©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Macroglobulin-mediated Clearance of Proteases from the Plasma of the American Horseshoe Crab, Limulus polyphemus(*)

Ralph Melchior (1) (2), James P. Quigley (2) (3), Peter B. Armstrong (2) (3)(§)

From the (1) Department of Molecular and Cellular Biology, University of California, Davis, California 95616-8755, the (2) Marine Biological Laboratory, Woods Hole, Massachusetts 02543, and the (3) Department of Pathology, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8691

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Because proteases free in the body are damaging to the tissues, animals have evolved various agents for their inactivation and clearance. Mammals, for instance, have a diverse array of active site protease inhibitors in the plasma. In addition, mammals have -macroglobulin (M), which binds active proteases, and the M-protease complex is then cleared from the plasma by a receptor-mediated endocytotic process. M is also present in the plasma of many invertebrates, and in the American horseshoe crab, Limulus polyphemus, it is the only protease inhibitor in the plasma. To search for a clearance process for proteases in Limulus, fluorescein isothiocyanate (FITC)-labeled proteins were injected into the blood, and the fluorescence in the plasma and associated with the blood cells was determined. FITC-labeled trypsin was cleared with an initial mixing period (0-10 min) and a rapid clearance period (10-30 min), followed by the reappearance of FITC in the plasma (45-90 min). Before and during the clearance process, the labeled trypsin was associated with a complex having a molecular mass identical to that of Limulus M, and that was precipitated by antibodies directed against Limulus M. The fluoresceinated material that reappeared in the plasma after 45 min was of low molecular mass (<10 kDa) and thus appears to have experienced degradation. The clearance of trypsin requires its protease activity, since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin was cleared only very slowly if at all (t > 180 min). FITC-labeled, trypsin-reacted Limulus M was cleared rapidly from the plasma of Limulus, whereas FITC-labeled, native Limulus M persisted undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus M, and the peak of recovery from the blood cells coincided with the minimum concentration of FITC-labeled protein in the plasma, suggesting that the blood cells participate in the clearance of M-protease complex from the plasma. Thus, we have demonstrated the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases and have shown that Limulus M is the probable agent for protease clearance. This is the first documentation of a protease clearance pathway in invertebrates and represents the first identified physiological function for M in invertebrates.


INTRODUCTION

Proteases are involved in the invasion and pathogenesis of bacteria, molds, and multicellular parasites (Breton et al., 1992; Hotez et al., 1990; McKerrow et al., 1993). Metazoans have evolved a variety of agents for the inactivation of proteases that help limit the destructive potential of these pathogens. The most important of these regulatory molecules are the polypeptide protease inhibitors, which can be classified into two groups, the active site-inhibitors and the -macroglobulin- (M)() -like inhibitors (Laskowski and Kato, 1980; Travis and Salvesen, 1983). The active site inhibitors bind to and inactivate the active sites of target proteases (Laskowski and Kato, 1980; Travis and Salvesen, 1983), whereas M enfolds the target protease molecule in a molecular cage that shields it from protein substrates (Sottrup-Jensen, 1987; Barrett and Starkey, 1973; Barrett, 1981). In marked contrast to the active site inhibitors, M-bound proteases retain the enzymatic activity of the active site and can still hydrolyze amide or ester substrates small enough to diffuse into the M cage (Bieth et al., 1978; Beatty et al., 1982; Berthillier et al., 1968). Protease binding begins with the proteolytic cleavage of the M polypeptide at a region known as the ``bait'' region (Barrett and Starkey, 1973; Barrett et al., 1979; Hall and Roberts, 1978; Sottrup-Jensen et al., 1981) and is immediately followed by a physical folding of M around the target protease to ``trap'' it within the M cage (Barrett et al., 1979; Cummings et al., 1984; Nelles et al., 1980; Hall and Roberts, 1978). The bait region contains peptide bonds susceptible to almost all endopeptidases, thereby conferring the broad reactivity of M and enabling it to react with proteases from endogenous sources involved in coagulation, fibrinolysis, and inflammation as well as with foreign proteases introduced by invading pathogens (Barrett and Starkey, 1973; Howell et al., 1983). M does not interact with enzymatically inactive proteases.

Homologues of M have recently been described from a variety of invertebrates, including mollusks and chelicerate and mandibulate arthropods (Armstrong and Quigley, 1995). Most of the unique structural and functional characteristics of M have been conserved over the 0.5-0.6 billion years of evolutionary divergence of the arthropod and vertebrate lineages, including significant sequence identity (Sottrup-Jensen et al., 1990), the internal thiol ester (Armstrong and Quigley, 1987; Spycher et al., 1987), the unique protease trapping mechanism (Armstrong and Quigley, 1985; Quigley et al., 1991), and the reactivity against a broad spectrum of proteases of diverse catalytic mechanisms (Quigley and Armstrong, 1985).

In mammals, the binding of M to endopeptidases is followed by the clearance of the M-protease complex from the circulation by hepatocytes (Van Leuven, 1984; Gliemann et al., 1983; Davidsen et al., 1985; Feldman et al., 1985). The conformational change of M that accompanies protease binding both entraps the protease and exposes a previously buried domain close to the carboxyl terminus (Holtet et al., 1994) that is then recognized by a cell surface receptor system that mediates the binding and endocytosis of the complex (Marynen et al., 1981, 1982; Van Leuven et al., 1983). The mammalian M receptor has been identified as being identical to the low density lipoprotein receptor-related protein and is specific for the protease-reacted form of M, lacking binding activity for unreacted M, which remains free in the plasma (Kristensen et al., 1990; Strickland et al., 1990). A conformational change in M similar to that produced by reaction with proteases can be produced by treatment with small primary amines such as methylamine and ammonium (Barrett et al., 1979). Like protease-reacted M, methylamine-reacted M is bound by cells (Kaplan and Nielsen, 1979; Kaplan et al., 1981; Debanne et al., 1976). Once internalized, the M-protease and M-methylamine complexes are degraded in secondary lysosomes (Sottrup-Jensen, 1987). Thus, in contrast to other protease inhibitors that bind to the active site of the protease resulting in immediate enzymatic inactivation, the receptor-mediated endocytotic clearance of M-protease complex from the circulation must also be regarded as part of its inactivation mechanism.

The definitive characterization of the essential functions of human M in vivo has proven difficult because mammalian plasma contains a diverse array of active site protease inhibitors that, in the aggregate, are able to bind most exogenous and endogenous proteases (Laskowski and Kato, 1980; Travis and Salvesen, 1983). In contrast, M is the only trypsin inhibitor in Limulus plasma (Quigley and Armstrong, 1983), making the horseshoe crab a much simpler system for its functional characterization. In general, the diversity of the plasma proteins in Limulus is relatively low, and the blood contains but a single cell type, the granular amebocyte, in the general circulation (Armstrong, 1985; 1991). Our aim is to investigate the fate of exogenous proteases introduced into the blood of Limulus and to characterize the molecules involved in protease clearance. In this report, we document a protease clearance pathway and demonstrate that M is the mediator for clearance. Furthermore, we show that the blood cells are capable of binding the M-protease complex.


MATERIALS AND METHODS

All experiments were conducted on adult male Limulus polyphemus (380-460 g) obtained from the Marine Resources Center of the Marine Biological Laboratory in Woods Hole, MA. Animals were used within a few days of their initial capture from the wild and were returned to the ocean unharmed at the completion of the experiment. No animal was used for more than one experiment.

Assay of Protein Clearance

For angiography the animals were restrained upside down on an x-ray table. 10 ml of Angiovist-282 (diatrizoate meglumine, 28% organically bound iodine) (Berlex Laboratories Inc., Wayne, NJ) was injected either into the blood space of one of the leg joints or into the heart at the cephalo-thoracic joint. The injection and distribution of the dye was observed by fluoroscopy and recorded onto video tape.

Labeled proteins were dissolved in endotoxin-free 3% NaCl (Travenol). The total blood volume was estimated as 30% of the total body volume (Armstrong, 1985). Solutions of labeled proteins as 1% of the total blood volume were introduced into a 50-ml syringe fitted with a 16-gauge needle, which was inserted through the cephalo-thoracic joint directly into the lumen of the heart. 30-40 ml of blood was aspirated into the syringe to mix with the sample of labeled protein, and then the entire syringe content was slowly injected back into the heart. At various times following injection, blood samples (2 ml volume) were taken from the blood spaces of the joints of the fourth pair of legs. Cells were removed from the blood samples by centrifugation at 1000 g for 5 min, and the fluorescence of the plasma was measured with a fluorometer (Turner Model 112; filters: emission: 47B, 2A, excitation 2A-12). Blood cell-associated fluorescence was measured after lysis of the pelleted blood cells in 10% sodium dodecyl sulfate and centrifugation (10,000 g, 20 min) to remove cell debris.

Fluoresceination of Proteins

Limulus M was purified as described previously (Armstrong et al., 1991). The trypsin-binding activities of the M preparations were determined by the soybean trypsin inhibitor protection assay (Armstrong et al., 1985). The thiol ester of Limulus M was inactivated by reaction with methylamine as described previously (Quigley and Armstrong, 1985), resulting in a loss of 96% of the trypsin binding activity. Limulus hemocyanin and Limulus M were labeled with fluorescein isothiocyanate (FITC) (Harlow and Lane, 1988). Trypsin (Sigma, catalog no. T-8003) was fluoresceinated with FITC in carbonate/bicarbonate buffer (pH 9.1) containing 20 mM benzamidine followed by dialysis against 1 mM HCl. To purify proteolytically active FITC-labeled trypsin, the pH of the sample was adjusted to 8.0 with Tris buffer, and the sample was applied to a column of benzamidine-Sepharose, which was then washed with Tris-buffered 0.1 M NaCl (pH 8.0) and eluted with 10 mM HCl (Hixson and Nishikawa, 1973). The labeling efficiency for all proteins, as measured by the ratio of optical absorbance (495/280 nm), ranged from 0.357 to 0.507. Biotinylated trypsin was purchased from Sigma (catalog no. T6640). To form the protease-Limulus M complex, a 2-fold excess of trypsin was exposed to Limulus M in 50 mM Tris (pH 8.0) for 3 min at room temperature, and the Limulus M was then separated from the unbound trypsin by gel filtration on a Sephadex G-50 column under lipopolysaccharide (LPS)-reduced conditions.

Association of Trypsin with M

To determine the association of FITC-labeled trypsin with blood proteins, the isolated plasma samples recovered from trypsin-injected animals were treated with 3% polyethylene glycol 8000 followed by centrifugation at 8000 g for 20 min to remove a majority of the hemocyanin, which interfered with molecular filtration. The molecular mass of the FITC-labeled material was estimated by centrifugation through molecular filters with defined size exclusion characteristics and by gel filtration chromatography. Aliquots of the supernatants were subjected to centrifugation for 4 h through Centricon filters (Amicon) with 10 or 100-kDa molecular mass retentions. The fluorescence of the flow-through and retained fractions was measured with the fluorometer. Gel filtration chromatography employed a calibrated 100 1.6-cm column of Sephacryl S-300 HR (Pharmacia Biotech Inc.) resin that was eluted at 1 ml/min. Pore-limit gel electrophoresis was conducted with 4-15% gradient gels (Mini-Protean II ready gels, Bio-Rad catalog no. 161-0902) with a Tris borate buffer system (84 mM Tris, 75 mM boric acid (pH 8.9)) for the upper and bottom chamber at 100 V for 3 h. Immunoblotting was conducted as described by Towbin et al. (1979). Immunoprecipitation was performed using an affinity-purified rabbit anti Limulus M antibody following the method of Harlow and Lane(1988). The antiserum was affinity purified on Limulus M-conjugated Sepharose with elution with 0.1 M glycine (pH 2.5).


RESULTS AND DISCUSSION

Angiogram of Limulus

The circulation of Limulus is very different from that of mammals, with a dorsally situated tubular heart that beats very slowly (30 beats/min at 23 °C). Blood is expelled into the frontal artery, one pair of anterior arteries, and four pairs of lateral arteries (Redmond et al., 1982; Shuster, 1978). From the arterial system, the blood flows into open sinuses. In addition to the heart pump, the gills participate in the venous system as accessory pumps (Freadman and Watson, 1989). The relatively lethargic circulation resulted in slow mixing with the blood of samples injected into the peripheral circulation (the leg joints; data not shown). Instead, good mixing of injected samples was obtained for tracer dye injected directly into the heart, with significant transfer of label to the peripheral circulation within 1 min and dissemination of the injected material by 5 min (Fig. 1, A-D).


Figure 1: Angiogram of adult male Limulus polyphemus. A, fluorography before injection of Angiovist. H, Limulus heart; N, needle introduced into the heart with connecting tubing to syringe containing 10 ml of Angiovist. B, distribution of the contrast medium during injection. The heart and the proximal arteries appear in much higher contrast. C, 1 min after injection of Angiovist-282. Contrast medium has reached the distal arteries but almost none has been transported to the blood sinuses peripheral to the heart. D, 5 min after injection of Angiovist. Contrast medium is still present in the heart and arteries, but it has also been transported from the heart to the vascular sinuses and the gills.



Stability of Endogenous Proteins in the Circulation of Limulus

Hemocyanin, which is the main oxygen transport protein of the blood and, at a concentration of 20-60 mg/ml, is the most abundant protein in the plasma (Volbeda and Hol, 1988; Van Holde and Miller, 1982), was selected for the determination of the behavior of an inert protein. Hemocyanin was collected from Limulus plasma by precipitation with 3% polyethylene glycol 8000 and was labeled with FITC. The protein was separated from the unbound FITC by gel filtration on Sephadex G-50 under LPS-reduced conditions and was then injected into the heart at 6 µg/g of body weight. To obtain a rapid mixing of the injected protein, 30-40 ml of blood was aspirated into the injection syringe to mix with the sample destined for injection, and the mixture was immediately reinjected into the heart. The Limulus blood cells react to mechanical stress and exposure to LPS by degranulation and clotting of the blood (Armstrong, 1979, 1980; Armstrong and Rickles, 1982; Armstrong, 1980). The LPS-reduced conditions ensured that no clotting of the blood cells occurred in the syringe during aspiration and reinjection. After 5 min, 90% mixing of the labeled protein with the peripheral blood was achieved, and a steady-state concentration was established by 10 min (Fig. 2). Over the observation period of 180 min, there was no significant clearance of FITC-labeled hemocyanin from the circulation (Fig. 2).


Figure 2: Distribution of FITC-labeled hemocyanin in the Limulus circulation following intracardiac injection. Fluorescence was measured in cell-free plasma samples collected from the blood spaces of the leg joints over a period of 180 min after injection. Break and scale change at 45 min. , fluorescence of plasma samples from an individual animal; , sample fluorescence (mean ± standard deviation) as fraction of the fluorescence at 10 min postinjection, with subtraction of background fluorescence of the t = 0 sample. Number of animals = 4. The introduced hemocyanin becomes uniformly mixed with the blood by 10 min following injection and then remains at a constant concentration during the observation period.



Clearance of Trypsin from the Plasma of Limulus

FITC-labeled, purified trypsin was introduced into the blood of Limulus as described above at 14.5 µg/g of body weight. The concentration of trypsin in the peripheral blood increased for 5-10 min and was then rapidly cleared from the plasma by 30-35 min after injection with a t for this clearance of 26.5 min. Clearance was followed by the reappearance of fluorescence in the plasma between 45-90 min to 52% of the amount of 10 min (Fig. 3). Clearance requires the proteolytic activity of trypsin. FITC-labeled, purified trypsin was inactivated by three sequential 1 mM additions of the irreversible active site inhibitor phenylmethylsulfonyl fluoride (PMSF) over a period of 90 min at room temperature (remaining activity < 4%) followed by dialysis against 1 mM HCl to remove free inhibitor. In marked contrast to the efficient clearance of active trypsin, inactivated enzyme was not removed from the circulation over the observation period of 180 min (Fig. 3).


Figure 3: Clearance of trypsin from Limulus plasma after intracardiac injection. FITC-trypsin () is removed from the plasma after 35 min (t = 26.5 min, n = 5, mean ± standard deviation). After 45 min, 52% of the fluorescence has reappeared in the plasma. , FITC-trypsin, inactivated by three sequential treatments with 1 mM PMSF. There is no detectable clearance of the inactivated protease over the 180-min observation period (t > 180 min, n = 4, mean ± standard deviation). , fluorescence associated with the blood cells following injection of FITC-trypsin into the Limulus heart. The maximum of the injected fluorescence associated with the blood cells, 51% of the concentration at 10 min, coincides approximately with the time of minimal fluorescence in the plasma (n = 3, mean ± standard deviation).



Clearance of Reacted and Unreacted Limulus-M from the Limulus Plasma

Limulus M was labeled with FITC as described above. In excess of 85% of the FITC-labeled Limulus M was reactive with trypsin, as determined by the soybean trypsin inhibitor protection assay using trypsin whose activity was determined by active site titration with p-nitrophenyl p`-guanidinobenzoate hydrochloride (Chase and Shaw, 1967). Unreacted FITC-labeled Limulus M was injected at 6.6 µg/g of body weight and became uniformly mixed with the blood by 10 min after injection and was not significantly cleared from the plasma over a period of 400 min (Fig. 4; data shown to 200 min after injection). Methylamine (MA)-reacted Limulus M was prepared by incubation with 0.1 M MA for 24 h at 4 °C followed by dialysis against 3% NaCl. FITC-labeled, MA-reacted Limulus M was injected at 6.4 µg/g of body weight and was cleared to 23% of the starting concentration by 24 min after injection. Clearance was followed by a reappearance of FITC in the plasma after 45 min (Fig. 4). FITC-labeled trypsin was reacted with Limulus M at pH 8.0 for 5 min followed by gel filtration to remove unbound trypsin from the complex. The trypsin-Limulus M complex experienced a clearance to 0.6% of the starting amount by 20 min after injection, followed by a reappearance in the plasma of 28% of the FITC at 30-90 min). Thus, the efficient clearance of the trypsin-M complex was similar to the clearance of MA-reacted M and distinct from the persistence in the plasma of native, unreacted M.


Figure 4: Clearance of Limulus -macroglobulin (Limulus-M) from the plasma of Limulus after intracardiac injection. , unreacted FITC-labeled Limulus M. Unreacted Limulus M was not cleared from the plasma during the observation period (t > 200 min, n = 5, mean ± standard deviation). , MA-treated Limulus M. MA-treated Limulus M was cleared from the plasma to 80% of its initial concentration by 25 min after injection (t = 20 min, n = 5, mean ± standard deviation). 47% of the fluorescence reappeared in the plasma by 45 min. , cell-associated fluorescence associated with the injection of unreacted FITC-Limulus M (n = 5, mean ± standard deviation). Very little unreacted Limulus M became associated with the blood cells.



The clearance of trypsin and trypsin-M complex resembles the patterns of clearance of proteases from mammals, where enzymatically active proteases are selectively eliminated. Protease-reacted M is cleared rapidly from the circulation of dogs (Ohlsson, 1971a), rabbits (Debanne et al., 1973), rats (Katayama and Fujita, 1974a 1974b), and mice (Imber and Pizzo, 1981) (t = 5-8 min). Trypsin-reacted Limulus M was injected at 6.3 µg/g of body weight. The clearance half-times for Limulus ranged from 18 min for trypsin-Limulus M complex (Fig. 5) to 26.5 min for trypsin (Fig. 3). Although the times for clearance are longer for Limulus than for mammals, this is largely attributable to the slower transport of the injected samples from the heart to the periphery in Limulus. The actual durations of clearance itself for mammals and for Limulus are similar after the lengthened mixing times in the latter are subtracted. The requirement for active enzyme (Fig. 3) is consistent with an involvement of protease inhibitors in clearance and not with the activity of a pathway that nonselectively clears any and all foreign proteins. For example, because proteolytic cleavage of the bait region is required for entrapment and binding (Hall and Roberts, 1978; Harpel, 1973; Sottrup-Jensen et al., 1989), M interacts only with active proteases and ignores inactive enzyme (Lanchantin et al., 1966; Starkey and Barrett, 1973).


Figure 5: Clearance of trypsin-reacted Limulus M from the Limulus plasma after intracardiac injection. , unreacted FITC-Limulus M persisted in the plasma for the duration of the observation period (for details, see Fig. 4). , FITC-labeled trypsin-reacted Limulus M was eliminated to 99.4% of the injected fluorescence after 20 min (t = 18 min, n = 5, mean ± standard deviation). , cell-associated fluorescence after injection of FITC-labeled trypsin-reacted Limulus M. The maximal peak of 48% of the injected fluorescence coincided approximately with the time of minimal fluorescence in the plasma (n = 5, mean ± standard deviation).



Blood cells from injected animals were solubilized with 10% SDS, and after centrifugation the cell-associated fluorescence of the solution was measured with the fluorometer. Whereas neither inactivated trypsin nor unreacted Limulus M associated with the blood cells at any time after injection (Fig. 4), 51% of the fluorescence of trypsin (Fig. 3) and 48% of the trypsin-Limulus M complex (Fig. 5) could be recovered in the cell-associated fraction at 30 and 20 min postinjection, respectively. The time course of the association of FITC with the blood cells was the inverse of the amount of fluorescence in the plasma ( Fig. 3and Fig. 5).

Association of Trypsin with Limulus M in Vivo

Microfiltration, gel filtration chromatography, and immunoprecipitation were used to characterize the association of the injected trypsin with plasma proteins. Postinjection plasma samples were precipitated with 3% polyethylene glycol 8000 and, after centrifugation to remove the precipitated hemocyanin, the supernatant was analyzed by microfiltration through Centricon filters (Amicon) with various molecular retention capabilities. After a 4-h centrifugation, the fluorescence of the flow-through and of the retained solution were measured with the fluorometer.

The molecular mass of trypsin is 23,800 Da (Desnuelle, 1960). When FITC-trypsin was applied to Centricon filters with 10- and 100-kDa molecular exclusions, less than 5% of the fluorescence appeared in the flow-through of the Centricon 10 kDa, but more than 97% of the fluorescence was found in the flow-through of the Centricon 100 kDa. The trypsin-associated fluorescence of plasma from preclearance stages (10 min after injection) was associated with a molecular complex larger than 100 kDa, because 84 and 82%, respectively, of the fluorescence was retained by filters with 10- and 100-kDa molecular exclusion limits (). However, the fluorescence that reappears in the plasma after clearance (40-45 min) is associated with small molecules since 82% of the fluorescence of plasma collected 40 min after injection and 89% of 45 min samples passed through the 10-kDa filter (). This indicates a degradation of the labeled trypsin following its clearance from the plasma. When samples of plasma recovered 10 min after injection of FITC-trypsin were applied to a calibrated 100 1.6-cm Sephacryl S-300 HR column, over 80% of the fluorescence was recovered in a high molecular mass complex that eluted at the same position on the column as purified Limulus M, indicating that trypsin binds to a molecule of the Limulus-plasma with a molecular mass identical to that of M (Fig. 6).


Figure 6: Gel filtration on a calibrated Sephacryl S-300 HR 100 1.6-cm column of a hemocyanin-depleted plasma sample 10 min after intracardiac injection of FITC-trypsin. , fluorescence of the sample; , optical absorbance at 280 nm of the sample. 80% of the fluorescence was associated with a high molecular mass complex that eluted at the same position as purified Limulus M.



Pore-limit polyacrylamide gel electrophoresis with Western blotting and immunoprecipitation further implicated M as the plasma protein that binds the injected trypsin prior to clearance. Biotinylated trypsin (active or inactivated by three sequential incubations with 1 mM PMSF) was injected into the animal, and plasma samples were taken 10 min after injection, a time of complete mixing but of minimal clearance. The samples were subjected to pore limit gel electrophoresis followed by electrophoretic transfer to nitrocellulose. One half of the nitrocellulose sheet was stained with affinity-purified rabbit anti-Limulus M and horseradish peroxidase-labeled anti-rabbit-IgG to localize M, and the other half was stained with horseradish peroxidase-labeled avidin to localize trypsin. The biotinylated trypsin (Fig. 7, lanes8 and 10) occupied the same position on the gel as did Limulus M (Fig. 7, lanes1-5). Lanes containing plasma of animals injected with PMSF-inactivated biotinylated trypsin did not contain trypsin (Fig. 7, lane9). Trypsin is a basic protein (pI = 10.5) (Desnuelle, 1960), which would be expected to migrate toward the cathode under the conditions of pore limit electrophoreses (electrophoresis buffering at pH 8.9) unless bound to an acidic protein. The migration into the pore limit gel only of enzymatically active trypsin and its localization at a position coincident with Limulus M is consistent with the suggestion that active trypsin introduced into the plasma becomes bound to M. The association of both active and PMSF-inactivated biotinylated trypsin with M in 10-min postinjection plasma samples was probed further by immunoprecipitation of M with an affinity-purified anti-Limulus M antiserum. Biotinylated enzymatically active trypsin co-immunoprecipitated with Limulus M (Fig. 8, lane8), whereas biotinylated PMSF-inactivated trypsin did not (Fig. 8, lane9). Taken together, these observations indicate that a majority of the injected active trypsin becomes associated with a molecule with the properties of Limulus M. Furthermore, it appears that the trypsin-M complex is degraded to small peptides following clearance. The blood cells appear to contribute to the clearance of the trypsin-M complex, since 50% of the cleared fluorescence of trypsin or M associates with the blood cells ( Fig. 3and Fig. 5 ).


Figure 7: Western blot of pore limit gel electropherogram of plasma sample of animal injected 10 min previously with biotinylated trypsin (pH 8.9 on a 4-15% polyacrylamide gradient gel). Lanes1-5, blot probed with rabbit anti-Limulus M antibody; lanes7-10, blot probed with horseradish peroxidase-avidin. Lane1, methylamine-reacted Limulus M; lane2, Limulus M reacted with excess biotinylated trypsin; lane3, hemocyanin-depleted plasma from an animal injected 10 min previously with biotinylated trypsin; lane4, hemocyanin-depleted plasma sample from an animal injected 10 min previously with PMSF-inactivated biotinylated trypsin; lane5, unreacted Limulus M; lane6, blank lane where the nitrocellulose blot was cut in two; lane7, biotinylated trypsin; lane8, hemocyanin-depleted plasma from an animal injected 10 min previously with biotinylated trypsin; lane9, hemocyanin-depleted plasma sample from an animal injected 10 min previously with PMSF-inactivated biotinylated trypsin; lane10, Limulus M reacted with biotinylated trypsin. Biotinylated trypsin is associated with a molecule that electrophoreses at the same position as Limulus M only in lanes8 and 10.




Figure 8: Immunoprecipitation with affinity-purified anti-Limulus M, followed by SDS-polyacrylamide gel electrophoresis and Western blotting, probed with horseradish peroxidase-avidin. Lanes1-5, samples were immunoprecipitated with preimmune serum; lanes7-9, samples were immunoprecipitated with rabbit anti-Limulus M antibody that had been affinity purified on Limulus M-conjugated Sepharose. Lane1, sample was methylamine-reacted Limulus M; lane2, biotinylated trypsin-reacted Limulus M; lane3, plasma collected 10 min after intracardiac injection of biotinylated trypsin; lane4, plasma collected 10 min after intracardiac injection of PMSF-inactivated biotinylated trypsin; lane5, unreacted Limulus M; lane6, biotinylated trypsin loaded directly onto the gel without immunoprecipitation; lane7, biotinylated trypsin-reacted Limulus M; lane8, plasma sample collected 10 min after intracardiac injection of biotinylated trypsin; lane9, plasma sample collected 10 min after intracardiac injection of PMSF-inactivated biotinylated trypsin. Biotinylated trypsin is present only in lanes6, 7, and 8. Unlike mammalian M, Limulus M fails to establish N(-glutamyl)-lysine bonds between the thiol ester glutamyl residue of M and lysine residues of the reacting protease (Quigley et al., 1991), so biotinylated trypsin bound to Limulus M migrates at the position of unreacted trypsin in the presence of SDS.



The mammalian receptor for the M-protease complex has been shown to be identical to the low density lipoprotein-related protein and belongs to the family of low density lipoprotein receptors (Zheng et al., 1994; Strickland et al., 1990). Low density lipoprotein receptor-related protein binds a variety of ligands in addition to the M-protease complex, including plasminogen activator-plasminogen activator inhibitor-1 complex (Kounnas et al., 1993), apoE-enriched very low density lipoprotein (Rebeck et al., 1993), lipoprotein lipase (Williams et al., 1994; Chappell et al., 1992), Pseudomonas exotoxin A (Kounnas et al., 1992), and the 40-kDa receptor-associated protein (Herz et al., 1991). We have not yet identified a specific cell surface receptor for Limulus M-protease complex from the blood cells, but detergent extracts of Limulus blood cells contain a protein that specifically binds mammalian receptor-associated protein and that may be a molecular homologue of low density lipoprotein receptor-related protein of mammals.()

It will be interesting to determine the processing pathway of M-protease complex in Limulus. After binding to its receptor, mammalian M is internalized via coated pits and sequestered in secondary lysosomes (Goldstein et al., 1979; Sottrup-Jensen, 1987), with the M-protease complex subsequently being degraded to low molecular weight products (Ohlsson, 1971b; Katayama and Fujita, 1974a, 1974b). The mechanism of endocytosis and the fate of the endocytosed protein seems to be similar to that of low density lipoprotein (Pastan et al., 1977; Brown and Goldstein, 1979; Goldstein et al., 1979) and of peptide hormones and other proteins that bind to specific cell surface receptors (Pastan and Willingham, 1981; Steinman et al., 1983; Besterman and Low, 1983). In Limulus, 40-50% of the fluorescence reappeared in the plasma after clearance from the plasma. The soluble postclearance fluorescence was associated with molecules smaller than 10 kDa, based on the free passage of fluorescence through molecular filters with a 10 kDa cutoff. This is consistent with the possibility of proteolytic degradation of the endocytosed complex in secondary lysosomes and with the suggestion that the entire pathway of inactivation of proteases by M has been conserved during the evolution of lineages as diverse as the vertebrates and the arthropods.

  
Table: Estimation of sizes of molecular complexes formed by FITC-trypsin with molecules of the plasma at different stages of the clearance cycle



FOOTNOTES

*
This work was supported by Grant MCB-9218460 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, University of California, Davis, CA 95616-8755. Tel.: 916-752-1565; Fax: 916-752-1449; E-mail: pbarmstrong@ucdavis.edu.

The abbreviations used are: M, -macroglobulin; FITC, fluorescein isothiocyanate; LPS, lipopolysaccharide; MA, methylamine; PMSF, phenylmethylsulfonyl fluoride.

D. K. Strickland, personal communication.


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