humoral immunity in long-lived arthropods

Pergamon 0022-1910(95)00082-8

J. lnsrct Physiol. Vol. 42, No. I, pp. 53-64, IYY6 Elsevier Science Ltd. Printed in Great Britain

Humoral Immunity in Long-lived Arthropods PETER B. ARMSTRONG,*yQ RALPH MELCHIOR,*t JAMES P. QUIGLEY’(-$

The innate immune system is represented by a complex of cellular and humoral factors and processes that protect the organism against pathogenic attack. Certain of these elements arose early in the evolution of multicellular animals and have been preserved throughout the evol- utionary divergence of the different animal phyla. Two mediators that have interested us are a,-macroglobulin and the pentraxins. Both function as elements in the plasma defense systems of arthropods and vertebrates. The ability to purify significant quantities of both proteins from the plasma of the American horseshoe crab Limulus polyphemus has enabled us to inves- tigate their functions at the molecular and organismal levels. In Limulus o,-macroglobulin functions as a broad-spectrum protease-binding molecule that mediates the clearance of proteases from the plasma. Limulus has at least two pentraxins in the plasma, an abundant form designated Limulus C-reactive protein, which is of unknown function, and limulin, a low- abundance form with sialic acid recognition capabilities, which mediates a cytolytic reactivity for the destruction of foreign cells that have contacted the plasma.

a,-Macroglobulin Pentraxin Plasma lectin Limulin Limulus

1NTKODUCTlON

All multicellular animals are subject to frequent challenge from potentially pathogenic microbes and multicellular parasites that penetrate the integument. Survival depends on the activities of host defense systems that eliminate or inactivate the challenging pathogens. The maximum life span for many invertebrate species is as long as it is for humans in spite of continual challenge from pathogens and without the benefit of medical care (Finch, 1990). Thus, long-lived invertebrates resist pathogenic attack and do so without the activity of the adaptive antibody-based immune system that is so important in the immune defenses of vertebrates (Marchalonis and Schluter, 1990). In the course of our characterization of the humoral immune systems of one of these long-lived invertebrates, the American horseshoe crab Limulus polyphemus, we have investigated two classes of immune mediators from the plasma of Limulus, a,-macroglobulin and members of the pentraxin family of proteins. Both agents are of ancient origin, since they are present in vertebrates and arthropods (Armstrong and Quigley, 1994a,b). The lineages of the vertebrates and the arthropods diverged 0.5-0.6 billion years ago, and the presence of cy,-macroglobulin and the pentraxins in modern representatives of both lineages indicates that the

*Department of Molecular and Cellular Biology, University of Califor- nia, Davis, CA 95616-8755, U.S.A.

tMarine Biological Laboratory, Woods Hole, MA 02543, U.S.A.

ZDepartment of Pathology, State University of New York, Stony

Brook, NY 11794-8691, U.S.A. gTo whom all correspondence should be addressed.

common ancestor of both lineages must have possessed these two classes of proteins. The preservation during this long period of evolution of a,-macroglobulin and the pentraxins in animals as diverse as humans and horseshoe crabs suggests that these are important proteins.

The cu,-macroglobulin family of proteins includes C3, C4, and C5 (Sot&up-Jensen, 1987, 1989; Tack, 1983) which are important components of the vertebrate complement system, pregnancy-zone protein (Christensen et al., 1989; Devriendt et al., 199 1 ), which is an acute phase protein of mammals, ovomacroglobulin from the eggs of reptiles (Ikai et al., 1983, 1990) and birds (Feldman and Pizza, 1984; Kitano et al., 1982; Nagase and Harris, 1983; Nagasc et al., 1983), and cY,-macroglobulin homologues found in the plasma of arthropods (Armstrong and Quigley, 1991,1994a,b; Armstrong et al., 1985), mollusks (Armstrong and Quigley, 1992; Bender et al., 1992; Thogersen et al., 1992) and all classes of vertebrates (Starkey and Barrett, 1982). In mammals, where it has received the most study, a,-macroglobulin functions to bind proteases and thereby mark them for removal from solution by a process of receptor-mediated endocytosis and intracellular degradation. In addition, cr,-macroglobulin binds a variety of cytokines and other proteins (Borth, 1992; Gonias, 1992; James, 1990).

The pentraxins of mammals are a family of plasma proteins that include C-reactive protein, serum amyloid protein, and TX-14 (Lee et al., 1993; Pepys and Baltz, 1983). The pentraxins are named for the native configur- ation of these proteins in mammals, which is that of a single or double stack of pentamers of non-glycosylated subunits of about 200 residues. Depending on the species

53

54 PETER B. ARMSTRONG et al.

of mammal, either C-reactive protein or serum amyloid protein is an acute-phase reactant. In humans, the C-reactive protein content of the blood increases by 2 to 3 orders of magnitude within 24 h after the onset of infection in response to a variety of inflammatory mediators, including IL-I, IL-6, TNF-a, LIF, TGF-P, and IFN-6 (Steel and Whitehead, 1994). The plasma concentration of C-reactive protein is widely used to monitor the pro- gression of a variety of diseases (Young et al., 1990). It binds a variety of macromolecular ligands in a Ca*+- dependent fashion, including C-polysaccharide of Strep- tococcus pneumoniae (from whence came its name), fibronectin (Tseng and Mortensen, 1988), laminin, and the complement component C lq (Kolb-Bachofen, 1991). C- reactive protein also binds to low molecular mass phosphate monoesters (Gotschlich et al., 1982). Binding to the cell wall of pneumococcal bacteria depends on the reactivity of C-reactive protein with phosphorylcholine. The usual isolation method for the pentraxins involves affinity chromatography with phosphorylcholine- or phosphorylethanolamine-derivatized agarose resins (Macintyre, 1988). Binding of C-reactive protein to fibronectin-coated surfaces or the bacterial cell wall promotes the activation of the classical complement pathway with C lq (Volankis, 1982) which in turn promotes the phagocytic clearance of the derivatized particles (Mold et al., 1982). Human C-reactive protein can protect mice from an otherwise lethal challenge of S. pneumoniae (Mold et al., 1981). Membrane-associated monomers of C-reactive protein promote the uptake by Kuppfer cells of particles expressing multiple galactosyl groups (Kolb- Bachofen, 1991). The identification of the essential functions of mammalian C-reactive protein in vivo has proven difficult because the opsonic functions described above can all be produced by other effecters of the plasma and serum. Clearly mammals possess multiple defense systems with redundant functional overlap with that of C- reactive protein. Nevertheless, C-reactive protein does appear to possess unique essential functions since, to date, viable humans genetically deficient for C-reactive protein have not been found, suggesting that the absence of this protein is lethal.

Both a,-macroglobulin and the pentraxins are soluble proteins of the plasma. Limulus offers several important advantages for the study of the plasma-based defense systems of invertebrates. In general, Limulus is probably the best choice of an invertebrate for hematological studies requiring large volumes of blood, since it is relatively easy for one investigator to prepare 1 or more liters of blood in a few hours. Additionally, the blood clotting system and the cellular immune systems of Limulus have been especially well characterized (Armstrong, 1985a, 1991; Iwanaga et al., 1992, 1994), making this species a favored invertebrate model for the investigation of immunity. The composition of the blood is simple, with a single blood cell, the granular amebocyte (Armstrong, 1985a, 1991), and only 3 abundant plasma proteins, hemocyanin, a,-macroglobulin, and the pentraxins (Fig. 1, lanes 1,2). The opportunity to obtain large quantities of

cc,-macroglobulin -

hemocyanin -

pentraxins -t

FIGURE I. SDS-PAGE (reducing conditions) of Limulus plasma that

has been fractionated to purify limulin and separate it from C-reactive

protein: Lane 1, starting sample (whole Limulus plasma). The three

most abundant proteins are cY,-macroglobulin (185 kDa), hemocyanin

(the very broad band at 67 kDa), and the pentraxins (25 kDa). Lane 2,

plasma that had been fractionated with polyethylene glycol (PEG) to

reduce the content of hemocyanin (3-10% PEG cut). The a,-macroglo-

bulin and pentraxin bands are more prominent because they are a larger

fraction of the total protein of this sample. Lane 3, non-bound fraction

from incubation with phosphorylethanolamine-agarose. This sample

lacks the pentraxin fraction. Lane 4, C-reactive protein isolated as the

pentraxin that failed to bind to fetuin-Sepharose in the presence of

CaZ+. Lane 5, limulin isolated as the pentraxin that bound to fetuin-

Sepharose and was eluted by 0.1 M citrate, pH 6.7. Affinity chromatog-

raphy was conducted with resins equilibrated in buffer A (0.15 M

NaCl, 50 mM Tris, pH 8.0, 10 mM CaCl), and elution of the bound

proteins accomplished with 0.1 M citrate, pH 6.7. Both C-reactive pro-

tein and limulin were dialyzed back into buffer A for subsequent func-

tional characterization. The purification scheme illustrated in Fig. 1

differs from that described in the Materials and Methods section in

the omission of the zinc acetate incubation to precipitate residual

hemocyanin.

plasma has facilitated the purification of the proteins of the plasma-based immune defense systems.

Our interest in the plasma-based immune systems of Limulus has focused on cY,-macroglobulin and the pentraxins. We, and others, have shown that cY,-macroglobulin from invertebrates presents the same unique suite of unusual functional properties as those found in vertebrate cY,-macroglobulin and functions in Limulus to eliminate proteases from the circulation, just as it does in mammals. We have identified limulin, one of the pentraxins of Limulus, as the principal mediator of a plasma-based cytolytic system in that animal.

MATERIALS AND METHODS

Blood was obtained from adult horseshoe crabs by car- diac puncture under sterile, lipopolysaccharide-free conditions as previously described (Armstrong, 1985b) from pre-chilled animals and the blood cells were immediately removed by centrifugation. It is important to avoid degranulation of the blood cells, since this releases proteases (Iwanaga et al., 1992) and active-site protease inhibitors (Armstrong and Quigley, 1985; Donovan and Laue, 1991; Nakamura et al., 1987) into the serum. Ani-

IMMUNITY IN ARTHROPODS 55

mals were released into the ocean unharmed after bleed- ing.

Hemocyanin was removed from the plasma by ultracentrifugation or by incubation with 3% polyethylene glycol-8000 with centrifugation at 30,000 g, 0.5 h, which removes most of the hemocyanin (Fig. 1, lane 2). The supernatant was then made 10% in polyethylene glycol and the precipitate redissolved in 0.1 M citrate, pH 7.0. The remainder of the hemocyanin was precipitated by the addition of zinc acetate to a final concentration of 0.1 M. The supernatant was then transferred to buffer A (0.15 M NaCl, 10 mM CaCl,, 50 mM Tris, pH 8.0). This fraction was depleted of several Sepharose-binding proteins by passage over a column of Sepharose 4B equilibrated with buffer A (0.2 vol resin/v01 3-10% polyethylene glycol cut). The unbound fraction was then incubated with gentle stirring with phosphorylethanolamine-agarose (Sigma Chemical Co., St Louis) to remove the pentraxins (0.1 vol resin/v01 plasma) (Fig. 1, lane 3). Exposure to the resin in bulk suspension was utilized because C-reactive protein precipitated onto the resin bed when plasma was passed over phosphorylethanolamine- agarose packed in a column. The phosphorylethanolamine-agarose was then packed in a column, washed with buffer A modified to contain 1 M NaCl, and eluted with 0.1 M citrate, pH 6.7 to recover the pentraxin fraction. Following dialysis back into buffer A, the pentraxin fraction was further fractionated by passage over a column of fetuin-Sepharose equilibrated with buffer A. The breakthrough fraction from the fetuin-Sepharose column is Limulus C-reactive protein (Fig. 1, lane 4). The bound fraction, which was eluted with 0.1 M citrate, pH 6.7, is the lectin limulin (Fig. 1, lane 5). The unbound fraction from the incubation with phosphorylethanolamine-agarose was dialyzed into 50 mM Tris, pH 7.4, applied to a column of Waters Protein Pak Q 15HR anion exchange resin, and eluted with a linear gradient of NaCl (0.0-0.5 M NaCl, 50 mM Tris, pH 7.4), using a Waters FPLC apparatus. The first major peak, at approx. 0.15-0.2 M NaCl, is slow-form cY,-macroglobulin. This was further purified by chromatography on a 100 x 1.6 cm column of Sephacryl S-300 gel filtration resin to remove aggre- gated cY,-macroglobulin and was stored in 0.1 M citrate, pH 6.7 at 4°C.

The hemolytic activity of the plasma was determined in duplicate or triplicate samples using sheep red blood cells (Gee, 1983; Kabat and Mayer, 1961; Sim, 1981). Unactivated sheep erythrocytes in Alsevers solution were obtained from Cappel, West Chester, PA (reference number 55875) and Becton Dickinson and Company, Cocke- ysville, MD (reference number 12388). The reaction mix- tures contained 3 x lo7 washed sheep red cells in a final volume of 800 ~1. The buffer system was modified DGVB (7 1 mM NaCl, 0.18 mM CaCl,, 0.5 mM MgCl,, 2.5% glucose, 0.1% gelatin, 2.5 mM sodium barbital, pH 7.3). The samples were incubated with stirring at 22- 23°C for 4 h, and the reaction was terminated by adding 2 ml of ice cold phosphate-buffered saline containing 5 mM ethylenediaminetetraacetic acid, followed by centri-

fugation to remove the red cells. The extent of hemolysis was determined by monitoring released hemoglobin in the supernatant by optical absorbance at 4 12 nm and was compared to full hemolysis produced by hypotonic lysis of the red cells.

RESULTS AND DISCUSSION

The cqmacroglobulins

The protease inhibitory mechanism of cY,-macroglobulin is unique amongst enzyme inhibitors, since it involves the physical entrapment of the protease molecule in a molecular cage that forms a stearic barrier which pre- vents subsequent contact of the entrapped protease molecule with protein substrates but that leaves intact the active site of the entrapped enzyme (Barrett and Starkey, 1973; Starkey and Barrett, 1977). Ester and amide substrates of the bound protease that are small enough to diffuse into the cY,-macroglobulin cage are readily hydrolyzed [Fig. 2(a)] (Bieth et al., 1978; Starkey and Barrett, 1977). During reaction with proteases, the LY>- macroglobulin polypeptide chain is proteolyzed at a spec- ialized region, the bait region (Hall and Roberts, 1978; Harpel, 1973; Sot&up-Jensen et al., 1981, 1989) and most forms of a,-macroglobulin undergo a significant compaction as a manifestation of the trapping process (Barrett et al., 1979; Bjork and Fish, 1982; Gonias et al., 1982). The protease-inhibitory mechanism of a,-macroglobulin stands in marked contrast to all other protease inhibitors, which bind to the active site and inhibit the activity of the target protease against both protein and low molecular mass amide and ester substrates (Laskowski and Kato, 1980; Travis and Salvesson, 1983). This is one of several diagnostic properties (Table 1, Fig. 2) that initially allowed us to identify the first example of c-r,-macroglobulin from the plasma of an invertebrate, the American horseshoe crab, L. poly-

phemus (Quigley and Armstrong, 1983; Quigley et al., 1982). Subsequently, molecular homologues of a2-macroglobulin have been found in the plasma of crustaceans (Armstrong et al., 1985; Hergenhahn and Soderhall, 1985; Hall et al., 1989; Hergenhahn et al., 1988; Liang et al., 1992; Spycher et al., 1987; Stocker et al., 1991) and mollusks (Armstrong and Quigley, 1992; Bender et

al., 1992; Thogersen et al., 1992) and probably are present in other phyla.

The cY,-macroglobulins from horseshoe crab, crayfish, lobster and octopus share many of the unique structural and functional characters of mammalian cY,-macroglobulin, including significant sequence identity (Hall et al.,

1989; Sottrup-Jensen et al., 1990a; Spycher et al., 1987; Thagersen et al., 1992), a reactive internal thiol ester bond (Armstrong and Quigley, 1987; Spycher et al., 1987) reactivity with proteases of differing enzymatic mechanism (Table 1; Quigley and Armstrong, 1983, 1985), the unique protease trapping mechanism (Fig. 3; Armstrong and Quigley, 1991; Armstrong et al., 1985) and the ability to participate in a clearance pathway for proteases introduced into the plasma (Melchior et al.,

PETER B. ARMSTRONG et al.

Effect of Limulus plasma inhibitor on trypsin hydrolysis of 14C-casein

56

(a)

1800

1400

g 1000

600

200 L

0

@I

@ -0 1 p8 Trypsin + 10 pl HSS _ H I pg Trypsio

/

.

/ /’

/, , -d __ _- -- -_ __

,

5 10 15 20 25 30 35

Time (min)

Protection of the active site of trypsin from SBTI by high speed supernatant of

Limulus plasma

0 10 20 30 40 50 60 70 80 90

Time (min)

0.800

0 <+ 0.600

Effect of Limulus ulasma inhibitor on trypsin hydroiysis of BAPNA

M 10 pg Trypsin

4 8 12 16 20 24 28

Time (min)

Methylamine inactivates the plasma protease inhibitor

z 2 ? 2 s

Sample

FIGURE 2. Evidence for a functional hmologue of cY,-macroglobulin in the plasma of Limulus: (a) Limulus plasma contains

a protease inhibitor that eliminates the caseinolytic activity of trypsin (left figure) but that fails to inhibit the activity of trypsin

against the low molecular mass amide substrate BAPNA (N”-benzoyl-DL-arginine p-nitroanilide) (right figure). Plasma was

cleared of hemocyanin by ultracentrifugation and is referred to as HSS (high-speed supernatant). Hydrolysis of [“‘Cl-casein

(left figure) was determined by the release of acid-soluble [“‘Cl. Hydrolysis of BAPNA (right figure) was determined by the

increase in optical absorbance at 410 nm. (b) Limulus plasma that has been cleared of hemocyanin by ultracentrifugation (high

speed supernatant = HSS) contains an agent capable of protecting trypsin from subsequent inactivation by the macromolecular

active site inhibitor, soybean trypsin inhibitor (SBTI) (Mr = 21,000). This assay employed saturating amounts of trypsin and

twice-saturating amounts of SBTI. The plasma sample was preincubated for 15 min with trypsin to allow binding by (Y?-

macroglobulin, and was then reacted with SBTI to inhibit any uncomplexed trypsin. The assay was initiated by adding the

low molecular mass substrate, BAPNA. The only fraction of trypsin capable of hydrolyzing BAPNA is that fraction that is

complexed with az-macroglobulin, and is thereby protected from SBTI. As the amount of plasma in the assay is increased,

the amount of trypsin that is in the protected state is correspondingly increased, as indicated by the increase in the rate of

hydrolysis of BAPNA. In the absence of plasma, there is no hydrolysis of BAPNA, since all of the trypsin is inhibited by

SBTI. Plasma itself does not hydrolyze BAPNA. The amount of ol,-macroglobulin-like activity in the sample, in fig trypsin bound/ml plasma, is estimated by comparison of the hydrolytic rates in the presence of plasma with the rates of hydrolysis

of BAPNA by different concentrations of trypsin in the absence of plasma and SBTI (dashed line), and from knowledge of

the fractional activity of the preparation of trypsin, as determined by titration with p-nitrophneyl p’-guanidinobenzoate hydroch-

loride (Chase and Shaw, 1970). As far as we are aware, the cy,-macroglobulins are the only protease-binding agents capable

of protecting proteases from active site inhibitors. If endogenous low-molecular mass active site trypsin inhibitors are present

in the plasma, they will reduce the amount of active trypsin and will cause artifactually low levels of a,-macroglobulin activity

in a SBTI-protection assay. Their effects can be estimated by the assay illustrated in the right panel of (a), which shows that such inhibitors are absent from Limulus plasma. The protease-inhibitory activity of the plasma is inactivated by exposure to

the small primary amine, methylamine (MA). Methylamine reacts (c) with the thiol ester of cY,-macroglobulin (see Fig. 5). thereby inactiving it. Sensitivity to methylanime is diagnostic for thiol ester proteins.


TABLE 1. Comparison of cY,-macroglobulin with active site protease

inhibitors

a,-macroglobulin Active site inhibitors

Inhibits the proteolytic activity

of proteases without inhibiting

the hydrolysis of low molecular

mass amide or ester substrates

Reacts with endopeptidases of

diverse catalytic mechanisms

and substrate specificities

Inhibit activity of target

proteases against polypeptide

and low molecular mass

substrates

React with a narrow spectrum

of related proteases

Shields bound proteases from

antibodies and high molecular

mass active site inhibitors

Bound proteases remain reactive

with antibodies

Presence of a unique internal

reactive thiol ester group

Internal thiol ester is found only

in proteins of the cr2-

macroglobulin family

1995). In this latter regard, fluorescein isothionate (FITC)-labeled trypsin was cleared from the plasma in a triphasic manner, with an initial lag period (10-20 min) and a rapid clearance period (20-30 min), followed by the reappearance of FITC in the circulation (45-90 min)[Fig. 4(A)]. FITC-labeled, protease-reacted Limulus a,-macroglobulin showed similar clearance kinetics, whereas unreacted Limulus cu,-macroglobulin persisted in the plasma and was not cleared [Fig. 4(B)]. Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass, as identified by gel filtration chromatography on Sephacryl S300 HR resin, identical to that of Limulus a,-macroglobulin, but the fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (< 10 kDa), and thus appears to have undergone proteolytic degradation, possibly in secondary lysosomes. Clearance requires proteolytic activity since enzymatically-inactive trypsin was not cleared [Fig. 4(A)] nor are non-proteases, such as hemocyanin. This demonstrates the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases. The Limulus homologue of aY,-macroglobulin appears to be capable of mediating this clearance, since protease-activated cr2- macroglobulin was cleared with similar kinetics to those of the introduced proteases. The blood cells appear to participate in the clearance, because FITC-labeled material could be isolated from detergent extracts of the blood cells at the very stages that protease-reacted (Y*- macroglobulin was being cleared from the plasma [Fig.

4(B)]. As mentioned above, the conformational changes that

are induced by the proteolysis of mammalian ac,-macroglobulin expose a domain at the carboxyl terminus of the polypeptide chains that is then recognized by the a2-macroglobulin-protease receptor on the cell surface (Holtet et al., 1994). The mammalian receptor for the a,-macroglobulin-protease complex has been identified as being identical to low density lipoprotein receptor-related protein (LRP) (Kristensen et al., 1990; Strickland et al.,

1990; Bu et al., 1992). Mammalian LRP is synthesized

as a 600 kDa protein with a single transmembrane

domain and is a member of the low density lipoprotein

receptor family. Mature LRP is proteolytically cleaved

in the extracellular domain to produce a 5 15 kDa extracellular heavy chain and a 85 kDa transcellular light

chain. In addition to cY,-macroglobulin-protease complex, LRP also appears to be involved in the cell surface bind-

ing of a variety of other ligands including plasminogen activator-plasminogen activator inhibitor- 1 complexes, lipoprotein lipase, lactoferrin, Pseudomonas exotoxin A, and apolipoprotein E-rich chylomicron remnant (Hussain

et al., 1991; Moestrup et al., 1993; Nykjaer et al., 1993). In mammals, LRP is associated with a 39 kDa protein, receptor-associated protein (RAP), in a 2:l RAP:LRP

ratio. RAP binds specifically to LRP and can be used as a probe in ligand blotting experiments to identify the

presence of LRP (Herz et al., 1991; Kounnas et al., 1992; Williams et al., 1992). Interestingly, mammalian RAP specifically binds to a protein in Western-blotted deter-

gent extracts of Limulus blood cells (Aimes et al., 1995). This is strong presumptive evidence that the receptor system in Limulus for protease-reacted a,-macroglobulin is

molecularly homologous to the LRP/RAP system of mammals.

At the molecular level, Limulus cY,-macroglobulin shows significant amino acid sequence identity with mammalian cY,-macroglobulin (Sottrup-Jensen et al.,

1990a) and, like mammalian cY,-macroglobulin, possesses an internal thiol ester bond (Armstrong and Quig-

ley, 1987; Sottrup-Jensen et al., 1990a)(Table 1, Fig. 5). Internal thiol esters are unique to proteins of the a2-macroglobulin family (Sot&up-Jensen, 1989; Tack, 1983). Proteolytic cleavage of the bait region of a,-macroglobulin is followed by rapid reaction of the thiol ester with

availablenucleophiles, which may be e-amino groups of

lysines of the reacting protease (Sottrup-Jensen et al.,

1990b) or of a,-macroglobulin itself (Quigley et al.,

199 1). Reaction of the y-carbonyl of the thiol ester gluta-

my1 residue with e-amino groups on the reacting protease allows cr,-macroglobulin to establish covalent y-glutamyl isopeptide bonds linking the protease to the peptide bear-

ing the thiol ester domain (Fig. S)(Chen et al., 1992; Sottrup-Jensen et al., 1990b). Bait region cleavage also initiates a rapid compaction of c+macroglobulin (Fig. 3) that allows it to physically entrap the target protease within the interior of an a,-macroglobulin cage and also exposes a previously hidden domain close to the carboxyl

terminus (Holtet et al., 1994) that is recognized by the integral membrane cell surface receptors that mediate the protease clearance pathway described above.

The a,-macroglobulins represent a conserved family of plasma proteins that have retained a number of unique features during the entire period of evolution of higher animals. Their retention over long periods of evolution of very different lineages suggests that they are essential to survival. It is anticipated that a comparative study of

their properties and functions in representatives of dis-

58

(a)

0.2

-.- 280 nM trypsin -.- 280 nM trypsin + .

_ -A- 280 nM trypsin +

930 nM SBTI / 930 nM SBTI + 135 nM azM

/’

5 IO 15

Min

(cl

1 human u?M (+CT)

1 human a,M (-CT)

1 hagflsh azM (+CT)

1 hagfish a2M (-CT)

1 ovomacroglobulin (+CT)

I(l) I(f) I($ ovomacroglobulin (chicken)

I\ Limulus azM (-CT)

I I

A Llmulus a2M (+CT)

excess 1 chymotrypsin

PETER B. ARMSTRONG et al.

V. = 7.0 ml V, = 18.0 ml

FIGURE 3. Evidence for the trap mechanism of protease binding by Limulus cYz-macroglobulin. (a) Reaction of trypsin with

purified Limulus a,-macroglobulin protects it from subsequent inactivation by the macromolecular active site trypsin inhibitor,

soybean trypsin inhibitor (SBTI) Mr = 21,000). Under these conditions, entrapment of trypsin within the a,-macroglobulin

cage shields it from large molecules, including macromolecular active site protease inhibitors. (b) Pore-limit polyacrylamide gel

electrophoresis of human and Limulus a,-macroglobulin reveals that Limulus a,-macroglobulin undergoes a major compaction

following reaction with proteases or the thiol ester reactant, methylamine, consistent with the proposal that protease binding

involves a major structural rearrangement of the a,-macroglobulin molecule. Unreacted tetrameric human cYz-macroglobulin

(lane I) shows a slower mobility in the gel than trypsin-reacted human cz-macroglobulin (lane 2). Similarly, unreacted dimeric

Limulus a,-macroglobulin (lane 3) shows a slower mobility than after reaction with trypsin (lane 4), chymotrypsin (lane 5)

or methylamine (lane 6). (c) Gel filtration chromatography of unreacted and chymotrypsin-reacted a,-macroglobulin also

provides evidence for compaction following reaction with proteases. Samples of unreacted and chymotrypsin-reacted Limulus

cY,-macroglobulin were applied to a TSK G4000SW gel permeation column and were eluted with 0.05 M phosphate buffer.

The protease-reacted sample was significantly retarded, indicative of a compaction of the molecule. The calibration bars over

the figure indicate the elution positions of native and chymotrypsin-reacted tetrameric (human crZM and ovomacroglobulin) and

dimeric (hagfish (Y*M) forms of cY,-macroglobulin. The column was further calibrated with tetrameric, dimeric, and monomeric ovomacroglobulin. The dimeric forms of az-macroglobulin (e.g. Limulus and hagfish a>-macroglobulin) show a more pro-

nounced compaction as estimated by the separation of the elution profiles of native and reacted forms, than do the tetrameric

varieties. (d) Transmission electron microscopy of individual molecules of negatively stained Limulus oz-macroglobulin shows

the dimeric character of Limulus a,-macroglobulin (upper two rows of micrographs of individual Limulus a?-macroglobulin

molecules) and the compaction experienced by the molecule after reaction with chymotrypsin (lower row of figures). The

native molecule of Limulus a,-macroglobulin is relatively extended and resembles a butterfly, whereas the chymotrypsin-

reacted molecule is significantly more compact. Bar = 20 nm.


120

y e

100

5 80

g 60

-1 40

r= 20

0

120 a

; 100 80

"0 g 60

5 40 ct

20

0

-.- FITC-LAM

+ FITC-Tiypsin-LAM

-c Cell-associated FITC

10 20 30 40 50 100 150 200

Time After Injection [min]

FIGURE 4. Clearance from the plasma of Limulus of: (A) fluorescein

(FlTC)-labeled samples of trypsin and (B) trypsin-reacted Limulus cv-

macroglobulin (LAM). Trypsin inactivated by prior reaction with phe-

nylmethylsulfonyl fluoride (A) and unreacted Limulus cu,-macroglobu-

lin (B) were not cleared from the circulation during the period of obser-

vation, whereas trypsin and activated Limulus cY,-macroglobulin were

cleared rapidly and efficiently (trypsin tm = 26 min; trypsin-reacted

Limdus cYz-macroglobulin t,,> = I7 min). The fraction of the fluor-

escein label that was associated with the blood cells of animals exposed

to FITC-labeled, trypsin-reacted Limulus c*,-macroglobulin was maxi-

mal at the time that the FITC label in the plasma was minimal (panel

B). The vertical line at 50 min indicates a change of scale of the

abscissa.

-Gly-Cys-Gly-Glu-Gln-Asn-

1 I

s- c=o

Thiol Ester

H,C-NH,

Methylamine

I

(Slow)

-Gly-Cys-Gly-Glu-Gln-Asn-

SH C=Cl

NH

C H,

tinct and widely separated lineages will help elucidate

these functions.

The pentraxins

The pentraxins of the arthropods have been characterized only in Limulus (Amatayakul-Chantler et al., 1993; Nguyen et al., 1986a,b; Robey and Liu, 1981; Tennent et al., 1993). Limulus has at least two pentraxins, Limulus C-reactive protein and limulin, a lectin with recognition capabilities for sialic acid and 2 keto-3-deoxyoc- tonate (Kehoe and Seide, 1986). Although it was previously assumed that limulin and Limulus C-reactive

protein were the same protein (cf. Robey and Liu, 1981), we have found them to be distinct but closely related proteins (Quigley et al., 1994). Limulin was purified by sequential chromatographic isolation (in either order) on fetuin-Sepharose (a sialo-conjugate-derivatized resin) and phosphorylethanolamine-agarose. Limulus C-reactive protein was purified as the unbound fraction when the total phosphorylethanolamine-binding fraction was subsequently chromatographed on fetuin-Sepharose (Fig. 1) or as the protein that bound to phosphorylethanolamine- agarose after plasma was passed over fetuin-Sepharose. Less than 1% of the phosphorylethanolamine-agarose- binding (e.g., total pentraxin) fraction is limulin. Clearly limulin and Limulus C-reactive protein are closely related: both proteins bind to phosphorylethanolamine- affinity resins; both have the same native molecular mass (approx. 300 kDa-by size exclusion chromatography on Sephacryl S-300 HR resin); both have the same amino

Activation ) -Gly-Cys-Gly-Glu-Gln-Asn-

(Rapid) 1

I SH *c=o

Free Sultbydryl Reactive Glutamyl

-Gly-Cys-Gly-Glu-GIn-Asn-

I I SH C==G

Methylamine-a,-macroglobulin complex s-Lysyl-y-Glutamyl protein cross-linking

FIGURE 5. Activation and cleavage of the internal thiol ester of ol,-macroglobulin exposes a new thiol group on the cysteine and a reactive y-carbonyl on the glutamyl residue. The reactive internal thiol ester of members of the cY,-macroglobulin protein

family is cleaved following proteolysis at the distantly-located protease bait region of the protein. Thiol ester cleavage generates an activated y-carbonyl at the glutamyl residue and a free thiol at the cystenyl residue (top line of the diagram). The reactive

glutamyl can form amide linkages with proteins (right arm of the diagram). The thiol ester can also react slowly with small

primary amines, such as methylamine (left arm of diagram), even in the absence of proteolytic cleavage at the bait region.

Methylamine treatment eliminates many of the functional activities of cu,-macroglobulin in parallel with its inactivation of the

thiol ester. Sensitivity of a molecular function to treatment with methylamine is a useful test for the possibility that the function is dependent on the activity of a protein of the cYz-macroglobulin family.


terminal peptide sequence (LEEGEGITSKV), and a polyclonal antiserum produced against purified Limulus C-reactive protein cross-reacted with limulin. The two proteins do, however, show important functional differences: limulin, but not Limulus C-reactive protein, binds to sialic acid, agglutinates sheep and rabbit erythrocytes, and, as will be discussed below, lyses sheep erythrocytes. In our attempts to discover the molecular basis for these significant functional differences between two closely related proteins, we have found that limulin and Limulus C-reactive protein show small, but reproducible differences in the pattern of protein bands seen by SDS-PAGE (Fig. 6). The two proteins differ in the extent of proteolytic fragmentation and the sizes of the peptide fragments

- 97.4

- 66.2

-45

31

- 21.5

- 14.4

FIGURE 6. SDS-PAGE (reducing conditions) of Limulus C-reactive

protein (lane 1) and limulin (lane 2). C-reactive protein was prepared by passage of a preparation of Limulus plasma that had been depleted

in hemocyanin by treatment with 3% polyethylene glycol-8000 over

phosphorylethanolamine agarose and depleted in limulin by passage

over fetuin-Sepharose. Limulin was prepared as the material that bound

to fetuin-Sepharose upon passage of the total pentraxin preparation

(the plasma fraction that bound to phosphorylethanolamine-agarose) and elution from fetuin-Sepharose with 0.1 M citrate, pH 6.7. Two

passages over fetuin-Sepharose were used to ensure that the limulin

preparation lacked C-reactive protein.

generated by proteolysis by variety of endopeptidases, suggesting that, while the amino termini of limulin and Limulus C-reactive protein are identical, the two proteins may possess differences in amino acid sequence in the interiors of the polypeptide chains. There are multiple

genes for the Limulus pentraxins (Nguyen et al., 1986 a,b), consistent with this possibility. About 253.3% of the molecular mass of limulin and Limulus C-reactive protein is neutral sugar (determined by the phenol-sulf- uric acid method), but Limulus C-reactive protein is recognized by a mannose-binding lectin from Artocarpus

integrifolia, indicating that its carbohydrate is of the polymannosyl type, whereas limulin fails to bind this lectin. These data are consistent with the possibility that limulin and Limulus C-reactive protein differ both in amino acid sequence and in glycosylation.

A major problem of comparative immunology is the identification and characterization of the internal defense systems that lyse foreign cells, such as bacteria and other microbial pathogens, that have gained entry into the body (Canicatti, 1990). Lysis must be selective to avoid col- lateral damage to the organism’s own tissues. Limulus shows a potent cytolytic activity in the plasma that has been assayed by its ability to lyse sheep erythrocytes (Armstrong et al., 1993; Day et al., 1970; Enghild et al., 1990; Noguchi, 1903). We have observed that limulin is responsible for the hemolytic activity of Limulus plasma. The sialic acid-binding lectins that have been found in the plasma of a variety of invertebrates (Mandal and Mandal, 1990) are poorly-characterized in function. Limulus possesses several sialic acid-binding lectins, of which limulin is the best characterized (Kehoe and Seide, 1986). Limulin is necessary for hemolysis by hemocyanin-depleted Limulus plasma since depletion in limulin, either by passage over phosphorylethanolamine-agarose or over fetuin-Sepharose, abolished its hemolytic activity. Hemolytic activity was restored to limulin- depleted plasma by the addition of purified limulin. Limulin is sufficient for hemolysis since purified limulin was hemolytic in a Ca2+-dependent manner at 10 nM (assuming a molecular mass of 300 kDa (Tennent et al., 1993)) in the absence of other plasma components. The hemolytic activity of purified limulin is dependent on its sialic acid-recognition capabilities, since limulin- mediated hemolysis was abolished by desialylation of the target erythrocytes with Vibrio cholerue neuraminidase, or by inclusion of 9 PM fetuin in the incubation medium, and was reduced 50% by 0.1 M N-acetyl neuraminic acid. Interestingly, complement-mediated hemolysis of sheep erythrocytes was potentiated by desialylation of the target cells (Fearon, 1978).

Limulin can be separated from the other sialic acid- binding lectins in the plasma of Limulus by its unique ability to bind to phosphorylethanolamine-agarose. Although the other sialic acid-binding lectins of Limulus plasma, which were isolated from the phosphorylethanolamine-agarose breakthrough fraction by their affinity to fetuin-Sepharose, agglutinated sheep erythrocytes, they showed no ability to lyse the cells when they were pre-


sent at hemagglutination titers equivalent to those of active concentrations of limulin. Thus hemolysis is not produced by any or all sialic acid-binding lectins.

Identification of the cytolytic protein in the plasma of Limulus as the lectin limulin is an important advance in our understanding of the mechanisms of immunity in this animal. The recognition of foreign cells for subsequent cytolytic destruction probably depends on the presen- tation of sugars recognized by limulin on the surfaces of the foreign cells. Although plasma lectins have been identified in a variety of animals, the functions of this class of proteins have been characterized in relatively few cases. To our knowledge, this is the first demonstration of a cytolytic function for a plasma lectin. Candi- date pentraxins have been identified in a variety of invertebrates (Elola and Vasta, 1994; Mandal et al., 1991; Vasta et al., 1986). It will be interesting to determine if any of these molecules participate in cytolytic defense pathways, analogous to the cytolytic activity of limulin in Limulus.

SUMMARY

The internal defenses against microbial pathogens can be divided into the innate immune responses and the adaptive responses. The latter includes the antibody- based immune system of vertebrates in which an enor- mous variety of antibodies with different recognition capabilities are produced by the V-J-D-C segment splic- ing in the construction of the mature genes encoding the immunoglobulins during the maturation of cells of the lymphopoietic lineage. Adaptive immune systems have the characteristics of highly specific target recognition, amplification of the response upon challenge, and immunologic memory. In contrast, the innate immune systems show less narrowly defined target recognition capabilities, lack immunologic memory, and the effecters may be consititutively expressed. Examples of innate immune systems and mediators of plasma and serum include the basic antimicrobial peptides of the secretory blood cells [e.g. the defensins of insects and mammals (Dimarcq et al., 1990; Gabay and Almeida, 1993), the several inducible antibacterial peptides of insects (Dunn, 1990; Hoffmann and Hoffmann, 1990) and the tachyplesins of Limulus (Nakamura et al., 1988)]; the prophenoloxidase system (Soderhall, 1992); lysozyme (Engstrom et al., 1985); lectins (Sastry and Ezekowitz, 1993); various antibody independent opsonins such as fibronectin and heparin (Hormann, 1985); the complement system of vertebrates (Law and Reed, 1988) and the varied blood clotting systems employed by different animals (Bohn, 1986; Iwanaga et al., 1992). Although some of these systems are restricted to particular taxa, others are found in phylo- genetically-diverse groups of animals. These latter must have appeared early in animal evolution and can be pre- sumed to be important for survival when they can be shown to have been retained during the evolution of a diverse array of species with very different body plans, habitats, and basic physiologies. The cr,-macroglobulins

and the pentraxins conform to this constraint since they are present, often at high abundance, in the plasma of animals as diverse as humans and horseshoe crabs. LYE- macroglobulin functions in protease clearance both in mammals and in Limulus. The best characterized of the Limulus pentraxins, limulin, functions as the principal cytolytic protein of the plasma. Undoubtedly other functions will be discovered for both of these proteins in the identification and clearance of potentially harmful cells and molecules that have entered the internal milieu.

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Acknowledgement-This research was supported by Grant No. MCB

9218460 from the National Science Foundation.

humoral immunity in long-lived arthropods

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