AAT is the prototypic member of the serpin super family, an acute phase protein, and a major inhibitor of neutrophil elastase and protease 3. AAT, like other serpins, forms denatured-stable complexes with its target proteases and behaves kinetically as an irreversible (suicide) inhibitor (23
). AAT is mainly synthesized in the liver, occurs in high concentrations in plasma (~ 26 μM), and transudes from plasma into the lung epithelial lining fluid, where its concentration is approximately 4 μM (24
). Under inflammatory conditions the concentration of AAT can rise by 3- to 5-fold above normal, and therefore the control of the excessive proteolytic activity by AAT has long been recognized to be crucial in protecting the lung parenchyma and other tissues in many inflammatory diseases (25
). AAT has also been shown to regulate alveolar fluid clearance because of its ability to inhibit trypsin-like protease (20–28 kD), which is secreted into alveolar fluid by epithelial and inflammatory cells and which can activate ENaC (26
). Furthermore, recent studies show that AAT can form complexes and inhibit activity of caspase-3 (a cysteine protease) in vitro
and in vivo
, and can protect various cells, including lung endothelial cells, against apoptosis (27
When the protein sequences of the three serine proteases, namely human neutrophil elastase (1B0F_A), porcine pancreatic elastase (1B0E_A), and catalytic domain of human matriptase (1EAX_A) were aligned with the ClustalW (21
) program, a high sequence alignment score of 1,124 was obtained for all three proteins. shows that the sequences are well aligned with the catalytic triad (His 57, Asp 102, and Ser 195 highlighted in yellow
in ) located in the active site of these enzymes well preserved. The catalytic triad is a coordinated structure consisting of these 3 amino acids located near the heart of the enzyme and each amino acid plays a key role in the cleaving ability of the proteases (28
). As shown in , crystal structure superimposition of the catalytic domains of human matriptase and neutrophil elastase structures confirms the alignment of the catalytic triad. The RMS score for this superimposition was 1.14 using the PyMOL molecular visualization tool suited for secondary structure alignments and production of high-quality three-dimensional images of proteins. The catalytic domain of human matriptase reveals the overall structural similarity to a (chymo)trypsin-like serine protease, specifically to elastase ( and ), but displays unique properties (such as the hydrophobic/acidic S2/S4 sub-sites) which considerably affect its substrate recognition and binding properties. Thus, the catalytic domain of human matriptase has a potential to bind to and form complexes with AAT. Therefore, in this study we examined for reaction between AAT and catalytic domain of matriptase.
Figure 1. ClustalW alignment of the sequences of human neutrophil elastase (1B0F_A), porcine pancreatic elastase (1B0E_A), and human matriptase (1EAX_A). Sequence identities are marked with an asterisk underneath and shown in red. The catalytic triad consisting (more ...)
Superimposed crystal structures of human neutrophil elastase (violet) and human matriptase (blue) with the catalytic triad amino acids (His 57, Asp 102, and Ser 195) highlighted in red (for matriptase) and green (for elastase).
Native AAT was incubated with matriptase at a molar ratio 1:2 for variable time periods before SDS-polyacrylamide gel electrophoresis. There was a clear evidence for the time-dependent formation of AAT/matriptase complex. shows that the native AAT/matriptase mixtures exhibited three major protein bands corresponding to a complex between AAT and matriptase (~ 78 kD), unreacted AAT (52 kD), and the cleaved AAT (45 kD). After 18 hours of AAT/matriptase incubation, all AAT appeared to be reacted with matriptase. Of note, the autoactivation process generated the mature form and a small pro-sequence of catalytic domain of matriptase (). In addition, AAT/matriptase mixtures (1:1), incubated for 18 hours at room temperature, were immunostained with a specific monoclonal antibody against AAT. As illustrated in , human AAT forms SDS-resistant complexes with catalytic domain of matriptase, suggestive of a covalent interaction. These findings indicate that AAT is a slow-binding inhibitor of matriptase catalytic domain that requires a certain time to build a inhibitor–protease complex. It is important to point out that the AAT/matriptase complex, similar to other AAT serine protease complexes, remained SDS-stable after incubation at room temperature for 24 and 48 hours, suggestive of a covalent interaction (data not shown).
Figure 3. (A) 7.5% SDS-PAGE and (B) Western blot of native AAT and matriptase catalytic domain mixtures. (A) Native AAT/matriptase mixtures were prepared by incubating native AAT (1 mg/ml, 19.2 μM) with matriptase (0.5 mg/ml, 19.2 μM) at 1:2 molar (more ...)
The structural properties that confer protease inhibitor activity of AAT make it also susceptible to post-translational modifications by oxidation, nitration, and polymerization (29
). For instance, the oxidation of AAT by the neutrophil-derived myeloperoxidase/H2
), activated neutrophils (31
), or oxidation by components of cigarette smoke (32
) result in oxidation of two of the nine methionines in AAT (358 and 351), leading to loss of inhibitory activity (33
). We showed that oxidation with N-chlorosuccinimide (0.05 to 1 mg/ml) abolished the ability of AAT to form SDS-stable complexes with both neutrophil elastase () and with the catalytic domain of matripase (). In addition, the intensity of the 52-kD band of oxidized AAT decreased with time, and lower-molecular-size fragments were detected, indicating that the oxidized AAT is a substrate of matriptase (). Since fragments of AAT show a variety of biological functions (34
), further studies are warranted to identify and characterize the fragments of oxidized AAT cleavage with matriptase.
Figure 4. SDS-PAGE (7.5%) of oxidized and native AAT incubated with human elastase. Lane 1, molecular size markers; lane 2, native (n) AAT; lane 3, native AAT + elastase incubated for 30 minutes at a molar ratio 1.2:1; lane 4, oxidized (ox) AAT; lane 5 (more ...)
Figure 5. SDS-PAGE (7.5%) of oxidized AAT and matriptase catalytic domain mixtures. Oxidized AAT/matriptase complex was prepared by incubating AAT with matriptase at 1:1 molar ratio in PBS, pH 7.4, for (A) 30 minutes and (B) 18 hours at room temperature. Figure (more ...)
Parallel studies for functional effects of AAT on matriptase catalytic domain activity have shown that native, but not oxidized, AAT inhibits enzyme activity. Bovine serum albumin used as a negative control showed no significant effect on matriptase activity (). Also, native but not oxidized AAT has been shown to inhibit amiloride-sensitive currents across Xenopus oocytes, heterologously transfected with α, β, and γ ENaC (35
Figure 6. Inhibition of matriptase activity by AAT. Effects of native (n) ad oxidized (ox) AAT, as well as bovine serum albumin (0.19 μM each), on the enzymatic activity of matriptase catalytic domain (2.5 nM). Results are shown as percent inhibition of (more ...)
From the relatively slow formation of AAT–matriptase SDS-stable complexes, it became clear that a large-molar excess of AAT over the matriptase catalytic domain is required to observe full inhibition of enzyme in relatively shorter time period. A plot of the residual matriptase activity versus AAT/matriptase molar ratios is shown in . Fitting the data with a straight line using linear regression (r2 = 0.882; P < 0.01) resulted in an x-intercept corresponding to the SI. Under our experimental conditions (18 h incubation of AAT and matriptase) the SI is equal to 1.
Figure 7. Stoichiometry of inhibition. Matriptase (19.2 μM) was pre-incubated with various concentrations of AAT (from 1:0.25 to 1:10, [Matriptase]:[AAT] molar ratio) for 18 hours before assay of matriptase inhibition. Data were fitted with linear regression (more ...)
We also calculated the reaction rate constant (k) by plotting the exponential free matriptase activity versus time of incubation with AAT, as described in Materials and Methods (). A good linear fit was obtained (R = 0.99812), and from the slope of the fitted line the k was calculated as 0.31 × 103 M−1s−1 (kobs = k × [AAT]0 = 0.01794 min−1).
Figure 8. Determination of reaction rate constant (k). (A) Kinetic curves of matriptase activity (cleavage of substrate) measured after pre-incubating matriptase catalytic domain (5 nM) with vehicle or with native AAT (960 nM) for 15 minutes, 30 minutes, 1 hour, (more ...)
This slow rate constant as well as our SDS-gel electrophoresis results confirm that AAT is a slow-binding matripase catalytic domain inhibitor. We speculate that this low inhibition rate, compared with other extracellular serine proteases, such as elastase (k = 6.5 × 107
), may reflect the requirement for specific microenvironments and effective (large) concentrations of the AAT at intracellular versus extracellular locations. A recent study, however, has shown matriptase complexes in human milk, which contain no HAI-1 (specific inhibitor of matriptase) and which are resistant to dissociation in boiling SDS in the absence of reducing agents. Proteomic and immunologic analysis identified the 45-kD fragment as the noncatalytic domains of matriptase and the 80-kD fragment as the matriptase serine protease domain covalently linked to serine protease inhibitors such as AAT, antithrombin III, and α2
-antiplasmin. This finding for the first time provides evidence in vivo
that the proteolytic activity of matriptase may be controlled by secreted serpins such as AAT (37
). These data are in agreement with our findings and point out a potentially new function of AAT. Furthermore, our kinetics data indicate that even the small amount of AAT in the extracellular space is sufficient to inhibit matriptase.
We would like to offer a few examples to illustrate the putative physiologic importance of AAT–matriptase interaction. For example, the leading hypothesis regarding the cause of airway disease in cystic fibrosis is that excessive Na+
absorption leads to inadequate hydration, resulting in mucus stasis and recurrent infection. Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol-anchored membrane serine protease believed to be critical for the regulation of epithelial sodium channel activity in cystic fibrosis. Prostasin is synthesized as an inactive zymogen that requires a site-specific endoproteolytic cleavage to be converted to an active protease. It has been recently reported that matriptase is necessary for this prostasin activation (11
). Therefore, the inhibition of matriptase by AAT may provide a pharmacologic means of improving Na+
current in cystic fibrosis airway. As mentioned above, AAT decreases amiloride-sensitive transport across both Xenopus oocytes, heterologously injected with ENaC, H441 cells (a human Clara cell line expressing native ENaC) and across the alveolar epithelium of rats in vivo
On the other hand, the overexpression of matriptase is a consistent feature of human epithelial tumors. It has been shown that matriptase possesses a strong oncogenic potential when unopposed by its endogenous inhibitor, HAI-1 (38
). Therefore, inhibition of proteolytic activity as a result of various kinds of proteinase–proteinase inhibitor interactions is an important aspect of tumor cell biology. Serpins such as maspin, AAT, α1
-antichymotrypsin, and Kunitz-type inhibitors such as urinary trypsin inhibitor (39
) have been previously implicated in suppression of cancer invasion. It has been reported that cultured human tumor cell lines produce serine protease inhibitors, including AAT, which may play a role in controlling tumor growth and invasion by regulating activity of specific enzymes.
In summary, our data provide new evidence for the formation of irreversible interactions among AAT, the most important serine-protease inhibitor, and matriptase, a serine-bound matriptase with diverse biological functions.