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

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
Alpha 1 -antitrypsin (α 1 -AT) and surfactant protein-A (SP-A) are major defense glycoproteins in the alveolar spaces of human lungs. α 1 -AT, a 52,000 D glycoprotein, is secreted mostly by hepatocytes, and, to a lesser extent, by lung epithelial cells and phagocytes. α 1 -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, α 1 -AT protects the connective tissue from HNE released by triggered neutrophils; as a result, subjects homozygous for the common deficiency variant Z α 1 -AT (associated with 15% of normal plasma α 1 -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][6][7][8][9][10]. SP-A, in particular, is able to bind several microorganisms 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 α 1 -AT. We hypothesized that, in analogy with what happens with infectious agents, SP-A could bind to α 1 -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 α 1 -AT towards HNE is significantly decreased in the presence of SP-A, probably as a consequence of SP-A binding to α 1 -AT. Such an interaction would represent a novel mechanism of regulating alveolar α 1 -AT. This could have relevance both for the pathogenesis of emphysema in patients with the Z α 1 -AT variant and for the lungs' defenses against infectious agents.

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 CaCl 2 , 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-Dmannopyranoside (MNOCH 3 ) (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 SucAlaAlaProPh-eNA (for αChy), and the irreversible inhibitors MeOSucA-laAlaProValCMK (for HNE) and TosPheCMK (for αChy) (all from Sigma) were dissolved in (CH 3 ) 2 SO. Wild-type α 1 -antitryspin (M α 1 -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 α 1 -AT variant. Z α 1 -AT variant was purified by covalent chromatography from subjects identified within the Italian screening program for α 1 -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 CaCl 2 , 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 CaCl 2 , 1 mM MgCl 2 , 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 recentrifuged 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 polymyxintreated 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 CaCl 2 , 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 1for more details.

1) Gel filtration HPLC
A mixture of SP-A (1.62 mg/ml) and α 1 -AT (1 mg/ml) in a 1:50 molar ratio was incubated for 24 hrs at 37°C in phosphate buffer. The SP-A/α 1 -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 Na 2 HPO 4, 2 mM CaCl 2 , pH 6.8 at a flow rate of 0.3 ml/ min, and monitored at 220 nm. The excluded (V 0 = 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: α 2 -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 α 1 -AT, purified SP-A, and SP-A/α 1 -AT complex collected from the Size Exclusion Chromatography experiments, were immobilized in 50 mM Na 2 CO 3 , 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 Na 2 CO 3 , 2% BSA pH 9.5, incubated for 2 hrs in the presence of primary antibodies diluted 1:500 (goat anti-human α 1 -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 H 2 SO 4 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 MeOSucAlaAl-aProValNA and SucAlaAlaProPheNA, respectively, at 37°C [22]. Product formation was monitored spectro-

Results
To investigate the interaction between SP-A and α 1 -AT we studied whether K ass values, derived from incubating HNE with α 1 -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 α 1 -AT and SP-A/α 1 -AT mixtures with increasing amounts of LPS, without measurable effect on the K ass of α 1 -AT or SP-A/α 1 -AT mixture (not shown). As expected, [24], we found that the K ass of Z α 1 -AT for HNE was 3.5 fold lower than that of the normal, M α 1 -AT. When Z α 1 -AT was coupled with increasing SP-A concentrations, a further decrease in K ass towards HNE was observed ( Table 2).
To exclude that the results were due to non-specific binding, we incubated 7.5 nM HNE with 0-100 nM α 1 -AT for 15 min at 37°C in microtiter plates or in glass tubes and then measured the residual HNE activity with 2 mM MeO-SucAlaAlaProValNA, finding no difference between plastics and glass. Furthermore, to exclude binding of SP-A to plastics we incubated 15 nM SP-A with I 125 α1-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 α 1 -AT to SP-A.
Gel filtration HPLC was then used to determine the molecular weight of the SP-A/α 1 -AT complex. As shown in Figure  immunochemical assay in which a polypropylene plate was probed with antisera anti α 1 -AT and anti SP-A. As shown in Figure 2B, the first peak in profile a of Figure 2A contained both α 1 -AT and SP-A.
The effect of SP-A on the K ass of α 1 -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 α 1 -AT, via the α 1 -AT carbohydrate chains. Consistent with these findings, the addition of 1 M mannopyranoside to the SP-A/α 1 -AT mixture almost totally reversed the reduction in the K ass (Figure 3), most likely by interfering with the binding of CRD to α 1 -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 α 1 -AT (Table 1).
α . To better clarify the role of the CRD in the binding of SP-A to α 1 -AT, we modified both proteins by enzymatic digestion, deglycosylation or boiling and then used them to calculate the K ass of α 1 -AT for HNE and to deduce the molar parts of α 1 -AT bound to SP-A from the number of turnovers per inactivation of α 1 -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 α 1 -AT since, when incubated with α 1 -AT, it retained the same K ass , as that of native SP-A ( Figure 4).
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 α 1 -AT was 24, i.e. one part of SP-A binds 23 molar parts of α 1 -AT and 24 SP-A plus α 1 -AT binds inhibit 1 part of enzyme ( Figure 5). The same binding pattern emerged when Z α 1 -AT was used instead of α 1 -AT, suggesting that the difference in the K ass between the two variants of α 1 -AT is independent of the number of molar parts of inhibitor bound to SP-A.
Deglycosylated α 1 -AT retains its ability to inhibit HNE (K ass 3.38 × 10 7 M -1 sec -1 ). We did, however, find that the inhibitory activity of α 1 -AT is greatly decreased in the presence of SP-A (K ass 1.1 × 10 7 M -1 sec -1 , Figure 4), indicating that binding of SP-A to the carbohydrate moiety of α 1 -AT is not the only mechanism involved. The turnover number of the SP-A/deglycosylated α 1 -AT is 12, half that displayed by native α 1 -AT (Figure 4, 5). To explore other mechanisms of binding between SP-A and α 1 -AT, we incubated boiled SP-A and α 1 -AT. We found that boiled SP-A/ native α 1 -AT displayed the same K ass and the same turnover number as native SP-A/deglycosylated α 1 -AT ( Figures  4, 5). We postulated that SP-A carbohydrate chains could bind α 1 -AT, possibly through the amino acid backbone. In fact, carbohydrate chains isolated from SP-A mixed with deglycosylated α 1 -AT resulted in the same K ass and turnover number as those of native SP-A/deglycosylated α 1 -AT (Figures 4, 5). Besides these mechanisms of binding of SP-A to α 1 -AT, a third mechanism, i.e. a carbohydrate/ carbohydrate interaction, probably exists since boiled SP-A and native α 1 -AT displayed a K ass of 1.9 × 10 7 M -1 sec -1 and ~6 turnovers (Figure 4, 5).
Finally, we studied the binding of deglycosylated SP-A to α 1 -AT. The K ass of native α 1 -AT mixed with deglycosylated SP-A was 1.2 × 10 7 M -1 sec -1 and the turnover number 18 (Figure 4, 5). Absence of SP-A/α 1 -AT binding, i.e. K ass 3.4 × 10 7 M -1 sec -1 , and a turnover number of 1, was achieved by two combinations: 1) SP-A deglycosylated and boiled with native α 1 -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 α 1 -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 α 1 -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 α 1 -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

-AT and Z α 1 -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.
α 1 -AT/SPA ratio α 1 -AT Z α 1 -AT Reaction condition K ass , (M -1 sec -1 ) decrease in K ass , n-fold Reaction condition K ass , (M -1 sec -1 ) decrease in K ass , n-fold Isolation and immunodetection of the α 1 -AT /SP-A complex  5 kDa). B: Immunodetection of the complex. α 1 -AT was added to wells a1 and a2, peak 1 (α 1 -AT /SP-A complex) of Figure 2A was added to wells b1 and b2, and SP-A to wells c1 and c2. Antiserum anti-α 1 -AT was added to wells a1, b1 and c1, antiserum anti-SP-A was added to wells a2, b2 and c2. Peak 1 (α 1 -AT /SP-A complex) is recognized by both antisera. http://respiratory-research.com/content/6/1/146 SP-A in the BALf of normal subjects is estimated to bẽ 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 α 1 -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 α 1 -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 α 1 -AT, binding between SP-A carbohydrates and the protein backbone of α 1 -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 α 1 -AT. Nevertheless, SP-A binds 11 molar parts of deglycosylated, fully active α 1 -AT (Figure 4, 5), thus suggesting a possible binding of SP-A carbohydrate chains to the amino acid backbone of α 1 -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 Nlinked 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 α 1 -AT. Such a type of linkage has been shown to operate in the calciummediated homotypic interaction between two Lewis (Le x ) determinants (Galβ1→4[Fucα 1 →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 ultraweak interactions are involved explains why this aspect is often underestimated [39][40][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 α 1 -AT. There are several known mechanisms that could explain the inactivation of α 1 -AT. Beside the physiologic irreversible suicide substrate mechanism by which α 1 -AT inhibits HNE [42], α 1 -AT may also be inactivated by oxidation of methionine residue(s) located at or near the active site [22,23]. Another mechanism of α 1 -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 α 1 -AT, that of Z α 1 -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 α 1 -AT is less efficient at inhibiting HNE, and has been demonstrated to be present in vivo, since Z α 1 -AT polymers have been detected in the BALf of Z α 1 -AT subjects with emphysema [45]. We found that SP-A binds Z α 1 -AT and that the binding further reduces the K ass , which is already impaired with respect to that of α 1 -AT. Were this binding to happen in vivo, it would further decrease the antiproteinase activity of Z α 1 -AT.
Effects of calcium removal and sugar addition on K ass M -1 sec -1 Figure 3 Effects of calcium removal and sugar addition on K ass M -1 sec -1 . Inhibition of HNE by α 1 -AT (7.5 nM), alone or coupled with 7.5 nM SP-A: (1) α 1 -AT alone, (2) α 1 -AT plus SP-A, The mechanism by which SP-A binding interferes with the α 1 -AT inhibitory mechanism is open to speculation. α 1 -AT inactivation taking place in vitro upon interaction between the two glycoproteins seems to occur because of the functional slowdown of α 1 -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 α 1 -AT and HNE progresses, through an acylenzyme intermediate resulting from peptide bond hydrolysis, to either a loop-inserted covalent complex (inhibitory pathway) or a cleaved serpin and free proteinase (noninhibitory or substrate pathway) [42]. The number of turnovers for native α 1 -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 α 1 -AT or Z α 1 -AT with native SP-A is 24 ( Figure 5), thus indicating that for α 1 -AT bound to SP-A the inhibitory pathway is precluded, and that the reaction inhibitor -HNE progresses mostly through the substrate pathway ( Figure 6B).
In spite of the detailed dissection of the binding mechanism of SP-A to α 1 -AT in vitro, an obvious limitation of the Turnover numbers per inactivation

SP-A degl./boil. SP-A SP-A sugar chains
Hypothetical mechanism of SP-A interference with α 1 -AT (simplification) Figure 6 Hypothetical mechanism of SP-A interference with Inhibitor pathway present paper is the lack of specific studies investigating a possible interaction between SP-A and α 1 -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 α 1 -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 α 1 -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 α 1 -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/α 1 -AT interaction taking place in vivo.

Conclusion
We have shown that SP-A binds α 1 -AT, and that this binding results in a significant decrease in the association rate constant of α 1 -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 α 1 -AT amino acid backbone or between carbohydrate chains of both glycoproteins. The presence of these complex binding mechanisms would exclude the hypothesis that the α 1 -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 α 1 -AT inhibitory activity. It might represent a physiologic mechanism of regulating α 1 -AT activity, especially in acute conditions (for example during defense against infections agents) [49], in which an excess of α 1 -AT would interfere with the physiologic role of proteinases. α 1 -AT is indeed a highly specialised proteinase inhibitor [50], but the presence in nature of several, robust mechanisms of α 1 -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/α 1 -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 α 1 -AT deficiency, since it would contribute significantly to the complex mechanisms of imbalance between Z α 1 -AT and HNE in the lungs. Obviously, all these speculations need further investigations, first of all to understand whether or not SP-A/α 1 -AT binding is a relevant down-regulatory mechanism of α 1 -AT inhibitory activity in vivo.