©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Microglobulin Destroys the Proteinase Inhibitory Activity of -Inhibitor-3 by Complex Formation (*)

(Received for publication, November 29, 1994)

Cecilia Falkenberg (§) Maria Allhorn Ida B. Thøgersen (1) Zuzana Valnickova (1) Salvatore V. Pizzo (1) Guy Salvesen (1) Bo Åkerström Jan J. Enghild (1)

From the Department of Medical and Physiological Chemistry, University of Lund, P. O. Box 94, S-221 00 Lund, Sweden and the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The immunoregulatory plasma protein alpha(1)-microglobulin (alpha(1)-m) and the proteinase inhibitor alpha(1)-inhibitor-3 (alpha(1)I(3)) form a complex in rat plasma. In the present work, it was demonstrated that the alpha(1)I(3)bulletalpha(1)-m complex has no inhibitory activity, the bait region was not cleaved by low amounts of proteinases, and it was unable to covalently incorporate proteinases. The results also indicated that the thiolester bond of the alpha(1)I(3)bulletalpha(1)-m complex was broken. The alpha(1)I(3)bulletalpha(1)-m complex was cleared from the circulation much faster than native alpha(1)I(3), with a half-life of approximately 7 min. Structurally, however, the alpha(1)I(3)bulletalpha(1)-m complex was similar to native alpha(1)I(3) rather than alpha(1)I(3) cleaved by proteinases. It is speculated that the role of alpha(1)-m is to destroy the function of alpha(1)I(3) by blocking the bait region and breaking the thiolester and causing its physical elimination by rapid clearing from the blood circulation. It is also possible that the formation of complexes between alpha(1)-m and alpha(1)I(3) may serve as a mean to regulate the function of alpha(1)-m since its complex with alpha(1)I(3) is taken up rapidly by cellular receptors for alpha-macroglobulins.


INTRODUCTION

alpha(1)-microglobulin (alpha(1)-m), (^1)also known as protein HC, is a low molecular weight plasma glycoprotein with various immunoregulatory properties (reviewed in Åkerström and Lögdberg, 1990 and Åkerström, 1992). alpha(1)-m is translated in the liver together with bikunin (Kaumeyer et al., 1986; Salier, 1990) as a precursor protein, which is cleaved late in the trans-Golgi network previous to secretion of free alpha(1)-m (Bratt et al., 1993). Part of the secreted alpha(1)-m is linked covalently to other plasma proteins, and circulating complexes with human IgA (Grubb et al., 1986), rat alpha(1)-inhibitor-3 (alpha(1)I(3)) (Falkenberg et al., 1990), and rat fibronectin (Falkenberg et al., 1994) have been isolated and characterized.

alpha(1)I(3)bulletalpha(1)-m is a 220-kDa 1:1 covalent complex circulating at a concentration of approximately 40 µg/ml (Falkenberg et al., 1994). Both alpha(1)-m and alpha(1)I(3) are produced by rat hepatocytes, but the site of formation of the complex is not known (Pierzchalski et al., 1992). alpha(1)I(3) is a 180-kDa proteinase inhibitor belonging to the same superfamily as human alpha(2)-macroglobulin (alpha(2)M), the alpha-macroglobulins (Sottrup-Jensen, 1987). Its binding and inhibition of proteinases is dependent on the ability to form covalent cross-links to the proteinases (Enghild et al., 1989a). During the reaction, alpha(1)I(3) is cleaved in the bait region by a variety of proteinases, leading to a change in conformation and a subsequent activation of an internal thiolester bond. A highly reactive glutamyl residue of the thiolester forms a covalent cross-link with a lysyl residue in the attacking proteinase. The thiolester, which is a common property of many of the alpha-macroglobulins, can also be broken by a small nucleophilic reagent such as methylamine (Barrett and Starkey, 1973). The conformational shift of alpha(1)I(3) leads to exposure of a domain with high affinity for receptors on hepatocytes and macrophages, resulting in a rapid clearance of the alpha(1)I(3)-proteinase complex, a property shared by many alpha-macroglobulins (Debanne et al., 1976; Van Leuven et al., 1979; Gliemann and Sottrup-Jensen, 1987).

The complex formation between alpha(1)-m and alpha(1)I(3) raised the question whether alpha(1)-m could influence the proteinase inhibitory activity or receptor recognition of alpha(1)I(3). In this work, the complex was isolated and compared with the native proteinase inhibitor, alpha(1)I(3).


EXPERIMENTAL PROCEDURES

Materials

Rat plasma was drawn from Sprague-Dawley rats (B& Universal AB, Sweden) or obtained from Pel-Freez Biologicals. Female CD-1 mice (20 weeks) were bought from Charles River Laboratories (Raleigh, NC). Monoclonal mouse anti alpha(1)-m BN 11.3 and BN 11.10, both binding to rat and human alpha(1)-m, were prepared as described (Babiker-Mohamed et al., 1991). The IgG fractions of rabbit antisera against rat alpha(1)I(3) and rat alpha(1)-m were prepared as described (Nilson et al., 1986). Hide powder azure, tosylphenylalanyl chloromethyl ketone-treated bovine trypsin, porcine pancreatic elastase (PPE), bovine chymotrypsin, papain, the serine proteinase inhibitor 3,4-dichloroisocoumarin (DCI), the cysteine proteinase inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), dithiothreitol (DTT), and methylamine hydrochloride were all from Sigma. Staphylococcus aureus V8 proteinase was purchased from Boehringer Mannheim. Cathepsin G was a kind gift from Dr. James Travis (University of Georgia). [^14C]Methylamine hydrochloride (59.4 mCi/mmol) was from DuPont NEN. Ultrapure urea was obtained from Life Technologies, Inc.

Protein Purification

Rat alpha(1)I(3) and human alpha(2)M were purified from plasma as described earlier (Enghild et al., 1989a; Salvesen and Enghild, 1993). alpha(1)I(3)bulletalpha(1)-m was purified by immunosorbent chromatography using monoclonal mouse antibodies BN 11.3 and BN 11.10 immobilized to Affi-gel Hz (60 mg/3 ml gel) according to instructions provided by the manufacturer (Bio-Rad). Plasma from Sprague-Dawley rats was centrifuged for 10 min at 10,000 times g, EDTA was added to a final concentration of 10 mM, and the plasma filtered through a 0.22-µm membrane. Before applying the sample to the affinity column, plasma was diluted twice with phosphate-buffered saline (10 mM phosphate buffer, pH 7.4, 0.12 M NaCl, 3 mM KCl including 10 mM EDTA). The column was rinsed and then eluted with 4 M MgCl(2). The eluate was dialyzed against 50 mM Tris, 50 mM NaCl, pH 7.4, followed by concentration. The final purification step was gel filtration on Sephacryl S-300 (Pharmacia LKB Biotechnology AB, Sweden) in 50 mM Tris, 150 mM NaCl, pH 7.4, after which the alpha(1)I(3)bulletalpha(1)-m containing fractions were concentrated by ultrafiltration.

Blue Hide Powder Assay

Increasing amounts of alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m (0-0.14 nmol) were allowed to inhibit 0.04 nmol of active site-standardized trypsin (Chase and Shaw, 1967) from digesting the high molecular weight substrate hide powder azure. The assay was done as described earlier (Enghild et al., 1989a). Briefly, trypsin and alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA were preincubated for 30 min at room temperature, and 0.4 ml of blue hide powder suspension (12.5 mg/ml in 0.6 M sucrose and 0.05% Triton X-100) were added. The reaction was incubated for another 30-60 min with agitation before terminating by adding 0.3 ml of glycine-HCl, pH 3.0. The undigested blue hide powder was pelleted by centrifugation, and absorbance was measured at 595 nm as a determination of proteolytic activity.

Protein Radiolabeling

Proteins were labeled with NaI (DuPont NEN) by the solid-state lactoperoxidase method of David and Reisfeld(1974).

Incubation with Radiolabeled Proteinases

I-Labeled papain was activated with 1 mM DTT in the presence of 1 mM EDTA for 15 min at 37 °C. 2 µg of alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m in 50 mM Tris-HCl, 100 mM NaCl, pH 7.4, were mixed with I-labeled trypsin (50 pmol), PPE (8 pmol), S. aureus V8 proteinase (3 pmol), chymotrypsin (33 pmol), cathepsin G (35 pmol), or papain (3 pmol) and incubated for 5 min. The reactions were stopped, with the exception of the papain reaction, by the addition of DCI to a final of concentration of 250 µM. The papain reaction was terminated with E-64 at a final concentration of 50 µM. The reaction products were separated by SDS-PAGE and examined by autoradiography.

Incubation of Plasma with Radiolabeled PPE

5 ml of rat plasma (Pel-Freez Biologicals) was incubated with I-labeled PPE (170 KBq/ml) for 20 min at room temperature. The reaction was stopped by adding DCI to a final concentration of 250 µM. The pH was corrected to 8.2 with 1 M Tris, and methylamine hydrochloride was added to 100 mM. After incubation for 2 h at room temperature, EDTA was added to a concentration of 10 mM; half of the plasma was applied to anti-alpha(1)-m, and the remainder was applied to anti-alpha-(1)I(3)-Sepharose. The columns were washed with phosphate-buffered saline, 10 mM EDTA and eluted with 0.1 M glycine-HCl, pH 2.5. Acidic fractions were neutralized by the addition of 1 M Tris-HCl, pH 8.0, analyzed for absorbance at 280 nm, and counted for radioactivity. Protein-containing fractions were dried in a Speed Vac concentrator, redissolved in 100 µl of deionized water, and precipitated overnight by adding 900 µl of EtOH.

Bait Region Cleavage

alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m (23 pmol) in 50 mM Tris-HCl, 100 mM NaCl, pH 7.4, were reacted with 2.3 pmol of PPE or papain for 5 min. Papain was first activated for 15 min with 1 mM DTT in the presence of 1 mM EDTA. The PPE reaction was stopped by adding DCI to 250 µM, and the papain reaction was stopped by adding E-64 to 50 µM. After another 5 min, the samples were made 2% in SDS and 1% in 2-mercaptoethanol before separating by SDS-PAGE (8.5%).

Incubation with Methylamine

alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m (10 µg in 20 µl) were treated with 0.05 µCi [^14C]methylamine hydrochloride for 2 h at room temperature after adjusting the pH to 8.2 with 1 M Tris. Unlabeled methylamine was added to 100 mM, and reactions were incubated overnight at room temperature, separated by SDS-PAGE, and analyzed by autoradiography. Alternatively, 5 ml of plasma (Pel-Freez) was adjusted to pH 8.2 with 1 M Tris, and 50 µCi [^14C]methylamine hydrochloride was added. After incubation for 2 h, unlabeled methylamine hydrochloride was added to 100 mM, and the mixture was incubated for another 2 h. Finally, EDTA was added, and half of the plasma was applied to anti-alpha(1)-m and the other half to anti-alpha(1)I(3)-Sepharose. For further details, see above.

Polyacrylamide Gel Electrophoresis

SDS-PAGE was performed in 5-15% linear gradient gels using the glycine/2-amino-2-methyl-1,3-propanediol/HCl system described by Bury (1981) or in 8.5% slab gels using the buffer system reported by Laemmli(1970). Samples that were separated under reducing conditions were made 50 mM in DTT or 1% in 2-mercaptoethanol. Molecular mass standards were rat alpha(1)I(3) (180 kDa), Bio-Rad low molecular mass standards, or high molecular mass Rainbow markers from Amersham International. Non-radioactive proteins were visualized by staining with Coomassie Brilliant Blue, and radioactive proteins were visualized by autoradiography. When analyzing [^14C]methylamine hydrochloride-conjugated proteins, gels were washed twice for 20 min each in deionized water after the destaining, equilibrated in 1 M sodium salicylate, dried, and fluorographed.

Transverse Urea Gradient Polyacrylamide Gel Electrophoresis

Transverse urea gradient polyacrylamide gel electrophoresis was performed by casting 5% polyacrylamide gels with a continuous 0-8 M urea gradient (Goldenberg 1989; Mast et al., 1991). The same buffer system, except SDS, was used as described for SDS-PAGE. alpha(1)I(3) (150 µg) or I-labeled alpha(1)I(3)bulletalpha(1)-m (20 KBq) in 25 µl was incubated with PPE at a molar ratio of 3:1 for 2 min. The reaction was terminated by the addition of DCI. Samples were diluted to 500 µl with phosphate-buffered saline and appropriate sample buffer, and radiolabeled samples were filtered through Microfilterfuge tubes (Rainin Instrument Co., Inc.), which were saturated with 0.1% bovine serum albumin. The gel was rotated 90 °, and alpha(1)I(3), I-labeled alpha(1)I(3)bulletalpha(1)-m, or PPE-treated alpha(1)I(3) orI-labeled alpha(1)I(3)bulletalpha(1)-m were loaded evenly across the top of the gel. The polyacrylamide gels were run for 2 h with a constant current of 13 mA. Proteins were visualized by staining with Coomassie Brilliant Blue or by autoradiography.

Plasma Clearance Studies

I-Labeled alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m (30 KBq) were injected into the lateral tail vein of CD-1 mice prior to and after a 5-min incubation with trypsin at a molar excess. I-labeled alpha(1)I(3)bulletalpha(1)-m was also injected together with a 1000-fold molar excess of methylamine-treated human alpha(2)M. Blood samples of 25 µl were collected at timed intervals via retroorbital puncture, and the radioactivity was determined. The radioactivity of the initial blood sample, taken 5-10 s after injection, was defined as representing 100% of the injected sample in circulation (Imber and Pizzo, 1981).

Electron Microscopy

The proteins were visualized by electron microscopy using negative staining or rotary shadowing. For negative staining, a drop of protein solution at 30 µg/ml was applied to carbon film made hydrophobic by glow discharge. The film was washed with several drops of uranyl acetate and drained. To prepare the proteins for electron microscopy, samples were sedimented through a 15-40% glycerol gradient (Fowler and Erickson, 1979). The proteins were observed in a Philips EM 301 at a magnification of 150,000.


RESULTS

Purification of alpha(1)I(3)bulletalpha(1)-m Complex

The purpose of this purification was to isolate alpha(1)I(3)bulletalpha(1)-m (Falkenberg et al., 1990) in a native form. alpha(1)-m-containing molecules were isolated from rat plasma by anti-alpha(1)-m affinity chromatography, using 4 M MgCl(2) for elution from the column. 0.1 M glycine, pH 2.0, which was used earlier, completely destroyed the proteinase inhibition activity of alpha(1)I(3) in blue hide powder assay (not shown). The alpha(1)I(3)bulletalpha(1)-m complex was separated from the two other forms of alpha(1)-m, alpha(1)-m/fibronectin (Falkenberg et al., 1994) and low molecular weight alpha(1)-m, by gel filtration on Sephacryl S-300 under native conditions. Gradient SDS-PAGE of the concentrated alpha(1)I(3)bulletalpha(1)-m, pooled narrowly, showed that the complex was pure and had a somewhat higher molecular mass compared with free alpha(1)I(3) (see Fig. 3A, lanes2 and 4). After separation by non-gradient SDS-PAGE (Fig. 2) the alpha(1)I(3)bulletalpha(1)-m complex was seen as a triplet band as previously described (Falkenberg et.al., 1990, 1994). N-terminal amino acid sequence analysis showed that all three bands contained alpha(1)I(3) and alpha(1)-m at a 1:1 molar ratio.


Figure 3: Incorporation of [^14C]methylamine into alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m. [^14C]methylamine was allowed to react with 10 µg of alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m for 2 h at pH 8.2, followed by incubation with unlabeled methylamine for 2 h. SDS-PAGE was performed on a 5-15% gradient gel of reduced alpha(1)I(3) (lane1) and alpha(1)I(3)bulletalpha(1)-m (lane2), and non-reduced alpha(1)I(3) (lane3) and alpha(1)I(3)bulletalpha(1)-m (lane4). Proteins are shown stained (A) and autoradiographed (B).




Figure 2: Bait region cleavage of alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m. alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m were incubated with PPE or papain at a molar ratio of 10:1 for 5 min. PPE incubations were terminated by adding DCI to a 250-µM final concentration and papain by adding E-64 to a 50-µM final concentration. Samples were run reduced in SDS-PAGE as follows: lane1, alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m; lane2, alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m + PPE; lane3, alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m + papain.



No Interaction between alpha(1)I(3)bulletalpha(1)-m and Proteinases

equal amounts of alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m (0.01 nmol) were incubated with six different I-labeled proteinases: papain, cathepsin G, chymotrypsin, S. aureus V8 proteinase, PPE, and trypsin, enzymes that are known to be inhibited by alpha(1)I(3). Depending on the rate of labeling, the radioactivity used in each case corresponded to absolute protein amounts from 3 to 50 pmol. The reactions were separated by SDS-PAGE under non-reducing conditions (Fig. 1). The autoradiogram shows that alpha(1)I(3) incorporates all six proteinases. However, no radioactivity was seen to be associated with the alpha(1)I(3)bulletalpha(1)-m band. This shows that alpha(1)I(3)bulletalpha(1)-m does not undergo covalent cross-linking with the proteinases.


Figure 1: Incorporation of I-labeled proteinases into alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m. 2 µg of alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m were incubated with different amounts (3-50 pmol) but with the same amount of radioactivity of I-labeled proteinases. After 5 min, the reactions were terminated by the addition of proteinase inhibitors. Non-reduced samples were separated by SDS-PAGE on a 5-15% gradient gel. Gels were dried and autoradiographed. Lane 1 of both gels represents incubation with 3 pmol of papain; lane 2, 35 pmol of cathepsin G; lane 3, 33 pmol of chymotrypsin; lane 4, 3 pmol of S. aureus V8 proteinase; lane 5, 8 pmol of PPE; lane 6, 50 pmol of trypsin. The position of uncleaved alpha(1)I(3)bulletalpha(1)-m as determined by staining of the gel is marked. Molecular mass markers are shown in kilodaltons.



To exclude the possibility that the purification procedure of the alpha(1)I(3)bulletalpha(1)-m complex caused the loss of the capability of the molecule to interact with various proteinases, I-labeled PPE was added directly to rat plasma. After stopping the reaction by adding the low molecular weight inhibitor DCI, half of the plasma volume was applied to anti-alpha(1)-m-Sepharose to isolate a pool including the alpha(1)I(3)bulletalpha(1)-m complex, and the other half was added to anti-alpha(1)I(3)-Sepharose to isolate alpha(1)I Equal amounts from each column were then separated by SDS-PAGE under non-reducing circumstances (not shown). Radioactive PPE had been incorporated to alpha(1)I(3) but not to alpha(1)I(3)bulletalpha(1)-m. This agrees well with the former experiment.

The ability of alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m to inhibit trypsin was also compared by using the high molecular weight substrate blue hide powder azure. As expected, alpha(1)I(3) inhibits the trypsinmediated degradation of the substrate, but alpha(1)I(3)bulletalpha(1)-m could not inhibit trypsin even at high concentrations (not shown).

alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m were incubated with two proteinases of different specificities, PPE and papain, in a molar ratio of 10:1. The reaction products were separated by reduced SDS-PAGE (Fig. 2). The cleavage of alpha(1)I(3) resulted in doublet bands migrating around 100 kDa, which previously have been shown to result from alpha(1)I(3) bait region cleavage (Enghild et. al., 1989a). alpha(1)I(3)bulletalpha(1)-m, on the other hand, was not cleaved by the proteinases using these conditions, as judged by SDS-PAGE (Fig. 2) and N-terminal amino acid sequence analysis (not shown), suggesting that the bait region was not accessible on the complex.

No Incorporation of [^14C]Methylamine into the alpha(1)I(3)bulletalpha(1)-m Thiolester

The nucleophile [^14C]methylamine was incubated with alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m, and the reactants were separated by SDS-PAGE under reducing or non-reducing conditions (Fig. 3). The results demonstrate that alpha(1)I(3) reacts with the radioactive methylamine, whereas no radioactive methylamine was associated with the alpha(1)I(3)bulletalpha(1)-m complex.

To exclude the possibility that the thiolester reactivity of alpha(1)I(3)bulletalpha(1)-m had been destroyed as a result of purification, [^14C]methylamine was also incubated with rat plasma. The plasma was then applied to either anti-alpha(1)-m or anti-alpha(1)I(3)-Sepharose. Equal amounts of proteins eluted from the two columns were then separated by non-reducing SDS-PAGE. Only very faint radioactivity could be seen in the alpha(1)I(3)bulletalpha(1)-m complex as compared with alpha(1)I(3) after elution from the anti-alpha(1)-m column (not shown). These experiments thus suggest that the thiolester of alpha(1)I(3) is intact and reactive with methylamine, whereas the thiolester of alpha(1)I(3)bulletalpha(1)-m is broken.

Rapid Clearance of alpha(1)I(3)bulletalpha(1)-m from Circulation

It has been shown that alpha-macroglobulins that are cleaved by proteinases undergo a conformational shift, leading to the exposure of receptor-binding domains and a rapid clearing from circulation (Gauthier et al., 1979). Consequently, it would be of interest to measure the clearance rate of alpha(1)I(3)bulletalpha(1)-m and alpha(1)I(3) before and after trypsin cleavage. As expected, proteinase-complexed alpha(1)I(3) was removed from the circulation quicker than the native form (Fig. 4A). Surprisingly, native alpha(1)I(3)bulletalpha(1)-m was cleared with a half-life of about 7 min, which is almost as quickly as native alpha(1)I(3) (Fig. 4B). The clearance of alpha(1)I(3)bulletalpha(1)-m was completely blocked by an excess of methylamine-treated alpha(2)M. This shows that native alpha(1)I(3)bulletalpha(1)-m is recognized by alpha-macroglobulin receptors and cleared from the circulation faster than native alpha(1)I(3). Furthermore, no increase in clearance rate was seen after incubation of the complex with trypsin, indicating that no significant structural difference exists between trypsin-treated and native alpha(1)I(3)bulletalpha(1)-m, which is completely in line with the inability of the alpha(1)I(3)bulletalpha(1)-m complex to interact with trypsin (see above).


Figure 4: Clearance of I-labeled alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m from mouse blood circulation. Native alpha(1)I(3), trypsin-treated alpha(1)I(3) (A), or native alpha(1)I(3)bulletalpha(1)-m, trypsin-treated alpha(1)I(3)bulletalpha(1)-m, and native alpha(1)I(3)bulletalpha(1)-m in competition with methylamine-treated alpha(2)M (B) were injected into the lateral vein of a mouse. The clearance of the radioactive proteins was determined by removing 25-µl aliquots of blood and counting them in a -counter.



Conformation of alpha(1)I(3)bulletalpha(1)-m

The conformational stability of the alpha(1)I(3)bulletalpha(1)-m complex was also investigated by transverse urea gradient-PAGE. alpha(1)I(3) or alpha(1)I(3)bulletalpha(1)-m, prior to and after proteinase treatment, were applied to the transverse urea gradient gels. Native alpha(1)I(3) migrated more slowly in the presence of increasing concentrations of urea (Fig. 5A). After treatment with the proteinase PPE, the migration of alpha(1)I(3) was independent of the urea concentration (Fig. 5B). This suggests that the conformation of native alpha(1)I(3) is less stable than proteinase-treated alpha(1)I(3), a characteristic trait of proteinase inhibitors of the alpha-macroglobulin family (Barrett et al., 1979). On the other hand, the migration of both native and proteinase-treated alpha(1)I(3)bulletalpha(1)-m was urea concentration dependent (Fig. 5A). This suggests that the conformations of native alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m are similar and that the conformation of alpha(1)I(3)bulletalpha(1)-m is not affected by proteinase treatment, most likely due to an inability of the enzyme to cleave the complex, as suggested above.


Figure 5: Transverse urea gradient-polyacrylamide gel electrophoresis of alpha(1)I(3) and alpha(1)I(3)bulletalpha(1)-m. A single sample was loaded along the top of each polyacrylamide (5%) gel and allowed to migrate toward the bottom, as indicated by the arrow. The gel was cast with a continuous urea gradient (0-8 M urea) running from left to right. alpha(1)I(3), I-labeled alpha(1)I(3)bulletalpha(1)-m, and I-labeled alpha(1)I(3)bulletalpha(1)-m incubated with PPE migrated identically, and only one of these samples, alpha(1)I(3), is shown in A. The migration of alpha(1)I(3) incubated with PPE is shown in B. Further details are described under ``Experimental Procedures.''



Finally, no major difference between alpha(1)I(3) and the alpha(1)I(3)bulletalpha(1)-m complex could be seen by electron microscopy. Both appeared as rounded molecules containing granular structures, suggesting the presence of intramolecular domains in both alpha(1)I(3) and the alpha(1)I(3)bulletalpha(1)-m complex.


DISCUSSION

In this work, the alpha(1)I(3)bulletalpha(1)-m complex was purified from rat plasma, and its proteinase inhibitory activities and some structural properties were compared with those of free alpha(1)I(3), the rat alpha(2)-macroglobulin homologue. The results demonstrate that the proteinase inhibitory activity of alpha(1)I(3)bulletalpha(1)-m is lost and its thiolester bond broken, and it was cleared from the circulation almost as quickly as proteinase-cleaved alpha(1)I(3). However, the structure of the alpha(1)I(3)bulletalpha(1)-m complex, both before and after proteinase treatment, is similar to native alpha(1)I(3) rather than proteinase-cleaved alpha(1)I(3).

The alpha(1)I(3)bulletalpha(1)-m complex lacked the proteinase inhibitory activity associated with alpha(1)I(3). Two observations can explain this finding. First, proteinases were unable to cleave the peptide backbone at the bait region of alpha(1)I(3)bulletalpha(1)-m (Fig. 2). As a result, there was no conformational shift of the alpha(1)I(3)bulletalpha(1)-m molecule (Fig. 5), which has previously been demonstrated to be the result of proteinase treatment of alpha(1)I(3) (Enghild et al., 1989a). It can be speculated that alpha(1)-m is bound closely to the bait region of the alpha(1)I(3) peptide, sterically blocking the attack of the proteinase. Second, methylamine was not incorporated into alpha(1)I(3)bulletalpha(1)-m as it was into alpha(1)I(3) (Fig. 3). This indicates that the thiolester bond of alpha(1)I(3)bulletalpha(1)-m is cleaved, making the complex unable to bind proteinases covalently via the glutamyl residue of the thiolester (see also Fig. 1). As a result, there can be no covalent binding of the attacking proteinase by alpha(1)I(3)bulletalpha(1)-m, which has been shown to be prerequisite for the efficient proteinase inhibition of alpha(1)I(3) (Enghild et al., 1989a).

The thiolester of native alpha-macroglobulins is believed to be hidden in a pocket that is too small for large molecules to enter (Barrett et al., 1979). The conformational shift induced by proteinase cleavage of the bait region leads to exposure of the thiolester, followed by a nucleophilic attack on the thiolester by a lysyl -amino group on the nearby located proteinase, and a subsequent Glu-Lys cross-linking of alpha(1)I(3) and the proteinase. However, the thiolester of the alpha(1)I(3)bulletalpha(1)-m complex is apparently cleaved, but since the conformation of the alpha(1)I(3)bulletalpha(1)-m complex is similar to native alpha(1)I(3), it is not likely that the thiolester has reacted with a proteinase. Instead, the thiolester could have reacted with a small nucleophilic molecule, such as methylamine, or with a structure on the alpha(1)-m molecule that somehow can reach the thiolester bond without disrupting the conformation of the native alpha(1)I(3). Consequently, it is possible that alpha(1)-m is cross-linked to alpha(1)I(3) via the glutamyl or thiol group involved in the thiolester bond. alpha(1)-m is not dissociated from the alpha(1)I(3)bulletalpha(1)-m complex by extensive boiling in the presence of reducing agents. (^2)This suggests that if the two proteins are cross-linked by a disulfide bond, it is most likely of an unusual type as in the IgAbulletalpha(1)-m complex in human plasma, which is also reduction resistant (Calero et al., 1994). The nature of the bond between alpha(1)-m and alpha(1)I(3) is the subject of present investigations at our laboratories.

Cleavage of alpha(1)I(3) by proteinases ultimately leads to an exposure of receptor-binding domains, and a rapid clearance of the proteinase-alpha(1)I(3) complex from the circulation (Enghild et al., 1989a). In agreement with this, the half-life of trypsin-treated alpha(1)I(3) was approximately 2 min (Fig. 4). Surprisingly, the alpha(1)I(3)bulletalpha(1)-m complex was also rapidly cleared, with a half-life of approximately 7 min in the mouse blood circulation. Trypsin treatment of alpha(1)I(3)bulletalpha(1)-m did not, as expected from the inability of the proteinase to induce conformational changes in the complex, further increase the clearance rate. Thus, despite the intact peptide backbone of alpha(1)I(3)bulletalpha(1)-m, its insusceptibility to bait region proteinase attacks, and the structural similarity between alpha(1)I(3)bulletalpha(1)-m and native alpha(1)I(3), it is possible that the receptor-binding domain of the alpha(1)I(3)bulletalpha(1)-m complex is exposed, resulting in a rapid clearing of the complex. The clearance of alpha(1)I(3)bulletalpha(1)-m could be inhibited by an excess of alpha(2)M, suggesting that it is in fact cleared by the same receptors that normally bind proteinase-cleaved alpha(1)I(3) and not by receptors with affinity for alpha(1)-m or new epitopes formed by the complex binding of alpha(1)-m and alpha(1)I(3). (^3)

The alpha(1)I(3)bulletalpha(1)-m complex is found at a relatively high and stable concentration in rat plasma. Thus, to match the rapid elimination from the circulation, the complex must be synthesized at a comparable rate. It has been shown previously that, although rat hepatocytes synthesize both alpha(1)-m and alpha(1)I(3), the complex is not synthesized at all or at a very low rate by rat hepatocytes (Pierzchalski et al., 1992). Moreover, a simple mixing and incubation of I-labeled alpha(1)-m with rat plasma or injection of I-labeled alpha(1)-m into living rats do not lead to the formation of I-labeled alpha(1)I(3)bulletalpha(1)-m (Falkenberg et al., 1994). Thus, despite a high rate of synthesis of the alpha(1)I(3)bulletalpha(1)-m complex, the site of formation of the complex is still unknown.

The consequences of complex formation between alpha(1)I(3) and alpha(1)-m are at present unknown. Complex formation may serve to regulate alpha(1)-m immunoregulatory activity. Recently, we have shown that covalent complexes of rat or human alpha-macroglobulins with lysozyme seem to augment antigen presentation of lysozyme by macrophages to T-cells (Chu and Pizzo, 1993; Chu et al., 1994). The alpha-macroglobulin-lysozyme covalent complexes were taken up very efficiently by macrophages via the low density lipoprotein-related protein-alpha(2)M receptor and then processed to present lysozyme peptides in complex with Ia antigen to T-cells that recognized the epitope. Whether uptake of alpha(1)-m via the low density lipoprotein-related protein-alpha(2)M receptor serves a functional role in macrophage regulation will require further study.

It has been shown in this work that alpha(1)I(3), the most abundant proteinase inhibitor in the rat, has lost its proteinase inhibitory activity when it is complex-bound to alpha(1)-m. Furthermore, alpha(1)I(3)bulletalpha(1)-m is cleared from the blood circulation at an unexpectedly high rate. A function of alpha(1)-m could be to ``kill'' alpha(1)I(3) by blocking the inhibitory activity and to cause its physical elimination from the circulation. Indeed, it can be speculated that alpha(1)-m ``kills'' its various complex partners, i.e. albumin, fibronectin, and IgA, by blocking one or more of their effector functions. It has, for example, been shown that alpha(1)-m is linked to IgA via the C-terminal part of the alpha-chain (Grubb et al., 1986), which is also involved in the binding to the secretory component (Mestecky and McGhee, 1987), potentially blocking the transport of IgA from blood to secretions. Thus, a scenario linking these observations would be for alpha(1)-m to rapidly down-regulate the activity of plasma proteins that are already in circulation before their concentration can be lowered by transcriptional down-regulation.


FOOTNOTES

*
This work was supported by the Swedish Medical Research Council (Project 7144), the Medical Faculty at the University of Lund, King Gustav V:s 80-year Foundation, the Foundations of Greta and Johan Kock, and Alfred Österlund, the Swedish Society for Medical Research, the Royal Physiographic Society in Lund, and NHLBI, National Institutes of Health Grants HL49542 and HL24066. 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. Tel.: 46-46-104509; Fax: 46-46-104022.

(^1)
The abbreviations used are: alpha(1)-m, alpha(1)-microglobulin; alpha(1)I(3), alpha(1)-inhibitor-3; alpha(2)M, alpha(2)-macroglobulin; PPE, porcine pancreatic elastase; DCI, 3,4-dichloroisocoumarin; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.

(^2)
C. Falkenberg, J. J. Enghild, I. B. Thøgersen, G. Salvesen, and B. Åkerström, unpublished observations.

(^3)
alpha(2)M was used instead of alpha(1)I(3) for blocking of the in vivo clearance of alpha(1)I(3)bulletalpha(1)-m. It has been shown previously that alpha-macroglobulins, including alpha(2)M and alpha(1)I(3), are bound by the same receptor on cultured macrophages and hepatocytes (Gliemann and Sottrup-Jensen, 1987; Enghild et al., 1989b).


ACKNOWLEDGEMENTS

We thank Dr. Harold Erickson for the electron micrographs.


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