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Fall 2003
Investigations into the digestive system proteases of Argiope aurantia (Araneae: Araneidae), hemolytic agents of spider venoms, and the role of both in necrotic arachnidism
Matthew Joseph Foradori University of New Hampshire, Durham
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Recommended Citation Foradori, Matthew Joseph, "Investigations into the digestive system proteases of Argiope aurantia (Araneae: Araneidae), hemolytic agents of spider venoms, and the role of both in necrotic arachnidism" (2003). Doctoral Dissertations. 178. https://scholars.unh.edu/dissertation/178
This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. INVESTIGATIONS INTO THE DIGESTIVE SYSTEM PROTEASES OF
ARGIOPE AURANT1A (ARANEAE: ARANEIDAE), HEMOLYTIC AGENTS OF
SPIDER VENOMS, AND THE ROLE OF BOTH IN NECROTIC ARACHNIDISM
BY
MATTHEW JOSEPH FORADORI
BS, Indiana University of Pennsylvania, 1996
MS, University of New Hampshire, 1999
DISSERTATION
Submitted to the University of New Hampshire
in Partial Fulfillment of
the Requirements for the Degree of
Doctor of Philosophy
In
Zoology
September, 2003
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Dissertation Director, Edward K. Tillinghast, Professor of Zoology, Emeritus
u , \ o4a)i?uA... Ji. S\^ahJbC% Larry G. Harris) Professor of Zoology
7 'flArt \j Marian K. Litvaitis, Associate Professor of Zoology
Myung-Jin Moon, Professor of Biological Sciences (Dankook University, South Korea)
Wells, DVM, MS, DACVP -'Senior Veterinary Pathologist
Z\ 2.063 Date
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION
For my parents, Alexander and Janice Foradori, who encouraged me to follow
dreams.
For my wife, Renee, without whose understanding and love I could not have
finished.
111
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS
I truly believe that I would not have gotten this far without the guidance of Dr.
Robert S. Prezant at the Indiana University of Pennsylvania. I had floundered at
Duquesne University, leaving with a bruised ego and battered self esteem. I had turned
things around at IUP. The real problem was, I was unsure of where my career was
heading, and Dr. Prezant advised me, “If you don’t apply anywhere, you wont have any
options.” It was a difficult time in my life. My father had just been diagnosed with Non-
Hodgkin's Lymphoma and fought insurmountable odds to beat it. I was graduating soon,
and I really didn’t want to leave home and my family. My mother and father, of.^ourse,
told me to go and do what I had to do to continue my education. I would like to thank
Dr. Prezant for his insistence that I get some graduate school applications sent out.
I really had no idea where I wanted to go school. I new that I would like the
northeast. I had been to Boston, Massachusetts and Bath, Maine and I really like the
area. The problem was, I couldn’t find a potential advisor that worked with arachnids. It
wasn’t until Renee showed me a guy from the University of New Hampshire who looked
like a priest in the picture. I hadn’t thought much about New Hampshire before, let alone
going to the university there. If Renee had not shown me that graduate catalog from the
Zoology Department, my life would have most certainly been different.
I wrote Dr. Tillinghast a letter, and he responded quickly, sending me reprints and
asking me how soon I could come and visit. I quickly applied to work with him. Renee
and I decided to use our spring break and visit some of the schools we applied to, and
iv
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. along the way, we came to UNH. It was picturesque; a beautiful campus covered in
snow. Shortly after we returned home I found out I was accepted into the Zoology
graduate program.
Dr. Tillinghast took me, a person with no molecular lab skills, and turned me into
an electrophoresis machine. My master’s took three and a half years, but it was good
work. We answered quite a few questions about the egg case of Argiope aurantia in
those years; it is work I am truly proud of. I decided to say for my Ph. D. after he asked
me to. We had gotten into some excited new research at the end of my master’s degree
that I wanted to stay and work on. I would like to thank Dr. T for believing in me and my
work. I would also like to thank him for taking me on those two week SDF power-
collecting trips. I have seen parts of the southeastern United States I would not have seen
otherwise.
While here at the University of New Hampshire, I have made many friendships,
but there are a few in particular I would like to acknowledge. Dr. Patrick Danley and I
have been friends since the first day we met during the TA training sessions, back in
1996. Pat is one of the most sincere and sarcastic people I know. A true friend, even
though he moved away, we still keep in touch and see each other from time to time. Dr.
James Sulikowski is another great person. James is one of the most driven people I
know. His athletic ability is amazing to me, and yet he truly breaks the “dumb jock”
stereotype. Dr. Rick Hochberg rounds out my close group of colleagues. He is one of
the few people I have ever met that can make me laugh all the time. These three men
have made this experience worthwhile for me.
v
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. There are a few other people I would like to mention. Lauren Keil, one of the
most meticulous lab workers I have ever met taught me how to be organized. Max Diem,
one of the most enthusiastic people I have ever met was always ready for a day in the
field with his “right on!” attitude. Last but not least, in one of the bumps I experienced
during my dissertation experience, Marian Litvaitis, told me to stick it out. I was so close
to being done that I could taste it. I’m glad I took her advice.
Thank you all.
vi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
DEDICATION ...... iii
ACKNOWLEDGEMENTS ...... iv
LIST OF TABLES...... ix
LIST OF FIGURES ...... ;...... x
ABSTRACT...... xii
CHAPTER PAGE
INTRODUCTION...... 1
I. AN EXAMINATION OF THE POTENTIAL ROLE OF SPIDER DIGESTIVE PROTEASES AS A CAUSATIVE FACTOR IN SPIDER BITE NECROSIS ...... 7
Abstract...... 7
Introduction ...... 7
Materials and Methods...... 9
Results ...... 14
Discussion ...... 22
II. SURVEY FOR POTENTIALLY NECROTIZING SPIDER VENOMS, WITH SPECIAL EMPHASIS ON CHEIRACANTHIUM MILDEI...... 27
Abstract ...... ,.27
Introduction ...... 28
Materials and Methods ...... 29
vii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results ...... 33
Discussion ...... ,...... 42
HI. FRACTIONATION OF SPIDER DIGESTIVE FLUID AND PARTIAL CHARACTERIZATION OF THE PROTEASES AND PROTEASE INHIBITORS...... 47
Abstract...... 47
Introduction ...... 47
Materials and Methods...... 49
Results ...... 54
Discussion ...... 65
LIST OF REFERENCES ...... 71
APPENDIX...... 80
viii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES
TABLE PAGE
2.1. Survey of the venom of selected spider species for their ability to cause hemolysis...... 34
3.1. N-terminal amino acid sequence of isolated protease from the digestive fluid of Argiope aurantia ...... 63
ix
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST GF FIGURES
FIGURE PAGE
1.1. Proteases in the digestive fluid (SDF) of A. aurantia using casein as a substrate. ...15
1.2. Effect of A. aurantia digestive fluid on (a) Type III collagen and (b) gelatin ...... 16
1.3. Effect of A. aurantia digestive fluid on (a) fibrinogen and (b) fibronectin ...... 17
1.4. Effect of A. aurantia digestive fluid on (a) fibrin and (b) elastin...... 18
1.5. The effect of EDTA on A. aurantia digestive fluid (SDF) proteases ...... 19
1.6. Inhibition of (a) A. aurantia digestive fluid (SDF) proteases and (b) L. reclusa venom proteases by A. aurantia hemolymph as shown by diffusion in agarose/ casein gels...... 20
1.7. Comparison of the effects of A. aurantia digestive fluid and L. reclusa venom on the skin of the rabbit 18 hours post-injection...... 21
1.8. A histological comparison of the effects of intradermal injection ofArgiope aurantia digestive fluid (II a and b; from site II, Fig. 7) and Loxosceles reclusa venom gland homogenate (III a and b; from site III, Fig. 7), 72 hours post injection ...... 23
2.1. The effects of spider venom gland homogenates (VGH) on RBC ghosts...... 37
2.2. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on individual purified phospholipids...... 38
2.3. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on phosphatidylcholine (/PC) fluorescently labeled on the sn-l (D-3771) or sn-2 (D-3803) position...... •...... 39
2.4. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on the skin of New Zealand white rabbits ...... 40
2.5. Histology of the skin of New Zealand white rabbit 48 hours after intradermal injection of 79pg protein Cheiracanthium mildei venom gland homogenate.*...... 41
x
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.6. Calcium dependency of Cheiracanthium mildei venom gland homogenate hemolytic factor...... 43
3.1. Fractionation of SDF by anion exchange chromatography ...... 55
3.2. Fractionation of fractions 1-10 on Sephacryl S-100 HR column...... 57
3.3. Fractionation of fractions 48-54 on cation exchange chromatography...... 58
3.4. Fractionation of fractions 26-32 on a Sephadex G-75 column ...... 59
3.5. Separation of SDF protease inhibitors by natural PAGE ...... 60
3.6. Separation of SDF protease inhibitors by SDS-PAGE ...... 62
3.7. Comparison of spider and crayfish digestive proteases...... 64
3.8. Fractionation of SDF by ultracentrifugation ...... 66
3.9. Whole SDF fractionation by sucrose density gradient ultracentrifugation examined by (A) zymography and (B) SDS-PAGE...... 67
xi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT
INVESTIGATIONS INTO THE DIGESTIVE SYSTEM PROTEASES OF
ARGIOPE AURANTIA (ARANEAE: ARANEIDAE), HEMOLYTIC AGENTS OF
SPIDER VENOMS, AND THE ROLE OF BOTH IN NECROTIC ARACHNIDISM
By
Matthew J. Foradori
University of New Hampshire, September, 2003
Necrotic arachnidism, or tissue necrosis following spider bites is a widespread
problem. Knowledge of the exact mechanism that produces such lesions is still
incomplete. Here I have examined spider digestive fluid proteases and spider venom
enzymes (e.g., sphingomyelinase D and phospholipase A2) for their potential to cause
necrotic lesions, as both are thought to be primary agents.
The digestive fluid of Argiope aurantia was examined for its ability to cleave a
variety of extra-cellular matrix proteins, including collagen, elastin, and fibronectin.
Having confirmed that the fluid has collagenases, the digestive fluid was injected into the
skin of rabbits to observe whether it would cause necrotic lesions. It did not. The data do
not support the suggestions that spider digestive collagenases have a primary role in
spider bite necrosis.
In an effort to design a simple, inexpensive screening method for identifying
spiders with necrotizing venoms, venom gland homogenates of a variety of spider species
were examined for their ability to cause hemolysis. Those venoms that were positive
xii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were further examined for the presence of sphingomyelinase D, and the ability to evoke
necrotic lesions in the skin of rabbits. Of 45 species examined, only the venom of
Loxosceles reclusa and Cheiracanthium mildei caused hemolysis. Unlike L. reclusa
venom, however, C. mildei venom did not possess sphingomyelinase D nor did it cause
necrotic lesions in the skin of rabbits. In an effort to determine the hemolytic agent in C.
mildei venom, I found evidence for a phospholipase A2.
Through a combination of ion exchange chromatography, size exclusion
chromatography, ultracentrifugation, and electrophoresis, an attempt was made to isolate
several digestive fluid proteases and serine protease inhibitors in milligram quantities
from the digestive fluid of Argiope aurantia. The proteases, however, eluted
anomalously during the size exclusion chromatography and ultracentrifugation trials. I
have hypothesized that spider proteases can undergo a reversible aggregation to account
for this behavior. I was able to isolate several proteases in quantities sufficient for N-
terminal amino acid sequences. These data revealed that spider proteases are related to
astacins and meprins, which are in the astacin family of metalloproteases.
xiii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION
Digestion is initiated externally in spiders. They are incapable of ingesting solid
food as they lack a chewing apparatus and have only a slender pharynx connecting with
the stomach. Moreover, bristles bordering the mouth opening serve as a sieve to prevent
particles in excess of 1 pm from clogging the opening. Their food must therefore be
liquefied. Typically, spiders envenomate prey and begin to crush it in their chelicerae.
Spiders then regurgitate digestive fluid from the intestinal tract onto the prey, wait a few
moments, then suck the solubilized products into their gut using the muscular pharynx
and sucking stomach (Foelix, 1996). The process is repeated until the prey is reduce to a
small chitinous mass.
Although there has been longstanding interest in the enzymatic content of spider
digestive fluid (SDF), research has been sporadic. One of the first demonstrations of
proteolytic activity from spiders was that of Plateau (1877). Using abdominal caeca
extracts from European araneomorph spiders, he discovered proteolytic, amylytic, and
lipolytic activities. Betkau (1885) confirmed the presence of proteolytic and other
enzyme activity. A quantitative investigation of tarantula (Avicularia) digestive enzymes
by Schlottke (1936) revealed a potent proteinase with caseinolytic ability, as well as
moderate amounts of peptidase and amylase activity. Pickford (1942) found fibrinase,
gelatinase (pH 1.5-12), amylase, phosphatases, lipase (pH 6-11) and proteinases in the
digestive fluids of Araneus trifolium, Araneus marmoreus, and Larinioides comutus.
Poliak (1966) and Houghton et al. (1967) found evidence of esterase activity in several
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. species of spider homogenates and histological sections, respectively. Couch and Benton
(1971) found general esterase and phosphatase activity in two species of spiders.
Tillinghast and Kavanagh (1977) confirmed the presence of alkaline proteases,
collagenase, and elastase activity in the digestive fluid of Argiope aurantia. They were
also the first to demonstrate spider web digestion in vitro. Mommsen’s (1979a, b, c)
systematic studies of the digestive enzymes of Tegenaria atrica is one of the most
complete inventories of spider digestive fluid to date. He found a single protease that
could hydrolyze native as well as denatured proteins, an amidase, and chymotrypsin-like
activity (Mommsen, 1979a), a chitinase and several a-amylases (Mommsen, 1979b), as
well as alkaline and acid phosphatases (Mommsen, 1979c). A later study (Mommsen,
1980) revealed the presence of chitinase and {3-N-acetylglucosaminidase in Cupiennius
salei. Rekow et al. (1983) found protease and lipase activity in the abdominal
homogenates of Loxosceles reclusa, but lacking in cephalothorax homogenates. Herrero
and Odell (1988) examined the digestive secretions of two Costa Rican tarantulas and
determined that they had high proteolytic activity. More recently, Joo et al. (2002) found
a prothrombin-activating protease in the isolated digestive tract homogenate ofNephila
clavata.
There are also numerous reports of proteases in spider venoms (see review by
Perret, 1977). However, Perret observed that proteases were absent from those venoms
which had been collected by methods which took precautions to avoid contamination by
SDF. Mebs (1970, 1972) for example, reported protease in tarantula (Pamphobetus
roseus) venom that he received from the Instituto Butantan (Sao Paolo, Brazil). He
isolated three proteases of approximately 11 kDaltons, that had pH optima of 8.5 and
2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cleaved casein, but did not cleave trypsin substrates, and were equally unaffected by
serine protease inhibitors. These properties are identical to those later reported for
proteases from SDF (Tillinghast and Kavanagh, 1977; Tugmon and Tillinghast, 1995). It
seems likely that the venom was contaminated by the spider’s digestive fluid.
The work I conducted for my master’s thesis examined the mechanism by which
Argiope aurantia spiderlings exit the egg case. The egg case is constructed of a tightly
woven outer cover, composed primarily of large-diameter cylindrical gland fibers and
small-diameter fibers, likely of aciniform gland origin; It must be sufficiently robust to
resist degradation for the many months between its formation in early fall and the
emergence of the spiderlings in late Spring. I demonstrated that spiderlings escape from
the egg case by a combination of enzymatic digestion and mastication with their
chelicerae to form a communal hole in the outer cover. I recorded the spiderlings
chewing the edges of the hole as they exited the cocoon using video equipment.
Electrophoretic analysis revealed that the proteases of the spiderlings were identical to
those found in washes of the edges of the exit holes (Foradori et al., 2002).
In the first chapter of this dissertation, I examined the possible involvement of
SDF proteases in the formation of lesions following spider bites. Atkinson and Wright
(1992) observed that gut extracts of spiders, including those of mygalomorphs and
araneomorphs, produced cellular disruption of mouse skin in cell culture. From their
research, they concluded that it was digestive proteases released at the area of the bite
rather than the constituents of the venom that were the cause of necrotic lesions. As gut
extracts contain a variety of tissues as well as digestive enzymes, I have, examined the
hypothesis of Atkinson and Wright (1992) using SDF alone. First, SDF was examined
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for its ability to cleave a variety of connective tissue proteins. Substrates including
casein and gelatin, and more importantly the basement membrane proteins fibrin,
fibrinogen, fibronectin, elastin, collagen were all cleaved by the proteolytic enzymes.
SDF was then injected into the skin of rabbits under the assumption that it would cause
necrotic lesions, but it evoked no response. By comparison, 3 pg of brown recluse
venom (Loxosceles reclusa) resulted in severe lesions. Thus, it does not appear that SDF
has a primary role in necrotic arachnidism (Foradori et al., 2001).
The venom of L. reclusa contains sphingomyelinase D, an enzyme that has been shown
to be responsible for the necrotic lesions which often follow this spider’s bite
(Kurpiewski, 1981).
In the second chapter of this dissertation, I attempted toidentify other spider
species that might cause necrotic lesions by analyzing venoms for sphingomyelinase D
activity. Sphingomyelinase D is known to lyse red blood cells (RBQ in addition to
causing necrotic lesions (Forrester et al., 1978). To identify other spiders that might
cause such lesions, I assayed homogenates of venom glands (VGH) for their ability to
cause RBC lysis (hemolysis). Venom gland homogenates of more than 260 spiders
representing 55 spider species from 18 families were examined. The VGH ofL. reclusa
always causes hemolysis, as reported by Forrester et al. (1979), and was therefore, used
as a positive control. Of the remaining 55 species, onlyCheiracanthium mildei VGH
caused hemolysis, however, further research revealed that it did not contain
sphingomyelinase D. To identify the agent causing RBC lysis, I subjected RBC ghosts to
C. mildei VGH. Chromatographic analysis of the products revealed that phospholipids
(glycerophospholipids) had been cleaved. I then evaluated the VGH for its ability to
4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cleave a variety of natural and synthetic phospholipids. These studies demonstrated the
presence of a phospholipase A2.
Cheiracanthium mildei is a common spider on the University of New Hampshire
campus, and the question arose as to whether its venom would cause necrotic lesions. A ./ review of the literature revealed that C. mildei venom caused necrotic lesions in some
laboratory animals (Spielman and Levi, 1970; Newlands et al., 1980; Hughes, 1981).
However, when I injected VGH of C. mildei into rabbits, no lesions resulted. Most
reports suggest that C. mildei venom produces only a mild effect in humans (Krinsky,
1987; Minton, 1972). Which was consistent with my own observations. Even Spielman
and Levi (1970), who reported necrosis in following a C. mildei bite, reported nothing
more than mild redness and swelling in humans.
The ability of the digestive enzymes of Argiope aurantia to cleave connective
tissue proteins suggests they would be valuable tools for medical research and a possible
therapeutic use. Previous efforts to isolate spider proteases, however, have resulted in
very low yield (Mebs, 1972; Kavanagh and Tillinghast, 1983). In the third chapter of this
dissertation I attempted to develop a more effective protocol for the purification of the
SDF proteases.
Digestive fluid was fractionated by a combination of ion-exchange
chromatography, gel filtration chromatography, and sucrose density gradient
ultracentrifugation. In none of theses investigations was I able to isolate an individual
protease excepting pl2 (a protein with a mass of 12 kDaltons). Protease 12 (pl2),
however, was found in all other fractions as well. These results led me to propose that
the spider digestive proteases are reversibly associated into higher aggregates. This
5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. assumption explains the anomalous elution times that I and Mebs (1972) observed with
gel filtration, and explains why earlier efforts to obtain individual proteases resulted in
low yields of a single protease (p i2).
6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1
AN EXAMINATION OF THE POTENTIAL ROLE OF SPIDER DIGESTIVE
PROTEASES AS A CAUSATIVE FACTOR IN SPIDER BITE NECROSIS1
ABSTRACT
Tissue necrosis following spider bites is a widespread problem. In the continental
United States the brown recluse {Loxosceles reclusa), hobo spider {Tegenaria agrestis),
garden spider {Argiope aurantia), and Cheiracanthium species among others reportedly
cause such lesions. The exact mechanism producing such lesions is controversial. There
is evidence for both venom sphingomyelinase and spider digestive collagenases.
I have examined the role of spider digestive proteases in spider bite necrosis. The
digestive fluid of A. aurantia was assayed for its ability to cleave a variety of connective
tissue proteins, including collagen. Having confirmed that the fluid has collagenases, the
digestive fluid was injected into the skin of rabbits to observe whether it would cause
necrotic lesions. It did not. The data do not support the suggestions that spider digestive
collagenases have a primary role in spider bite necrosis.
1.1. Introduction
Tissue necrosis resulting from spider bites is a widespread problem (Bettini and Brignoli,
1978). Whereas Loxosceles species are the best known spider producing
1. Foradori, M.J., Keil, L.M., Wells, R.E., Diem, M., Tillinghast, E.K., 2001. An examination of the potential role of spider digestive proteases as a causative factor in spider bite necrosis. Comp. Biochem. Physiol. 130 C: 209-18.
7
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. necrotic lesions, other spider species are thought to do so as well. Thus Vest (1987) has
reported that the bite of Tegenaria agrestis (hobo spider) in the Pacific Northwest is
responsible for necrotic lesions, and Spielman and Levi (1970) have reported that the bite
of Cheiracanthium inclusum is also capable of producing such lesions. Russell and
Gertsch (1983) have compiled a list of other spider species suspected of causing necrotic
arachnidism.
At this time there is a lack of agreement as to the exact mechanisms that cause
tissue necrosis (Futrell, 1992). Among the existing theories, Forrester et al. (1978) and
Kurpiewski et al. (1981) have proposed that the venom enzyme sphingomyelinase D is
the responsible agent. Atkinson and Wright (1992) observed that gut extracts of 13
different spider species, including mygalomorphs and araneomorphs, produced cellular
disruption of mouse skin in cell culture. They also observed high collagenase activity in
the gut extracts of these spiders. They concluded that the digestive system collagenases,
rather than any venom component, were responsible for the development of tissue
necrosis. As gut homogenates very likely contain a variety of enzymes and
pharmacologically active compounds, I have tested their hypothesis using spider
digestive fluid.
Argiope aurantia was chosen to test the hypothesis of Atkinson and Wright
(1991, 1992) because preliminary research from the laboratory detected collagenases in
the digestive fluid of this species. In addition, the digestive fluid is easily collected from
A. aurantia in large quantity (an average of 100 pi per spider) and thus I was able to
examine the fluid for its ability to cleave a variety of connective tissue proteins.
Moreover, A. aurantia hemolymph contains inhibitors that effectively block digestive
8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fluid proteases (Tugmon and Tillinghast, 1995). This provided me with a tool to block
spider digestive proteases and thereby test the hypothesis that the digestive proteases
have a role in spider bite necrosis. Had the proteases caused necrosis, hemolymph
inhibitors would have provided insight into the mechanisms that cause necrosis as well as
serve as a potential antidote.
My results confirm the observation of Atkinson and Wright (1992) that the gut of
spiders contains collagenases. In addition, spider digestive fluid had the ability to cleave
a variety of other connective tissue proteins such as elastin and fibrinogen. In spite of the
presence of these proteases, intradermal injections of this fluid into rabbits did not result
in necrosis. My results do not support the hypothesis that the spider digestive proteases
have a primary role in spider bite necrosis.
1.2. Materials and Methods
1.2.1. Collection of spider fluids
A. aurantia were collected in Maine, New Hampshire, North Carolina, South
Carolina, Georgia and Virginia during the summers of 1998/99. Twenty microliter
micropipettes were used to collect spider digestive fluid (henceforth referred to as SDF),
which spiders regurgitated freely when handled. Approximately 4 ml was collected from
100 spiders. SDF was pooled, placed on ice, and frozen within 24 hours of its collection.
Hemolymph was collected fromA. aurantia narcotized by carbon dioxide. The tip of the
fourth left leg was cut off with surgical scissors and the exuding hemolymph was
collected with a micropipette. Hemolymph was pooled and stored at -20° C.
9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2. Zymography
To visualize the major proteases in the digestive fluid of A. aurantia as well as
establish their apparent molecular weights. SDF from three A. aurantia spiders (0.5 pi
quantities) were examined separately for caseinolytic activity. The samples where
electrophoresed on 4-16% Zymogram (blue casein) gels (Invitrogen, EC# 6415) under
non-reducing conditions. Molecular weight standards ran concurrently were from Gibco-
BRL (Benchmark prestained protein ladder #10748-010). Following the electrophoretic
separation, gels were renatured and developed according to the manufacturer's
recommendations.
1.2.3. Degradation o f extracellular matrix proteins
The degradation of collagen was visualized by the method of Phillips and Dresden
(1973). Three milligrams of collagen (Sigma, C-3511, type in calf skin) was solubilized
in 1.5 ml 0.1 mM acetic acid for two days at 15° C then dialyzed overnight against 0.01M
Tris, pH 7.8 containing 0.15 M NaCl and 0.5 mM CaCl2. Ten microliters SDF was then
added, mixed, and incubated at 20° C. Thirty microliter aliquots were removed at 0, 0.5,
2, 4, and 23 hours. These were mixed immediately with an equal volume of sample
buffer, and boiled to stop the reaction. Controls lacking SDF were incubated
concurrently for 23 hours. The samples were electrophoresed (SDS-PAGE) on 4-20%
Tris Glycine gels (Invitrogen, EC6028) under reducing conditions with constant 1,25 V
and stained with a 0.02% Coomassie R-250,40% Methanol (v/v), and 1% Acetic acid
(v/v) solution overnight to observe degradation products. The degradation of gelatin was
performed in an identical manner except that the collagen (Sigma, C-3511, calf skin) was
heated to 70° C for 15 minutes and cooled to 20° C prior to the addition of SDF.
10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The effect of SDF on extracellular matrix proteins was visualized by the method
of Feitosa et al. (1998). Two microliters of SDF (89 pg protein) was added to 0.5 mg of
fibrinogen (Sigma, F-8630) or fibronectin (Sigma, F-4759) suspended in 200 pi 25 mM
Tris buffer, pH 8.1, containing 5 mM CaCl2 (henceforth referred to as buffer A) at 37° C.
Thirty microliter aliquots of the reaction mixtures were removed at 0 and 30 min, 1, 2, 4,
8, and 23 h and frozen promptly. Controls lacking SDF were incubated for 23 h. The
fibrinogen and fibronectin samples were electrophoresed (SDS-PAGE) on 10% Tris-
Glycine gels (Invitrogen, EC# 6078) under non-reducing and reducing conditions,
respectively, with constant 125 V and stained with a 0.02% Coomassie Blue R-250, 25%
2-propanol, and 10% acetic acid solution to observe degradation products.
The degradation of elastin was examined by the radial-diffusion method of
Schumacher, and Schill (1972). A gel was prepared by adding 100 mg agarose to 6 ml
buffer A and boiling for 5 min. Upon cooling to 60°C, 50 mg of elastin (Sigma, E-1625)
suspended in 4 ml buffer A was added and poured into a diffusion plate. When the gel
solidified at room temperature, a 6 mm diameter well was placed in the center. A 20 pi
sample containing 2 pi SDF (89 pg protein) and 18 pi buffer A was added to the well.
Fibrinolytic activity was observed by the method of Yang and Ru (1997). A fibrin plate
was prepared by adding 75 mg agarose to 10 ml 0.02M Tris buffer, pH 8.0, containing 85
mg NaCl and boiling for 5 min. When the temperature declined to 58°C, 3 ml of a
fibrinogen solution (150 mg Sigma, F-8630 in 3 ml of the above buffer) and thrombin
(100 pi containing 200 units, Sigma T-4648) were added, and the mixture was poured
into a diffusion plate and allowed to solidify for 10 min. Samples of 2 pi SDF (89 pg
protein) made to 20 pi with buffer A were placed in the wells. The plates were incubated
11
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. overnight and the diameter of the ring of digestion of elastin and fibrin was measured in mm.
All protein concentrations were estimated by the method of Lowry et al. (1951)
using bovine serum albumin as a standard.
1.2.4. The effect of EDTA on spider proteases.
To observe whether SDF proteases are metalloproteases, 100 pi SDF (4.45 mg
protein) was made to 3.0 ml with 25 mM Tris buffer, pH 8.1, containing 20 mM EDTA
and set at room temperature for 1 h. SDF was then dialyzed against the same buffer
without EDTA for an hour. 100 pi of SDF (4.45 mg protein) made to 3.0 ml with 25 mM
Tris, pH 8.1, and containing 2 mM CaCl2 served as the control. One hundred microliter
aliquots of EDTA-treated enzyme were made to 1 ml with increasing quantities of 2.0
mM ZnS04 and incubated for 30 min at room temperature. One ml of 1% Hammersten
casein (Schwarz/Mann) was then added and all tubes were incubated for 15 min at 37°C.
The reactions were stopped by the addition of 3 ml 10% TCA. After 30 min the samples
were filtered through Whatman No.l filter paper and the optical density of the eluant was
read at 275 nm (Kunitz, 1947).
1.2.5. Inhibition of spider digestive proteases by spider hemolymph
To determine the quantity of hemolymph necessary to inhibit the SDF proteases,
90 mg of agarose and 90 mg Carnation dry milk were added to 9 ml of buffer A. The
mixture was boiled for 5 min and poured into a diffusion plate. Wells were cut when the
gel solidified. Two microliters of SDF (89 pg protein) mixed with 18 pi buffer A was
added to one well and 2 pi SDF (89 pg protein) mixed with 18 piA. aurantia
12
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hemolymph was added to a second well. The plate was incubated overnight at room
temperature. .
1.2.6. The effect o f spider digestive fluid on the skin o f rabbits
To observe if SDF from A. aurantia is capable of causing necrotic lesions samples
were injected into the skin of rabbits. Because it is the best documented example of
spider-induced necrotic lesions on rabbit skin (Atkins et al., 1958; Smith and Micks,
1968; Geren et al., 1976; Barbara et al., 1992, 1996; Gomez et al., 1999; Elston et al.,
2000), the venom of L. reclusa served as a positive control. A. aurantia venom has not
been examined in the skin of rabbits. Three adult female New Zealand white rabbits
were injected in a shaved area on the back with the following solutions: site I: 200 pi
PBS, pH 7.4; site II: SDF (20 pg protein) in 200 pi PBS; site III:L. reclusa venom gland
homogenate (10 pg protein) in 200 pi PBS; site IV:A. aurantia hemolymph (100 pg
protein) in 200 pi PBS; site V: SDF (20 pg protein) +A. aurantia hemolymph (100 pg
protein) in 200 pi PBS; and site VI:L. reclusa venom gland homogenate (10 pg protein)
+ A. aurantia hemolymph (100 pg protein) in 200 pi PBS. All preparations were filtered
through a 0.2 micron filter prior to injection. The injections were administered
intradermally with a 26 gauge needle approximately 6-8 cm apart. The rabbits were
observed hourly for the first six hours and then every six hours for 72 hours. The lesions
were photographed with a digital camera and scored according to the intracutaneous test
described in the USP 24-NF19 (2000). The experiments were carried out in compliance
with the Public Health Service Policy, the Guide for the Care and Use of Laboratory
Animals (1996), and University Policy (see Appendix).
13
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The rabbits were euthanized 72 hours after injection. Tissues samples including
skin and subcutis were taken from all injection sites and fixed for 48 hours in 10% neutral
buffered formalin, dehydrated in an ethanol series, cleared in Histoclear II (National
Diagnostics), then embedded in Paraplast Plus (Fisher Scientific) using a Citadel 2000
(Shandon) carousel style automatic tissue processor. Sections were cut at 6 pm, mounted
on glass slides, and stained with Harris’ hematoxylin and eosin (Luna, 1979) and
examined under light microscopy.
1.3. Results
There are at least four major proteases in the SDF. They have apparent molecular
weights of 120, 38, 30, and 12 kDa when observed by zymography (Fig. 1.1). In addition
to their ability to cleave casein, the SDF proteases are capable of cleaving native collagen
and gelatin (Fig. 1.2), fibrinogen and fibronectin (Fig. 1.3), as well as elastin and fibrin
(Fig. 1.4). The exposure of 100 pi SDF to 20 mM EDTA for 1 h reduced the proteolytic
activity to about 35% of that of the control. The activity was largely restored by
exposure to 1.4 mM ZnS04, above which the activity was reduced (Fig. 1.5).
The spider proteases in 2 pi SDF were effectively blocked by 18 pi hemolymph
(Fig. 1.6a). The hemolymph of A. aurantia also inhibited proteases from the venom
gland homogenates of L. reclusa (Fig. 1.6b).
The effect of SDF on New Zealand white rabbit skin was examined by
intradermal injection. The effectiveness of inhibition by spider hemolymph was observed
by mixing a pre-established volume of hemolymph with each of the fluids immediately
prior to injection. The injection of SDF (20 pg protein) produced a well defined oval
area of redness approximately 1.0 x 0.7 cm in diameter and 1.0 mm in height (Fig. 1.7,
J 14
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.1. Proteases in the digestive fluid (SDF) of A. aurantia using casein as a substrate.
Spider digestive fluid (0.5 pi) from 3 different Argiope aurantia were electrophoresed on 4-16% zymogram (blue casein) gels under non-reducing conditions. The gels were renatured, developed, then stained according to the manufacturer's recommendations.
15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ;.-iA («&$*» la# M ikm?’h i® ;||i® lfP|; J i t UpP Jfif§! lo u if' <1 2417 l'J ? iy t
1} :•
■«m, .■■!'►. :
1 2 3-4 5 67 MPa
im# m 4 m s # u 4fJ 3fi4 347 ifj .13.1 §j
Figure 1.2. Effect of A. aurantia digestive fluid on (a) Type III collagen and (b) gelatin
Nine microliters of spider digestive fluid was added to a solution containing 3 mg collagen or 4 pi of spider digestive fluid was added to a solution containing 1.2 mg of gelatin. The mixtures were incubated for 23 h at room temperature (20 ° C). Samples (20 gl) were removed at: 0 time (lane 2), 30 min (lane 3), 1 h (lane 4), 2 h (lane 5), 4 h (lane 6), and 23 h (lane 7). Lane 1 in each case was a control (con.) containing substrate and buffer only. The fibrinogen and fibronectin samples were electrophoresed on 4-20% SDS-PAGE gradient gels under reducing conditions. The gels were stained with Coomassie blue to visualize substrate fragments.
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MDa 172.6 1IJW 79.6 61.3 f l H B I 49,0
con, 0 0.5 1
Figure 1.3. Effect of A. aurantia digestive fluid on (a) fibrinogen and (b) fibronectin.
Two microliters of spider digestive fluid was added to a solution containing 0.5 mg fibrinogen or fibronectin in 200 pi 25 mM Tris buffer, pH 8.1. The mixtures were incubated for 23 h at 37°C. Samples (20 pi) were removed at: 0 time (lane 2), 30 min (lane 3), 1 h (lane 4), 2 h (lane 5), 4 h (lane 6), 8 h (lane 7), and 23 h (lane 8). Lane 1 in each case was a control (con.) containing substrate and buffer only. The samples were added to an equal volume of sample buffer and electrophoresed on 10% SDS-PAGE gels under non-reducing and reducing conditions respectively. The gels were stained with Coomassie blue to visualize substrate fragments.
17
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.4. Effect of A. aurantia digestive fluid on (a) fibrin and (b) elastin.
Fibrin and elastin gels were prepared with Tris buffer, pH 8.1, as described in the text. Two microliters of A. aurantia digestive fluid made to 20 pi with the same buffer, then added to the wells. The plates were incubated overnight.
18
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ZiiKoaaaiiifiMcii. (mM)
Figure 1.5. The effect of EDTA on A. aurantia digestive fluid (SDF) proteases.
One hundred microliters SDF was made to 3 ml with 25 mM Tris buffer, pH 8.1, containing 20 mM EDTA incubated for 1 hour, then dialyzed to remove the chelator. One hundred microliter aliquots of treated SDF were incubated with increasing concentrations of ZnS04 then assayed for protease activity. The results were compared to the protease activity of spider digestive fluid in the above buffer, but lacking EDTA and containing 2 mM CaCl2.
19
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SDF + Hemolymph
^ m m w i
/ aurantsa Hemolymph L teclusa VGH
Figure 1.6. Inhibition of (a) A. aurantia digestive fluid (SDF) proteases and (b) L. reclusa venom proteases by A. aurantia hemolymph as shown by diffusion in agarose/casein gels.
(a) Two microliters (89 pg protein)A. aurantia digestive fluid (SDF) mixed with 18 pi Tris buffer (left well), and 2 pi SDF mixed with 18 pi A. aurantia hemolymph (right well), (b) Twenty five microliters A. aurantia hemolymph (left well) and 10 piL. reclusa venom gland homogenate (VGH) made to 25 pi with PBS (right well). Note the inhibition on the left side of the right well of b.
20
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.7. Comparison of the effects of A. aurantia digestive fluid and L. reclusa venom on the skin of the rabbit 18 hours post-injection.
New Zealand White rabbits were injected intradermally at the numbered sites shown on the dorsum with the following solutions made to 200 pi with PBS. II. A. aurantia digestive fluid (20 pg protein), III. brown recluse venom (10 pg protein), V. A. aurantia digestive fluid (20 pg protein) plus A. aurantia hemolymph (100 pg protein), and VI. brown recluse venom (10 pg protein) plus A. aurantia hemolymph (100 pg protein). Sites I and IV were saline and hemolymph controls respectively, and are not shown.
21
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. site Et). It was most intense after 6 h, but stabilized thereafter, decreasing in color
intensity after 24 h. Mixing A. aurantia hemolymph (100 pg protein) with the SDF
almost completely negated these effects (Fig. 1.7, site Y).
Histological sections of skin and subcutis from site I (PBS) showed no observable
changes and served as a negative control. Although SDF is rich in connective tissue
degrading proteases, sections of tissue from site II (SDF) showed only minimal reaction
characterized by low numbers of heterophilic leukocytes (Fig. 1.8). Tissues from site IV
(A. aurantia hemolymph) showed little or no observable reaction and were comparable to
site I. There was negligible to minimal reaction in the tissues from site V, injected with a
mixture of A. aurantia hemolymph and SDF. The microscopic appearance of the
injection sites was consistent in all three rabbits.
1.4. Discussion
While a number of spider species have been listed as potentially causing necrotic
lesions (Russell and Gertsch, 1983), the agent responsible for these lesions remains a
subject of debate. There is strong evidence that the venom enzyme, sphingomyelinase
D, is the causative agent (Kurpiewski et al., 1981) in the bite of the L. reclusa. However,
Atkinson and Wright (1991, 1992) have presented evidence that spider digestive
collagenases rather than venom enzymes are the cause. I have tested their hypothesis
with the digestive fluid from A. aurantia.
The digestive fluid of A. aurantia contains casein cleaving proteases ranging in
molecular weights from 12 to 140 kDa. The most evident of these had apparent
molecular weights of 12, 30, 38, and 120kDa (Fig. 1.1). The proteases proved capable of
22
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.8. A histological comparison of the effects of intradermal injection of Argiope aurantia digestive fluid (II a and b; from site II, Fig. 1.7) and Loxosceles reclusa venom gland homogenate (III a and b; from site III, Fig. 1.7), 72 hours post-injection. Note edema and heavy infiltration of heterophils (HI a) and degeneration of connective tissue and panniculus muscle (arrow, site III b).
23
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cleaving native collagen (Fig. 1.2), fibrinogen and fibronectin (Fig. 1.3), as well as
elastin and fibrin (Fig.4), Feitosa et al. (1998) have reported that the venom of L.
intermedia has proteolytic activity against gelatin, fibrinogen, and fibronectin, but is
unable to cleave types I and Et collagen. This is consistent with the observations of
Kaiser and Raab (1967) who reported that the venom of both Phoneutriafera and Lycosa
erythrognatha can readily digest gelatin, but neither had the ability to cleave native
collagen. By contrast, A. aurantia digestive proteases can not only cleave all of these
substrates, but native collagen as well (Fig. 1.2). The presence of eollagenases in spider
digestive fluid is in agreement with the report of collagenase activity in spider midgut
extracts (Atkinson and Wright, 1992).
Most of the spider digestive proteases appear to be metalloproteases. SDF
protease activity is greatly inhibited by EDTA, but not serine, cysteine or aspartate
protease inhibitors (Tillinghast and Kavanagh, 1977: Tugmon and Tillinghast, 1995).
The inhibition by EDTA is relieved by the addition of zinc ions (Fig. 1.5). Spider venom
proteases also appear to be metalloproteases. They are inhibited by EDTA (Jong et al.,
1979; Feitosa et al., 1998), and not by serine protease inhibitors (Mebs, 1972). Both
spider digestive proteases (Tillinghast and Kavanagh, 1977; Mommsen, 1978) and spider
venom proteases (Mebs, 1972; Jong et al., 1979; Veiga et al., 2000) have alkaline pH
optima.
Whereas the preceding observations suggest a strong similarity between digestive
fluid proteases and those of venom, there is one important difference: neither Kaiser and
Raab (1967), Wright et al. (1973), nor Feitosa et al. (1998) could find evidence of
eollagenases in spider venom. The digestive fluid of A. aurantia, by contrast, has
24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. collagenase activity. Perret (1977) has observed that spider venom collected by
electrostimulation can be contaminated by digestive fluids. My observations suggest that
spider venom collected in this manner might first be screened for eollagenases as a
precaution against possible contamination from digestive fluid.
In spite of the fact that SDF has the ability to cleave a variety of connective tissue
proteins, this fluid produced only a minimal effect in the skin of rabbits (Fig. 1.7, Site II).
By contrast, brown recluse venom caused all the classical features associated with its bite
(Fig. 1.7, site III). These observations are consistent with those of Smith and Micks
(1968) who found that homogenates of the abdomens of L. reclusa do not evoke necrotic
lesions in the skin of rabbits. In addition, Geren et al. (1973) reported that brown recluse
spiders often regurgitate when biting, however, when the regurgitated material was tested
for toxicity, none was found. Taken together, these observations suggest that the
digestive proteases of spiders have no primary role in necrotic lesion formation.
The role Of venom proteases in spider bite necrosis remains uncertain.
Preliminary studies in the laboratory suggest that proteases are not a major component of
this fluid, and Kuhn-Nentwig et al. (2000) have estimated that the venom glands have
only one-ten thousandth of the protease activity of the digestive fluid. However, if
proteases had a primary role in spider bite necrosis, one would have expected that the
effects of the brown recluse venom in the skin of rabbits would have been reduced by the
simultaneous injection of hemolymph as this fluid neutralized brown recluse venom
proteases in vitro (Fig. 1.6a). However, it did nothing to suppress the effect of the brown
recluse venom (Fig. 1.7, site VI). At this time the most convincing agent associated with
spider bite necrosis appears to be sphingomyelinase D. The isolated agents appear to
25
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cause both necrosis and hemolysis (Kurpiewski et al., 1981; Tambourgi et al., 1998) and
antibodies to these enzymes effectively prevent necrosis (Rees et al., 1984). At this time
there is no corresponding set of evidence in support of a primary role for venom or
digestive fluid proteases in necrosis. It is conceivable, however, that the proteases could
enhance the effect of venom in spider bites.
26
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2
SURVEY FOR POTENTIALLY NECROTIZING SPIDER VENOMS, WITH SPECIAL
EMPHASIS ON CHEIRACANTHIUM MILDEI
Abstract
It has proven difficult to identify those spiders which cause necrotic lesions. In an
effort to design a simple, inexpensive screening method for identifying spiders with
necrotizing venoms, I have examined the venom gland homogenates of a variety of spider
species for their ability to cause red blood cell lysis. Those venoms which were positive
were further examined for the presence of sphingomyelinase D, and their ability to evoke
necrotic lesions in the skin of rabbits. Sphingomyelinase D is known to be the causative
agent of necrosis and red blood cell (RBC) lysis in the venom of the brown recluse spider
(.Loxosceles reclusa), and my assumption was that this would be the same agent in other
spider venoms as well. This did not prove to be the case. Of 45 species examined, only
the venom of L. reclusa and Cheiracanthium mildei lysed sheep red blood cells. Unlike
L. reclusa venom, however, C. mildei venom did not possess sphingomyelinase D nor did
it cause necrotic lesions in the skin of rabbits. In an effort to determine the hemolytic
agent in C. mildei venom, I found evidence for a phospholipase A2.
27
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1. Introduction
It has proven difficult to identify which spider species produce necrotic lesions.
Only rarely does a person seeking medical attention for a spider bite have the specimen in
their possession. Moreover, if a culprit is not present, it is nearly impossible to
distinguish spider bites from those produced by other arthropods (Russell and Gertsch,
1983) or even some disease states (Vetter, 2000). It is also difficult for arachnologists to
link particular spider species directly with clinically significant bites. First, it is
necessary to inspect the area where suspected spider bites occurred to confirm that the
spider exists in sufficient numbers as to suggest a probable cause. In addition, it is
necessary to demonstrate that the injection of the venom will produce the appropriate
lesions (Spielman and Levi, 1970; Vest, 1987). And, whereas reviews listing spider
species that potentially cause lesions have been assembled (Russell and Gertsch, 1967;
White et al., 1995; Vetter and Vischer, 1998; Sams et al., 2002), the lists continue to be
modified. The continuing lack of consensus has led White (1999) to remark that "a
concerted effort to determine which species can cause necrotic arachnidism should
become a prime focus for research in this field".
It is now well established that Loxosceles spp. are capable of causing severe
necrotic lesions (Macchiavello, 1937; Atkins et al., 1958). In a systematic search for the
necrotizing agent, Forrester and coworkers (1978) discovered sphingomyelinase D, a
hemolytic constituent in the venom of L. reclusa. One microgram of the isolated enzyme
was later shown to cause necrotic lesions in rabbits (Kurpiewski et al., 1981). Antibodies
to the purified enzyme are able to prevent necrosis (Rees et al, 1984; Gomez et al., 1999).
28
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These observations have since been extended to South American Loxosceles species
(Barbara et al., 1994: Tambourgi et al, 1998; Guilherme et al., 2001).
In an effort to develop a simple, inexpensive screening method to identify those
spiders which are capable of causing necrotic lesions, I have examined the venom of a
variety of spiders for the ability to cause sheep red blood cell hemolysis. Those which
were positive were further screened for sphingomyelinase D and dermonecrotic activity.
My survey was based upon the assumption that those spider venoms which caused
necrosis would also possess sphingomyelinase D and, therefore, cause sheep rbc lysis. I
recognize that this assumption is not completely valid: there are antimicrobial peptides in
the venom of some spiders that are also hemolytic (Yan and Adams, 1998; Corzo et al.,
2002; Kuhn-Nentwig et al., 2002a,b). By using sheep red blood cell suspensions and by
keeping the venom extracts in high dilution I hoped to avoid any antimicrobial peptide
influence with the assay.
2.2. Materials and Methods
2.2.1. Reagents
Defibrinated sheep blood was obtained from Northeast Laboratory (Waterville,
ME), co-trinitrophenylaminolauric acid (TNPAL)-sphingomyelin (T 1014),
sphingomyelinase D from Staphylococcus aureus (S 8633), snake venom from Crotalus
atrox (V 7000), and ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid
(EGTA, E 3889) were purchased from Sigma (St. Louis, MI). Silica Gel 60 TLC plates
(5721-7) were purchased from EM Sciences (Cincinnati, OH). Purified
phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),
phosphatidylinositol (PI), and sphingomyelin (SPM) were acquired as a
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phosphoglycerides kit (#1053) from Matreya, Inc. (State College, PA). BODIPY-labeled
phosphatidylcholines 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-
pqntanoyl)-1 -hexadecanoyl- sn-glycero-3-phosphocholine (D-3803) and 2-decanoyl-l-
(0-(l l-(4,4-difluoro- 5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-
propionyl)amino)undecyl)-sn- glycero-3-phosphocholine) (D-3771) were purchased from
Molecular Probes (Eugene, OR).
2.2.2. Preparation of venom gland homogenates
All spiders were collected between June 2001 and November 2002 in the vicinity
of Durham, New Hampshire, unless otherwise noted. The spiders were fasted for at least
three days post-capture to ensure venom glands were not depleted. Spiders were
narcotized by carbon dioxide, and venom glands were removed by micro-dissection. The
glands were either used immediately or frozen at -20° C for future use.
2.2.3. Hemolysis assay
Hemolytic activity was determined using the spectrophotometric method of
Forrester et al. (1978). Individual venom glands were homogenized in 50 pi of a 20 mM
Tris buffered saline with 8 mM CaCl2, pH 7.4 (TBSC; after Babcock et al., 1981). The
entire venom gland homogenate (VGH) was added to 1.0 ml suspension of 2% sheep red
blood cells in a TBSC suspension and incubated at 37° C for 1.5 hours with gentle
agitation. The suspension was then diluted with 3.0 ml TBSC and centrifuged at
approximately 760 X g for 5 minutes. The optical densities of the supernatants were
measured at 540 nm with a Spectronic 601 spectrophotometer (Milton-Roy). Within
each assay, a 1 ml aliquot of sheep red blood cells was incubated as stated above, then
30
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lysed with 3 nils distilled water, and used as the standard for 100% hemolysis. Percent
hemolysis was determined against total hemolysis.
2.2.4. Sphingomyelinase Assay
Sphingomyelinase D activity was determined using the spectrophotometric
method of Gatt et al. (1978) as modified by Young and Pincus (2001) using (TNPAL)-
sphingomyelin as substrates. Each venom gland was homogenized in 30 pi 20 mM
HEPES with 8 mM CaCl2 pH 7.1 then added to 170 pi solution of 0.3 mM TNPAL-
sphingomyelin containing 1.5 mM Triton X-100 in the above buffer. All samples were
incubated at 37° C for 2 hours. The positive control substituted 1.0 unit of
sphingomyelinase D from Staphylococcus aureus in place of spider VGH. Reactions
were stopped by the addition of 750 pi of isopropanol: heptane: 5M sulfuric acid (40:10:1
v/v). The mixtures were extracted with 450 pi heptane, followed by 400 pi distilled H 20.
All samples were vortexed then centrifuged for ten minutes. The upper heptane phase
was removed and its absorbance was read at 330 nm.
2.2.5. Phospholipase Assays
Defibrinated sheep red blood cell ghosts were prepared according to Dodge et al.
(1963). Individual venom glands from spiders were homogenized in 50 pi of 20 mM
HEPES buffer pH 7.1 with 8 mM CaCl2. The VGH was then added to 500 pi of a 17%
RBC ghost suspension in the same buffer, and incubated for 3 hrs at 37° C. Eight pg
protein of snake venom from Crotalus atrox were incubated with the same amount of the
RBC ghost suspension and used as a positive control. Membrane phospholipids and
cleavage products were extracted with chloroform: methanol (2:1 v/v) and separated on
Silica Gel 60 TLC using Solvent B (chloroform: methanol: 2-propanol:0.25% potassium
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chloride: triethylamine (30:9:25:6:18 v/v)) of Touchstone et al. (1980). Results were
visualized by staining with a 3% cupric acetate, 8% phosphoric acid solution then heating
at 180° C for 10 minutes, or by using a 1% Rhodamine 6G stain (for quantitative use).
Phospholipase A activity was determined by the method of Farber et al. (1999).
BODIPY®-FL labeled phosphatidylcholine (Molecular Probes, WA) was used to
determine whether the enzyme cleaved the fatty acid from the sn-l (D-3771) or sn-2 (D-
3803) position. Cleavage products were separated by TLC methods using solvent B of
Touchstone et al. (1980) and visualized with a 488 nm laser on a Bio-Rad Molecular
Imager FX Pro (Bio-Rad).
2.2.6. The effect of C. mildei venom gland homogenate on the skin o f rabbits
To observe if venom from C. mildei is capable of causing necrotic lesions VGH
was injected into the skin of rabbits. The experiments were carried out in compliance
with the Public Health Service Policy, the Guide for the Care and Use of Laboratory
Animals (1996), and University Policy (see Appendix). Three adult female New Zealand
white (NZW) rabbits were injected in a shaved area on the back with the following
solutions: site I: 200 pi PBS, pH 7.4; site II: 15 pg of C. mildei VGH protein in 200 pi
PBS. All preparations were filtered through a 0.2 micron filter prior to injection. The
injections were administered intradermally with a 26 gauge needle approximately 10-12
cm apart. The rabbits were observed hourly for the first six hours and then every twelve
hours for the next 138 hrs. The injection sites were photographed with a digital camera
and scored according to the intracutaneous test described in the USP 24-NF19 (2000).
Inasmuch as no necrosis was observed, the same rabbits were administered a 5-fold
increase in concentration of C. mildei VGH protein seven days later. Site I again served
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. as the control, being 200 jil PBS. Site II was moved to the opposite side of the freshly
shaved rabbit. The injectate contained 79 pg of C. mildei VGH protein (approximately
4.4 venom glands) in 200 pi PBS. The rabbits were observed hourly for the first six
hours and then every twelve hours for 48 hours. Rabbits were euthanized and skin
samples were taken as described in the methods of Foradori et al. (2001).
2.2.7. The calcium dependency of the hemolytic factor in C. mildei venom
To observe whether calcium was essential to the hemolytic factor, individual
venom glands were homogenized in 1.0 ml 2% sheep RBC in 20 mM Tris buffered
saline, pH 7.4 (300 mOsm) with or without 8mM CaCl2. Hemolysis was evaluated as
described above (part 2.3). The comparison was repeated three times and the data was
evaluated using a t-test.
2.3. Results
2.3.1. Hemolysis assay
Cheiracanthium mildei was the only VGH in this survey found to cause sheep red
blood cell hemolysis (Table 2.1), other than Loxosceles reclusa, which was used as the
positive control. C. mildei females caused an average of 65.4% hemolysis; males 38.9%
hemolysis. L. reclusa females caused 37.5% hemolysis. The VGH of all other spiders
surveyed showed little (< 0.5%) or no ability to cause hemolysis.
2.3.2. Sphingomyelinase Assay
Sphingomyelinase D activity was not observed in female C. mildei VGH using
the modified method of Gatt (1978). L. reclusa VGH homogenate was rich in this
enzyme. See also figure 2.1 where L. reclusa VGH completely removed sphingomyelin,
but C. mildei VGH had no effect on sphingomyelin concentration.
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Family Species Sheep RBG Hemolysis
Loxoscelidae Loxosceles reclusa (3 )1 ++
Miturgidae Cheiracanthium mildei female (9) ++ male (8) +
Gnaphosidae Herpyllus ecclesiastices (2) Micaria longipes (2) - Unidentified Gnaphosidae (6)
Dtsderidae Dysderia crocata (1) "
Salticidae Phidippus audax <8) Phidippus clarus (2) Platycryptus undatus (4) Salticus scenicus (1) Unidentified Salticidae (5)
Tetragnathidae Tetragnatha elongata (6) Nephila madigascari (3) Nephila clavipes (4 )f
Araneidae Argiope aurantia (4) Argiope trifasciata (6) - Larinioides cornutus (10) p Neoscona arabesca (5) Araneus cavaticus (4) Araneus diadematus (3) Araneus marmoreus (4) Araneus trifolium (8) Gastercantha cancriformis (8 )r Micrathena sagittata (4 )8 Micrathena gracilis (3 )8 - Metazyga sp. (5) Verrucosa sp. (3) f - unidentified Areneidae (9)
Table 2.1. Survey of the venom of selected spider species for their ability to cause hemolysis. One venom gland was homogenized from each spider and assayed for its ability to cause hemolysis in 1.0 ml of 2% sheep RBC suspension. Number of spiders tested is indicated in parentheses. All spiders tested were female unless noted otherwise. All spiders were collected in New Hampshire unless otherwise noted (f = Florida; g = Georgia; k=Kansas; n = North Carolina; p = Pennsylvania; t = Texas; v = Virginia).
34
Reproduced with permission of the copyright owner. Fudher reproduction prohibited without permission. Family Species Sheep RBC Hemolysis
Agelenidae Agelenopsis aperta (4 )v - Unidentified Agelenopsis (30+) -
Filistatidae Kukulcania hibemalis (6 )f -■
Scytodidae Scytod.es thoracica (2) -
Pholcidae Pholcus phalangoides (4) -
Mimetidae Mimetus sp. (5) -
Theridiidae Steatoda borealis (7) Theridionfrondeum (3) - Achaearanea tepidariorum (8) - Latrodectus geometricus (4) r - Latrodectus maetans (3) -
Pisanridae Pisaurina mira (7) Dolomedes tenebrosus (3) - Dolomedes triron (3) Dolomedes vittatus (3) “ Unidentified Pisauridae
Thomisidae Misumena vatia (3) . Misumena asperatus (1) - Tibellus sp (5) “ Unidentified Thomisidae Yellow crab spider (7)
Oxyopidae Oxyopes salticus salticus (3 )k - Peucetia viridans (2 )f "
Linyphiinae Frontinella sp (5) -
Lycosidae Gladicosa gulosa (1) - Unidentified Lycosidae (3) -
Table 2.1. Continued.
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.3. Phospholipase assay
The analysis of cleavage products from RBC ghosts exposed to venom gland
homogenates of C. mildei revealed the release of free fatty acids signaling the presence of
a phospholipase (Figure 2.1). Phosphatidylcholine (PC), phosphatidylethanolamine (PE),
and phosphatidylserine (PS) all released a free fatty acid and lysophospholipid when
exposed to VGH of C. mildei (Fig. 2.2) suggesting the presence of a Phospholipase A.
Phosphatidylcholine fluorescently labeled on the sn-1 position released fluorescent
labeled lysophosphatidylcholine; phosphatidylcholine fluorescently labeled on the sn-2
(D-3803) position released fluorescently labeled free fatty acid upon exposure to C.
mildei venom. These observations suggest the presence of a phospholipase A2 (Fig. 2.3).
2.3.4. The effect of C. mildei venom gland homogenate on the skin o f rabbits
Histological sections of skin and subcutis of NZW rabbits injected intradermally
with C. mildei VGH showed a mild to moderate reaction in comparison with the lesions
which I observed earlier in rabbits injected with Loxosceles reclusa VGH (Foradori et al.,
2001). Microscopic examination of the 5-fold increases of C. mildei VGH protein
injection site revealed a well demarcated and focal reaction with edema and mixed
inflammatory cell infiltrates (Fig. 2.4). In some instances myofibril necrosis was
observed (Fig. 2.4). A 6mm diameter red flare appeared within hours of the C. mildei
VGH injection, but slowly disappeared within 24 hours (Fig. 2.5). PBS was used as a
control and produced no observable changes in the skin and subcutis. The microscopic
appearance of the injection sites was consistent in all three rabbits.
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. f a ■ : IK
■vIKv^
PE 9 ^ m m m
PI PS LP
SM • •
RBC ghost RBC ghost RBC ghost RBC ghost RBC ghost blank + ::^Z; R 't+:\/ \/;.G:Y. v : r C. mildei P.andax L. geometriais L.reclusa VGH VG-H VGH VGH
Figure 2.1. The effects of spider venom gland homogenates (VGH) on RBC ghosts.
Extracts of RBC ghosts treated with VGH were chromatographed in the one dimensional system of Touchstone et al. (1980). The main components were phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI), and sphingomyelin (SM). RBC ghosts treated with Cheiracanthium mildei VGH had a decreased density of PC, PE, and PS, and the appearance of free fatty acids (FA), as well as lysoforms (LP) of the previously mentioned phospholipids. Note the complete disappearance of SM withLoxosceles reclusa VGH. (Spiders in order are: C. mildei, Phidippus audax, Latrodectus geometricus, and L. reclusa)
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ffa *
t
PE i PS PC • • m y ' W"- m : m
* # * * , ■v: -i PE+ PE+ PE + PS + PS+ PS + PC + PC + PC + Cjnidei Cjnildei CMrax Cmiciei Cjnildei C.atrox C.mildei Cjnildei Catrcsc 0 mm 120 min 120 min. Omin. 120 min. 120 mini Omin. 120 min. 120 mm.
Figure 2.2. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on individual purified phospholipids: Phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PC). Snake venom {Crotalus atrox) was used as a positive control.
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. /FA
/PC /LPC
W
Figure 2.3. The effect of Cheiracanthium mildei venom gland homogenate (YGH) on phosphatidylcholine (/PC) fluorescently labeled on the sn-l (D-3771) or sn-2 (D-3803) position. The release of fluorescent fatty acid (/FA) from D-3803 and fluorescent lysophosphatidylcholine (/LPC) from D-3771 indicate the presence of phospholipase A2 in the VGH.
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.4. The effect of Cheiracanthium mildei venom gland homogenate (VGH) on the skin of New Zealand white rabbits. Approximately 79pg protein of C. mildei VGH was injected intradermally. Picture taken 48 hours post-injection. Note: unlabelled dark spots are permanent marker used to mark coordinates of injection sites.
40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.5. Histology of the skin of New Zealand white rabbit 48 hours after intradermal injection of 79pg protein Cheiracanthium mildei venom gland homogenate. A. View of the dermis and epidermis reveals edema and mixed inflammatory cell infiltrates (25x); B. Hair follicle showing heavy heterophil infiltration (100X); C. The panniculus (p) is beginning to lose banding, signaling degeneration and mild necrosis (200x)
41
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.5. The calcium dependency o f the hemolytic factor in C. mildei venom gland
homogenate
The absence of calcium significantly reduced the hemolytic capacity of the
enzyme by almost 3-fold (p > 0.001) as seen in figure 2.6.
2.4. Discussion
Venom survey
The VGH of a variety of spiders has been surveyed for their ability to cause
hemolysis of sheep erythrocytes as a potentially simple and inexpensive way to screen for
necrotizing spider venoms. This survey was based upon the assumption that such
venoms would contain sphingomyelinase D, the necrotizing component in the venom of
the brown recluse (Kurpiewski et al, 1981), which is also known to cause red RBC lysis
(Forrester et al., 1978). Of the 45 spider species examined, representing 20 spider
families, only the VGH of Cheiracanthium mildei and L. reclusa were observed to cause
hemolysis (Table 2.1). The results accord with those of Croucamp and Veale (1999),
who observed hemolytic activity in the venom of Cheiracanthium furculatum. However,
in contrast to L. reclusa, C. mildei VGH did not possess sphingomyelinase D.
There are hemolytic factors in the venom of some spiders other than
sphingomyelinase D. Lycosids (Yan and Adams, 1998) and Oxyopids (Corzo et al.,
2002) have amphipathic peptides that are both antimicrobial and hemolytic. Their
effectiveness on red blood cells, however, seems to depend upon the phosphatidylcholine
(PC) content of the membranes. The PC content of sheep erythrocytes is less than 1%
(See Fig. 2.1) and higher concentrations of the amphipathic peptides are required for lysis
than is the case for the guinea pig and pig (Corzo et al., 2002). One venom gland in 1.0
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80
7 0 -
6 0 -
20 -
TBS + Ca TBS w/o Ca
Figure 2.6. Calcium dependency of Cheiracanthium mildei venom gland homogenate hemolytic factor. Homogenates of individual venom glands were incubated with 1.0 ml 2% sheep RBC in 20 mM Tris buffered saline, pH 7.4 (300 mOsm) with or without 8 mM CaCl2. (n=3; error bars = SEM)
43
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ml of 2% sheep red blood cell suspension was used for an assay, it is likely that the
amphipathic peptides were too dilute to observe an effect with those species (Table 2.1).
Cupiennius salei (Family Ctenidae) venom has also been shown to contain antimicrobial
peptides (Kuhn-Nentwig et al., 2002a,b), so it may be that antimicrobial peptides are
universal components in the venom of spiders. Their likely function is that of disrupting
ion and voltage gradients across the membranes of excitable cells (Yan and Adams,
1998).
Cohen and Quistad (1998) observed that the venom of Phidippus audax contained
a potent cytolytic factor that disrupted cells in culture. However, neither lysis of
erythrocytes nor cleavage of products when the VGH of P. audax was incubated with
RBC ghosts were observed (Fig. 2.1).
Hemolytic factor in the venom o f Cheiracanthium mildei
To establish the factor present in the VGH of C. mildei responsible for RBC lysis,
filtered VGHs were incubated with sheep RBC ghosts. Thin layer chromatography of
incubation extracts revealed a decreased density of PE, PS, and PI as well an appearance
of fatty acids and lysoforms of the lipids, suggesting the presence of a phospholipase
(Fig. 2.1). Incubation of C. mildei VGH with commercially available PE, PS, and PC
confirmed the cleavage of these compounds (Fig. 2.2). The reduction in activity of the
hemolytic agent by the removal of calcium (Fig. 2.6) also indicates the presence of
phospholipase A2(PLA2), as Ca2+ is required for optimal activity (Hanahan, 1997). To
identify the site of cleavage of the phospholipids, VGH was incubated with PC
fluorescently labeled on the sn-l (D-3771) or sn-2 position (D-3803). Whereas
fluorescently labeled fatty acid was released from D-3803, fluorescently labeled
44 '
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lysophospholipid was released from D-3771 (Fig. 2.3) suggesting the presence of a
phospholipase A2 in the venom gland homogenates ofC. mildei.
PLA2 has been observed in the venom of the spider Eresus cinnaberinus (= niger)
and has been shown to produce an irreversible blockage of frog neuromuscular junctions
as well as anticoagulant activity (Usmanov and Nuritova, 1994). The enzyme is also
present in the venom of most snakes (Tu, 1977), and like the snake venom PLA2, C.
mildei venom gland homogenate PLA2 has a calcium requirement (Fig. 2.6).
The venom of C. mildei has been observed to cause necrotic lesions in the skin of
guinea pigs (Spielman and Levi, 1970) and hamsters (Hughes, 1981), and
Cheiracanthium lawrencei venom has been shown to cause necrosis in the skin of rabbits
(Newlands et al., 1980). We, however, observed only a mild erythema on the skin of
rabbits following the injection of the equivalent of 4 glands of protein (Fig. 2.4). Whereas
Spielman and Levi (1970), Hughes (1981), and Newlands et al. (1980) allowed spiders to
bite their prey, I injected the rabbits intradermally with VGH. The difference in the
technique may account for the differences in the results obtained. And whereas Newlands
et al. (1980) observed ulcerating lesions in humans resulting from the envenomation of C.
lawrencei, most observations suggest that the effects of Cheiracanthium spider bites in
humans are mild. Spielman and Levi (1970) observed only a mild reddening of the skin
of a human volunteer resulting from C. mildei spider bite. Minton (1972) and Krinsky
(1987) also observed only redness, swelling, and itching in humans resulting from the
bite of C. mildei. Similar observations have been made for the venom of C. inclusum
(Furman and Reeves, 1957; Gorham and Rheney, 1968; Leech and Brown, 1994), C.
punctorium (Maretic, 1982), and C. japonicum (Owri, 1978).
45
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The foregoing experiments support the theory that severely necrotizing spider
venoms are rare. Of all the spider venoms examined, only two produced hemolysis, and
of these, only the brown recluse produced necrotic lesions in the skin of New Zealand
white rabbits. Although the venom of the local yellow sac spider, Cheiracanthium
mildei, produced hemolysis, its effect upon the skin of New Zealand white rabbits at
normal levels was mild. My results suggest that the widespread fear of spider bites, for
the most part, is unwarranted.
46
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3
FRACTIONATION OF SPIDER DIGESTIVE FLUID AND PARTIAL
CHARACTERIZATION OF THE PROTEASES AND PROTEASE INHIBITORS
ABSTRACT
Digestive fluid proteases and serine protease inhibitors play an important role in
the life of the spider. The proteases allow for external digestion of prey, as well serving
as a tool to recycle silk. Through a combination of ion exchange chromatography, size t exclusion chromatography, ultracentrifugation, and electrophoresis, I have tried to isolate
several digestive fluid proteases and serine protease inhibitors in milligram quantities
from the digestive fluid of Argiope aurantia. The proteases, however, eluted
anomalously during the size exclusion chromatography and migrated anomalously during
ultracentrifugation trials. I have hypothesized that proteases can undergo a reversible
aggregation to account for this behavior. I have also examined the possibility that spider
digestive enzymes are related to crayfish gastric proteases. While my evidence supports
this in part, I found major differences between the two groups of enzymes. I found
evidence for 10 or more serine protease inhibitors in Argiope aurantia digestive fluid.
3.1. Introduction
The digestive proteases of orb weaving spiders play an important role not only in
the consumption of prey, but also in the recycling of their orb web (Tillinghast and
Kavanagh, 1977; Townley and Tillinghast, 1988) and the escape of spiderlings from the
47
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. egg sac (Foradori et al., 2002). Little is known about the biochemical characteristics of
spider digestive enzymes which must function during the initial stages of digestion
without the aid of a controlled environment (e.g., stomach of mammals). The
evolutionary relationship of spider digestive proteases to those of other animals has not
been established either.
Bertkau (1885), Schlottke (1936), and Pickford (1942) were among the first to
study the digestive hydrolases of spiders. A more complete inventory was provided by
Mommsen (1978 a,b,c) who fractionated the digestive fluid by polyacrylamide gel
electrophoresis. Mommsen (1978a) identified one protease capable of cleaving natural
proteins and several enzymes capable of cleaving synthetic substrates typically employed
for studying the functioning of mammalian proteases. Tillinghast and Kavanagh (1977)
brought two spider digestive proteases to apparent homogeneity. They had masses of
approximately lOkDa and were metalloproteases with alkaline pH optima. One of these,
Argiope protease B, proved capable of cleaving major ampullate silk (Kavanagh and
Tillinghast, 1983). More recently, serine protease inhibitors have also been demonstrated
in the digestive fluid of A. aurantia (Tugmon and Tillinghast, 1995).
Proteases have also been reported in a number of spider venoms (reviewed by
Perret, 1977). In one of these reports, Mebs (1972) isolated three proteases with
molecular weights of approximately 11 kDa. They had pH optima of 8.5, did not cleave
trypsin substrates, and were equally unaffected by serine protease inhibitors. These
characteristics are essentially identical to proteases later described in spider digestive
fluid (Kavanagh and Tillinghast, 1977). Mebs also noted that the spider proteases were
48
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. similar in molecular weight and catalytic properties to a protease isolated from crayfish
gastric fluid (Pleiderer et al., 1967).
Previous efforts to isolate individual spider digestive proteases resulted in a very
low yield (Kavanagh and Tillinghast, 1983). In this study, I have attempted to develop a
more effective protocol for fractionating digestive fluid and purifying its proteases and
serine protease inhibitors for biochemical characterization. In these experiments, I have
used the spider digestive fluid amylases and N-acetylglucosaminidase as internal
indicators of the effectiveness of my methods. Both are major enzymes in the spider’s
digestive fluid and have established molecular weights (Mommsen, 1978). I have also
examined the possibility that spider digestive proteases are related to crayfish digestive
proteases
3.2. Materials and Methods
3.2.1. Reagents
Casein substrate was Carnation Dry Milk or Hammersten casein (Schwarz-Mann,
Orangeburg, NJ). Tris (15504-020) was purchased from GIBCO-BRL. All dialysis was
performed using 3500MWCO Slide-A-lyzer dialysis cassettes (Pierce). Size exclusion
columns were calibrated with various combinations of alcohol dehydrogenase (Sigma,
A8656, MW 150kDa), bovine serum albumin (A8531, MW 66 kDa), carbonic anhydrase
(C 7025, MW 29kDa), cytochrome C (C7150, MW 12.4 kDa). All other reagents were
products of the Sigma Chemical Company, St. Louis MO.
3.2.2. Collection of biological fluids.
Spider digestive fluid (SDF) was collected from Argiope aurantia by holding the
animal in one hand and putting a micropipette to its mouthparts. Under these
49
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. circumstances A. aurantia freely released digestive fluid (SDF) which was collected and
frozen until used. The SDF was then centrifuged for 2 min at 14,000 rpm in an
Eppendorf centrifuge 5415 to remove particulate matter and then dialyzed overnight
against the buffer in which it would be fractionated.
Crayfish gastric fluid (CGF) was obtained by inserting a 5 ml syringe into the
stomach of an anesthetized Astacus sp. and withdrawing fluid. Hemolymph was obtained
from both animals by clipping a leg and drawing fluid into micropipettes.
3.2.3. Isolation o f SDF Proteases
SDF was dialyzed against 25 mM Tris, pH 8.1 containing 2 mM CaCl2 (buffer
A). A 1.5 ml sample was injected into a 5 ml Q econo-cartridge (Bio-Rad) and was
eluted with a 0 to 0.25 M NaCl gradient in the same buffer. The fractions were
monitored at OD 280nm for protein. Fractions were pooled and assayed for protein,
proteases, amylase, N-acetylglucosaminidase, and protease inhibitors by methods
described below.
The first ten fractions from anion-exchange chromatography were pooled and
dialyzed against 50 mM Tris buffer, pH 7.6 and the dialysate was applied to a column
(1.4x100 cm) of Sephacryl S-100 HR pre-equilibrated with the same buffer. The sample
was eluted at a flow rate of 12.8 ml per hr and 3.2 ml fractions were collected. The
fractions were monitored at 280nm for protein. Samples were assayed for amylase
activity and proteases. The proteases were Visualized by zymography (3.2.3 below).
Fractions 48-54 from the Sephacryl S-100HR column were pooled and concentrated by
lyophilization, dialyzed against 20 mM HEPES buffer (H7006), pH 7.5 and applied to a
5ml High S econo-cartridge (Bio-Rad) pre-equilibrated with the same buffer. The
50
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. proteins were eluted with a gradient of 0 to 0.25M NaCl in the same buffer. Samples
were assayed for protease activity. Active fractions were pooled, lyophilized, and
aliquots electrophoresed to observe the proteases. Selected bands were electroblotted for
sequencing.
^ 3.2.4. Fractionation of whole SDF
In other studies, whole SDF was fractionated on Sephacryl S-100 HR column
(1.4x100cm). The SDF was first dialyzed against 50 mM Tris buffer, pH 7.6, with 100
mM KC1 and eluted with the same buffer at a flow rate of 13.6 ml per hour with 3.4 ml
fractions being collected per tube. Fractions 26-32 were further fractionated on a column
(1.5X75 cm) of Sephadex G-75 using a 50 mM Tris buffer, pH 7.6 containing 100 mM
KC1. Proteins were eluted at a flow rate of 20 min/tube and 3.0 ml collected per tube.
3.2.5. Isolation of SDF serine protease inhibitors
One ml of SDF was applied to a Q-Sepharose anion-exchange column (1.4x20.3
cm) and eluted with 100 ml 20 mM Bis-Tris (B7535), pH 6.8, followed by 400 ml of a 0
to 500 mM NaCl gradient in the same buffer. This was followed by an additional 100 ml
of the buffer containing 500 mM NaCl. The flow rate was 1.3 ml /min and 3.4 ml
fractions were collected. The eluate was monitored continuously at 280 nm. Peak
fractions and regions between were pooled separately, lyophilized to reduce volume and
de-salted with Sephadex PD-10 columns. The high molecular weight fractions were
reduced in volume in a Savant speed-vac concentrator, boiled for 4 min, and centrifuged
at 14,000 rpm for 5 min to remove inactivated proteases. The fractions were reduced to
250 pi in the Savant concentrator and 20pl volumes were used to locate protease inhibitor
activity by radial diffusion. Pooled fractions containing the highest inhibitor activity
51
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were examined by native and denaturing PAGE with sample aliquots of 7.5 to 15 pi per
lane.
3.2.6. N-terminal sequencing
Proteins were transferred from SDS-PAGE gels to polyvinylidene difluoride
(PVDF) membranes at 30 V (constant) for 45 min to 1 hr using the Nu-PAGE system
(Invitrogen). The PVDF membrane was stained with 50% methanol, 10% acetic acid and
0.1% Coomassie blue solution briefly to visualize proteins of interest, then destained with
50% methanol (Matsiduria, 1993). Selected bands were cut out and sent to the University
of Texas Medical Branch Protein Chemistry Laboratory for sequencing.
3.2.7. Comparison of spider and crayfish proteases
Electrophoresis-horizontal; 1% argarose (A6013) gels were prepared with barbital
buffer, pH 8.6 (B6632). Samples of SDF or CGF were electrophoresed at 30 mA
(constant) for 1 hr. To visualize protease activity, selected lanes were removed and
placed on gels prepared with 1% agarose (A6013) and 1% casein (Carnation dry milk) in
buffer A. Other lanes were stained for protease inhibitor activity as described below.
Hemolymph protease inhibitor studies (radial diffusion): Gels composed of 1%
argarose (A6013) and 1% casein were prepared with buffer A. Mixtures were prepared
containing 5pi SDF or CGF and 15 pi spider hemolymph buffer A or 15 pi buffer A.
These were vortexed and transferred to wells cut into the gels. The gels were incubated
overnight and the diameter of digestion was recorded.
3.2.8. Sucrose Density Centrifugation
SDF was centrifuged at 3000 rpm for 3 min, then 15pl aliquots were mixed with
180 pi buffer (50 mM Tris-HCl, pH 7.5) and carefully applied to a 5 ml linear gradient
52
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (5-30% w/v) of sucrose in the same buffer. The samples were centrifuged at 4°C for 21
hr at 160,000 x g in a Beckman model L8-70 ultracentrifuge using a SW65 rotor. Spun
tubes were placed in a holder and the bottom of the tube was punctured with a needle and
0.25 ml fractions collected. The fractions were assayed for proteases, protease inhibitors,
amylase, and N-acetylglucosaminidase.
3.2.9. Electrophoresis
Native PAGE: The high pH discontinuous system described in Hames and
Rickwood (1981) was used with 8.5% resolving gels containing AcrylAide (EMC
BioProducts , Rockland, ME) as the cross-linker.
SDS-PAGE: Small format, Novex Tris Glycine gels (Invitrogen) of different
percentages were employed and run at 125 V (constant) for 2 hrs. For large format
electrophoresis, the discontinuous system of Laemmli (1970) was used with 13% gels
resolving gels, again containing AcrylAide, and samples were boiled for 1 min after
addition of sample buffer.
Zymography: 4-16% Novex Zymogram gels (Invitrogen; EC 6415) were used to
visualize proteases according to the manufacturer’s instructions.
SDF serine protease inhibitors were visualized in gels by the method of Broadway
(1993). Briefly, following SDS-PAGE, SDS was removed from the gels by washing the
gels sequentially in 40 mM Tris HC1 (T8529), pH 9.0 for 15 min, distilled water for 5
min, the same Tris buffer for 25 min and distilled water for 10 min at 37°C. The gels
were then covered with a Trypsin, (TPCK treated; T8642) solution (0.04 mg/ml PBS, pH
7.4) for 10 min at 37°C. The solution was then decanted and the residual trypsin allowed
to penetrate the gel for 30 min at 37°C. The gel was then covered with tow solutions
53
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mixed just prior to staining. The first was 50 mg of the tryptic substrate N-Acetyl-DL-
phenylalanine (Tnaphthyl ester (A7512) dissolved in 20 ml N ,N-dimethylformamide
(D9654); the other waslOO mg O-dianisidine, tetrazotized (D3502) dissolved in 180 ml
PBS, pH 7.4. The stain was decanted and the gels stored in 2% acetic acid.
Elution: Proteins were eluted from agarose and polyacrylamide gel slices in
volatile NH4HC03 buffer using Bio-Rad Model 422 electro-eluter. Electroelution was at
a constant 8 mA per tube for 6 hr. The apparatus was placed on ice to prevent
overheating.
3.2.10. Enzyme Assays
Protease activity was assayed by radial diffusion agarose/casein gels as described
by Schumacher and Schill (1972) or the method of Kunitz (1947) using a 1%
Hammersten casein solution as the substrate. SDF serine protease inhibitors were
assayed using radial diffusion in argarose/casein gels as described by Tugmon and
Tillinghast (1995). Amylase was assayed according to the procedure of Bernfeld (1951)
using dinitrosalycylic acid as the color reagent. Maltose was used to construct a standard
curve. N-acetylglucosaminidase was assayed by the method of Peters et al. (1998) using
4-Nitrophenyl N-acetyl-(TD-glucosaminide as substrate.
3.3. Results
3.3.1. Isolation o f Proteases
The fractionation of SDF by anion-exchange chromatography is presented in
Figure 1. The bulk of the proteases eluted prior to the application of the salt gradient
(Fig. 3.1 A). The amylase also appeared with unbound material. Protease inhibitors and
N-acetylglucosaminidase were both released early in the salt gradient (Fig. 3. IB).
54
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A
3.0 r 0.50 ■OD 280 2.5 ■[NaCl] - 0.40
2.0 0.30 COo Q 1.5 O 0.20 <3 1.0 O Z 0.0 0.00 o in o in o in o in co co it Fraction Number B 1 0 0 -i El% amylase D % protein ■% protease 80 - P % inhibitor 70 - E0% glucosaminidase 60 - c O4) <0 CL O o O lO o o CM CO CO Tj- lO in Pooled Fraction Fig. 3.1. Fractionation of SDF by anion exchange chromatography A. SDF (0.5ml) dialyzed against 0.25 mM Tris buffer, pH 8.1 containing 5 mM CaCl2. A 1.5 ml sample was applied to a 5 ml high Q econo-cartridge (Bio-Rad) pre- equilibrated with the same buffer. The sample was eluted with a gradient of 0 to 0.25 M NaCl. B. Pooled fractions were assayed for protease, amylase, N-acetylglucosaminidase, protein and protease inhibitor. The specific activities of the enzymes were determined and the percent f the total calculated for each pooled fraction. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. When the first 10 tubes from anion-exchange chromatography were pooled and applied to Sephacryl S-100 HR amylase appeared in fraction 36 whereas pl2 and pl5 eluted prior to it and p27 and p35 eluted later. Protease pl2 appeared in all fractions (Fig. 3.2). To further purify p27, SDF fractions 48-54 were pooled and passed through a High S Bio- Rad econo-column at pH 7.5 and eluted with a Salt gradient. A doublet appeared in fractions 26-40 with apparent molecular weights of 24 and 28 kDa. These two isolated proteases were electroblotted and sent out for sequencing (Fig. 3.3). 3.3.2. Whole SDF fractionation on Sephacryl S-100 HR and Sephadex G-75 When whole SDF was passed through Sephacryl S-100 HR column, N- acetylglucosaminidase (MW108kDa; Mommsen, 1980) appeared in fractions 21-25; proteases pl5, p20 and p i 10 appeared in fractions 26-32; proteases p27 and p35 appeared in fractions 33-38 along with amylase (MW approx. 60kDa; Mommsen, 1978d). Protease pl2 appeared in all fractions including all of those after fraction 38. When fractions 26-32 were further subject to size exclusion chromatography on Sephadex G-75, pl5 and pllO emerged following p20. pl2 appeared in all fractions (Fig 3.4) 3.3.3. SDF serine protease inhibitors The bulk of the protease inhibitors eluted in fractions 56-91 during anion- exchange chromatography. Ten serine protease inhibitor bands were seen when pooled fractions 56-67 and 68-91 were examined by 8.0% PAGE. These were designated A, B, C, D, E, E’, F, G and G’ with E ’ and G’, appearing in fractions 68-91 and not in 56-67. Likewise A and B occurred in 56-91 but not in 68-91 (Fig. 3.5). Individual inhibitors C, D, E, F, and G were eluted from unstained parallel lanes of gels containing fractions 56- 67 and re-electrophoresed on 8% PAGE gel (Fig. 3.5). Three of these were sufficiently 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.6 25 27 33 35 37 38 40 42 45 Fraction # 0 .4 0.2 i i i i i 1"I I i I i TTTT'f I 'I "1 l"'T'"T I I M TTTTTTT i "i i m i i r~ oocvi Fig. 3.2. Fractionation of fractions 1-10 on Sephacryl S-100 HR column. The first ten fractions from anion exchange chromatography (Fig. 3.1) were pooled, lyophilized to reduce volume, dialyzed against 0.25 mM Tris buffer, pH 8.1 and applied to a 1.4x100 cm column of Sephacryl S-100 HR pre-equilibrated with the same buffer. The sample was eluted at a flow rate of 12.8 ml per hr and 2.8 ml fractions were collected. Selected fractions were examined for protease activity by zymography (inset) using 20pl aliquots. Note that pl2 occurs in every fraction. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. kDa fraction 20.5 Fig. 3.3. Fractionation of fractions 48-54 on cation exchange chromatography Fractions 48-54 from the S-100 HR column (Fig. 3.2) were pooled, lyophilized to 3.0 ml, then dialyzed overnight against 25 mM HEPES, pH 7.5. The dialysate was applied to 5ml High S econo-cartridge (Bio-Rad) pre-equilibrated with the same buffer. The sample was eluted at a flow rate of 2.0ml per minute with a gradient of 0 to 0.25 M NaCl. Fractions 30-36 containing high protease activity were subject to 10% SDS-PAGE and stained for protein. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. kDa 178 0.8 111 0.7 0.6 0.5 0.4 m 0.3 32 37 0.2 fraction # 0.1 0 j | — CD co CD CO lO N- CO h - CD T- T— CM CM CM CM CO CO CO CO 't f Fraction # Figure 3.4. Fractionation of fractions 26-32 on a Sephadex G-75 column. Whole SDF was fractionated on Sephacryl S-100 HR column using a 50 mM Tris buffer, pH 7.6 containing 100 mM KC1. Intense protease activity was found in fractions 26-32, which included pl5, p20, and pi 10. The pooled fractions were placed on a Sephadex G- 75 column, with the above mentioned buffer. Selected fractions were examined for proteases activity by zymography (inset) using 20 pi aliquots. Note that p 15 and pi 10 emerged ahead of p20 and that a majority of the fractions contain pl2. 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58-67 68-91 C D E F G I I I Fig. 3.5. Separation of SDF protease inhibitors by natural PAGE. SDF was fractionated by anion-exchange chromatography and those fractions containing serine protease inhibitors were separated by natural electrophoresis on a large format 8% PAGE gel. Ten serine protease inhibitors are seen in the two samples of pooled fractions (Gel I). C, D, E, F, and G were electroeluted individually from parallel lanes of Gel I and electrophoresed on a second gel (Gel II). 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. concentrated (C, E, F) to again elute from parallel unstained lanes and re-electrophorese on a 13% SDS-PAGE gel. All inhibitors now appeared as doublets (Fig. 3.6). When 2- mercaptoethanol was included during electrophoresis no inhibitors were observed. 3.3.4. N-terminal amino acid sequences o f proteases A pattern emerged from the N-terminal amino acid sequences I observed for several proteases (Table 3.1). The N-terminal amino acid sequence pattern for the first 20 amino acids was as follows: NAIPGQHYR-PGAKVPY-I-. This generic amino acid sequence, as well as each of the individual sequences were subjected to a protein homology and similarity search using FAST A (Pearson and Lipman, 1988; Pearson, 1990). All of the sequences had a 52.94% identity (52.94% ungapped) to chitinase. Protease SDF 25 (S) A 20-25 was unlike any of the other protease, and when subjected to a FAST A search, had 61.111% identity (61.111% ungapped) to an astacin precursor and a 40.0% identity (40.0% ungapped) to Astacin family metalloendopeptidase, both of which are found in crayfish digestive fluid. 3.3.5. Comparison o f spider and crayfish digestive proteases When SDF and CGF were co-electrophoresed at pH 8.6, the crayfish proteases moved rapidly toward the anode, whereas the spider proteases migrated toward the cathode (Fig 3.7A). Radial diffusion studies showed that whereas spider hemolymph readily inhibits SDF proteases, the spider’s hemolymph had only a little effect upon crayfish protease (Fig 3.7B). The converse experiment showed that whereas crayfish hemolymph readily inhibited crayfish proteases their hemolymph had little effect upon spider digestive proteases. 3.3.6. Ultracentrifugation of SDF 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -a tm ttMM C E F Fig. 3.6. Separation of SDF protease inhibitors by SDS-PAGE. C, E and F were eluted from parallel lanes in gel II (Fig 3.5). These were subject to denaturing electrophoresis using a 13% SDS-PAGE gel. Note that all three eluted inhibitors now appear as doublets. No bands appeared if 2-mercaptoethanol was included in the gel 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amino acid residue sample 10 SDF 25 (S) A 20-25 Kd E E SDF 25 (S)B 20-25 Kd SDF 08 01-10 15 Kd SDF 08 Q l-10 19 Kd X X SDF 2003 Series A 15KD SDF 2003 Series A 23KD SDF 2003 Series A 27KD SDF 2003 Series A 28KD SDG-15-15 W ! F I ! 1 SDG-15-15.2 SDG-15-36 STB-15-12 Table 3.1. N-terminal amino acid sequence of isolated protease from the digestive fluid of Argiope aurantia, i Digestive fluid enzymes were isolated by ion exchange chromatography and SDS- PAGE, blotted to PVDF for determination of N-terminal amino acid sequence; All of the proteins sequences possess the same core sequence (except for SDF 25 (S) A 20-25 Kd): NAIPGQHYR-PGAKVPY-I-. Queries using a FAST A protein homology and similarity search (Pearson and Lipman,1988) revealed ho significant matches. (“X” denotes no amino acid given, shaded amino acids are identical) 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A 'CGF+"SH OOF Fig. 3.7. Comparison of spider and crayfish digestive proteases A. 20 pi samples of spider digestive fluid (SDF) and crayfish gastric fluid (CGF) were electrophoresed in a 1% agarose gel, pH 8.6 for 1 hr at 20 mA (constant). Selected lanes were removed and placed on gels composed of 1% casein and 1% agarose,'pH 8.1 to visualize proteases. Note that the bulk of the spider proteases move slowly toward the cathode, whereas the crayfish proteases move rapidly toward the anode. B. Five pi samples of SDF or CGF was mixed with 15 pi spider hemolymph or 15 pi buffer A (25 mM Tris, pH 8.1 containing 2 mM CaCl2). These were placed in wells of gels composed of 1% agarose and 1% casein, pH 8.1 and allowed to incubate overnight. 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To observe if the proteases clustered into higher aggregates, SDF was subjected to sucrose density gradient centrifugation at 160,000 x g for 21 hr, Whereas the spider N- acetylglucosaminidase, amylase and protease inhibitors separated from each other as rather narrow peaks in accordance with their masses, the proteases formed a single broad peak with protease activity appearing in every fraction (Fig. 8). In part this broad peak might be explained by the widely differing masses of the individual proteases (Fig. 9b), however, proteases pi 10, p32, p27 and pl2 were still seen together in every fraction (Fig. 9a). 3.4. Discussion This study was initiated with the goal of isolating spider digestive proteases in milligram quantities for biochemical characterization. Although the digestive fluid of A. aurantia contains approximately 35mg protein per ml (Tillinghast and Kavanagh, 1977), and at least six major proteases (pi2, pl5, p27, p35, and pi 10), I encountered more difficulty than anticipated. In no chromatographic separation did a protease, other than p i2, ever elute as a single protein species and pl2 eluted with almost every fraction. My experience was much the same as Mebs (1972) in his attempt to purify spider venom proteases. In passing venom through a series of Sephadex gels, Mebs observed anomalous elution times such as to suggest that the proteases were aggregating and disassociating. He isolated proteases from three very different chromatographic fractions and yet all had masses of approximately 1 lkDa, identical catalytic properties, did not cleave trypsin substrates, and were equally unaffected by serine protease inhibitors. He concluded that they were the same enzyme and that the reversible aggregation of the proteases accounted for their differing elution rates. The data for spider gut proteases are 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.600 ■0 “ —=>pratease activity OD 275 60 -A— amylase activity OD 540 1.400 ~e—-N-acetyl glucosaminidase OD 410 •B—-percent inhibition 50 1.200 S ' 1 .0 0 0 40 0.600 3 0 0.600 20 0.400 0 .2 0 0 0.000 1 2 3 4 5 6 10 1 1 12 13 1 4 1516 17 18 Fraction # Figure 3.8. Fractionation of SDF by ultracentrifugation 15 |il SDF was applied to 5 ml of a 5-30% sucrose density gradient and centrifuged for 21 hr at 160,000 x g (SW65 rotor) at 4°C. Fractions were assayed for the distribution of amylase, proteases, N-acetylglucosaminidase and protease inhibitors. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fractions 2 4 6 8 10 12 14 16. 18 kDa iPllilgi 182 9 113.7 80 9 63 8 HIS 37 4 26 0 ilplSlP 149 3BMItey _ — IlilliS MHM pBBliMIp I fractions- 1-5 6-9 10-13 14-18 Figure 3.9. Whole SDF fractionation by sucrose density gradient ultracentrifugation examined by (A) zymography and (B) SDS-PAGE. A. Five pi of the even fractions from SDF ultracentrifugation were examined for protease activity by 4-16% SDS-PAGE zymography. Note the persistence of pl2, p27, p32, and pllO throughout the fractions. B. Fractions from SDF ultracentrifugation were pooled into groups of four or five. Twenty pi of each pooled fraction were examined for protein content by 4- 20% SDS-PAGE. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. consistent with Mebs (1972) interpretation and I believe that it explains why only low molecular weight proteases have been purified to date and why the yield has been so small (Tillinghast and Kavanagh, 1977, Kavanagh and Tillinghast, 1983). It has long been known that related proteins such as hemocyanin undergo reversible association and dissociation depending on protein concentration, pH and the ionic environment (Pedersen, 1950). Future efforts to isolate individual proteases may find it useful to first identify conditions of pH, temperature and ionic environment such as to reduce the association of the proteases more fully prior to column chromatography. High pH, for example, has proven a very effective way of dissociating spider hemocyanin monomers (Sugita and Sekiguchi, 1975). It is possible that the reversible association of the spider proteases may have a functional importance in the digestion of insect prey. Having two or more proteases with differing cleavage specificities in close proximity may help to more rapidly inactivate the proteases of the prey during extra-oral digestion. Or it may be that differing catalytic rates occur in the associated and dissociated forms as Hipps and Nelson (1974) suggested for cockroach digestive esterases. And, whereas I did not find conclusive evidence for aggregates by sucrose density gradient centrifugation, this may be explained by the high dilution (15pl SDF in 5ml sucrose) used in these experiments. However, the appearance of the p32, p27 and pl2 in all fractions (Fig. 6) does suggest that these proteases maintained an affinity for one another. The SDF would not become so dilute when the spider consumes its prey. It is often easy, for example, to obtain 100 pi or more of the SDF from a single spider depending on the state of hydration of the animal. If this volume were injected into an insect of equal size the dilution would only be about 1:2, 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and this would likely favor the equilibrium towards the association of the proteases. At first, I thought that the serine protease inhibitors would be linked with the hypothetical proteases aggregates. This does not seem to be the case. During ultracentrifugation experiments the inhibitors separated discretely from the bulk of the proteases (Fig. 6). Mebs (1972) noted a similarity of the masses and enzymatic properties of the isolated spider venom proteases compared to those of the crayfishAstacus fluviatilis. I examined the possibility that the spider and crayfish digestive proteases are closely related by determining the N-terminal sequences of isolated spider proteases. I found, however, only one set of data indicating a close relationship (SDF 25 (S) A 20-25 Kd; Table 3.1). There must however be major differences as: 1) the spider hemolymph exerted only a slight inhibition of crayfish gastric proteases and likewise the crayfish hemolymph exerted only a slight inhibition of spider digestive prbteases (Fig.5b) and whereas the bulk of the spider digestive proteases appear to be cationic at pH 8.6, the major crayfish proteases are anionic (Fig.5a). My observations are consistent with Zwilling and Neurath (1981) who noted the low isoelectric point of the LMW proteases of the crayfish gastric fluid. I found 10 serine protease inhibitors when SDF was fractionated by natural PAGE (Fig 3.4). They had masses ranging from 14 to 43 kDa. When three of these were isolated and subject to SDS-PAGE, each separated into two bands. The additional bands may indicate that the pairs of inhibitors had co-migrated with natural PAGE or possibly that the inhibitors are heterodimers. In any case no inhibitors were seen when 2- mercaptoethanol was used during electrophoresis suggesting that disulfide bonds are essential to their structure and function. The SDF serine protease inhibitors are 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reminiscent of those found in nematodes (Zang and Maizel, 2001). They are also of low molecular weight, and have disulfide bonds which are thought to render them heat resistant. Presumably the inhibitors protect the nematodes from proteolytic degradation by the host’s digestive enzymes (Zang and Maizels, 2001). The serine protease inhibitors of the spider digestive fluid are also of relatively low molecular weight and are heat stable. They may serve to protect the spider digestive enzymes during the digestion of prey. Note: For the following reasons I believe that Mebs (1972) inadvertently described spider digestive proteases rather than venom proteases. It seems likely to me that his venom sample was contaminated by SDF. 1) He acknowledged that the venom was a gift to him; he did not collect it. 2) Very little protease activity has ever been observed in any spider venom which has been collected carefully so as to avoid contamination by SDF (see Perret, 1977 and Kuhn-Nentwig et al., 1994 for reviews of this problem). 3) The proteases he described are essentially identical in their biochemical features to those described for SDF proteases (Kavanagh and Tillinghast, 1983). The results of this study demonstrate that SDF is a complex fluid containing both proteases and protease inhibitors. The proteases appear to self-assemble into clusters, making their purification difficult. A greater knowledge of the protein-protein interaction should help to develop a more effective means of purification. My results suggest that SDF proteases are members of the astacin family of metalloproteases, and this accords with earlier observations of the ion requirements of spider digestive enzymes. My research has also revealed at least ten serine protease inhibitors in the SDF, whose function remains unknown. 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF REFERENCES Atkins, J.A., Wingo, C.W., Sodeman, W.A., Flynn, J.E., 1958. Necrotic arachnidism. Am. J. Trop. Med. Hyg. 7, 165-184. Atkinson, R.K., Wright, L.G., 1991. Studies on the necrotic actions of the venoms of several Australian spiders. Comp. Biochem. Physiol. 98C, 441-444. Atkinson, R.K., Wright, L.G., 1992. The involvement of collagenases in the necrosis induced by the bites of some spiders. Comp. Biochem. Physiol. 102C, 125-128. Babcock, J.L., Civello, D.J., Geren, C.R., 1981. 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Invertebrate Proteases In: Colowick, P.S.; Kaplan, N.O., (Eds) Methods in Enzymology, vol. 80. Academic Press, New York. pp. 633-664. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. JjU §*», NIYERSITY y OF-JNIEW T v i ■v? & t JrlAMPSHIRE 1 . jT a ~sl *&*yyr*sr~%, Tg^T Office o tSponsored Research mm mum « if c g t*y FIRST MAME {Stew* man a pp'l da te a g a i n OWJMMR® IACBC # . s m s A»aiss {if «ppMM«; « f iK W L E m m RR©«€T TITLE t e s t e in tabbiis and the |te ® fe ta ^ « te B of ifcg t e h » i ^ |p s ^ r pretease M t e i s lt e t aM f m hamoiymptt 4 1 ««#«} j m M M W a f l w t t wrecords must meted# yettrM CiJC P roiom i# « flrlwtabm e. The Imtitatioiial Animal Care and Use Committee has reviewed and approved the protocol submitted for this study under Category 2 on Page 3 Of the “Application far Review of Animal Use or Instmaion ProtoeolH - the research potentially involves minor tb a f c to m pats, discomfort or dlsttess which w il l* treated with appropriate anesthetics/analgesics. Approval is pantedfor a period ofte e yeas fpa»the approval date above. Continued approval thma^aoat the te e yearperiod is contingent upon com plete ofannual tspoas on the use of animals. At the end o f the te e year approvalperiod you may submit a new application and request for extension to continue te fta p e i. -fepeste for extension most be filed prior to the expiration of tire original approval. For the Institutional Animal Cate aid Use Commi ttee, Kara L. BMy, MJLA, Manager, Regulatory Compliance 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U n iv er sity o f N e w H a m p sh ir e Gffies «f Spoil Wed .Research SetvfajeBuading 51 College Road Durham, Nev. Hampshire 03S24-35e5 w y m a m F A X ' IAS7 NAME RRSTNAME 0EPT NEXT REVIEW DATE OFF-CAMPUS lACUC f ADDRESS (If applicable) REVIEW LEVEL I DATE OF NOTICE PROJECT i t spiders wife necrotizing venoms TITUs The instiHrfopai Animat Care and Use Committee {iACUC} has reviewed and approved your request for a time extension fcr this protocol. Approval is granted until the "Next Review Date" indicated above. You will be asked to subih't s orcjcct roport witn regard to tie mvoivument cr animals before that date. If your project Is still active, you may apply fot exteusmfi of IACUC approval w ougb ihis olnce. The appropriate use and care of animats In your project is an ongoing process for which you hold primary responsibility. Changes in your protocol must be submitted to the IACUC tor review and approval prior to their implementation. If you nave questions or concerns about your project or this approval, please feel free to contact the Regulatory Compliance Office at 862-2003 or 882-3536. Please note; Use of anraais in research and instruction is approved contingent upon participation in fee UNH Occupational Health Program for persons handling animate. Participation is mandatory ior all principal investigators and tneir affiliated paraon'wl, employees of fee University and students alike. A Medical History Questionnaire accompanies tnis approval, ploasa copy and oistnoute to sll listed project staff who have not completed thisfcrrr. already. Completed questionnaires should be sent to Dr. Giadi Porsche, UNH Health Services. Please refer to fee IACUC # above in ail correspondence related to this project. The IACUC wishes you success wife your research. Foofee IACUC, f t i Jbhn A, Utvaitfe, Ph.D. Chair cc: File . .. grig apfl ti»8ip#ea 82 Reproduced with permission of the copyright owner. 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