Alpha 1-antichymotrypsin is the human plasma inhibitor of macrophage ectoenzymes that cleave pro-Macrophage Stimulating Protein

Macrophage stimulating protein (MSP) is secreted as 78-kDa single chain pro-MSP, which is converted to biologically active, disulfide-linked alphabeta chain MSP by cleavage at Arg(483)-Val(484). Murine resident peritoneal macrophages have two cell surface proteolytic activities that cleave pro-MSP. One is a pro-MSP convertase, which cleaves pro-MSP to active MSP; the other degrades pro-MSP. The degrading protease is inhibited by soybean trypsin inhibitor or by low concentrations of blood plasma, which allows the convertase to cleave pro-MSP to MSP. Using pro-MSP cleavage as the assay, we purified the inhibitor from human plasma. The bulk of the plasma protein was removed by salting out and by isoelectric precipitation of albumin. Highly purified inhibitor was then obtained in three steps: dye-ligand binding and elution, ion exchange chromatography, and high performance liquid chromatography gel filtration. After SDS-polyacrylamide gel electrophoresis and transfer to a polyvinylidene membrane, N-terminal sequencing of the product identified it as alpha(1)-antichymotrypsin. The mean concentration of alpha(1)-antichymotrypsin in human plasma is 7 micrometer. At this concentration, alpha(1)-antichymotrypsin inhibits both macrophage enzymes. A concentration of 0.4 micrometer, which is in the expected concentration range in extracellular fluid, preferentially inhibits the degrading enzyme, which allows for cleavage to active MSP by the pro-MSP convertase.


Introduction
Macrophage Stimulating Protein (MSP) is a pleiotropic 78 kDa growth and motility factor that is structurally related to several proteins of the coagulation system (1,2). MSP acts on a number of cell types including tissue macrophages, epithelia, and hematopoietic cells (3). Actions on macrophages include stimulation of motility (4), induction of phagocytosis of serum complement-coated erythrocytes (1), and inhibition of the upregulation of NO synthase by inflammatory stimuli (5,6). MSP induces adhesion, motility and replication of epithelial cells, and it can prevent the apoptosis that occurs when epithelial cells are prevented from attachment to a substrate (7). MSP mediates its effects by binding to and activating a cell receptor tyrosine kinase known as RON in humans (8,9) and STK in mice (10,11).
Human MSP is a disulfide-linked heterodimer comprising an α and β chain with molecular masses (not including N-linked carbohydrate) of 53 and 25 kDa, respectively. It is a member of a plasminogen-related family of proteins characterized by multiple copies in the α chain of a highly conserved triple disulfide loop structure (kringle). The kringle protein family includes plasminogen (12), prothrombin (13), urokinase (14), and hepatocyte growth factor/scatter factor (HGF/SF) (15,16). These proteins are secreted as single-chain precursors, which have no biological activity until the protein is cleaved into α and β chains by specific serine proteases at an Arg-X bond (17), in which X is most frequently Val. The αβ chain junction of MSP is Arg 483 -Val 484 (18) . The β chain of MSP is homologous to the β chain catalytic domain of the serine protease members of the kringle family.
However MSP and HGF/SF are devoid of proteolytic activity, because of amino acid substitutions of the three β chain protease active site residues His, Asp and Ser. They are thought to have evolved from an ancient coagulation protein (19) to become growth and motility factors, while retaining the protease-dependent activation mechanism of the zymogens of the family.
Like kringle proteins of the coagulation system, MSP is constitutively synthesized by hepatic parenchymal cells and is secreted into the circulating blood as biologically inactive pro-MSP. The mean concentration of pro-MSP in the plasma of a series of normal human subjects is 5 nM (20). The level is not changed in the course of an acute phase reaction (21). To act on target cells in extravascular sites, pro-MSP must diffuse into tissues and be proteolytically cleaved to the biologically active disulfide-linked αβ-chain heterodimer (22). The EC 50 for the action of the MSP heterodimer on macrophages is 0.25 nM (1).
The R 483 -V 484 scissile bond of pro-MSP is a typical cleavage site for trypsin-like serine proteases. Several such proteases of the coagulation system, including factors XIa and XIIa and serum kallikrein cleave pro-MSP to active MSP in vitro (23). However, cleavage is minimal when blood clots, indicating that pro-MSP is not a preferred substrate for these enzymes (22). We have described two pro-MSP convertase activities in extravascular sites, one in wound fluid exudates (20) and the other associated with murine resident peritoneal macrophages is present, the degrading enzyme is preferentially inhibited, and the pro-MSP convertase cleaves the protein to active MSP, as shown by SDS-PAGE of the products and by induction of characteristic shape changes in the macrophages.
The effect of STI could be reproduced by adding human or mouse serum to the cultures. A number of human serum protease inhibitors, including α1-antitrypsin, C1-inhibitor, anti-thrombin III and α2-macroglobulin, had no effect on macrophage cleavage of pro-MSP (22). Identification of the serum inhibitor and approaches to determining its physiological role are the subjects of this communication.  MSP induced macrophages to become elongated in shape (Fig. 6B). Similar macrophage morphology was observed in wells with pro-MSP in the presence of STI (Fig. 6C) or α1-antichymotrypsin (Fig. 6D). These findings correlate with the data in Figure 4, showing that inhibition of the degrading enzyme by STI or

Discussion
We reported that murine resident peritoneal macrophages have two cell surface proteolytic activities that can cleave pro-MSP. One cleaves pro-MSP to active MSP, the other degrades pro-MSP (22). The degrading protease was inhibited by STI and by normal human or mouse serum It was not inhibited by a number of plasma protease inhibitors, including α1-antitrypsin, C1-inhibitor, anti-thrombin III and α2-macroglobulin (22). We have now shown that the inhibitor in human plasma is α1-antichymotrypsin. It is therefore probable that the macrophage pro-MSP degrading enzyme is a serine protease with chymotrypsin or cathepsin-G like substrate preferences, which cleaves at sites with aromatic or large aliphatic residues. Inasmuch as the initial major macrophage inactivating cleavage product of pro-MSP is a 46 kDa fragment ((22) and Fig. 4, lane 1), our results suggest that this fragment is formed by cleavage at Phe 418 , which would yield a polypeptide with a calculated mass of 45.7 kDa.
Phe 418 is located within the triple disulfide loop structure of kringle 4, and therefore release of the fragment would require reduction. Consistent with this prediction, the amount of 46 kDa fragment was minimal after SDS-PAGE under non-reducing conditions (data not shown).
Inhibition of the macrophage pro-MSP degrading enzyme by STI (Fig. 4, lane 2) led to the expectation that the degrading enzyme would be a trypsin-like serine protease. It was thus a surprise to find that the serum inhibitor of the enzyme is α1-antichymotrypsin. A review of the literature showed that STI can bind and inhibit chymotrypsin as well as trypsin. In fact, the inhibitor has two binding sites, one for trypsin or chymotrypsin and another for chymotrypsin (26) Since the macrophage pro-MSP convertase generates active MSP, the cleavage site is expected to be Arg 483 -Val 484, the αβ chain junction (18). This is a preferred cleavage site for trypsin-like serine proteases. However, the inhibitor profile for the macrophage pro-MSP convertase is unusual, in that it is not inhibited by various inhibitors of trypsin-like serine proteases, including leupeptin, aprotinin, FPR-chloromethylketone, FFR-chloromethylketone (data not shown), and STI. And it is completely inhibited, along with the pro-MSP degrading enzyme, by 5 µM α1-antichymotrypsin, a preferential inhibitor of chymotrypsin-like proteases. Purification and characterization of the macrophage pro-MSP convertase would therefore be of interest. It is possible that the convertase and the degrading activities reflect dual specificities of a single protein. An example is human cathepsin G, which has both trypsin-and chymotrypsin-like specificities (27), and binds both trypsin and chymotrypsin inhibitors (28). In addition to its well-known location in leukocyte azurophilic granules, cathepsin G with dual specificity has been detected on cell membranes isolated from the U-937 human promonocytic cell line (29). These cells, with or without the maturational stimulus of phorbol esters, had no protease activity for pro-MSP (data not shown).
Like the coagulation factors from which MSP is thought to have evolved (19), regulation of its activity depends on proteolytic cleavage of the single chain precursor pro-MSP, which is present in human circulating blood at a concentration of 5 nM (20), about 20x the EC 50 of 0.25 nM (1). If pro-MSP and α1-antichymotrypsin diffuse from the circulation into the extravascular space, inhibition of pro-MSP degradation by tissue macrophages might allow the macrophage pro-MSP convertase to generate active MSP, which could act on cells expressing the RON MSP receptor, including macrophages themselves as well as epithelial cells (30).
Using the murine peritoneal cavity as an example of extravascular space, we showed that peritoneal fluid from normal or serotonin-treated mice could preferentially inhibit the macrophage pro-MSP degrading enzyme and allow cleavage by the convertase to MSP (Fig. 5, lanes 3 and 4). From the A 280 of peritoneal washouts and determination of pre-washout peritoneal fluid volume by radioactive dextran dilution (as described in Methods), we estimated that the mean A 280 in peritoneal fluid of normal and serotonin-treated mice was 6.8 ± 0.6 and 4.4 ± 0.3 respectively for a series of three mice each. The A 280 of the fluids tested in the experiment illustrated in Figure 5  In contrast, since the concentration of plasma proteins in acute, severe inflammatory exudates approaches that of proteins in the circulation, the mean α1-antichymotrypsin concentration of 7 µM (25) is in the range that completely inhibits macrophage pro-MSP convertase activity (Fig. 4, lane 5). Mature MSP is present in these exudates (20), generated by a unique fluid phase pro-MSP convertase, purification of which is in progress. In contrast to the exudate fluid phase convertase, the potential significance of the macrophage pro-MSP convertase may be to generate MSP locally to act on cells expressing the RON receptor under physiological conditions, in the absence of inflammation.