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Protease inhibitors in plasma of the softshell clam Mya arenaria: identification and effects of disseminated sarcoma
Comparative Biochemistry and Physiology Part B 123 (1999) 427 – 435 www.elsevier.com/locate/cbpb
Protease inhibitors in plasma of the softshell clam Mya arenaria: identification and effects of disseminated sarcoma Ehab E. Elsayed a,1, Shawn M. McLaughlin b, Mohamed Faisal a,* a
Department of En6ironmental Science, Virginia Institute of Marine Science/School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062, USA b National Oceanic and Atmospheric Administration, National Ocean Ser6ice, Center for Coastal En6ironmental Health and Biomolecular Research, Oxford, MD 21654, USA Received 8 January 1999; received in revised form 11 May 1999; accepted 25 May 1999
Abstract Despite the ecologic and economic significance of the softshell clam (Mya arenaria), little is known about the humoral factors involved in its host defense mechanisms. Protease inhibitors, a group of proteins believed to play a role in host defense mechanisms against infections and proliferative diseases, have recently been identified in bivalve molluscs. In the present study we provide evidence for the presence of protease inhibitors in softshell clam plasma. Levels of protease inhibitory activities against the enzymes tested varied greatly, e.g. 1 mg of plasma protein inhibited 35.39 9.69 ng pepsin (aspartic protease), 4.99 1.45 ng papain (cysteine protease) and 3.1 90.88 ng trypsin (serine protease). On the contrary, the level of anti-metalloprotease (thermolysin) activities was much lower. The sensitivity to methylamine and the ability to protect trypsin from active site trypsin inhibitors provided evidence for the presence of an a2-macroglobulin-like molecule in softshell clam plasma. In the Chesapeake Bay widespread epizootics of disseminated sarcoma have been described in M. arenaria populations. The impact of this lethal proliferative disorder on clam defense responses has received little attention. In this study the effects of sarcoma progression on plasma protease inhibitory activities were, therefore, assessed. Clams with early stages of sarcoma showed a non-significant decrease in protease inhibitor levels. Clams with advanced stages of sarcoma showed a significant decrease in the ability to inhibit trypsin and papain, while the protease inhibitory activity levels against aspartic and metalloprotease were completely exhausted. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Softshell clam; Protease inhibitors; Sarcoma; Proteases; a2-Macroglobulin
1. Introduction The softshell clam (Mya arenaria) is an important molluscan filter feeder in the Chesapeake Bay and along the northern Atlantic coast of the USA. Despite the ecologic and economic significance of the softshell clam, little is known about its host defense mechanisms especially humoral factors. This lack of knowledge undermines the ability to respond to increasing num
Disclaimer: reference to trade names does not imply endorsement by NOAA. * Corresponding author. Tel.: + 1-804-684-7231; fax: + 1-804-6846186. E-mail address: [email protected] (M. Faisal) 1 Present address: Department of Fish Medicine and Management, College of Veterinary Medicine, Cairo University, Giza, Egypt.
bers of reports on diseases and parasites affecting this species [6,13,15,21,22]. Protease inhibitors are a group of humoral factors found in several animal species [10,12,26] that neutralize unwanted proteases including those of microbial origin and maintain internal homeostasis [25]. Recent studies revealed the presence of plasma protease inhibitors (PI) in some marine bivalve molluscs including the eastern (Crassostrea 6irginica) and Pacific (Crassostrea gigas) oysters [14] and the surf clam (Spisula solidissima) [2]. In this study, PI activities in plasma of M. arenaria have been assessed. Among the known protease inhibitors, a2-macroglobulin (a2-M) occupies a central position. This high molecular weight protein possesses a bait region that is susceptible to proteolytic attack by proteases of all known catalytic mechanistic classes. Cleavage of the
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bait region triggers conformational changes in a2-M that physically trap proteases thus allowing small, but not large, molecular weight substrates to access the entrapped protease active site [25]. Therefore, the second objective of this study was to investigate the presence of a2-M-like activities in the plasma of softshell clams. Epizootics of disseminated sarcoma (DS) have been reported in softshell clams from the Chesapeake Bay and New England [7,8,11,16,17,24,27]. The disease is characterized by the presence of actively dividing large circulating cells with high nucleus to cytoplasm ratios. The proliferative condition is progressive and usually fatal. Previous studies have associated the progression of DS with several biochemical, physiological and immunological changes in the affected clams. For example, hemocytes of sarcomatous clams exhibited reduced phagocytic abilities, distortion of the cytoskeleton and loss of enzyme activities [4,23]. In this study, we wished to investigate if sarcoma progression would affect the levels of plasma protease inhibitory activities in M. arenaria.
non-sarcomatous and sarcomatous clam groups were analyzed for inhibitory activities against four proteases representing all mechanistic classes: papain from Papaya latex (cysteine protease), pepsin from porcine stomach mucosa (aspartic protease), thermolysin from Bacillus thermoproteolyticus rokko (metalloprotease), and trypsin from bovine pancreas (serine protease). Stock solutions of the different proteases used were prepared on ice at concentrations of 1 mg/ml in 100 mM Tris–HCl (pH 8.2) for trypsin, 50 mM phosphate buffer saline (PBS) (pH 7.5) for thermolysin, distilled water for papain and 10 mM HCl for pepsin. Protease inhibition activities by softshell clam plasma were measured using the substrate hide powder azure (HPA) in a microtiter plate as described by Bender et al. [5]. The substrate was prepared as 2.5% w/v in assay buffer consisting of 150 mM Trizma base, 30 mM CaCl2, 0.05% (w/v) Brij35 and 20% sucrose, pH 8.2, 7.5 and 2.0 for trypsin, thermolysin and pepsin, respectively. In the case of papain the assay buffer consisted of 150 mM Trizma base, 0.05% (w/v) Brij 35, 20% (w/v) sucrose, 2 mM EDTA, 5 mM cysteine and 300 mM NaCl (pH 6.2).
2. Materials and methods
2.4. Estimation of protein content of softshell clam plasma
2.1. Clam Softshell clams were collected from Swan Point in the Chester River, MD and maintained in recirculating aquaria at 8 ppt and 12°C for approximately 1 month. The clams ranged in length from 59 mm to 89 mm (69.6398.9 mm) and were diagnosed for sarcomas using histocytology [16] and histology [19] techniques. Hemolymph and tissues were fixed in 1% glutaraldehyde –4% formaldehyde in half ambient seawater and stained with Fuelgen picromethyl blue (FPM) or hematoxylin and eosin (H&E), respectively. Preparations were examined and the stage of sarcoma was determined according to criteria described by Farley et al. [17].
2.2. Hemolymph collection and plasma separation Hemolymph samples were collected by inserting a sterile 5-ml syringe with a 26-gauge needle into the posterior adductor muscles of the clams. Hemolymph was withdrawn, held on ice, and centrifuged at 400 rpm for 10 min at 4°C. The cell-free hemolymph (plasma) was then collected, filtered, aliquoted and stored at − 20°C until examined.
2.3. Chemicals and stock solutions All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise mentioned. Plasma samples from
Plasma samples from non-sarcomatous and sarcomatous clam groups were analyzed for protein content using a Pierce BCA protein Analysis Kit (Pierce, Rockford, IL).
2.5. Assessment of proteolytic inhibitory acti6ities in plasma of non-sarcomatous clams To determine the levels of protease inhibitory (PI) activities, a volume of 50 ml plasma previously diluted 1:1 in 0.2 M sodium chloride solution was incubated at room temperature for 15 min in a 96 well microtiter plate with 4 ml protease (trypsin at a concentration of 2.5 ng/ml, thermolysin at a concentration of 10 ng /ml, papain at a concentration of 62.5 ng/ml, and pepsin at a concentration of 1 mg/ml). Each sample was analyzed in triplicate. After 15 min of preincubation, a volume of 100 ml HPA in assay buffer specific for each enzyme was added and the plates were incubated for 2 h at 37°C with continuous shaking. After incubation, 100 ml cold Tris–HCl pH 8.2 (in the case of trypsin), phosphate buffered saline pH 7.5 (in the case of thermolysin), papain assay buffer, or pepsin assay buffer was added to each well of the plate. The plate was centrifuged at 1000×g at 4°C for 5 min to remove the insoluble substrate and the supernatant carefully transferred to another 96-well plate. Absorbance at 570 nm (A570) was determined using a Dynatech plate reader MR 5000 (Dynatech, Chantilly, VA). A standard curve
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consisting of serial dilutions of protease was prepared in triplicate, simultaneously with each enzyme assay, as follows: thermolysin 40 – 2.5 ng, trypsin, 20 – 1.25 ng, pepsin, 2 mg–125 ng and papain 125 – 7.826 ng. In addition, controls containing 50 ml plasma with no protease were prepared for each sample as well as triplicate blanks with no protease or plasma for each assay set. Standard curve, control and blank samples were treated in the same way as plasma samples. A standard curve of absorbance versus protein concentration was generated for each protease using the following calculation for triplicate samples of each serial dilution: (Average Protease Optical Density (OD))− (Average Blank OD). Each plasma sample’s (protease +plasma sample, and plasma sample only) triplicate values were then averaged and the level of protease activity was then determined by comparison to the standard curve absorbance (protease only), giving an activity value equivalent to protease enzyme ng/ sample. Total protease ng inhibited/50 ml plasma was determined by subtraction of the protease activity value, determined as above, for plasma sample (control), and protease + plasma sample, from the activity value obtained for protease (at the concentration used for co-incubation with plasma). The specific activity of protease inhibition, ng inhibited/mg plasma protein, was then calculated for each sample.
2.6. Determination of the presence of a2 -M-like acti6ities in the plasma of non-sarcomatous clams
2.6.1. Protection of trypsin from soybean trypsin inhibitor (SBTI) Increasing volumes of plasma (50, 100 and 200 ml) were pre-incubated with 50 ml Tris – HCl, pH 8.2, containing 1 mg trypsin for 15 min and then incubated with 50 ml Tris–HCl pH 8.2 containing 2 mg soybean trypsin inhibitor (SBTI) for 15 min prior to assay using the small molecular weight substrate a-N-Benzoyl-DLarginine-P-nitroanilide (BAPNA). The reaction was terminated by the addition of 300 ml 30% cold acetic acid. The optical densities of the samples were measured at 410 nm (A410) every 10 min in a Shimadzu spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) against a blank composed of BAPNA and buffer without trypsin. Controls consisted of trypsin at different concentrations (1, 2 and 5 mg) with BAPNA (without plasma) and plasma with BAPNA (without trypsin). Controls and blanks were treated in the same manner as samples. Similar plasma samples pre-incubated with trypsin were assayed using HPA as previously mentioned.
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2.6.2. Methylamine treatment of the plasma Inhibition of plasma a2-M-like activity by methylamine (MA) was assayed by incubation of equal volumes (200 ml) of plasma with either 100 mM Tris–HCl (pH 8.5) (control), or the same buffer containing 500 mM methylamine hydrochloride and incubated for 2 and 24 h at 25°C, respectively. The treated plasma was assayed, using HPA, for the inhibitory activities against both trypsin and thermolysin. 2.7. a2 -M-like acti6ities in clams affected with sarcoma Plasma samples from non-sarcomatous and sarcomatous clam groups were assayed for the level of a2-Mlike activities. Plasma (100 ml) were pre-incubated with 50 ml Tris–HCl, pH 8.2, containing 1 mg trypsin for 15 min and then incubated with 50 ml Tris–HCl (pH 8.2) containing 2 mg soybean trypsin inhibitor (SBTI) prior to assay using the small molecular weight substrate BAPNA as mentioned previously.
2.8. Effects of sarcoma on total protease inhibitory acti6ities Plasma samples from non-sarcomatous and sarcomatous clam groups were assayed for the level of inhibitory activities against proteases representing different mechanistic classes using the previously described HPA assay.
2.9. Statistical analysis The data were first tested for normality. Nonparameteric data were analyzed using Kruskal–Wallis One Way Analysis of Variance on Ranks followed by Dunn’s Pairwise Multiple Comparison Procedures at P5 0.05. Parameteric data were analyzed using One Way Analysis of Variance (ANOVA) followed by The Pairwise Multiple Comparison Procedures (Student– Newman–Keuls Methods) at P50.05. Analyses were performed using SigmaStat computer software (Jandel Scientific, San Rafael, CA).
3. Results
3.1. Diagnosis of clam sarcoma Examination of hemolymph preparations stained with FPM resulted in identification of nine non-sarcomatous clams, seven clams with early sarcomas, seven with moderate sarcomas, and 10 with advanced stages of sarcomas (Fig. 1). These findings were confirmed by examination of H&E stained tissue sections. The severity of the sarcoma was determined by the ratio of sarcoma cells to normal hemocytes. In early sarcoma
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stages, up to 5% of the cells were sarcomatous. The number of sarcoma cells rose to 49% in clams with moderate sarcomas. Sarcoma cells predominated the hemolymph preparations in clams with advanced sarcomas (Fig. 1c).
Fig. 2. Protein concentration of cell-free plasma from different clam groups. Values represent mean ( 9S.E.M.) of nine non-sarcomatous clams, seven clams with early or moderate sarcoma stages and 10 clams with advanced sarcoma.
3.2. Protein concentration of clam plasma samples Plasma protein of non-sarcomatous clams showed an average concentration of 0.7890.39 mg/ml. There were no significant differences between plasma proteins of non-sarcomatous clams and clams diagnosed with early (0.7090.19 mg/ml) and moderate (0.619 0.33 mg/ml) stages of sarcoma. Clams with advanced sarcoma stages exhibited significantly lower plasma protein concentrations (0.57 90.51 mg/ml) as compared to those of non-sarcomatous clams (P= 0.002) (Fig. 2).
3.3. Protease inhibitory acti6ities in plasma of non-sarcomatous clam samples Inhibitory activities were found in plasma of non-sarcomatous clams against all tested proteases representing different mechanistic classes. The maximum inhibition by the plasma was against pepsin (35.3 9 9.69 ng pepsin inhibited/mg plasma protein) and the minimum inhibition was against thermolysin (1.629 1.35 ng thermolysin inhibited/mg plasma protein). Inhibitory activities in the plasma of M. arenaria could also be detected against trypsin at levels of 3.19 0.88 ng enzyme/mg plasma protein and papain at levels of 4.99 1.45 ng enzyme/mg plasma protein.
Fig. 1. Modulation contrast photomicrograph of unfixed hemolymph preparations from non-sarcomatous and sarcomatous softshell clams, Mya arenaria. (a) Normal hemocytes of a healthy clam; (b) the hemolymph from an infected clam showing hemocytes and sarcoma cells (arrow); and (c) hemolymph preparation predominated by sarcoma cells. Scale bars = 10 mm.
3.4. Effect of sarcoma on plasma protease inhibitory acti6ities Our data suggest the presence of modulatory effects of sarcoma on PI activities in clam plasma (Fig. 3). The highest PI modulatory effect of sarcoma occurred with
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pepsin and thermolysin. Plasma inhibitors against pepsin decreased significantly in clams with early stages of sarcoma, to reach 23.1 ng/mg plasma protein (35.33 ng/mg protein in non-sarcomatous clams). Plasma inhibitors against thermolysin showed a significant suppression in clams with early stages of sarcoma (0.44 ng/mg plasma protein) compared to non-sarcomatous clams (1.62 ng/mg plasma protein). Plasma of clams with moderate or advanced sarcoma totally lost their ability to inhibit pepsin and thermolysin (Fig. 3a and b). There were noticeable decreases in plasma PI levels against trypsin and papain in early and moderate sarcomatous clams that became significant in clams with advanced sarcoma (Fig. 3c and d).
3.5. a2 -M-like acti6ities in plasma of non-sarcomatous clams 3.5.1. Trypsin protection by plasma As shown in Fig. 4a, 1 mg trypsin was inhibited by 50 ml M. arenaria plasma when high molecular weight HPA was used as a substrate. On the contrary, when small molecular weight BAPNA was used as a substrate, trypsin retained most of its hydrolytic activity after incubation with the same volume of clam plasma (Fig. 4b). In another set of assays, the presence of a2-M-like activity was suggested due to the ability of plasma to protect trypsin from its active site inhibitor, SBTI. Incubation of trypsin with increasing plasma volume (50–200 ml) was accompanied by increasing the amount of trypsin protected from SBTI. This protected trypsin is able to hydrolyze increasing amounts of BAPNA. Trypsin incubated directly with SBTI shows complete inhibition of its amidolytic activity against BAPNA (Fig. 5). 3.5.2. Inhibition of plasma a2 -M-like acti6ity by methylamine Upon incubation with 250 mM MA for 2 h, non-sarcomatous clam plasma showed a slight decrease in its activity against thermolysin and trypsin. However, such activities were significantly inhibited (PB 0.001 for trypsin and PB 0.05 for thermolysin) when the incubation time was extended to 24 h (Table 1). 3.6. Effects of sarcoma on a2 -M-like acti6ities Our data suggest the presence of modulatory effects of sarcoma on a2-M-like activities in the plasma of M. arenaria. While non-sarcomatous clam plasma was able
Fig. 3.
Fig. 3. Modulation of plasma protease inhibitors by sarcoma. Protease inhibition by plasma of different clam groups was performed using HPA assay. (a) Pepsin inhibition; (b) thermolysin inhibition; (c) trypsin inhibition; and (d) papain inhibition. Values represent mean ( 9S.D.) of nine non-sarcomatous clams, seven clams with early or moderate sarcoma stages and 10 clams with advanced sarcoma.
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to protect trypsin from inhibition by SBTI (A410 = 0.859 0.06), clams with early sarcoma stages showed a significant decrease in the ability of plasma to protect trypsin from the active site inhibitor SBTI (A410 = 0.139 0.12). Moreover, plasma of clams in moderate and advanced sarcoma stages completely lost its ability to protect trypsin from inhibition by SBTI (Table 2).
4. Discussion The data presented in this study clearly demonstrate the presence of protease inhibitory activities in the plasma of softshell clams. The constitutive levels of PI
activities in M. arenaria for papain and pepsin were lower than those detected in the plasma of eastern and Pacific oysters [14]. In contrast, the softshell clam possesses higher levels of plasma trypsin inhibitors than oysters. Despite the relatively low level of thermolysin protease inhibitors detected in the plasma of clams, this level is far higher than that detected in Crassostrea spp. [14]. The differences in inhibitory activity could be attributed to species and habitat differences among the bivalve species tested. The data presented in Table 1 provide evidence that the plasma protease inhibitors can be attributed, at least in part, to a2-M-like activities. Sensitivity to methylamine and the ability to inhibit proteases from all classes are
Fig. 4. Effect of clam plasma on trypsin activity. Trypsin, previously preincubated with clam plasma for 15 min, was assessed for its activity using (a) the high molecular weight substrate HPA (A570) and (b) the small molecular weight substrate BAPNA (A410). The X-axis represents the time of incubation with the substrates. Points represent means ( 9 S.D.) of nine non-sarcomatous clams.
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Fig. 5. Protection of trypsin from SBTI by clam plasma. Trypsin was preincubated with clam plasma followed by a further incubation with SBTI. The assay was initiated by adding the small molecular weight substrate BAPNA. The X-axis represents the time of incubation with BAPNA. Points represent means ( 9 S.D.) of nine non-sarcomatous clams.
among the major characteristics of a2-M molecules [1,20]. Suppression of the inhibitory activity of M. arenaria plasma by methylamine indicates that the integrity of the thiol ester bond is essential for a2-M of this species. Also, the decrease in the inhibitory activity of methylamine-treated plasma of M. arenaria against trypsin and thermolysin further confirms that a2-M of M. arenaria acts on both proteases. Protease inhibition by a2-M results from the entrapment of the bound protease within the molecular cage of the a2-M molecule. This mechanism leaves the active site free to react with low molecular weight but not the large molecular weight substrates [2,3]. Trypsin activity was greatly reduced by clam plasma when large molecular weight HPA was used as a substrate, however, this activity was retained when a small molecular weight substrate was used. Moreover, the protection of trypsin from a large molecular weight active site inhibitor (SBTI) by plasma confirms the presence of a2-M-like molecules in M. arenaria plasma. Clam a2-M-like activities were suppressed in clams diagnosed with any of the sarcoma stages, particularly, moderate and late stages. There are several possibilities to explain this phenomenon. Firstly, a2-M could be depleted or bound to proteases secreted by sarcoma cells. Secondly, malnutrition due to disease may have contributed to decreased production of plasma proteins including a2-M. Finally, enzymes and other lytic factors
produced by sarcoma cells could have degraded plasma proteins including a2-M molecules. PI activities in clam plasma have also been influenced by sarcoma. Data shown in Fig. 3(a–d) provide evidence of the modulatory effects of sarcoma on PI levels in affected clams with prominent negative correlation between disease stage and plasma PI levels. The discrepancies of modulatory effects of sarcoma on different PI
Table 1 Inhibition of a2-M-like activities in the plasma of softshell clams using methylaminea 2h
Control Methylamine P
24 h
Trypsin
Thermolysin
Trypsin
Thermolysin
29.5 9 12.4 26.5 96.7
14.8 96.1 13.9 97.2
31.4 910.6 18.3 98.1
12.6 95.8 9.4 9 2.1
B0.001
B0.05
NS
NS
a Clam plasma was incubated with an equal volume of 500 mM methylamine (MA) in 100 mM Tris–HCl (pH 8.5) for 2 and 24 h at 25°C. Treated plasma was then assayed for the inhibitory activities against trypsin and thermolysin. Control plasma samples were incubated with 100 mM Tris–HCl (pH 8.5). Data are expressed as mean (9 S.D.) of the amount of enzyme (pg) inhibited/ml plasma. Nine non-sarcomatous clams were used in this experiment. NS, statistically non-significant.
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Table 2 Effect of sarcoma on a2-M-like activities in the plasma of M. arenaria a Treatment
Absorbance (A410)
Trypsin alone Trypsin+SBTI Non-sarcomatous (trypsin+plasma)+SBTI Early sarcoma (trypsin+plasma)+SBTI Moderate sarcoma (trypsin+plasma)+SBTI Late sarcoma (trypsin+plasma)+SBTI
0.939 0.06 0.059 0.01 0.85 9 0.06 0.139 0.12 0 0
A volume of 100 ml plasma of different clam groups was preincubated with 1 mg trypsin for 15 min then incubated with 2 mg SBTI. Trypsin activity was assayed using BAPNA. Nine non-sarcomatous clams, seven clams with early and moderate sarcoma stages and 10 clams with advanced sarcoma were used in this experiment. a
levels are difficult to interpret since there is a scarcity of knowledge on the effects of sarcoma on humoral immune responses. Natural variations may exist in the sensitivity of clam PI to sarcoma or its secretions. One could also speculate that some PI molecules are involved in host defense against sarcoma, and are thereby severely conformed during neutralization of sarcoma secretions. In fact, the role played by PI in malignancy progression remains to be elucidated. However, the possibility of the involvement of PI in surveillance against sarcoma is expected. In vertebrates there is increasing data regarding the involvement of PI in the body’s defense against tumor progression [9,18]. However, such roles have not been investigated in invertebrates. Further studies are needed to better understand the mechanisms of PI modulation and its possible role in clam immune responses against various endogenous and exogenous proteases. In conclusion, our study provided evidence for PI in softshell clams (M. arenaria) which belong to both active site and a2-M activities. Moreover, we found that PI activities are modulated by different sarcoma stages.
Acknowledgements This research was supported by a grant from the National Oceanic and Atmospheric Administration (NOAA), Virginia Sea Grant College Program, grant NA 56RGO141. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies. Virginia Institute of Marine Science contribution c 2220
References [1] Armstrong P, Rossner MT, Quigley JP. An a2-macroglobulin activity in the blood of chelicerate and mandibulate arthropods. J Exp Zool 1985;236:1–9.
[2] Armstrong P, Quigley J. Humoral immunity: a2 macroglobulin activity in the plasma of mollusks. Veliger 1992;35:161–4. [3] Barrett AJ, Starkey PM. The interaction of a2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J 1973;133:709 – 24. [4] Beckmann N, Morse MP, Moore CM. Comparative study of phagocytosis in normal and diseased hemocytes of the bivalve mollusc Mya arenaria. J Invertebr Pathol 1992;59:124 –32. [5] Bender RC, Fryer SE, Bayne CJ. Proteinase inhibitory activity in the plasma of a mollusc: evidence for the presence of amacroglobulin in Biomphalaria glabrata. Comp Biochem Physiol 1992;102B:821– 4. [6] Bower S, McGladdery SE, Price IA. Synopsis of infectious diseases and parasites of commercially exploited shellfish. Annu Rev Fish Dis 1994;4:1 – 199. [7] Brousseau DJ, Baglivo JA. Field and laboratory comparisons of mortality in normal and neoplastic Mya arenaria. J Invertebr Pathol 1991;57:59 – 65. [8] Brown RS, Wolke RE, Saila SB, Brown CW. Prevalence of neoplasia in 10 New England populations of the soft-shell clam (Mya arenaria). Ann New York Acad Sci 1977;298:522–34. [9] Calkins C, Sloane B. Mammalian cysteine protease inhibitors: biochemical properties and possible role in tumor progression. Biol Chem Hoppe-Seyler 1995;376:71 – 80. [10] Christensen S, Sottrup-Jensen L. Characterization of two serpins from bovine plasma and milk. Biochem J 1994;303:383–90. [11] Cooper KR, Brown RS, Chang PW. The course and mortality of a hematopoietic neoplasm in the soft-shell clam, Mya arenaria. J Invertebr Pathol 1982;39:149 – 57. [12] Eguchi M. Protein protease inhibitors in insects and comparison with mammalian inhibitors. Comp Biochem Physiol 1993;105B: 449 – 56. [13] Elston RA, Moore JD, Brooks K. Disseminated neoplasia of bivalve molluscs. Rev Aquat Sci 1993;6:405 – 66. [14] Faisal M, MacIntyre EA, Adham KG, Tall BD, Kothary MH, LaPeyre JF. Evidence for the presence of protease inhibitors in Eastern (Crassostrea 6irginica) and Pacific (Crassostrea gigas) oysters. Comp Biochem Physiol 1998;121B:161– 8. [15] Faisal M, McLaughlin S, Elsayed E, MacIntyre E, Kotob S, El-Gawady, H. Isolation of a Labyrinthuloides sp. (Protozoa:Labyrinthomorpha) with unique properties from the softshell clam (Mya arenaria). The 3rd Int. Symp. on Aquatic Animal Health, Baltimore, MD, USA, 30 August – 3 September, 1998, p. 174. [16] Farley CA, Otto SV, Reinisch CL. New occurrence of epizootic sarcoma in Chesapeake Bay softshell clams, Mya arenaria. Fish Bull 1986;84:851 – 7. [17] Farley CA, Plutschak DL, Scott RF. Epizootiology and distribution of transmissible sarcoma in Maryland soft-shell clams, Mya arenaria, 1984 – 1988. Environ Health Perspect 1991;90:35–41. [18] Howard E, Bullen E, Banda M. Preferential inhibition of 72 kDa and 92 kDa gelatinases by TIMP-2. J Biol Chem 1991;266:13070– 5. [19] Howard DW, Smith CS. Histological techniques for marine bivalve mollusks. US Department of Commerce, National Marine Fisheries Service, NOAA Technical Memo NMFS-F/ NEC-25, 1983, 97 pp. [20] Laskowski M, Kato I. Protein inhibitors of proteases. Annu Rev Biochem 1980;49:593 – 626. [21] McLaughlin S, Faisal M. Histopathological alterations associated with Perkinsus spp. infections in the softshell clam, Mya arenaria. Parasite 1998;5:263 – 71. [22] McLaughlin S, Faisal M. Comparison of tissues in fluid thioglycolate assays for diagnosis of Perkinsus spp. in the softshell clam, Mya arenaria. Aquaculture 1999;172:197 – 204. [23] Moore CA, Beckmann N, Morse MP. Cytoskeletal structure of diseased and normal hemoyctes of Mya arenaria. J Invertebr Pathol 1992;60:141 – 7.
E.E. Elsayed et al. / Comparati6e Biochemistry and Physiology, Part B 123 (1999) 427–435 [24] Reinisch CL, Charles AM, Stone A. Epizootic neoplasia in soft shell clams collected from New Bedford Harbor. Hazard Waste 1984;1:73 – 81. [25] Sottrup-Jensen L. 2 Macroglobulin structure, shape and mechanism of proteinase complex formation. J Biol Chem 1989;264:11539– 42.
.
435
[26] Sto¨ker W, Breit S, Sottrup-Jensen L, Zwilling R. 2-Macroglobulin from the hemolymph of the freshwater crayfish Astacus astacus. Comp Biochem Physiol 1991;98B:501 – 9. [27] Yevich PP, Barszcz CA. Neoplasia in soft shell clam (Mya arenaria) collected from oil-impacted sites. Ann New York Acad Sci 1977;298:409 – 26.
Protease inhibitors in plasma of the softshell clam Mya arenaria: identification and effects of disseminated sarcoma Ehab E. Elsayed a,1, Shawn M. McLaughlin b, Mohamed Faisal a,* a
Department of En6ironmental Science, Virginia Institute of Marine Science/School of Marine Science, The College of William and Mary, Gloucester Point, VA 23062, USA b National Oceanic and Atmospheric Administration, National Ocean Ser6ice, Center for Coastal En6ironmental Health and Biomolecular Research, Oxford, MD 21654, USA Received 8 January 1999; received in revised form 11 May 1999; accepted 25 May 1999
Abstract Despite the ecologic and economic significance of the softshell clam (Mya arenaria), little is known about the humoral factors involved in its host defense mechanisms. Protease inhibitors, a group of proteins believed to play a role in host defense mechanisms against infections and proliferative diseases, have recently been identified in bivalve molluscs. In the present study we provide evidence for the presence of protease inhibitors in softshell clam plasma. Levels of protease inhibitory activities against the enzymes tested varied greatly, e.g. 1 mg of plasma protein inhibited 35.39 9.69 ng pepsin (aspartic protease), 4.99 1.45 ng papain (cysteine protease) and 3.1 90.88 ng trypsin (serine protease). On the contrary, the level of anti-metalloprotease (thermolysin) activities was much lower. The sensitivity to methylamine and the ability to protect trypsin from active site trypsin inhibitors provided evidence for the presence of an a2-macroglobulin-like molecule in softshell clam plasma. In the Chesapeake Bay widespread epizootics of disseminated sarcoma have been described in M. arenaria populations. The impact of this lethal proliferative disorder on clam defense responses has received little attention. In this study the effects of sarcoma progression on plasma protease inhibitory activities were, therefore, assessed. Clams with early stages of sarcoma showed a non-significant decrease in protease inhibitor levels. Clams with advanced stages of sarcoma showed a significant decrease in the ability to inhibit trypsin and papain, while the protease inhibitory activity levels against aspartic and metalloprotease were completely exhausted. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Softshell clam; Protease inhibitors; Sarcoma; Proteases; a2-Macroglobulin
1. Introduction The softshell clam (Mya arenaria) is an important molluscan filter feeder in the Chesapeake Bay and along the northern Atlantic coast of the USA. Despite the ecologic and economic significance of the softshell clam, little is known about its host defense mechanisms especially humoral factors. This lack of knowledge undermines the ability to respond to increasing num
Disclaimer: reference to trade names does not imply endorsement by NOAA. * Corresponding author. Tel.: + 1-804-684-7231; fax: + 1-804-6846186. E-mail address: [email protected] (M. Faisal) 1 Present address: Department of Fish Medicine and Management, College of Veterinary Medicine, Cairo University, Giza, Egypt.
bers of reports on diseases and parasites affecting this species [6,13,15,21,22]. Protease inhibitors are a group of humoral factors found in several animal species [10,12,26] that neutralize unwanted proteases including those of microbial origin and maintain internal homeostasis [25]. Recent studies revealed the presence of plasma protease inhibitors (PI) in some marine bivalve molluscs including the eastern (Crassostrea 6irginica) and Pacific (Crassostrea gigas) oysters [14] and the surf clam (Spisula solidissima) [2]. In this study, PI activities in plasma of M. arenaria have been assessed. Among the known protease inhibitors, a2-macroglobulin (a2-M) occupies a central position. This high molecular weight protein possesses a bait region that is susceptible to proteolytic attack by proteases of all known catalytic mechanistic classes. Cleavage of the
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bait region triggers conformational changes in a2-M that physically trap proteases thus allowing small, but not large, molecular weight substrates to access the entrapped protease active site [25]. Therefore, the second objective of this study was to investigate the presence of a2-M-like activities in the plasma of softshell clams. Epizootics of disseminated sarcoma (DS) have been reported in softshell clams from the Chesapeake Bay and New England [7,8,11,16,17,24,27]. The disease is characterized by the presence of actively dividing large circulating cells with high nucleus to cytoplasm ratios. The proliferative condition is progressive and usually fatal. Previous studies have associated the progression of DS with several biochemical, physiological and immunological changes in the affected clams. For example, hemocytes of sarcomatous clams exhibited reduced phagocytic abilities, distortion of the cytoskeleton and loss of enzyme activities [4,23]. In this study, we wished to investigate if sarcoma progression would affect the levels of plasma protease inhibitory activities in M. arenaria.
non-sarcomatous and sarcomatous clam groups were analyzed for inhibitory activities against four proteases representing all mechanistic classes: papain from Papaya latex (cysteine protease), pepsin from porcine stomach mucosa (aspartic protease), thermolysin from Bacillus thermoproteolyticus rokko (metalloprotease), and trypsin from bovine pancreas (serine protease). Stock solutions of the different proteases used were prepared on ice at concentrations of 1 mg/ml in 100 mM Tris–HCl (pH 8.2) for trypsin, 50 mM phosphate buffer saline (PBS) (pH 7.5) for thermolysin, distilled water for papain and 10 mM HCl for pepsin. Protease inhibition activities by softshell clam plasma were measured using the substrate hide powder azure (HPA) in a microtiter plate as described by Bender et al. [5]. The substrate was prepared as 2.5% w/v in assay buffer consisting of 150 mM Trizma base, 30 mM CaCl2, 0.05% (w/v) Brij35 and 20% sucrose, pH 8.2, 7.5 and 2.0 for trypsin, thermolysin and pepsin, respectively. In the case of papain the assay buffer consisted of 150 mM Trizma base, 0.05% (w/v) Brij 35, 20% (w/v) sucrose, 2 mM EDTA, 5 mM cysteine and 300 mM NaCl (pH 6.2).
2. Materials and methods
2.4. Estimation of protein content of softshell clam plasma
2.1. Clam Softshell clams were collected from Swan Point in the Chester River, MD and maintained in recirculating aquaria at 8 ppt and 12°C for approximately 1 month. The clams ranged in length from 59 mm to 89 mm (69.6398.9 mm) and were diagnosed for sarcomas using histocytology [16] and histology [19] techniques. Hemolymph and tissues were fixed in 1% glutaraldehyde –4% formaldehyde in half ambient seawater and stained with Fuelgen picromethyl blue (FPM) or hematoxylin and eosin (H&E), respectively. Preparations were examined and the stage of sarcoma was determined according to criteria described by Farley et al. [17].
2.2. Hemolymph collection and plasma separation Hemolymph samples were collected by inserting a sterile 5-ml syringe with a 26-gauge needle into the posterior adductor muscles of the clams. Hemolymph was withdrawn, held on ice, and centrifuged at 400 rpm for 10 min at 4°C. The cell-free hemolymph (plasma) was then collected, filtered, aliquoted and stored at − 20°C until examined.
2.3. Chemicals and stock solutions All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise mentioned. Plasma samples from
Plasma samples from non-sarcomatous and sarcomatous clam groups were analyzed for protein content using a Pierce BCA protein Analysis Kit (Pierce, Rockford, IL).
2.5. Assessment of proteolytic inhibitory acti6ities in plasma of non-sarcomatous clams To determine the levels of protease inhibitory (PI) activities, a volume of 50 ml plasma previously diluted 1:1 in 0.2 M sodium chloride solution was incubated at room temperature for 15 min in a 96 well microtiter plate with 4 ml protease (trypsin at a concentration of 2.5 ng/ml, thermolysin at a concentration of 10 ng /ml, papain at a concentration of 62.5 ng/ml, and pepsin at a concentration of 1 mg/ml). Each sample was analyzed in triplicate. After 15 min of preincubation, a volume of 100 ml HPA in assay buffer specific for each enzyme was added and the plates were incubated for 2 h at 37°C with continuous shaking. After incubation, 100 ml cold Tris–HCl pH 8.2 (in the case of trypsin), phosphate buffered saline pH 7.5 (in the case of thermolysin), papain assay buffer, or pepsin assay buffer was added to each well of the plate. The plate was centrifuged at 1000×g at 4°C for 5 min to remove the insoluble substrate and the supernatant carefully transferred to another 96-well plate. Absorbance at 570 nm (A570) was determined using a Dynatech plate reader MR 5000 (Dynatech, Chantilly, VA). A standard curve
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consisting of serial dilutions of protease was prepared in triplicate, simultaneously with each enzyme assay, as follows: thermolysin 40 – 2.5 ng, trypsin, 20 – 1.25 ng, pepsin, 2 mg–125 ng and papain 125 – 7.826 ng. In addition, controls containing 50 ml plasma with no protease were prepared for each sample as well as triplicate blanks with no protease or plasma for each assay set. Standard curve, control and blank samples were treated in the same way as plasma samples. A standard curve of absorbance versus protein concentration was generated for each protease using the following calculation for triplicate samples of each serial dilution: (Average Protease Optical Density (OD))− (Average Blank OD). Each plasma sample’s (protease +plasma sample, and plasma sample only) triplicate values were then averaged and the level of protease activity was then determined by comparison to the standard curve absorbance (protease only), giving an activity value equivalent to protease enzyme ng/ sample. Total protease ng inhibited/50 ml plasma was determined by subtraction of the protease activity value, determined as above, for plasma sample (control), and protease + plasma sample, from the activity value obtained for protease (at the concentration used for co-incubation with plasma). The specific activity of protease inhibition, ng inhibited/mg plasma protein, was then calculated for each sample.
2.6. Determination of the presence of a2 -M-like acti6ities in the plasma of non-sarcomatous clams
2.6.1. Protection of trypsin from soybean trypsin inhibitor (SBTI) Increasing volumes of plasma (50, 100 and 200 ml) were pre-incubated with 50 ml Tris – HCl, pH 8.2, containing 1 mg trypsin for 15 min and then incubated with 50 ml Tris–HCl pH 8.2 containing 2 mg soybean trypsin inhibitor (SBTI) for 15 min prior to assay using the small molecular weight substrate a-N-Benzoyl-DLarginine-P-nitroanilide (BAPNA). The reaction was terminated by the addition of 300 ml 30% cold acetic acid. The optical densities of the samples were measured at 410 nm (A410) every 10 min in a Shimadzu spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) against a blank composed of BAPNA and buffer without trypsin. Controls consisted of trypsin at different concentrations (1, 2 and 5 mg) with BAPNA (without plasma) and plasma with BAPNA (without trypsin). Controls and blanks were treated in the same manner as samples. Similar plasma samples pre-incubated with trypsin were assayed using HPA as previously mentioned.
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2.6.2. Methylamine treatment of the plasma Inhibition of plasma a2-M-like activity by methylamine (MA) was assayed by incubation of equal volumes (200 ml) of plasma with either 100 mM Tris–HCl (pH 8.5) (control), or the same buffer containing 500 mM methylamine hydrochloride and incubated for 2 and 24 h at 25°C, respectively. The treated plasma was assayed, using HPA, for the inhibitory activities against both trypsin and thermolysin. 2.7. a2 -M-like acti6ities in clams affected with sarcoma Plasma samples from non-sarcomatous and sarcomatous clam groups were assayed for the level of a2-Mlike activities. Plasma (100 ml) were pre-incubated with 50 ml Tris–HCl, pH 8.2, containing 1 mg trypsin for 15 min and then incubated with 50 ml Tris–HCl (pH 8.2) containing 2 mg soybean trypsin inhibitor (SBTI) prior to assay using the small molecular weight substrate BAPNA as mentioned previously.
2.8. Effects of sarcoma on total protease inhibitory acti6ities Plasma samples from non-sarcomatous and sarcomatous clam groups were assayed for the level of inhibitory activities against proteases representing different mechanistic classes using the previously described HPA assay.
2.9. Statistical analysis The data were first tested for normality. Nonparameteric data were analyzed using Kruskal–Wallis One Way Analysis of Variance on Ranks followed by Dunn’s Pairwise Multiple Comparison Procedures at P5 0.05. Parameteric data were analyzed using One Way Analysis of Variance (ANOVA) followed by The Pairwise Multiple Comparison Procedures (Student– Newman–Keuls Methods) at P50.05. Analyses were performed using SigmaStat computer software (Jandel Scientific, San Rafael, CA).
3. Results
3.1. Diagnosis of clam sarcoma Examination of hemolymph preparations stained with FPM resulted in identification of nine non-sarcomatous clams, seven clams with early sarcomas, seven with moderate sarcomas, and 10 with advanced stages of sarcomas (Fig. 1). These findings were confirmed by examination of H&E stained tissue sections. The severity of the sarcoma was determined by the ratio of sarcoma cells to normal hemocytes. In early sarcoma
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stages, up to 5% of the cells were sarcomatous. The number of sarcoma cells rose to 49% in clams with moderate sarcomas. Sarcoma cells predominated the hemolymph preparations in clams with advanced sarcomas (Fig. 1c).
Fig. 2. Protein concentration of cell-free plasma from different clam groups. Values represent mean ( 9S.E.M.) of nine non-sarcomatous clams, seven clams with early or moderate sarcoma stages and 10 clams with advanced sarcoma.
3.2. Protein concentration of clam plasma samples Plasma protein of non-sarcomatous clams showed an average concentration of 0.7890.39 mg/ml. There were no significant differences between plasma proteins of non-sarcomatous clams and clams diagnosed with early (0.7090.19 mg/ml) and moderate (0.619 0.33 mg/ml) stages of sarcoma. Clams with advanced sarcoma stages exhibited significantly lower plasma protein concentrations (0.57 90.51 mg/ml) as compared to those of non-sarcomatous clams (P= 0.002) (Fig. 2).
3.3. Protease inhibitory acti6ities in plasma of non-sarcomatous clam samples Inhibitory activities were found in plasma of non-sarcomatous clams against all tested proteases representing different mechanistic classes. The maximum inhibition by the plasma was against pepsin (35.3 9 9.69 ng pepsin inhibited/mg plasma protein) and the minimum inhibition was against thermolysin (1.629 1.35 ng thermolysin inhibited/mg plasma protein). Inhibitory activities in the plasma of M. arenaria could also be detected against trypsin at levels of 3.19 0.88 ng enzyme/mg plasma protein and papain at levels of 4.99 1.45 ng enzyme/mg plasma protein.
Fig. 1. Modulation contrast photomicrograph of unfixed hemolymph preparations from non-sarcomatous and sarcomatous softshell clams, Mya arenaria. (a) Normal hemocytes of a healthy clam; (b) the hemolymph from an infected clam showing hemocytes and sarcoma cells (arrow); and (c) hemolymph preparation predominated by sarcoma cells. Scale bars = 10 mm.
3.4. Effect of sarcoma on plasma protease inhibitory acti6ities Our data suggest the presence of modulatory effects of sarcoma on PI activities in clam plasma (Fig. 3). The highest PI modulatory effect of sarcoma occurred with
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pepsin and thermolysin. Plasma inhibitors against pepsin decreased significantly in clams with early stages of sarcoma, to reach 23.1 ng/mg plasma protein (35.33 ng/mg protein in non-sarcomatous clams). Plasma inhibitors against thermolysin showed a significant suppression in clams with early stages of sarcoma (0.44 ng/mg plasma protein) compared to non-sarcomatous clams (1.62 ng/mg plasma protein). Plasma of clams with moderate or advanced sarcoma totally lost their ability to inhibit pepsin and thermolysin (Fig. 3a and b). There were noticeable decreases in plasma PI levels against trypsin and papain in early and moderate sarcomatous clams that became significant in clams with advanced sarcoma (Fig. 3c and d).
3.5. a2 -M-like acti6ities in plasma of non-sarcomatous clams 3.5.1. Trypsin protection by plasma As shown in Fig. 4a, 1 mg trypsin was inhibited by 50 ml M. arenaria plasma when high molecular weight HPA was used as a substrate. On the contrary, when small molecular weight BAPNA was used as a substrate, trypsin retained most of its hydrolytic activity after incubation with the same volume of clam plasma (Fig. 4b). In another set of assays, the presence of a2-M-like activity was suggested due to the ability of plasma to protect trypsin from its active site inhibitor, SBTI. Incubation of trypsin with increasing plasma volume (50–200 ml) was accompanied by increasing the amount of trypsin protected from SBTI. This protected trypsin is able to hydrolyze increasing amounts of BAPNA. Trypsin incubated directly with SBTI shows complete inhibition of its amidolytic activity against BAPNA (Fig. 5). 3.5.2. Inhibition of plasma a2 -M-like acti6ity by methylamine Upon incubation with 250 mM MA for 2 h, non-sarcomatous clam plasma showed a slight decrease in its activity against thermolysin and trypsin. However, such activities were significantly inhibited (PB 0.001 for trypsin and PB 0.05 for thermolysin) when the incubation time was extended to 24 h (Table 1). 3.6. Effects of sarcoma on a2 -M-like acti6ities Our data suggest the presence of modulatory effects of sarcoma on a2-M-like activities in the plasma of M. arenaria. While non-sarcomatous clam plasma was able
Fig. 3.
Fig. 3. Modulation of plasma protease inhibitors by sarcoma. Protease inhibition by plasma of different clam groups was performed using HPA assay. (a) Pepsin inhibition; (b) thermolysin inhibition; (c) trypsin inhibition; and (d) papain inhibition. Values represent mean ( 9S.D.) of nine non-sarcomatous clams, seven clams with early or moderate sarcoma stages and 10 clams with advanced sarcoma.
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to protect trypsin from inhibition by SBTI (A410 = 0.859 0.06), clams with early sarcoma stages showed a significant decrease in the ability of plasma to protect trypsin from the active site inhibitor SBTI (A410 = 0.139 0.12). Moreover, plasma of clams in moderate and advanced sarcoma stages completely lost its ability to protect trypsin from inhibition by SBTI (Table 2).
4. Discussion The data presented in this study clearly demonstrate the presence of protease inhibitory activities in the plasma of softshell clams. The constitutive levels of PI
activities in M. arenaria for papain and pepsin were lower than those detected in the plasma of eastern and Pacific oysters [14]. In contrast, the softshell clam possesses higher levels of plasma trypsin inhibitors than oysters. Despite the relatively low level of thermolysin protease inhibitors detected in the plasma of clams, this level is far higher than that detected in Crassostrea spp. [14]. The differences in inhibitory activity could be attributed to species and habitat differences among the bivalve species tested. The data presented in Table 1 provide evidence that the plasma protease inhibitors can be attributed, at least in part, to a2-M-like activities. Sensitivity to methylamine and the ability to inhibit proteases from all classes are
Fig. 4. Effect of clam plasma on trypsin activity. Trypsin, previously preincubated with clam plasma for 15 min, was assessed for its activity using (a) the high molecular weight substrate HPA (A570) and (b) the small molecular weight substrate BAPNA (A410). The X-axis represents the time of incubation with the substrates. Points represent means ( 9 S.D.) of nine non-sarcomatous clams.
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Fig. 5. Protection of trypsin from SBTI by clam plasma. Trypsin was preincubated with clam plasma followed by a further incubation with SBTI. The assay was initiated by adding the small molecular weight substrate BAPNA. The X-axis represents the time of incubation with BAPNA. Points represent means ( 9 S.D.) of nine non-sarcomatous clams.
among the major characteristics of a2-M molecules [1,20]. Suppression of the inhibitory activity of M. arenaria plasma by methylamine indicates that the integrity of the thiol ester bond is essential for a2-M of this species. Also, the decrease in the inhibitory activity of methylamine-treated plasma of M. arenaria against trypsin and thermolysin further confirms that a2-M of M. arenaria acts on both proteases. Protease inhibition by a2-M results from the entrapment of the bound protease within the molecular cage of the a2-M molecule. This mechanism leaves the active site free to react with low molecular weight but not the large molecular weight substrates [2,3]. Trypsin activity was greatly reduced by clam plasma when large molecular weight HPA was used as a substrate, however, this activity was retained when a small molecular weight substrate was used. Moreover, the protection of trypsin from a large molecular weight active site inhibitor (SBTI) by plasma confirms the presence of a2-M-like molecules in M. arenaria plasma. Clam a2-M-like activities were suppressed in clams diagnosed with any of the sarcoma stages, particularly, moderate and late stages. There are several possibilities to explain this phenomenon. Firstly, a2-M could be depleted or bound to proteases secreted by sarcoma cells. Secondly, malnutrition due to disease may have contributed to decreased production of plasma proteins including a2-M. Finally, enzymes and other lytic factors
produced by sarcoma cells could have degraded plasma proteins including a2-M molecules. PI activities in clam plasma have also been influenced by sarcoma. Data shown in Fig. 3(a–d) provide evidence of the modulatory effects of sarcoma on PI levels in affected clams with prominent negative correlation between disease stage and plasma PI levels. The discrepancies of modulatory effects of sarcoma on different PI
Table 1 Inhibition of a2-M-like activities in the plasma of softshell clams using methylaminea 2h
Control Methylamine P
24 h
Trypsin
Thermolysin
Trypsin
Thermolysin
29.5 9 12.4 26.5 96.7
14.8 96.1 13.9 97.2
31.4 910.6 18.3 98.1
12.6 95.8 9.4 9 2.1
B0.001
B0.05
NS
NS
a Clam plasma was incubated with an equal volume of 500 mM methylamine (MA) in 100 mM Tris–HCl (pH 8.5) for 2 and 24 h at 25°C. Treated plasma was then assayed for the inhibitory activities against trypsin and thermolysin. Control plasma samples were incubated with 100 mM Tris–HCl (pH 8.5). Data are expressed as mean (9 S.D.) of the amount of enzyme (pg) inhibited/ml plasma. Nine non-sarcomatous clams were used in this experiment. NS, statistically non-significant.
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Table 2 Effect of sarcoma on a2-M-like activities in the plasma of M. arenaria a Treatment
Absorbance (A410)
Trypsin alone Trypsin+SBTI Non-sarcomatous (trypsin+plasma)+SBTI Early sarcoma (trypsin+plasma)+SBTI Moderate sarcoma (trypsin+plasma)+SBTI Late sarcoma (trypsin+plasma)+SBTI
0.939 0.06 0.059 0.01 0.85 9 0.06 0.139 0.12 0 0
A volume of 100 ml plasma of different clam groups was preincubated with 1 mg trypsin for 15 min then incubated with 2 mg SBTI. Trypsin activity was assayed using BAPNA. Nine non-sarcomatous clams, seven clams with early and moderate sarcoma stages and 10 clams with advanced sarcoma were used in this experiment. a
levels are difficult to interpret since there is a scarcity of knowledge on the effects of sarcoma on humoral immune responses. Natural variations may exist in the sensitivity of clam PI to sarcoma or its secretions. One could also speculate that some PI molecules are involved in host defense against sarcoma, and are thereby severely conformed during neutralization of sarcoma secretions. In fact, the role played by PI in malignancy progression remains to be elucidated. However, the possibility of the involvement of PI in surveillance against sarcoma is expected. In vertebrates there is increasing data regarding the involvement of PI in the body’s defense against tumor progression [9,18]. However, such roles have not been investigated in invertebrates. Further studies are needed to better understand the mechanisms of PI modulation and its possible role in clam immune responses against various endogenous and exogenous proteases. In conclusion, our study provided evidence for PI in softshell clams (M. arenaria) which belong to both active site and a2-M activities. Moreover, we found that PI activities are modulated by different sarcoma stages.
Acknowledgements This research was supported by a grant from the National Oceanic and Atmospheric Administration (NOAA), Virginia Sea Grant College Program, grant NA 56RGO141. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies. Virginia Institute of Marine Science contribution c 2220
References [1] Armstrong P, Rossner MT, Quigley JP. An a2-macroglobulin activity in the blood of chelicerate and mandibulate arthropods. J Exp Zool 1985;236:1–9.
[2] Armstrong P, Quigley J. Humoral immunity: a2 macroglobulin activity in the plasma of mollusks. Veliger 1992;35:161–4. [3] Barrett AJ, Starkey PM. The interaction of a2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J 1973;133:709 – 24. [4] Beckmann N, Morse MP, Moore CM. Comparative study of phagocytosis in normal and diseased hemocytes of the bivalve mollusc Mya arenaria. J Invertebr Pathol 1992;59:124 –32. [5] Bender RC, Fryer SE, Bayne CJ. Proteinase inhibitory activity in the plasma of a mollusc: evidence for the presence of amacroglobulin in Biomphalaria glabrata. Comp Biochem Physiol 1992;102B:821– 4. [6] Bower S, McGladdery SE, Price IA. Synopsis of infectious diseases and parasites of commercially exploited shellfish. Annu Rev Fish Dis 1994;4:1 – 199. [7] Brousseau DJ, Baglivo JA. Field and laboratory comparisons of mortality in normal and neoplastic Mya arenaria. J Invertebr Pathol 1991;57:59 – 65. [8] Brown RS, Wolke RE, Saila SB, Brown CW. Prevalence of neoplasia in 10 New England populations of the soft-shell clam (Mya arenaria). Ann New York Acad Sci 1977;298:522–34. [9] Calkins C, Sloane B. Mammalian cysteine protease inhibitors: biochemical properties and possible role in tumor progression. Biol Chem Hoppe-Seyler 1995;376:71 – 80. [10] Christensen S, Sottrup-Jensen L. Characterization of two serpins from bovine plasma and milk. Biochem J 1994;303:383–90. [11] Cooper KR, Brown RS, Chang PW. The course and mortality of a hematopoietic neoplasm in the soft-shell clam, Mya arenaria. J Invertebr Pathol 1982;39:149 – 57. [12] Eguchi M. Protein protease inhibitors in insects and comparison with mammalian inhibitors. Comp Biochem Physiol 1993;105B: 449 – 56. [13] Elston RA, Moore JD, Brooks K. Disseminated neoplasia of bivalve molluscs. Rev Aquat Sci 1993;6:405 – 66. [14] Faisal M, MacIntyre EA, Adham KG, Tall BD, Kothary MH, LaPeyre JF. Evidence for the presence of protease inhibitors in Eastern (Crassostrea 6irginica) and Pacific (Crassostrea gigas) oysters. Comp Biochem Physiol 1998;121B:161– 8. [15] Faisal M, McLaughlin S, Elsayed E, MacIntyre E, Kotob S, El-Gawady, H. Isolation of a Labyrinthuloides sp. (Protozoa:Labyrinthomorpha) with unique properties from the softshell clam (Mya arenaria). The 3rd Int. Symp. on Aquatic Animal Health, Baltimore, MD, USA, 30 August – 3 September, 1998, p. 174. [16] Farley CA, Otto SV, Reinisch CL. New occurrence of epizootic sarcoma in Chesapeake Bay softshell clams, Mya arenaria. Fish Bull 1986;84:851 – 7. [17] Farley CA, Plutschak DL, Scott RF. Epizootiology and distribution of transmissible sarcoma in Maryland soft-shell clams, Mya arenaria, 1984 – 1988. Environ Health Perspect 1991;90:35–41. [18] Howard E, Bullen E, Banda M. Preferential inhibition of 72 kDa and 92 kDa gelatinases by TIMP-2. J Biol Chem 1991;266:13070– 5. [19] Howard DW, Smith CS. Histological techniques for marine bivalve mollusks. US Department of Commerce, National Marine Fisheries Service, NOAA Technical Memo NMFS-F/ NEC-25, 1983, 97 pp. [20] Laskowski M, Kato I. Protein inhibitors of proteases. Annu Rev Biochem 1980;49:593 – 626. [21] McLaughlin S, Faisal M. Histopathological alterations associated with Perkinsus spp. infections in the softshell clam, Mya arenaria. Parasite 1998;5:263 – 71. [22] McLaughlin S, Faisal M. Comparison of tissues in fluid thioglycolate assays for diagnosis of Perkinsus spp. in the softshell clam, Mya arenaria. Aquaculture 1999;172:197 – 204. [23] Moore CA, Beckmann N, Morse MP. Cytoskeletal structure of diseased and normal hemoyctes of Mya arenaria. J Invertebr Pathol 1992;60:141 – 7.
E.E. Elsayed et al. / Comparati6e Biochemistry and Physiology, Part B 123 (1999) 427–435 [24] Reinisch CL, Charles AM, Stone A. Epizootic neoplasia in soft shell clams collected from New Bedford Harbor. Hazard Waste 1984;1:73 – 81. [25] Sottrup-Jensen L. 2 Macroglobulin structure, shape and mechanism of proteinase complex formation. J Biol Chem 1989;264:11539– 42.
.
435
[26] Sto¨ker W, Breit S, Sottrup-Jensen L, Zwilling R. 2-Macroglobulin from the hemolymph of the freshwater crayfish Astacus astacus. Comp Biochem Physiol 1991;98B:501 – 9. [27] Yevich PP, Barszcz CA. Neoplasia in soft shell clam (Mya arenaria) collected from oil-impacted sites. Ann New York Acad Sci 1977;298:409 – 26.