SP-A binds alpha₁-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase

Abstract

Background

α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction.

Methods and results

At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.

Conclusion

We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.

Background

Alpha₁-antitrypsin (α₁-AT) and surfactant protein-A (SP-A) are major defense glycoproteins in the alveolar spaces of human lungs. α₁-AT, a 52,000 D glycoprotein, is secreted mostly by hepatocytes, and, to a lesser extent, by lung epithelial cells and phagocytes. α₁-AT inhibits a variety of serine proteinases by its active site (Met358-Ser359), but its preferential target is human neutrophil elastase (HNE) as demonstrated by the high association rate constant (K _ass) for this proteinase [1]. In the lungs, α₁-AT protects the connective tissue from HNE released by triggered neutrophils; as a result, subjects homozygous for the common deficiency variant Z α₁-AT (associated with 15% of normal plasma α₁-AT levels) develop pulmonary emphysema early in life, especially if they smoke [2].

SP-A, a member of the collectin (collagen-lectin) family [3], is one of the proteins of surfactant. Structurally, it comprises an N-terminal collagen-like domain connected by a neck to a C-terminal carbohydrate recognition domain (CRD) [4]. Six trimers are linked by disulfide bridges in an octadecamer of 650,000 D, in a "flower bouquet" alignment pattern [4, 5]. A complex, predominantly triantennary, carbohydrate chain of ~4,000 D [6] is attached to the asparagine at position 187 of the CRD [7]. SP-A is mainly present in the alveoli in association with phospholipids, only 1% being present in the free form [8, 9]. The primary function of surfactant is to reduce alveolar surface tension at end expiration. It is now however clear that SP-A, together with SP-D, another hydrophilic surfactant protein, plays a major role in the innate defenses of lung [5–10]. SP-A, in particular, is able to bind several micro-organisms and enhance their uptake by phagocytes, stimulate the production of free oxygen radicals, and induce phagocyte chemotaxis [11].

Most binding to micro-organisms, including influenza and herpes simplex viruses, Gram-positive and Gram-negative bacteria, mycobacteria, fungi, and Pneumocystis carinii, occurs via the CRD and is inhibited by sugars or calcium chelators [12].

Since some SP-A is present in the alveoli in the free form, it has a chance of coming into contact with α₁-AT. We hypothesized that, in analogy with what happens with infectious agents, SP-A could bind to α₁-AT, which carries 3 biantennary or triantennary asparagine-linked carbohydrate chains [13].

In this paper we provide in vitro evidence that the inhibitory activity of α₁-AT towards HNE is significantly decreased in the presence of SP-A, probably as a consequence of SP-A binding to α₁-AT. Such an interaction would represent a novel mechanism of regulating alveolar α₁-AT. This could have relevance both for the pathogenesis of emphysema in patients with the Z α₁-AT variant and for the lungs' defenses against infectious agents.

Methods

Preparative procedures

All reagents were of analytical grade, unless otherwise specified. The buffer used in all experiments was 0.2 M Na-K phosphate, with 0.5 M NaCl, 2 mM CaCl₂, and 0.05% w/w Triton × 100, pH 8.0 (phosphate buffer), unless otherwise specified. Lipopolysaccharide (LPS) from E. coli serotype 026:B6 (Sigma) and methyl-α-D-mannopyranoside (MNOCH₃) (Sigma) were dissolved in phosphate buffer. HNE and human α chymotrypsin (αChy) (ART, Athens, GA) were dissolved in 50 mM sodium acetate, 150 mM NaCl, pH 5.5 and diluted with phosphate buffer. N-glycocosidase F from Flavobacterium meningosepticum (PNGase F; EC 3.5.1.52) was purchased from Roche Diagnostics (Monza, Italy). Clostridium histolyticum collagenase type III (EC 3.4.24) came from Calbiochem (La Jolla, CA). The chromogenic substrates MeOSucAlaAlaProValNA (for HNE) and SucAlaAlaProPheNA (for αChy), and the irreversible inhibitors MeOSucAlaAlaProValCMK (for HNE) and TosPheCMK (for αChy) (all from Sigma) were dissolved in (CH₃)₂SO. Wild-type α₁-antitryspin (M α₁-AT) was either from ART or purified from human serum by covalent chromatography. Capillary isoelectric focusing (CIEF) with bare fused-silica capillaries filled with polyethylene oxide and carrier ampholyte solutions in the pH 3.5–5.0 range [14] was applied to confirm the presence of the common M α₁-AT variant. Z α₁-AT variant was purified by covalent chromatography from subjects identified within the Italian screening program for α₁-AT deficiency [15]. SP-A was isolated as described [16] from surfactant obtained from 3 patients affected by pulmonary alveolar proteinosis (PAP), subjected to therapeutic whole lung lavage [17] and from adult New Zealand rabbits. To isolate surfactant, the bronchoalveolar lavage fluid was filtered through gauze and centrifuged at 150 g for 10 minutes. The supernatant was centrifuged for 30 minutes at 80,000 × g and the resulting pellet was suspended in 10 mM Tris-HCl pH 7.4, 145 mM NaCl, 1.25 mM CaCl₂, 1 mM MgCl_2, 2.2 M sucrose (solution A), overlaid with 10 mM Tris-HCl pH 7.4, 145 mM NaCl, 1.25 mM CaCl₂, 1 mM MgCl₂, 2 M sucrose (solution B) and ultracentrifuged overnight at 85,000 × g in a Ti 60 rotor (Beckman). The floating material was dispersed in water and centrifuged for 30 minutes at 100,000 × g and the pellet recovered was stored at -70°C (purified surfactant). To obtain SP-A, surfactant was injected into a 50-fold excess by volume of 1-butanol and stirred at room temperature for 30 minutes. After centrifugation, the pellet was suspended in 1-butanol and re-centrifuged at 4,000 × g for 1 hour at room temperature. The final precipitate was dried under nitrogen and then resuspended in 5 mM Tris-HCl, 145 mM NaCl, 20 mM octyl β-D-glucopyranoside, pH 7.4 (solution C). After centrifugation at 100,000 × g for 1 hour, the pellet was resuspended in 5 mM Tris-HCl pH 7.4 (solution D) and dialyzed against solution D for 48 hours with at least six changes. The final solution was centrifuged at 100,000 × g for 1 hour and the resulting supernatant, containing purified SP-A, was stored. Endotoxin-free SP-A was obtained by treatment with polymyxin-B (Sigma). Small aliquots of SP-A in solution D were incubated in a 1:1 ratio for 6 hours at 4°C with polymyxin-agarose previously equilibrated with 5 mM Tris-HCl, 100 mM octyl β-D-glucopyranoside and 2 mM EDTA, pH 7.4. Polymyxin-agarose was removed by centrifugation at 14,000 × g for 15 minutes, and the supernatant was then dialyzed against 5 mM Tris-HCl pH 7.4 for 48 hours with at least six changes and lyophilized [17, 18]. For some experiments polymyxin-treated SP-A was further purified by D-mannose sepharose 4B chromatography. SP-A was added to a small column containing D-mannose sepharose 4B (Pharmacia) previously equilibrated with 5 mM HEPES, 0.4% Triton × 100 and 1.5 mM CaCl₂, pH 7.2 (solution E), and the column was washed extensively with solution E. SP-A was finally eluted with 5 mM HEPES, 0.4% Triton × 100 and 2.5 mM EDTA, pH 7.2 (solution F).

Modification of the native proteins

Native and modified proteins used in our experiments were at high degree of purification (Figure 1). See additional file 1 for more details.

Figure 1

SDS-PAGE under reducing conditions. A: α₁-AT; B: SP-A. Lane 1: molecular weights; lane 2: native protein; lane 3: deglycosylated protein. The two bands in gel B, lanes 2 and 3 correspond to dimers (57 and 50 kDa, respectively) and monomers (33 and 27 kDa, respectively) of SP-A.

Identification of the SP-A/ α₁-AT complex

1) Gel filtration HPLC

A mixture of SP-A (1.62 mg/ml) and α₁-AT (1 mg/ml) in a 1:50 molar ratio was incubated for 24 hrs at 37°C in phosphate buffer. The SP-A/α₁-AT mixture and single proteins were loaded in a Jasco PU 980 HPLC system (Japan Spectroscopic, Tokyo, Japan) equipped with two Biosep-SEC-S 4000 columns (300 × 7.80 mm each, Phenomenex, Torrence, CA, USA) connected in series. Samples were eluted with 100 mM Na₂HPO_4, 2 mM CaCl₂, pH 6.8 at a flow rate of 0.3 ml/ min, and monitored at 220 nm. The excluded (V₀ = 12.43 ml) and total (V_t = 24.82 ml) volumes were determined using dextran and creatinine, respectively; a calibration curve was obtained by running through the column a set of standard proteins: α₂-macroglobulin (725 kD), aldolase (158 kD), bovine serum albumin (67 kD), chymotrypsinogen (25 kD), and cytochrome C (12.5 kD). The results were reported as mean ± SD of three separate experiments. 2

2) Qualitative immunodetection by ELISA

250 ng of standard α₁-AT, purified SP-A, and SP-A/α₁-AT complex collected from the Size Exclusion Chromatography experiments, were immobilized in 50 mM Na₂CO₃, pH 9.5 overnight at 4°C in a polypropylene plate (Corning, New York, USA). Plates were then brought at room temperature, washed with 150 mM NaCl, 0.1% Tween 20 (ELISA buffer), blocked for 1 h with 50 mM Na₂CO₃, 2% BSA pH 9.5, incubated for 2 hrs in the presence of primary antibodies diluted 1:500 (goat anti-human α₁-AT and rabbit anti-human SP-A; ICN, Aurora, OH, USA), washed and finally reacted for 2 hrs with the appropriate biotinylated secondary antibodies diluted 1:5000 (Chemicon, Temecula, CA, USA). After washing, 100 μL of avidin diluted 1:2000 were added, and samples were incubated for 30 min. Color development was achieved by incubating the samples with 1,2-phenylenediamine dihydrochloride (Dako, Bucks, UK). The reaction was stopped by addition of 100 μl of 0.5 M H₂SO₄ and OD was read at 490 nm with a Bio-Rad 680 Microplate Reader (Bio-Rad Laboratories, CA, USA).

Kinetics studies

Rate constants were derived by competition experiments of HNE and αChy. Kinetic parameters were determined as described [20, 21]. The active sites of HNE and αChy were titrated using a procedure based on the measurement of pNa released after enzymatic cleavage of MeOSucAlaAlaProValNA and SucAlaAlaProPheNA, respectively, at 37°C [22]. Product formation was monitored spectrophotometrically at a wavelength of 405 nm using a Bio Rad Microplate Reader model 3550. To titrate the different forms of α₁-ATs (α₁-AT, deglycosylated α₁-AT and Z α₁-AT), 7.5 nM HNE was incubated for 15 min at 37°C with 0–100 nM inhibitor, in the presence of 2 mM MeOSucAlaAlaProValNA. All following kinetic experiments were derived from α₁-ATs and SP-A/α₁-ATs complexes (obtained by incubation of α₁-ATs, from 0 to 25 nM, with SP-A 15, 7.5, 1.5, 0.15 mM for 15 min at 37°C). See additional file 1 for more details.

Results

To investigate the interaction between SP-A and α₁-AT we studied whether K _ass values, derived from incubating HNE with α₁-AT, were modified by SP-A. Indeed we found a progressive decrease in the K _ass as the SP-A concentrations increased (Table 1), irrespective of the animal source of SP-A. To exclude that the observed effect was due to LPS co-purified with SP-A [23], we repeated the assay using endotoxin-free SPA, but found no differences with native SP-A (Table 1). To reinforce this finding, in separate experiments we spiked α₁-AT and SP-A/α₁-AT mixtures with increasing amounts of LPS, without measurable effect on the K _ass of α₁-AT or SP-A/α₁-AT mixture (not shown). As expected, [24], we found that the K _ass of Z α₁-AT for HNE was 3.5 fold lower than that of the normal, M α₁-AT. When Z α₁-AT was coupled with increasing SP-A concentrations, a further decrease in K _ass towards HNE was observed (Table 2).

Table 1 Association rate constant (K _ass M^-1sec^-1) for inhibition of HNE by α₁-AT with SP-A. SP-A employed was both from humans affected by PAP or from rabbit, polymyxin treated and polymyxin-mannose treated. Data are means ± SD of three different experiments.

Table 2 Association rate constant for inhibition of HNE by α₁-AT and Z α₁-AT with SP-A. Data are means ± SD of experiments performed in triplicate with 3 different batches of human SP-A and1batch of rabbit SP-A.

To exclude that the results were due to non-specific binding, we incubated 7.5 nM HNE with 0–100 nM α₁-AT for 15 min at 37°C in microtiter plates or in glass tubes and then measured the residual HNE activity with 2 mM MeOSucAlaAlaProValNA, finding no difference between plastics and glass. Furthermore, to exclude binding of SP-A to plastics we incubated 15 nM SP-A with I¹²⁵α1-AT (from 0 to 100 nM) at 37°C. The number of Cpm of the samples with SP-A were the same of wells without proteins. We concluded that our data were compatible with binding of α₁-AT to SP-A.

Gel filtration HPLC was then used to determine the molecular weight of the SP-A/α₁-AT complex. As shown in Figure 2A, profile a, a mixture of SP-A and α₁-AT (1 mg/ml), gave two peaks, one corresponding to free ₁-AT (unreacted α₁-AT) and one, with a theoretical molecular weight of 1,642 kD ( ₁-AT/SP-A complex), possibly corresponding to a complex made by one molecule of SP-A (670 kD) and 18 molecules of α₁-AT (54 kD), suggesting that, under the experimental conditions applied, each monomer of SP-A bound one molecule of α₁-AT. Further evidence that the first peak of profile a (Figure 2A) contained the complex SP-A/α₁-AT was obtained by using an immunochemical assay in which a polypropylene plate was probed with antisera anti α₁-AT and anti SP-A. As shown in Figure 2B, the first peak in profile a of Figure 2A contained both α₁-AT and SP-A.

Figure 2

Isolation and immunodetection of the α ₁ -AT /SP-A complex. A: Isolation of the complex by gel filtration chromatography on two Biosep SEC – S 4000 columns connected in series using HPLC. Gel filtration profiles: commercial α₁-AT (in profile c; 19.32 ± 0.1 mL); purified SP-A (in profile b; 16.49 ± 0.07 mL); α₁-AT /SP-A complex (in profile a; 15.31 ± 0.04 mL) and unreacted α₁-AT (in profile a; 19.35 ± 0.09 mL). Inset: calibration curve obtained using the following standards: A = α₂-macroglobulin (725 kDa), B = aldolase (158 kDa), C = bovine serum albumin (67 kDa), D = chymotrypsinogen (25 kDa), E = cytocrome C (12.5 kDa). B: Immunodetection of the complex. α₁-AT was added to wells a1 and a2, peak 1 (α₁-AT /SP-A complex) of Figure 2A was added to wells b1 and b2, and SP-A to wells c1 and c2. Antiserum anti-α₁-AT was added to wells a1, b1 and c1, antiserum anti-SP-A was added to wells a2, b2 and c2. Peak 1 (α₁-AT /SP-A complex) is recognized by both antisera.

The effect of SP-A on the K _ass of α₁-AT for HNE was calcium-dependent, being abrogated by EDTA (Figure 3). Since the calcium-binding domain of SP-A lays at the COOH terminus, next to the CRD [25], we supposed that this part of SP-A could be involved in the binding of SP-A to α₁-AT, via the α₁-AT carbohydrate chains. Consistent with these findings, the addition of 1 M mannopyranoside to the SP-A/α₁-AT mixture almost totally reversed the reduction in the K _ass (Figure 3), most likely by interfering with the binding of CRD to α₁-AT carbohydrate chains [26, 27]. The fact that the lipid recognition domain of SP-A is located in the neck region of the molecule, far from the CRD [23], could explain the lack of influence of LPS on the binding of SP-A to α₁-AT (Table 1).

Figure 3

Effects of calcium removal and sugar addition on K _ass M^-1sec^-1. Inhibition of HNE by α₁-AT (7.5 nM), alone or coupled with 7.5 nM SP-A: (1) α₁-AT alone, (2) α₁-AT plus SP-A, (3) α₁-AT plus SP-A with 5 mM EDTA and (4) α₁-AT plus SP-A with 1 M MNOCH₃. (Data are means ± SD of experiments performed in triplicate).

To better clarify the role of the CRD in the binding of SP-A to α₁-AT, we modified both proteins by enzymatic digestion, deglycosylation or boiling and then used them to calculate the K _ass of α₁-AT for HNE and to deduce the molar parts of α₁-AT bound to SP-A from the number of turnovers per inactivation of α₁-AT not bound to SP-A. Thus we found that the CRD of SP-A appears to contain all the putative SP-A binding sites for α₁-AT since, when incubated with α₁-AT, it retained the same K _ass, as that of native SP-A (Figure 4).

Figure 4

K _ass M^-1sec^-1for inhibition of HNE by modified proteins (7.5 nM), alone or in combination. K_ass data are means ± SD of experiments performed in triplicate. SI values of the associations in bold at the top of the figure.

Turnover per inactivation (also referred to as stoichiometry of inhibition (SI) or partition ratio + 1) defines the number of moles of irreversible inhibitor required to completely inhibit 1 mole of target proteinase. The turnover number resulting from the interaction between unmodified SP-A and α₁-AT was 24, i.e. one part of SP-A binds 23 molar parts of α₁-AT and 24 SP-A plus α₁-AT binds inhibit 1 part of enzyme (Figure 5). The same binding pattern emerged when Z α₁-AT was used instead of α₁-AT, suggesting that the difference in the K _ass between the two variants of α₁-AT is independent of the number of molar parts of inhibitor bound to SP-A.

Figure 5

Turnover numbers per inactivation. Turnover numbers were determined plotting residual enzyme activity/initial enzyme activity versus initial inhibitor concentration/initial enzyme activity. A: (1) native α₁-AT, (2) deglycosylated α₁-AT, (3) Z α₁-AT, (4) native α₁-AT coupled with deglycosylated and boiled SP-A, (5) deglycosylated α₁-AT coupled with deglycosylated SP-A, (6) deglycosylated α₁-AT coupled with deglycosylated and boiled SP-A, (7) native α₁-AT coupled with boiled SP-A and (8)native α₁-AT coupled with SP-A sugar chains. B: (9) native α₁-AT coupled with native SP-A and (10) Z α₁-AT coupled with native SP-A, (11) deglycosylated α₁-AT coupled with native SP-A, (12) deglycosylated α₁-AT coupled with boiled SP-A, (13) deglycosylated α₁-AT coupled with SP-A sugar chains and (14) native α₁-AT coupled with deglycosylated SP-A.

Deglycosylated α₁-AT retains its ability to inhibit HNE (K _ass 3.38 × 10⁷ M^-1sec^-1). We did, however, find that the inhibitory activity of α₁-AT is greatly decreased in the presence of SP-A (K _ass 1.1 × 10⁷ M^-1sec^-1, Figure 4), indicating that binding of SP-A to the carbohydrate moiety of α₁-AT is not the only mechanism involved. The turnover number of the SP-A/deglycosylated α₁-AT is 12, half that displayed by native α₁-AT (Figure 4, 5). To explore other mechanisms of binding between SP-A and α₁-AT, we incubated boiled SP-A and α₁-AT. We found that boiled SP-A/native α₁-AT displayed the same K _ass and the same turnover number as native SP-A/deglycosylated α₁-AT (Figures 4, 5). We postulated that SP-A carbohydrate chains could bind α₁-AT, possibly through the amino acid backbone. In fact, carbohydrate chains isolated from SP-A mixed with deglycosylated α₁-AT resulted in the same K _ass and turnover number as those of native SP-A/deglycosylated α₁-AT (Figures 4, 5). Besides these mechanisms of binding of SP-A to α₁-AT, a third mechanism, i.e. a carbohydrate/carbohydrate interaction, probably exists since boiled SP-A and native α₁-AT displayed a K _ass of 1.9 × 10⁷ M^-1sec^-1 and ~6 turnovers (Figure 4, 5).

Finally, we studied the binding of deglycosylated SP-A to α₁-AT. The K _ass of native α₁-AT mixed with deglycosylated SP-A was 1.2 × 10⁷ M^-1sec^-1 and the turnover number 18 (Figure 4, 5). Absence of SP-A/α₁-AT binding, i.e. K _ass 3.4 × 10⁷ M^-1sec^-1, and a turnover number of 1, was achieved by two combinations: 1) SP-A deglycosylated and boiled with native α₁-AT, and 2) both proteins deglycosylated (Figures 4, 5). In the former case, absence of SP-A carbohydrates and denaturation of CRDs hindered any possible binding of SP-A to native α₁-AT. In the latter case, the binding was precluded by the absence of carbohydrates on both proteins, in spite of the presence of intact CRDs in the SP-A.

Discussion

The present data provide evidence for an in vitro interaction between SP-A and α₁-AT. These glycoproteins belong to two systems of the lung that are supposed to act independently: the surfactant system and the proteinase/proteinase inhibitor system. Nevertheless, evidence for possible links between the two systems does exist. As an example, it has been shown that SP-A may be digested by elastolytic enzymes [28, 29], and that inhalation of α₁-AT in patients with cystic fibrosis may result in an increase of SP-A levels in bronchoalveolar lavage fluid (BALf) [30]. In addition, SP-D induces the production of matrix metalloproteinases by human alveolar macrophages [31], whereas the cysteine proteinase cathepsin H is involved in the first N-terminal processing step of SP-C [32]. The two systems may therefore interact in the lungs, both in physiologic and in pathologic pathways. The concentration of SP-A in the BALf of normal subjects is estimated to be ~277 nM [33]. Since approximately 1% of total SP-A is present in the free form [8, 9], its concentration in BALf would be ~2.8 nM. Given that the concentration of α₁-AT is ~5 μM [34], we reasoned that the two glycoproteins have a good chance of coming into contact during their life cycle.

Indeed our in vitro experiments indicate that the interaction between SP-A and α₁-AT results in binding between them. This binding, which is calcium-dependent, appears to be complex since it could involve binding between the CRD of SP-A and carbohydrates on α₁-AT, binding between SP-A carbohydrates and the protein backbone of α₁-AT, and binding between the carbohydrate chains of both proteins.

Turnover per inactivation suggests that one part of SP-A binds 23 molar parts of α₁-AT. Nevertheless, SP-A binds 11 molar parts of deglycosylated, fully active α₁-AT (Figure 4, 5), thus suggesting a possible binding of SP-A carbohydrate chains to the amino acid backbone of α₁-AT. Asn, to which carbohydrates of the native glycoprotein are linked [35], is a likely candidate. This hypothesis was confirmed by the results obtained with boiled SP-A and with isolated SP-A carbohydrate chains (Figure 4, 5). In support of this hypothesis, it has been reported that the binding of SP-A to influenza virus [36], herpes virus type 1 infected cells [37], and M. tuberculosis [38], involves N-linked carbohydrate chains on SP-A. Interestingly, there may be multiple binding sites on individual micro-organisms [12].

Our experiments also suggest a possible carbohydrate/carbohydrate interaction between SP-A and α₁-AT. Such a type of linkage has been shown to operate in the calcium-mediated homotypic interaction between two Lewis (Le^x) determinants (Galβ1→4[Fucα₁→3]GlcNAc) involved in cell adhesion during murine embryogenesis [39]. Interestingly Le^x-Le^x interactions appear to be calcium-dependent [40], by involving van der Waal forces. The fact that ultra-weak interactions are involved explains why this aspect is often underestimated [39–41].

It is difficult to postulate whether the three proposed mechanisms of binding take place simultaneously between native proteins. It may be that the CRD plays the main role and that the other two mechanisms are less important or take place only as artificial mechanisms once the proteins have been manipulated.

The binding with SP-A results in a decrease in the inhibition of HNE by α₁-AT. There are several known mechanisms that could explain the inactivation of α₁-AT. Beside the physiologic irreversible suicide substrate mechanism by which α₁-AT inhibits HNE [42], α₁-AT may also be inactivated by oxidation of methionine residue(s) located at or near the active site [22, 23]. Another mechanism of α₁-AT inactivation is proteolytic degradation at or near the active site by a number of host and non-host, mostly microbial, proteinases [42]. Whether these mechanisms may act in vivo, thereby contributing to the imbalance between proteinases and inhibitors in the pathogenesis and progression of pulmonary emphysema, is still a debated issue.

With respect to the inhibitory activity of α₁-AT, that of Z α₁-AT is further impaired by this latter's enhanced tendency to undergo spontaneous polymerization [2]. This phenomenon, also known as loop-sheet polymerization, likely accounts for why Z α₁-AT is less efficient at inhibiting HNE, and has been demonstrated to be present in vivo, since Z α₁-AT polymers have been detected in the BALf of Z α₁-AT subjects with emphysema [45]. We found that SP-A binds Z α₁-AT and that the binding further reduces the K _ass, which is already impaired with respect to that of α₁-AT. Were this binding to happen in vivo, it would further decrease the antiproteinase activity of Z α₁-AT.

The mechanism by which SP-A binding interferes with the α₁-AT inhibitory mechanism is open to speculation. α₁-AT inactivation taking place in vitro upon interaction between the two glycoproteins seems to occur because of the functional slowdown of α₁-AT in the presence of SP-A, the turnover number shifting from 1 to 24. After an initial, non-covalent, Michaelis-like complex, the reaction between α₁-AT and HNE progresses, through an acyl-enzyme intermediate resulting from peptide bond hydrolysis, to either a loop-inserted covalent complex (inhibitory pathway) or a cleaved serpin and free proteinase (non-inhibitory or substrate pathway) [42]. The number of turnovers for native α₁-AT is 1 (Figure 5), indicating that the reaction inhibitor-HNE progresses towards the inhibitory pathway on the other side (Figure 6A). The number of turnovers after the incubation of native α₁-AT or Z α₁-AT with native SP-A is 24 (Figure 5), thus indicating that for α₁-AT bound to SP-A the inhibitory pathway is precluded, and that the reaction inhibitor – HNE progresses mostly through the substrate pathway (Figure 6B).

Figure 6

Hypothetical mechanism of SP-A interference with α ₁ -AT (simplification). A: interaction of α₁-AT (I) with HNE (E). After an initial non-covalent Michaelis-like complex (EI), the interaction progresses through a tetrahedral intermediate (EI ♠), forming a covalent acyl-enzyme intermediate (EI ♥). The substrate pathway results in free HNE and cleaved α₁-AT (I*); the inhibitory pathway results in a, about 100%, kinetically trapped loop-inserted covalent complex (E-I*). B: the SP-A (here shown as a trimer) interacts with α₁-AT. In the presence of HNE, the formation of a covalent complex E-I* almost precluded (about 4%), and the reaction progresses through the substrate pathway towards free E and I* (cleaved α₁-AT) – SP-A (96%). SI = stoichiometry of inhibition

In spite of the detailed dissection of the binding mechanism of SP-A to α₁-AT in vitro, an obvious limitation of the present paper is the lack of specific studies investigating a possible interaction between SP-A and α₁-AT in vivo. Nevertheless, some indirect evidence suggesting that such an interaction might take place is available, although it is not possible to address a plausible expectation of physiologic or pathophysiologic relevance of these findings. For example, a recent report has shown that in human sputum supramolecular complexes with heparan sulfate/Syndecan-1 and proteinase and inhibitors are present [46]. These complexes contain the proteinase inhibitors SLPI and α₁-AT, NE as well, whose proteolytic activity is however not decreased . The large MW of SP-A makes difficult to highlight the occurrence of such supramolecular complexes including α₁-AT by standard techniques [47]. Nevertheless, a report focusing on two-dimensional electrophoretic characteristics of BALf proteins in subjects affected by interstitial lung diseases [48] has intriguingly shown that some α₁-AT fragments were superimposed on spots of SP-A, in its upper, acidic position. These findings, confirmed by mass spectrometric MALDI-TOF analysis, would suggest a possible SP-A/α₁-AT interaction taking place in vivo.

Conclusion

We have shown that SP-A binds α₁-AT, and that this binding results in a significant decrease in the association rate constant of α₁-AT for HNE. The mechanism of the binding seems to be predominantly mediated by the SP-A CRDs, as indicated by the calcium dependence and by the turnovers for inactivation, but other mechanisms may be involved, such as an interaction between SP-A carbohydrates and the α₁-AT amino acid backbone or between carbohydrate chains of both glycoproteins. The presence of these complex binding mechanisms would exclude the hypothesis that the α₁-AT inhibition occurred simply due to steric inhibition of the large SP-A molecule, but it would rather suggest a programmed, coordinated mechanism.

The in vitro interaction described here, if present in vivo, would be a novel mechanism of impairment of α₁-AT inhibitory activity. It might represent a physiologic mechanism of regulating α₁-AT activity, especially in acute conditions (for example during defense against infections agents) [49], in which an excess of α₁-AT would interfere with the physiologic role of proteinases. α₁-AT is indeed a highly specialised proteinase inhibitor [50], but the presence in nature of several, robust mechanisms of α₁-AT downregulation (i.e. inherited deficiency, susceptibility to oxidative stress and proteolysis, polymerization) would imply the occurrence of intrinsic risks related to the overexpression of a nearly perfect and immortal inhibitor. Therefore, the formation of supramolecular complexes SP-A/α₁-AT might be a sort of reserve mechanism, taking place in case of need.

On the other hand, the interaction with SP-A would be of particular relevance in the pathogenesis of pulmonary emphysema associated with α₁-AT deficiency, since it would contribute significantly to the complex mechanisms of imbalance between Z α₁-AT and HNE in the lungs. Obviously, all these speculations need further investigations, first of all to understand whether or not SP-A/α₁-AT binding is a relevant down-regulatory mechanism of α₁-AT inhibitory activity in vivo.