elastase, 465#1 0.1M Tris-HC1, pH 8.3, containing 0.96M NaC1 + 100/~1 water or protease inhibitor + 5/~1 elastase (equivalent to 0.154).25 #g pancreatic elastase) +30/~1 S-2484 (16 mM in DMSO). For the binding studies 10#1 MAPVA (25 mM in DMSO) was used as substrate. Binding studies were performed with 3 concentrations of enzyme (0.1,0.2 and 0.4 units per assay), using the method described by Green and Work (1952). Protease activity for any of the enzymes described above was measured as an increase of absorbance at 410nm caused by release of paranitroaniline from the chromogenic substrate. The rate of change in absorbance was measured spectrophotometrically in a Zeiss model M4QIII at room temperature. In routine studies the reaction mixture was incubated at 37°C, then 0.2 ml 50% glacial acetic acid was added at various times and the absorbance read at 410 nm (Murer et al., 1984). The unit of protease inhibitory activity is calculated as/~g of protease inhibited 100% and specific activity as units per mg protease inhibitor protein. Units of granulocyte elastase activity were determined on the basis of a titration against purified elastase inhibitor from Ascaris lumbricoides (Peanasky et al., 1984; Murer and Weinbaum, 1984). The Ascaris inhibitor is active against both granulocyte and pancreatic elastase, and its inhibitory capacity
was determined against purified porcine pancreatic elastase (prepared by E. Murer). Column chromatography The chromatography methods described by Seemuller et al. (1977) were followed with minor modifications. Nephelopsis obseura: Crude inhibitor from 56 g dehydrated leeches (95 mg protein with 250 units chymotrypsin inhibitor) was applied to a 40 x 1.6cm Sephadex G-75 column in 0.5M NaC1 buffered to pH 7.8 with 50mM Tris HC1 and eluted with the same buffer. All column fractions were assayed for protease inhibitor activity against trypsin and chymotrypsin. The fractions with activity against chymotrypsin (fractions 12 27) were collecled. The latter ~ of the active fractions (corresponding to a molecular weight below 18,000 dalton) were combined in two fractions (the early fraction with tubes 17-19 and the late fraction with tubes 20-27). These fractions were concentrated against Aquacide 1I and each applied to a 20 × 1 cm DE52 column and eluted with 30 mM ammonium acetate of ptt 6.4 followed by a gradient of 25 ml of the elution butler and 25ml of 0.4M NaC1 with 60mM of the same buffer. Activities were determined by testing each fraction against trypsin and chymotrypsin, then the active fractions combined to four peaks. The first peak was designated peak 1 + 2 since the activity against trypsin was eluted slightly slower than the activity against ch3motrypsin, indicating separate anti-chymotrypsin and anti-trypsin peaks. Peaks 1 + 2 were eluted with the application buffer, peak 3 (the main anti-trypsin peak) with a salt gradient of 90 150 mM NaCI, peak 4 with 150 170mM NaCI and peak 5 with 195 290 mM NaC1. The respective peaks from the two runs were combined and the relative activity of granulocyte elastase, trypsin, and chymotrypsin inhibition determined in each fraction, based on l~g protease inhibited. Erpohdella punctata: Crude inhibitor from 10 leeches (15.4 g dehydrated leeches) containing 46 units elastase inhibitor was applied to the DE52 column in 30raM ammonium acetate, pH 6.4, without previous gel filtration. Five protease inhibitor peaks were obtained as described tk~r Nephelopsis, and total protease inhibitory capacity against trypsin, chymotrypsin and granulocyte elastase determined for each peak. Hemopis marmorata: Crude inhibitor from four leeches (26.5 g dehydrated leeches) was applied to Sephadex G75 in 50 mM Tris-HC1, pH 7.8 with 0.5 M NaCh The fractions with activity against trypsin and chymotrypsin were combined, concentrated and applied in 30 mM ammonium acetate, pH 6.4 to the DE52 column. Only one peak of protease inhibitor activity was obtained which eluted with 90 to 150 mM NaCI. Protein was determined by the Lowry method (Lowry et al., 1951). Samples were concentrated against Aquacide II (Calbiochem) in Ultraspec 6 dialysis tubing with cut off point of 1000 daltons. This tubing was also used when the crude leech material was dialyzed.
RESULTS Gel chromatography q f crude Nephelopsis inhibitors
A b a t c h of 56 g d e h y d r a t e d Nephelopsis leeches was p r e p a r e d by the m e t h o d of M a r k w a r d t (1970) a n d the crude inhibitors applied to a Sephadex G75 c o l u m n as described in Materials and Methods. The distribution of protease inhibitors against trypsin and c h y m o t r y p s i n in the eluate is given in Fig. 1. The active fractions were c o m b i n e d to three pooled fractions, a high molecular weight (H) fraction consisting of tubes 12-16, a m e d i u m molecular weight (M) fraction, tubes 17-19, a n d the low molecular weight (L) fraction, tubes 20-27. Each pooled fraction was
Leech serine protease inhibitors
119
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Fig. 1. Elution pattern of gel filtration of protease inhibitors from Nephelopsis obscura. A crude extract from 56 g leeches was applied to a Sephadex G75 column and eluted with 0.5 M NaC1 buffered to pH 7.8 with 50 mM Tris-HCl. Fractions of I ml were collected. Samples (100 pl) were tested against I unit trypsin or 0.04 unit chymotrypsin. Then fractions 12-16, fractions 17-19 and fractions 20-27 were pooled and 100 yl samples tested against 0.3 units granulocyte elastase. Solid lines represent trypsin inhibition, broken lines chymotrypsin inhibition and shaded area elastase inhibition. 100.
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Fig. 2. Elution pattern for anion exchange of fractions 17-19 from Fig. 1. The fractions were concentrated and equilibrated against 30 mM ammonium acetate and applied to a DE52 column in this buffer. Fractions of 1 ml were collected. After fraction No. 30 a salt gradient was applied, consisting of 25 ml of the application buffer and 25 ml 0.4 M NaC1 buffered to pH 6.4 with 60 mM ammonium acetate. Inhibition of 1 unit trypsin was tested with 50 pl samples before fraction 30 and with 100 pl samples from fraction 30 on. Inhibition of 0.04 units chymotrypsin was tested with 10 # 1samples. Dotted lines represent trypsin inhibition, broken lines chymotrypsin inhibition and shaded area protein concentration.
120
GOLDSTE1N et
ALLAN M.
tested for activity against granulocyte elastase. Fraction H contained the bulk of the anti-trypsin activity and showed no activity against granulocyte elastase. Fraction M was rich in both anti-trypsin and antichymotrypsin activity, and contained a moderate amount of granulocyte elastase activity. Fraction L contained the bulk of anti-chymotrypsin and antigranulocyte elastase activity. Fraction M was applied to a DE52 column and eluted with a salt gradient as described in Materials and Methods. The elution pattern as given in Fig. 2 presented five peaks of protease inhibitor activity, as described in Materials and Methods. The fraction which was eluted with the application buffer contained anti-chymotrypsin and anti-trypsin activity, with the anti-trypsin activity trailing the anti-chymotrypsin activity. We therefore designated the activities in two peaks, peak 1 with primarily anti-chymotrypsin activity, and peak 2 with primarily anti-trypsin activity. The first protease inhibitor peak appearing with the salt gradient contained the bulk of anti-trypsin activity from Fraction M, but no anti-chymotrypsin activity. With higher salt concentration appeared a peak (peak 4) with both anti-chymotrypsin and anti-trypsin activity, and with 0.2 M salt concentration above 0.2 M a small anti-chymotrypsin peak (peak 5). The material in Fraction L was also applied to the DE52 column and five peaks were obtained. Each peak from the two runs were combined, and the amount of protease inhibitor capacity against trypsin, chymotrypsin and granulocyte elastase determined in each combined peak. No activity was detected against pancreatic elastase. The results are tabulated in Table I. Peaks 1, 2 and 5 were rich in anti-elastase activity, with no anti-elastase in peak 3 and little in peak 4. The anti-elastase activity did therefore not correspond to the anti-chymotrypsin activity, which was prevalent in peak 4. Dissociation constant between granulocyte elastase and the peak 1 inhibitor was 9 x 10 to for the peak 5 inhibitor 2 x 10 ,7.
Effect of chymotrypsin on the inhibitory capaci O, of the anti-elastase Table 2 shows that inhibition of granulocyte elastase exerted by the compounds in peaks 1 + 2 could be prevented by preincubation of the inhibitor with a 10-30-fold excess of chymotrypsin, while chymotrypsin had no effect on the inhibition of elastase exerted by peak 5 compounds. This suggests that the anti-chymotrypsin and anti-elastase activities in peaks 1 and 2 reside in the same molecule, but that
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Table 2. Reversal of Chvmotrvpsin
Nephelopsis elastase inhibition by chymotrypsin % Elastase Peak i+2
(units)
0 (I. !~
b5 h]
77
1 4
~2 10
79 74
Protease m B Protein
77
A 100 ~1 sample of peak I + 2 or peak 5 was incubated for 5 rain with increasingconcentrations of chymotrypsin Then 0.17 unils granulocyte elastase was added, followed by incubation for 5 min more, and finally the elastase substrate was added, and development of color followed at 410 n m
the two inhibitory activities in peak 5 reside in different loci.
Gel chromatography ~?/ crude Erpobdella inhibitors Ten specimens of Erpobdella punctata (15 g) were prepared by the method of Markwardt (1970) and the crude inhibitors applied directly to a DE52 column and eluted with a salt gradient as described in Materials and Methods. The elution pattern as given in Fig. 3 was characterized by a lack of a distinct anti-trypsin peak (peak 2) in the material which eluted with the application buffer. Peak 1 contained both anti-chymotrypsin and anti-elastase activity, but in contrast to Nephelopsis the prevailing activity was against chymotrypsin. A strong anti-trypsin peak appeared at the site of peak 3 (110-170 mM NaC1). The anti-chymotrypsin did not present a sharp peak, but appeared roughly in the area of peak 4 (140-180 mM NaCI). A second peak of anti-chymotrypsin activity appeared at above 200 mM NaCI, thus representing peak 5 in the Nephelopsis pattern. Peak 5 contained anti-elastase, but again anti-chymotrypsin was the prevailing inhibitor activity (Table 3).
Gel chromatography of crude Hemopis inhibitors Four dehydrated Hemopis leeches were prepared by the method of Markwardt (1970) and the crude inhibitors applied to a Sephadex G75 column as described in Materials and Methods. The distribution of protease inhibitors against trypsin and chymotrypsin in the eluate is given in Fig. 4. The anti-trypsin and anti-chymotrypsin activity could not be separated by gel filtration. The active fractions were combined, concentrated and equilibrated against 30 mM ammonium acetate, pH 6.4, applied to a DE52 column and eluted with a salt gradient as described in Materials and Methods (Fig. 5). The entire anti-trypsin and anti-chymotrypsin activity was eluted in the area characteristic for Peak 3 in the elution pattern for
Table 1. Protease inhibitor capacity of the different peaks of the DE52 column for Nephelopsis material Peak
Inhibition Peak 5
Trypsin
Granulocyt e Elastaae
ChTmot ryps in
1+2
2.1
33
12,8
3.9
3 4 5
1.5 3.9 1.7
230 130 71
0 2.9 25.5
3.9 52.0 17.0
The peaks are given in Fig. 2, but the results involve fractions 17-27, while the results in the Figure only involve fractions 17 19 from the Sephadex G-75 column. Peaks I + 2 are fractions 6-11 from the ion exchange eluate, peak 3 fractions 43 52, peak 4 fractions 53-57 and peak 5 fractions 58-66. Numbers represent units of inhibitory capacity. One unit is the capacity to neutralize I ,ug protease.
Leech serine protease inhibitors
121
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Fig. 3. Elution pattern for anion exchange of protease inhibitors from Erpobdella punctata. A crude extract from 15.4g leeches contained 46 units anti-elastase activity and 15 mg protein. This sample was applied to a DE52 column and eluted with a salt gradient as described in the legend to Fig. 2. 1.4 ml fractions were collected. Samples (50 #1) were tested against chymotrypsin and 100 #1 samples against trypsin and granulocyte elastase. Symbols as described in legend to Fig. 2. Solid lines represent elastase inhibition.
Nephelopsis protease inhibitors (with peak at about 120 mM NaCI). No anti-elastase activity was detected in the preparation from Hemopis leeches. The inhibition of 40ng chymotrypsin was almost completely prevented by preincubation with 1 pg trypsin (Table 4). Protease inhibitor localization Nephelopsis and Hemopis leeches were divided into a frontal and a hind part (head and tail), and crude inhibitor mixes prepared by the method of Markwardt (1970). Table 5 shows that both chymotrypsin and trypsin inhibitors were abundant in both head and tail segments. The anti-trypsin capacity was about 20 x that of chymotrypsin in the hind part of Nephelopsis, and about 10 x that of chymotrypsin in the front part, while Hemopis had roughly equal inhibitor capacity against the two proteases in both front and hind parts (Table 5). DISCUSSION The previous studies of protease inhibitors from leeches (other than the thrombin inhibitor hirudin) have concentrated on the material obtained from the section of Hirudo medicinalis and Hementeria species
which contains the salivary glands. Thus protease inhibitors which appear in other parts of the body may have been overlooked or underestimated. A comparison between the results obtained by Seemuller et al. (1977) with our results involving extracts of whole leeches may therefore not be completely valid. However, we believe our findings will contribute significantly to our knowledge of the nature of the protease inhibitors in leeches. Our findings will also be helpful in showing that leeches available to researchers in this country contain protease inhibitors which in some cases are similar to those described from Hirudo medicinalis, and in other cases are unique. Seemuller et al. (1977) have previously described the elution pattern by gel filtration of protease inhibitors from Hirudo medicinalis. The material included a higher molecular weight fraction with protease inhibitor activity against trypsin, and a fraction with mol. wt between 11,000 and 5000 daltons, which contained activity against trypsin, chymotrypsin, thrombin and granulocyte elastase. When this material was applied to a DE52 column in 30 mM ammonium acetate and eluted with a salt gradient six peaks of protease inhibitor activities were obtained, the first two peaks (with activity against trypsin,
Table 3. Proteas¢ inhibitorycapacity of the different peaks from DE52 chromatography of Erpobdella material Peak 1+2
3 4 5
Protein (m~)
Trypsin
1.2 0.8 -0.4
0 47,2 0 0
GranuLocyte Elastase 4.5
0 0 1.3
Chymot rypsin S.0 I0.0 i,I 1.5
46 unitsErpobdellaelastaseinhibitorin 15mgwereappliedto a smallcolumn (20 x 1cm) of DE52 celluloseas seen in Fig. 3.
ALLAN M. GOLDSTE1Net al.
122
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Fig. 4. Elution pattern of gel filtration of protease inhibitors from Hemopis marmorata. A crude extract from 26.5 g leeches was applied to a Sephadex G75 column and eluted with 0.5 M NaC1 buffered to pH 7,8 with 50ram Tris HCI. Fractions 1 ml were collected. Samples (100/~1) were tested against 1 unit trypsin and 50 #1 samples against 0.04 unit chymotrypsin. Solid lines represent trypsin inhibition, broken lines chymotrypsin inhibition and shaded area protein concentration.
chymotrypsin and granulocyte elastase) eluted with the application buffer, the latter four peaks with increasing salt concentration. Peak 3 contained the bulk of anti-trypsin activity, peak 4 was active against trypsin and chymotrypsin, and peak 5 was active against trypsin, chymotrypsin and granulocyte elastase. Peak 6, which eluted with 0.4 M NaCl, contained hirudin, the specific thrombin inhibitor which is characteristic for Hirudo leeches. Using their methods we found five peaks from Nephelopsis which corresponded to the five first peaks of Seemuller et al. (1977), but a peak corresponding to the hirudin peak was not found in Nephelopsis, in agreement with the lack of inhibition against thrombin. Erpobdella leeches contained protease inhibitors which eluted at the same salt concentrations as the protease inhibitors from Nephelopsis, but the ratio between the different inhibitor activities in each peak was quite different from that found in Nephelopsis. In contrast the ratio of inhibitor activity in the different peaks described for Hirudo (Seemuller et al., 1977) seems to be rather similar to that found in Nephelopsis, although a direct correlation cannot be made both because the material collected by these workers was obtained from a narrow segment of the leech and because they did not provide a quantitation of their findings. The finding that chymotrypsin blocks the elastase inhibitor in peak 1 + 2, but not that in peak 5, suggests that peak 1 + 2 contain an elastase inhibitor with anti-chymotrypsin activity, while peak 5 contains one inhibitor which is specific for granulocyte elastase and another inhibitor against chymotrypsin. This would suggest that at least in
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Fig. 5. Elution pattern for anion exchange of fractions
22-28 from Fig. 4. The fractions (3mg protein) were concentrated and equilibrated against 30mM ammonium acetate and applied to a DE52 column in this buffer. Fractions of 1 ml were collected. After fraction No. 40 a salt gradient was applied, consisting of 25 ml of the application buffer and 25 ml 0.4 M NaC1 buffered to pH 6.4 with 60 mM ammonium acetate. Inhibition of 1 unit trypsin was tested with 100 ~1 samples. Inhibition of 0.04 units chymotrypsin was tested with 20/tl samples. Solid lines represent trypsin inhibition and broken lines chymotrypsin inhibition.
Leech serine protease inhibitors
123
Table 4. Reversal of Hemopis chymotrypsin inhibition by trypsin Chymotrypsln Added
Trypsin Adde_dd
Yes Yes
No Yes
No
Yes No
Yes (no inhibitor)
OD 410 Chanse 0.025 0.304 0.017 0.452
A 5 #g sample of crude Hemopisinhibitor was incubated 5 min at room temperature with or without 1/~g of trypsin, then for 5 min at 37°C with 40 ng chymotrypsin.
Table 5. Correlation between protease inhibition in head and tail portion of Nephelopsis and Hemopis leeches. Protease Source o f Inhibitor
Trypsin
Nephelopsis (I) Head Tail
13.1 12.8
6.3 18.8
Nephelopsis (II) Head Tail
28.0 16.9
9.9 11.6
Hemops i s Head Tail
8.2 10.4
0.8 1.3
Chymotrypsln
Two 20 g batches of dried Nephelopsisleeches and one 12.3g batch of dried Hemopis leeches were cut in half, and the head and tail end of the leeches extracted separately by the Markwardt method (1970). The crude protease inhibitor was tested for activity against 1 #g trypsin and 40 ng cbymotrypsin. Numbers given are #g crude extract protein needed to give 100% inhibition of protease activity. Results are mean of three determinations.
Nephelopsis there is one inhibitor t h a t is primarily
REFERENCES
anti-granulocyte elastase, one t h a t is primarily antic h y m o t r y p s i n a n d one which is a specific elastase inhibitor. In c o n t r a s t Erpobdella seems to contain one i n h i b i t o r which is mainly anti-chymotrypsin. The seco n d peak with anti-elastase activity contains material which m a y be similar to t h a t f o u n d in peak 5 for
Budzynski A. Z., Olexa S. A. and Sawyer R. T. (1981a) Composition of salivary gland extracts from the leech
Nephelopsis. The protease inhibitor from Hemopis is unique in being evidently a single inhibitor species with activity against b o t h trypsin a n d chymotrypsin, but lacking inhibition against granulocyte elastase. We suggest therefore t h a t Hemopis contains a single inhibitor species with activity against b o t h trypsin a n d chymotrypsin, a n d w i t h o u t activity against elastase. O u r findings suggest t h a t the protease inhibitors f o u n d in Nephelopsis obscura a n d Erpobdella punctata are qualitatively similar to those described in Hirudo medicinalis, but t h a t the serine protease inhibitor from Hemopis marmorata is distinctly different, alt h o u g h Hemopis a n d Hirudo are classified in one s u b o r d e r G n a t h o b d e l l a e a n d Erpobdellae a n d Nephelopsis in a separate suborder, Pharyngobdellae. Nephelopsis m a y be a useful source for b o t h eglintype granulocyte elastase/chymotrypsin inhibitors a n d low-molecular-weight inhibitors with great specificity towards elastase.
Acknowledgements--We are grateful to Dr Donald J. Klemm of the USEPA for the identification of leech species. The work was supported by NRS fellowship HL06703 (E.H.M.), BRSG RR05874 and a grant from Hoffmann-La Roche Inc., Nutley, NJ. Allan Goldstein was a recipient of a 1985 Westinghouse Fellowship for work reported here.
Haementeria ghilianii. Proc Soc. exp. Biol. Med. 168, 259-265. Budzynski A. Z., Olexa S. A., Brizuela B. Z., Sawyer R. T. and Stent G. S. (1981b) Anticoagulant and fibrinolytic properties of salivary gland proteins from the leech
Haementeria ghilianii. Proc. Soc. exp. Biol. Med. 168, 266-275. DNR Report No. 52, Minnesota Department of Natural Resources, 1979. Fritz H. and Krejci K. (1976) Trypsin-plasmin inhibitors (bdellins) from leeches. Meth. Enzymol. 45, 797-805. Green N. M. and Work E. (1952) Pancreatic trypsin inhibitor. 2. Reaction with trypsin. Biochem. J. 54, 347-352. Jochum M., Duswald K.-H., Neumann S., Witte J., Fritz H. and Seemuller U. (1983) Proteinases and their inhibitors in inflammation: Basic concepts and clinical implications. In Proteinase Inhibitors: Medical and Biological Aspects (Edited by Katunuma N., Umezama H. and Holzer H.), pp, 85-95. Japan Sci. Soc. Press; Springer Verlag, Tokyo-Berlin. Klemm D. J. (1982) Leeches (Annelida: Hirudinea of North America.) US Department of Commerce National Technical Information Service 1982; PB 82-208679: 1-177. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Markwardt F. (1970) Hirudin as an inhibitor of thrombin. Meth. Enzymol. 19, 924-932. Murer E. H. and Weinbaum G. (1984) Purification of an inhibitor of human granulocyte elastase from Ascaris
lumbricoides suum. Fedn. Proc. Fedn. Am. Socs. exp. Biol. 43, 1510. Murer E. H., James H. L., Budzynski A. Z., Malinconico S. M. and Gasic G. J. (1984) Protease inhibitors in
124
ALLAN M. GOLDSTEIN et al.
Haementeria leech species. Thromb. Haemostas. 51, 24-26. Peanasky R. J., Bentz Y., Paulson B., Graham D. L. and Babin D. R. (1984) The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: isolation by affinity chromatography and association with the enzyme. Arch. Biochem. 232, 127-134. Seemuller U., Meier M., Ohlsson K., Muller H.-P. and Fritz H. (1977) Isolation and characterization of a low molecular weight inhibitor (of chymotrypsin and human gran-
ulocyte elastase and cathepsin G) from leeches. HoppeSeyler's Z. Physiol. Chem. 358, 1105-1117. Shotton D. M. (1976) Elastase. Meth. Enzymol. 45, 113-140. Sloan B., Abrams W. R., Maranze D. R., Kimbel P. and Weinbaum G. (1981) Emphysema induced in z~itro and in vivo in dogs by a purified elastase from homologous leukocytes. Am. Rev. Resp. Dis. 124, 295-301. Stone P. J. (1983) The elastase-antielastase hypothesis of the pathogenesis of emphysema. Clin. Chest Med. 4, 405-412.
0305-0491/86 $3.00+ 0.00 © 1986 Pergamon Press Ltd
SERINE PROTEASE INHIBITORS OF NORTH AMERICAN LEECHES ALLAN M. GOLDSTEIN, ERIK H. MURER and GEORGE WEINBAUM Research Division, Department of Medicine, The Graduate Hospital, Philadelphia, PA 19146, USA (Tel.: 215-893-7628)
(Received 19 August 1985)
Abstract--1. Serine protease inhibitors in extracts from three North American leeches, Nephelopsis obscura, Erpobdella punctata and Hemopis marmorata have been separated by anion exchange chromatography and the activity pattern against human granulocyte elastase and porcine chymotrypsin and trypsin determined. 2. All three leech species contained a major peak with anti-trypsin activity, but Hemopis was unique in that the trypsin inhibitor was equally active against chymotrypsin. 3. Nephelopsis was rich in anti-elastase activity of two types, one which was also active against chymotrypsin, and one which was a specific elastase inhibitor. 4. Erpobdella contained inhibitors against elastase and chymotrypsin but with major activity against the latter.
INTRODUCTION
of protease inhibitors of granulocyte elastase. The interest in granulocyte elastase inhibitors is related to the possible utilization of such inhibitors therapeutically for the arrest or prevention of emphysema (Stone, 1983).
Since the demonstration that the anticoagulant of Hirudo medicinalis was a specific thrombin inhibitor (Markwardt, 1970) the serine protease inhibitors of this leech have been the subject of extensive studies (Fritz and Krejci, 1976; Seemuller et al., 1977; Jochum et al., 1983). Four different types of protease inhibitors have been characterized, (a) hirudin (Markwardt, 1970) with specificity against thrombin and trypsin, (b) bdellin, a low-molecular-weight protein with inhibitory activity against trypsin and plasmin (Fritz and Krejci, 1976), (c) a highmolecular-weight protein with inhibitory activity against trypsin (Seemuller et al., 1977), and (d) eglins, two low-molecular-weight polypeptides with inhibitory activity against chymotrypsin, granulocyte elastase, subtilisin and cathepsin G (Seemuller et al., 1977). The interest in the protease inhibitors of H, medicinalis has not caused analysis of protease inhibitors from other types of leeches. One exception is the South American bloodsucker Haementeria ghilianii, which was shown by Budzynski et al. (1981a, b) to contain an enzyme in its salivary glands that degraded fibrin and fibrinogen, thus preventing coagulation of the host's blood. This leech did not contain a hirudin-type protease inhibitor. Murer et al. (1984) showed that salivary gland extracts from H. ghilianff and the related species H. officinalis contained inhibitors of trypsin, chymotrypsin, plasmin and granulocyte elastase. One result of the interest in the protease inhibitors of Hirudo medicinalis is that it is now difficult to obtain this leech in large quantities. We have therefore undertaken a study of protease inhibitors from leeches which are available in North America, one of the most available being the "bait leech" Nephelopsis obscura. Our primary aim has been to determine whether any of these leeches may be a good source
MATERIALS AND METHODS
Source of inhibitors Leeches were obtained from bait stores in Minnesota. Nephelopsis obscura (the ribbon leech) is the primary species sold at bait stores. It is distinguished from other leeches by behavior (it will stay in a bucket half full with water, while the horse leech Hemopis will climb out.) Nephelopsis prefers shallow, stagnant water and is bred commercially in ponds (DNR Report 1979). Erpobdella punctata (the dog leech) was found occasionally among the purchased Nephelopsis leeches (I0 Erpobdella leeches in 1 lb Nephelopsis leeches). Hemopis marmorata (the horse leech) is discarded by the bait salesmen, but we were able to obtain four big specimens, which were classified by family and used for protease inhibitor preparation. The leeches were identified by Dr D. J. Klemm of the Environmental Protection Agency, Cincinnati, OH (Klemm, 1982). Preparation of leech material (a) Leeches were prepared by the method used by Markwardt for Hirudo medicinalis (Markwardt, 1970). The leeches were fixed twice for 24 hr each in 95% ethanol, then cut into 0.5-1 cm wide slices. The sliced tissue was extracted twice for 30 min each with 10 volumes of 40% aqueous acetone, followed by centrifugation at 500 g for 5 min. The extract was diluted with 1/2 volume 80% acetone (pH 4.3-4.5) and the formed precipitate removed by centrifugation. This precipitate was inactive as a protease inhibitor. The pH of the supernatant was readjusted to 6 with dilute ammonia, and the acetone removed under vacuum. The extract volume was reduced 90% by freeze drying and acidified to pH 2-2.5 with 20% trichloroacetic acid. Nine volumes of pure acetone was added, and the formed precipitate collected by centrifugation. In many preparations heavy drops of active material appeared below the acetone layer, dark brown of color. These were collected with a 117
118
ALLAN M. GOLDSTEIN et al.
Pasteur pipette. The active material was dialysed against water using a membrane with a mol. wt cut-off of 1000 daltons (crude extract). The crude extract was dialyzed against the equilibrium buffer for application to chromatography columns. Proteases
Chymotrypsin a (3 x chrystallized) and porcine trypsin were purchased from Sigma Chemical Company, St. Louis, MO. Porcine trypsin type lI from Sigma was used to prepare purified pancreatic elastase by the method of Shotton (1976). The preparation was free from trypsin activity and had less than 1% chymotrypsin. Granulocyte elastase was prepared from human leukocytes. Human peripheral blood was collected by venipuncture, anticoagulated with EDTA and separated by dextran sedimentation and 30 sec lysis of red blood cells with distilled water (Sloan et al., 1981). The isolated neutrophils ( > 80% pure) were frozen and thawed 3 times in 0.l% Triton X100 in water or saline. The pellets were sedimented by centrifugation at 18,000g in an Eppendorf centrifuge and extracted with 0.96 M NaCI buffered to pH 8.3 with 0.1 M Tris HCI. The pellet from this high salt extraction was discarded, while the supernatant was used directly as soluble granulocyte elastase. Protease substrates. The substrate for chymotrypsin was Meo-Suc-Arg-Pro-Tyr-paranitroanilide (pNA) (Kabi substrate S-2586), Helena Laboratories, Beaumont, TX and for granulocyte elastase L-pyroglutamyl-Pro-Val-pNA (Kabi substrate S-2484), also from Helena Laboratories, Beaumont, TX. For determination of the binding constant was used Bzoxy-Suc-Ala-Ala-Pro-Vat-pNA (MAPVA) (Sigma Chemical Co., St. Louis, MO). Substrate for pancreatic elastase was Suc-Ala-Ala-Ala-pNA (SAPNA) (Peptide Research Institute, Osaka, Japan) and for trypsin benzoylarginyl-pNA (BAPNA) (Sigma Chemical Co). Protease assays
Reaction mixtures for measurement of protease activity were: (a) chymotrypsin, 425/d 0.1 M Tris-HC1, pH 8.3, containing 0.96 M NaC1 + 100/~1 water or protease inhibitor + 50 #1 chymotrypsin (0.8/~g/ml) + 25 #1 S-2586 (2.4mM in water); (b) pancreatic elastase, 600/d 0.2M Tris-HC1, pH 8.0 + 100 #1 water or protease inhibitor+2.5#1 elastase (0.4mg/ml)+ 100#1 SAPNA (2mg/ml in 0.2M Tris-HC1, pH 8.0); (c) trypsin, 680#1 0.1 M Tris-HCl, pH 8.0, containing 10 mM CaC12 + 100 #1 water or protease inhibitor+ 10#1 trypsin (0.1 mg/ml)+ 10 #I BAPNA (0.1 M in DMSO); (d) granulocyte elastase, 465#1 0.1M Tris-HC1, pH 8.3, containing 0.96M NaC1 + 100/~1 water or protease inhibitor + 5/~1 elastase (equivalent to 0.154).25 #g pancreatic elastase) +30/~1 S-2484 (16 mM in DMSO). For the binding studies 10#1 MAPVA (25 mM in DMSO) was used as substrate. Binding studies were performed with 3 concentrations of enzyme (0.1,0.2 and 0.4 units per assay), using the method described by Green and Work (1952). Protease activity for any of the enzymes described above was measured as an increase of absorbance at 410nm caused by release of paranitroaniline from the chromogenic substrate. The rate of change in absorbance was measured spectrophotometrically in a Zeiss model M4QIII at room temperature. In routine studies the reaction mixture was incubated at 37°C, then 0.2 ml 50% glacial acetic acid was added at various times and the absorbance read at 410 nm (Murer et al., 1984). The unit of protease inhibitory activity is calculated as/~g of protease inhibited 100% and specific activity as units per mg protease inhibitor protein. Units of granulocyte elastase activity were determined on the basis of a titration against purified elastase inhibitor from Ascaris lumbricoides (Peanasky et al., 1984; Murer and Weinbaum, 1984). The Ascaris inhibitor is active against both granulocyte and pancreatic elastase, and its inhibitory capacity
was determined against purified porcine pancreatic elastase (prepared by E. Murer). Column chromatography The chromatography methods described by Seemuller et al. (1977) were followed with minor modifications. Nephelopsis obseura: Crude inhibitor from 56 g dehydrated leeches (95 mg protein with 250 units chymotrypsin inhibitor) was applied to a 40 x 1.6cm Sephadex G-75 column in 0.5M NaC1 buffered to pH 7.8 with 50mM Tris HC1 and eluted with the same buffer. All column fractions were assayed for protease inhibitor activity against trypsin and chymotrypsin. The fractions with activity against chymotrypsin (fractions 12 27) were collecled. The latter ~ of the active fractions (corresponding to a molecular weight below 18,000 dalton) were combined in two fractions (the early fraction with tubes 17-19 and the late fraction with tubes 20-27). These fractions were concentrated against Aquacide 1I and each applied to a 20 × 1 cm DE52 column and eluted with 30 mM ammonium acetate of ptt 6.4 followed by a gradient of 25 ml of the elution butler and 25ml of 0.4M NaC1 with 60mM of the same buffer. Activities were determined by testing each fraction against trypsin and chymotrypsin, then the active fractions combined to four peaks. The first peak was designated peak 1 + 2 since the activity against trypsin was eluted slightly slower than the activity against ch3motrypsin, indicating separate anti-chymotrypsin and anti-trypsin peaks. Peaks 1 + 2 were eluted with the application buffer, peak 3 (the main anti-trypsin peak) with a salt gradient of 90 150 mM NaCI, peak 4 with 150 170mM NaCI and peak 5 with 195 290 mM NaC1. The respective peaks from the two runs were combined and the relative activity of granulocyte elastase, trypsin, and chymotrypsin inhibition determined in each fraction, based on l~g protease inhibited. Erpohdella punctata: Crude inhibitor from 10 leeches (15.4 g dehydrated leeches) containing 46 units elastase inhibitor was applied to the DE52 column in 30raM ammonium acetate, pH 6.4, without previous gel filtration. Five protease inhibitor peaks were obtained as described tk~r Nephelopsis, and total protease inhibitory capacity against trypsin, chymotrypsin and granulocyte elastase determined for each peak. Hemopis marmorata: Crude inhibitor from four leeches (26.5 g dehydrated leeches) was applied to Sephadex G75 in 50 mM Tris-HC1, pH 7.8 with 0.5 M NaCh The fractions with activity against trypsin and chymotrypsin were combined, concentrated and applied in 30 mM ammonium acetate, pH 6.4 to the DE52 column. Only one peak of protease inhibitor activity was obtained which eluted with 90 to 150 mM NaCI. Protein was determined by the Lowry method (Lowry et al., 1951). Samples were concentrated against Aquacide II (Calbiochem) in Ultraspec 6 dialysis tubing with cut off point of 1000 daltons. This tubing was also used when the crude leech material was dialyzed.
RESULTS Gel chromatography q f crude Nephelopsis inhibitors
A b a t c h of 56 g d e h y d r a t e d Nephelopsis leeches was p r e p a r e d by the m e t h o d of M a r k w a r d t (1970) a n d the crude inhibitors applied to a Sephadex G75 c o l u m n as described in Materials and Methods. The distribution of protease inhibitors against trypsin and c h y m o t r y p s i n in the eluate is given in Fig. 1. The active fractions were c o m b i n e d to three pooled fractions, a high molecular weight (H) fraction consisting of tubes 12-16, a m e d i u m molecular weight (M) fraction, tubes 17-19, a n d the low molecular weight (L) fraction, tubes 20-27. Each pooled fraction was
Leech serine protease inhibitors
119
Chymo
Trypsin
I
' 80 I I I I | I |
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40
I I
o
--I
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Fraction
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t
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Fig. 1. Elution pattern of gel filtration of protease inhibitors from Nephelopsis obscura. A crude extract from 56 g leeches was applied to a Sephadex G75 column and eluted with 0.5 M NaC1 buffered to pH 7.8 with 50 mM Tris-HCl. Fractions of I ml were collected. Samples (100 pl) were tested against I unit trypsin or 0.04 unit chymotrypsin. Then fractions 12-16, fractions 17-19 and fractions 20-27 were pooled and 100 yl samples tested against 0.3 units granulocyte elastase. Solid lines represent trypsin inhibition, broken lines chymotrypsin inhibition and shaded area elastase inhibition. 100.
IV
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Fig. 2. Elution pattern for anion exchange of fractions 17-19 from Fig. 1. The fractions were concentrated and equilibrated against 30 mM ammonium acetate and applied to a DE52 column in this buffer. Fractions of 1 ml were collected. After fraction No. 30 a salt gradient was applied, consisting of 25 ml of the application buffer and 25 ml 0.4 M NaC1 buffered to pH 6.4 with 60 mM ammonium acetate. Inhibition of 1 unit trypsin was tested with 50 pl samples before fraction 30 and with 100 pl samples from fraction 30 on. Inhibition of 0.04 units chymotrypsin was tested with 10 # 1samples. Dotted lines represent trypsin inhibition, broken lines chymotrypsin inhibition and shaded area protein concentration.
120
GOLDSTE1N et
ALLAN M.
tested for activity against granulocyte elastase. Fraction H contained the bulk of the anti-trypsin activity and showed no activity against granulocyte elastase. Fraction M was rich in both anti-trypsin and antichymotrypsin activity, and contained a moderate amount of granulocyte elastase activity. Fraction L contained the bulk of anti-chymotrypsin and antigranulocyte elastase activity. Fraction M was applied to a DE52 column and eluted with a salt gradient as described in Materials and Methods. The elution pattern as given in Fig. 2 presented five peaks of protease inhibitor activity, as described in Materials and Methods. The fraction which was eluted with the application buffer contained anti-chymotrypsin and anti-trypsin activity, with the anti-trypsin activity trailing the anti-chymotrypsin activity. We therefore designated the activities in two peaks, peak 1 with primarily anti-chymotrypsin activity, and peak 2 with primarily anti-trypsin activity. The first protease inhibitor peak appearing with the salt gradient contained the bulk of anti-trypsin activity from Fraction M, but no anti-chymotrypsin activity. With higher salt concentration appeared a peak (peak 4) with both anti-chymotrypsin and anti-trypsin activity, and with 0.2 M salt concentration above 0.2 M a small anti-chymotrypsin peak (peak 5). The material in Fraction L was also applied to the DE52 column and five peaks were obtained. Each peak from the two runs were combined, and the amount of protease inhibitor capacity against trypsin, chymotrypsin and granulocyte elastase determined in each combined peak. No activity was detected against pancreatic elastase. The results are tabulated in Table I. Peaks 1, 2 and 5 were rich in anti-elastase activity, with no anti-elastase in peak 3 and little in peak 4. The anti-elastase activity did therefore not correspond to the anti-chymotrypsin activity, which was prevalent in peak 4. Dissociation constant between granulocyte elastase and the peak 1 inhibitor was 9 x 10 to for the peak 5 inhibitor 2 x 10 ,7.
Effect of chymotrypsin on the inhibitory capaci O, of the anti-elastase Table 2 shows that inhibition of granulocyte elastase exerted by the compounds in peaks 1 + 2 could be prevented by preincubation of the inhibitor with a 10-30-fold excess of chymotrypsin, while chymotrypsin had no effect on the inhibition of elastase exerted by peak 5 compounds. This suggests that the anti-chymotrypsin and anti-elastase activities in peaks 1 and 2 reside in the same molecule, but that
al.
Table 2. Reversal of Chvmotrvpsin
Nephelopsis elastase inhibition by chymotrypsin % Elastase Peak i+2
(units)
0 (I. !~
b5 h]
77
1 4
~2 10
79 74
Protease m B Protein
77
A 100 ~1 sample of peak I + 2 or peak 5 was incubated for 5 rain with increasingconcentrations of chymotrypsin Then 0.17 unils granulocyte elastase was added, followed by incubation for 5 min more, and finally the elastase substrate was added, and development of color followed at 410 n m
the two inhibitory activities in peak 5 reside in different loci.
Gel chromatography ~?/ crude Erpobdella inhibitors Ten specimens of Erpobdella punctata (15 g) were prepared by the method of Markwardt (1970) and the crude inhibitors applied directly to a DE52 column and eluted with a salt gradient as described in Materials and Methods. The elution pattern as given in Fig. 3 was characterized by a lack of a distinct anti-trypsin peak (peak 2) in the material which eluted with the application buffer. Peak 1 contained both anti-chymotrypsin and anti-elastase activity, but in contrast to Nephelopsis the prevailing activity was against chymotrypsin. A strong anti-trypsin peak appeared at the site of peak 3 (110-170 mM NaC1). The anti-chymotrypsin did not present a sharp peak, but appeared roughly in the area of peak 4 (140-180 mM NaCI). A second peak of anti-chymotrypsin activity appeared at above 200 mM NaCI, thus representing peak 5 in the Nephelopsis pattern. Peak 5 contained anti-elastase, but again anti-chymotrypsin was the prevailing inhibitor activity (Table 3).
Gel chromatography of crude Hemopis inhibitors Four dehydrated Hemopis leeches were prepared by the method of Markwardt (1970) and the crude inhibitors applied to a Sephadex G75 column as described in Materials and Methods. The distribution of protease inhibitors against trypsin and chymotrypsin in the eluate is given in Fig. 4. The anti-trypsin and anti-chymotrypsin activity could not be separated by gel filtration. The active fractions were combined, concentrated and equilibrated against 30 mM ammonium acetate, pH 6.4, applied to a DE52 column and eluted with a salt gradient as described in Materials and Methods (Fig. 5). The entire anti-trypsin and anti-chymotrypsin activity was eluted in the area characteristic for Peak 3 in the elution pattern for
Table 1. Protease inhibitor capacity of the different peaks of the DE52 column for Nephelopsis material Peak
Inhibition Peak 5
Trypsin
Granulocyt e Elastaae
ChTmot ryps in
1+2
2.1
33
12,8
3.9
3 4 5
1.5 3.9 1.7
230 130 71
0 2.9 25.5
3.9 52.0 17.0
The peaks are given in Fig. 2, but the results involve fractions 17-27, while the results in the Figure only involve fractions 17 19 from the Sephadex G-75 column. Peaks I + 2 are fractions 6-11 from the ion exchange eluate, peak 3 fractions 43 52, peak 4 fractions 53-57 and peak 5 fractions 58-66. Numbers represent units of inhibitory capacity. One unit is the capacity to neutralize I ,ug protease.
Leech serine protease inhibitors
121
| | il
| I |
//7 400
ii
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li'
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30
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Fig. 3. Elution pattern for anion exchange of protease inhibitors from Erpobdella punctata. A crude extract from 15.4g leeches contained 46 units anti-elastase activity and 15 mg protein. This sample was applied to a DE52 column and eluted with a salt gradient as described in the legend to Fig. 2. 1.4 ml fractions were collected. Samples (50 #1) were tested against chymotrypsin and 100 #1 samples against trypsin and granulocyte elastase. Symbols as described in legend to Fig. 2. Solid lines represent elastase inhibition.
Nephelopsis protease inhibitors (with peak at about 120 mM NaCI). No anti-elastase activity was detected in the preparation from Hemopis leeches. The inhibition of 40ng chymotrypsin was almost completely prevented by preincubation with 1 pg trypsin (Table 4). Protease inhibitor localization Nephelopsis and Hemopis leeches were divided into a frontal and a hind part (head and tail), and crude inhibitor mixes prepared by the method of Markwardt (1970). Table 5 shows that both chymotrypsin and trypsin inhibitors were abundant in both head and tail segments. The anti-trypsin capacity was about 20 x that of chymotrypsin in the hind part of Nephelopsis, and about 10 x that of chymotrypsin in the front part, while Hemopis had roughly equal inhibitor capacity against the two proteases in both front and hind parts (Table 5). DISCUSSION The previous studies of protease inhibitors from leeches (other than the thrombin inhibitor hirudin) have concentrated on the material obtained from the section of Hirudo medicinalis and Hementeria species
which contains the salivary glands. Thus protease inhibitors which appear in other parts of the body may have been overlooked or underestimated. A comparison between the results obtained by Seemuller et al. (1977) with our results involving extracts of whole leeches may therefore not be completely valid. However, we believe our findings will contribute significantly to our knowledge of the nature of the protease inhibitors in leeches. Our findings will also be helpful in showing that leeches available to researchers in this country contain protease inhibitors which in some cases are similar to those described from Hirudo medicinalis, and in other cases are unique. Seemuller et al. (1977) have previously described the elution pattern by gel filtration of protease inhibitors from Hirudo medicinalis. The material included a higher molecular weight fraction with protease inhibitor activity against trypsin, and a fraction with mol. wt between 11,000 and 5000 daltons, which contained activity against trypsin, chymotrypsin, thrombin and granulocyte elastase. When this material was applied to a DE52 column in 30 mM ammonium acetate and eluted with a salt gradient six peaks of protease inhibitor activities were obtained, the first two peaks (with activity against trypsin,
Table 3. Proteas¢ inhibitorycapacity of the different peaks from DE52 chromatography of Erpobdella material Peak 1+2
3 4 5
Protein (m~)
Trypsin
1.2 0.8 -0.4
0 47,2 0 0
GranuLocyte Elastase 4.5
0 0 1.3
Chymot rypsin S.0 I0.0 i,I 1.5
46 unitsErpobdellaelastaseinhibitorin 15mgwereappliedto a smallcolumn (20 x 1cm) of DE52 celluloseas seen in Fig. 3.
ALLAN M. GOLDSTE1Net al.
122
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Fig. 4. Elution pattern of gel filtration of protease inhibitors from Hemopis marmorata. A crude extract from 26.5 g leeches was applied to a Sephadex G75 column and eluted with 0.5 M NaC1 buffered to pH 7,8 with 50ram Tris HCI. Fractions 1 ml were collected. Samples (100/~1) were tested against 1 unit trypsin and 50 #1 samples against 0.04 unit chymotrypsin. Solid lines represent trypsin inhibition, broken lines chymotrypsin inhibition and shaded area protein concentration.
chymotrypsin and granulocyte elastase) eluted with the application buffer, the latter four peaks with increasing salt concentration. Peak 3 contained the bulk of anti-trypsin activity, peak 4 was active against trypsin and chymotrypsin, and peak 5 was active against trypsin, chymotrypsin and granulocyte elastase. Peak 6, which eluted with 0.4 M NaCl, contained hirudin, the specific thrombin inhibitor which is characteristic for Hirudo leeches. Using their methods we found five peaks from Nephelopsis which corresponded to the five first peaks of Seemuller et al. (1977), but a peak corresponding to the hirudin peak was not found in Nephelopsis, in agreement with the lack of inhibition against thrombin. Erpobdella leeches contained protease inhibitors which eluted at the same salt concentrations as the protease inhibitors from Nephelopsis, but the ratio between the different inhibitor activities in each peak was quite different from that found in Nephelopsis. In contrast the ratio of inhibitor activity in the different peaks described for Hirudo (Seemuller et al., 1977) seems to be rather similar to that found in Nephelopsis, although a direct correlation cannot be made both because the material collected by these workers was obtained from a narrow segment of the leech and because they did not provide a quantitation of their findings. The finding that chymotrypsin blocks the elastase inhibitor in peak 1 + 2, but not that in peak 5, suggests that peak 1 + 2 contain an elastase inhibitor with anti-chymotrypsin activity, while peak 5 contains one inhibitor which is specific for granulocyte elastase and another inhibitor against chymotrypsin. This would suggest that at least in
too.
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ao-
\
60-
,. .o 40. ,,",a 20 ® o " 50
60
70
Gradient
Fig. 5. Elution pattern for anion exchange of fractions
22-28 from Fig. 4. The fractions (3mg protein) were concentrated and equilibrated against 30mM ammonium acetate and applied to a DE52 column in this buffer. Fractions of 1 ml were collected. After fraction No. 40 a salt gradient was applied, consisting of 25 ml of the application buffer and 25 ml 0.4 M NaC1 buffered to pH 6.4 with 60 mM ammonium acetate. Inhibition of 1 unit trypsin was tested with 100 ~1 samples. Inhibition of 0.04 units chymotrypsin was tested with 20/tl samples. Solid lines represent trypsin inhibition and broken lines chymotrypsin inhibition.
Leech serine protease inhibitors
123
Table 4. Reversal of Hemopis chymotrypsin inhibition by trypsin Chymotrypsln Added
Trypsin Adde_dd
Yes Yes
No Yes
No
Yes No
Yes (no inhibitor)
OD 410 Chanse 0.025 0.304 0.017 0.452
A 5 #g sample of crude Hemopisinhibitor was incubated 5 min at room temperature with or without 1/~g of trypsin, then for 5 min at 37°C with 40 ng chymotrypsin.
Table 5. Correlation between protease inhibition in head and tail portion of Nephelopsis and Hemopis leeches. Protease Source o f Inhibitor
Trypsin
Nephelopsis (I) Head Tail
13.1 12.8
6.3 18.8
Nephelopsis (II) Head Tail
28.0 16.9
9.9 11.6
Hemops i s Head Tail
8.2 10.4
0.8 1.3
Chymotrypsln
Two 20 g batches of dried Nephelopsisleeches and one 12.3g batch of dried Hemopis leeches were cut in half, and the head and tail end of the leeches extracted separately by the Markwardt method (1970). The crude protease inhibitor was tested for activity against 1 #g trypsin and 40 ng cbymotrypsin. Numbers given are #g crude extract protein needed to give 100% inhibition of protease activity. Results are mean of three determinations.
Nephelopsis there is one inhibitor t h a t is primarily
REFERENCES
anti-granulocyte elastase, one t h a t is primarily antic h y m o t r y p s i n a n d one which is a specific elastase inhibitor. In c o n t r a s t Erpobdella seems to contain one i n h i b i t o r which is mainly anti-chymotrypsin. The seco n d peak with anti-elastase activity contains material which m a y be similar to t h a t f o u n d in peak 5 for
Budzynski A. Z., Olexa S. A. and Sawyer R. T. (1981a) Composition of salivary gland extracts from the leech
Nephelopsis. The protease inhibitor from Hemopis is unique in being evidently a single inhibitor species with activity against b o t h trypsin a n d chymotrypsin, but lacking inhibition against granulocyte elastase. We suggest therefore t h a t Hemopis contains a single inhibitor species with activity against b o t h trypsin a n d chymotrypsin, a n d w i t h o u t activity against elastase. O u r findings suggest t h a t the protease inhibitors f o u n d in Nephelopsis obscura a n d Erpobdella punctata are qualitatively similar to those described in Hirudo medicinalis, but t h a t the serine protease inhibitor from Hemopis marmorata is distinctly different, alt h o u g h Hemopis a n d Hirudo are classified in one s u b o r d e r G n a t h o b d e l l a e a n d Erpobdellae a n d Nephelopsis in a separate suborder, Pharyngobdellae. Nephelopsis m a y be a useful source for b o t h eglintype granulocyte elastase/chymotrypsin inhibitors a n d low-molecular-weight inhibitors with great specificity towards elastase.
Acknowledgements--We are grateful to Dr Donald J. Klemm of the USEPA for the identification of leech species. The work was supported by NRS fellowship HL06703 (E.H.M.), BRSG RR05874 and a grant from Hoffmann-La Roche Inc., Nutley, NJ. Allan Goldstein was a recipient of a 1985 Westinghouse Fellowship for work reported here.
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