Skip to main content

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



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.


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.


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 [510]. 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.


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 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 MgCl2, 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
figure 1_384

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 Na2HPO4, 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 (Vt = 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.


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
figure 2_384

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
figure 3_384

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
figure 4_384

K ass M-1sec-1for inhibition of HNE by modified proteins (7.5 nM), alone or in combination. Kass 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
figure 5_384

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.


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 (Lex) determinants (Galβ1→4[Fucα1→3]GlcNAc) involved in cell adhesion during murine embryogenesis [39]. Interestingly Lex-Lex 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 [3941].

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
figure 6_384

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.


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.





α chymotrypsin




carbohydrate recognition domain


collagenase-resistant fragment


human neutrophil elastase






surfactant protein-A


  1. Travis J, Salvesen GS: Human plasma proteinase inhibitors. Annu Rev Biochem 1983, 52:655–709.

    Article  CAS  PubMed  Google Scholar 

  2. Carrell RW, Lomas DA: Alpha 1 -antitrypsin deficiency. A model for conformational diseases. N Engl J Med 2002, 346:45–53.

    Article  CAS  PubMed  Google Scholar 

  3. Sastry K, Ezekowitz RA: Collectins: pattern recognition molecules involved in the first line of host defense. Curr Opin Immunol 1993, 5:59–63.

    Article  CAS  PubMed  Google Scholar 

  4. Haagsman HP, White RT, Schilling J, Lau K, Benson BJ, Golden J, Hawgood S, Clements JA: Studies of the structure of lung surfactant protein SP-A. Am J Physiol 1989, 257:L421-L429.

    CAS  PubMed  Google Scholar 

  5. McCormack FX, Whitsett JA: The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J Clin Invest 2002, 109:707–712.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bhattacharyya SN, Lynn WS: Structural studies and oligosaccharides of glycoprotein isolated from alveoli of patients with alveolar proteinosis. J Biol Chem 1977, 252:1172–1180.

    CAS  PubMed  Google Scholar 

  7. Munakata H, Nimberg RB, Snider GL, Robins AG, Van Halbeek H, Vliegenthart JF, Schmid K: The structure of the carbohydrate units of the 36K glycoprotein derived from the lavage of a patient with alveolar proteinosis by high-resolution 1 H-NMR spectroscope. Biochem Biophys Res Commun 1982, 108:1401–1405.

    Article  CAS  PubMed  Google Scholar 

  8. Baritussio A, Alberti A, Quaglino D, Pettenazzo A, Dal Zoppo D, Sartori L, Pasquali-Ronchetti I: SP-A, SP-B, and SP-C in surfactant subtypes around birth: reexamination of alveolar life cycle of surfactant. Am J Physiol 1994, 266:L436-L447.

    CAS  PubMed  Google Scholar 

  9. Savov J, Wright JR, Young SL: Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and Clara cells. Am J Physiol 2000, 279:L118-L126.

    CAS  Google Scholar 

  10. Wright JR: Immunomodulatory functions of surfactant. Physiol Rev 1997, 77:931–962.

    CAS  PubMed  Google Scholar 

  11. Korfhagen TR: Surfactant protein A (SP-A)-mediated bacterial clearance. SP-A and cystic fibrosis. Am J Respir Cell Mol Biol 2001, 25:668–672.

    Article  CAS  PubMed  Google Scholar 

  12. Mason RJ, Greene K, Voelker DR: Surfactant protein A and surfactent protein D in health and disease. Am J Physiol 1998, 275:L1-L13.

    CAS  PubMed  Google Scholar 

  13. Mega T, Lujan E, Yoshida A: Studies on the oligosaccharide chains of human α1-proteinase inhibitor. I. Isolation of glycopeptides. J Biol Chem 1980, 255:4053–4056.

    CAS  PubMed  Google Scholar 

  14. Lupi A, Viglio S, Luisetti M, Gorrini M, Coni P, Faa G, Cetta G, Iadarola P: α1-antitrypsin in serum determined by capillary isoelectric focusing. Electrophoresis 2000, 21:3318–3326.

    Article  CAS  PubMed  Google Scholar 

  15. Ferrarotti I, Baccheschi J, Zorzetto M, Tinelli C, Corda L, Balbi B, Campo I, Pozzi E, Faa G, Coni P, Massi G, Stell G, Luisetti M: Prevalence and phenotype of subjects carrying rare variants in the Italian Registry for alpha1-antitrypsin deficiency. J Med Genet 2005, 42:282–287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Howgood S, Benson BJ, Shilling J, Damm D, Clements JA, White RT: Nucleotide and amino acid sequence of pulmonary surfactant protein SP18 and evidence for co-operation between SP18 and SP28–36 in surfactant lipid adsorption. Pro Natl Acad Sci USA 1987, 84:66–70.

    Article  Google Scholar 

  17. Alberti A, Luisetti M, Braschi A, Rodi G, Iotti G, Sella D, Poletti V, Benori V, Baritussio A: Broncho-alveolar lavage fluid composition in alveolar proteinosis. Early changes after therapeutic lavage. Am J Respir Crit Care Med 1996, 154:817–820.

    Article  CAS  PubMed  Google Scholar 

  18. Wright JR, Zlogar DF, Taylor JC, Zlogar TM, Restepo CI: Effect of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages. Am J Physiol 1999, 276:L650-L658.

    CAS  PubMed  Google Scholar 

  19. Meloni F, Alberti A, Bulgheroni A, Lupi A, Paschetto E, Marone Bianco A, Rodi G, Fietta A, Luisetti M, Baritussio A: Surfactant apoprotein A modulates interleukin-8 and monocyte chemotactic peptide-1 production. Eur Respir J 2002, 19:1128–1135.

    Article  CAS  PubMed  Google Scholar 

  20. Vincent J-P, Lazdunski M: Trypsin-pancreatic trypsin inhibitor associations. Dynamics of the interaction and role of disulfide bridges. Biochemistry 1972, 11:2967–2977.

    Article  CAS  PubMed  Google Scholar 

  21. Beatty K, Bieth J, Travis J: Kinetics of association of serine proteinases with native and oxidized α-1-proteinase inhibitor and α-1-antichymotrypsin. J Biol Chem 1980, 255:3931–3934.

    CAS  PubMed  Google Scholar 

  22. Gorrini M, Lupi A, Viglio S, Pamparana F, Cetta G, Iadarola P, Powers JC, Luisetti M: Inhibition of human neutrophil elastase by erythromycin and flurythromycin, two macrolide antibiotics. Am J Respir Cell Mol Biol 2001, 25:492–499.

    Article  CAS  PubMed  Google Scholar 

  23. Creuwels LAJM, van Golde LMG, Haagsman H: The pulmonary surfactant system: biochemical and clinical aspects. Lung 1997, 175:1–39.

    Article  CAS  PubMed  Google Scholar 

  24. Ogushi F, Fells GA, Hubbard RC, Straus SD, Crystal RG: Z-type α1-antitrypsin is less competent than M1-type α1-antitrypsin as an inhibitor of neutrophil elastase. J Clin Invest 1987, 80:1366–1374.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Haagsman HP, Sargeant T, Hauschka VH, Benson BJ, Hawgood S: Binding of calcium to SP-A, a surfactant-associated protein. Biochemistry 1990, 29:8894–8900.

    Article  CAS  PubMed  Google Scholar 

  26. Haurum JS, Thiel S, Haagsman HP, Laursen SB, Larsen B, Jensenius JC: Studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein A and comparison with two other collectins: mannan-binding protein and conglutinin. Biochem J 1993, 293:873–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Khubchandani KR, Oberley RE, Snyder JM: Effects of surfactant protein A and NaCl concentration on the uptake of Pseudomonas aeruginosa by THP-1 cells. Am J Respir Cell Mol Biol 2001, 25:699–706.

    Article  CAS  PubMed  Google Scholar 

  28. Rubio F, Cooley J, Accurso FJ, Remold-O'Donnell E: Linkage of neutrophil serine proteases and decreased surfactant protein-A (SP-A) levels in inflammatory lung disease. Thorax 2004, 59:318–323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Beatty AL, Malloy JL, Wright JR: Pseudomonas aeruginosa degrades pulmonary surfactant and increases conversion in vitro . Am J Respir Cell Mol Biol 2005, 32:128–134.

    Article  CAS  PubMed  Google Scholar 

  30. Griese M, von Bredow C, Birrer P: Reduced proteolysis of surfactant protein A and changes of the bronchoalveolar lavage fluid proteome by inhaled alpha 1-protease inhibitor in cystic fibrosis. Electrophoresis 2001, 22:165–171.

    Article  CAS  PubMed  Google Scholar 

  31. Crippes Trask B, Malone MJ, Lum EH, Welgus HG, Crouch EC, Shapiro SD: Induction of macrophage matrix metalloproteinase biosynthesis by surfactant protein D. J Biol Chem 2001, 276:37846–37852.

    Google Scholar 

  32. Brasch F, ten Brinke A, Johnen G, Ochs M, Kapp N, Müller KM, Beers MF, Fehrenbach H, Richter J, Batenburg JJ, Bühling F: Involvement of cathepsin H in the processing of the hydrophobic surfactant-associated protein C in type II pneumocytes. Am J Respir Cell Mol Biol 2002, 26:659–670.

    Article  CAS  PubMed  Google Scholar 

  33. Van de Graaf EA, Jansen HM, Lutter R, Alberts C, Kobsen J, de Vries IJ, Out TA: Surfactant protein A in bronchoalveolar lavage fluid. J Lab Clin Med 1992, 120:252–263.

    CAS  PubMed  Google Scholar 

  34. Rennard SI, Ghafouri M, Thompson AB, Linder J, Vaughan W, Jones K, Ertl RF, Christensen K, Prine A, Stahl MG, et al.: Fractional processing of sequential bronchoalveolar lavage to separate bronchial and alveolar samples. Am Rev Respir Dis 1990, 141:208–217.

    Article  CAS  PubMed  Google Scholar 

  35. Sprio RG: Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 2000, 12:43R-56R.

    Article  Google Scholar 

  36. Benne CA, Kraaijeveld CA, Van Strijp JAG, Brouwer E, Harmsen M, Verhoef J, van Golde LMG, van Iwaarden JF: Interactions of surfactant protein A with influenza A viruses: binding and neutralization. J Infect Dis 1995, 171:335–341.

    Article  CAS  PubMed  Google Scholar 

  37. Van Iwaarden JF, Van Strijp JAG, Visser H, Haagsman HP, Verhoef J, van Golde LMG: Binding of surfactant protein A (SP-A) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of SP-A. J Biol Chem 1992, 267:25039–25043.

    CAS  PubMed  Google Scholar 

  38. Gaynor CD, McCormack FX, Voelker DR, McGowan SE, Schlesinger LS: Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages. J Immunol 1995, 155:5343–5351.

    CAS  PubMed  Google Scholar 

  39. Pincet F, Le Bouar T, Zhang Y, Esnault J, Mallet J-M, Perez E, Sinaÿ P: Ultraweak sugar-sugar interactions for transient cell adhesion. Biophys J 2001, 80:1354–1358.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Henry B, Desvaux H, Pritstchepa M, Berthault P, Zhang Y, Mallet J-M, Esnault J, Sinaÿ P: NMR study of a Lewis x pentasaccharide derivative: solution structure and interaction with cations. Carbohydr Res 1999, 315:48–62.

    Article  CAS  PubMed  Google Scholar 

  41. Sears P, Wong C-H: Intervention of carbohydrate recognition by proteins and nucleic acids. Proc Natl Acad Sci USA 1996, 93:12086–12093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettings PGW, Irving JA, Lomas DA, Luke CJ, Moyer RW, Pemberton PA, Remold-O'Donnell E, Salvesen GS, Travis J, Whisstock JC: The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. J Biol Chem 2001, 276:33293–33296.

    Article  CAS  PubMed  Google Scholar 

  43. Taggart C, Cervantes-Laurean D, Kim G, McElvaney NG, Wehr N, Moss J, Levine RL: Oxidation of either methionine 351 or methionine 358 in α1-antitrypsin causes loss of anti-neutrophil elastase activity. J Biol Chem 2000, 275:27258–27265.

    CAS  PubMed  Google Scholar 

  44. Travis J, Shieh B-H, Potempa J: The functional role af acute phase plasma proteinase inhibitors. Tokai J Exp Clin Med 1988, 13:313–320.

    CAS  PubMed  Google Scholar 

  45. Elliott P, Bilton D, Lomas DA: Lung polymers in Z α 1 -antitrypsin related emphysema. Am J Respir Cell Mol Biol 1998, 18:670–674.

    Article  CAS  PubMed  Google Scholar 

  46. Chan SCH, Shum DKY, Ip MSM: Sputum sol neutrophil elastase activity in bronchiectasis. Differential modulation by Syndecan-1. Am J Respir Crit Care Med 2003, 168:192–198.

    Article  PubMed  Google Scholar 

  47. Madsen J, Kliem A, Nielsen O, Koch C, Steinhilber W, Holmskov U: Expression and localization of lung surfactant protein A in human tissues. Am J Respir Cell Mol Biol 2003, 29:591–597.

    Article  CAS  PubMed  Google Scholar 

  48. Magi B, Bini L, Perari MG, Fossi A, Sanchez JC, Hochstrasser D, Paesano S, Raggiaschi R, Santucci A, Pallini V, Rottoli P: Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study. Electrophoresis 2002, 23:3434–3444.

    Article  CAS  PubMed  Google Scholar 

  49. Matthay MA, Zimmerman GA: Centennial Review. Acute lung injury and the acute respiratory distress syndrome. Four decades of Inquiry into pathogenesis and rational management. Am J Respir Cell Mol Biol 2005, 33:319–327.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Otlewski J, Jelen F, Zakreweska M, Oleksy A: The many faces of protease-protein inhibitor interaction. EMBO J 2005, 24:1303–1310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank Dr. I. Ferrarotti for her contribution to this research, and Dr R. Stenner for editing the manuscript. This work was in part supported by the IRCCS Policlinico San Matteo Ricerca Corrente and by the Fondazione Cariplo

Author information

Authors and Affiliations


Corresponding author

Correspondence to Maurizio Luisetti.

Additional information

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

MG participated in the study design, performed most experiments, and helped to draft the manuscript. AL participated in the deglycosylation experiments and carbohydrate chains isolation. PI designed the experiments for the α1-AT/SP-A complex identification, and helped to draft the manuscript. CDS participated in the kinetic studies. PR performed the experiments for the α1-AT/SP-A complex identification. DD performed the purification of SP-A and CRF. NC took part to some kinetic experiments. EP participated in the coordination of the study. AB performed the purification of SP-A and CRF, helped to draft the manuscript and critically reviewed it. ML conceived the study, participated in its design, and coordinated the manuscript final version. All authors read and approved the final manuscript.

Electronic supplementary material


Additional File 1: contain portion of Methods' section and include details on modification of native proteins used in the experiments and details of kinetic procedures. (DOC 30 KB)

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Gorrini, M., Lupi, A., Iadarola, P. et al. SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase. Respir Res 6, 146 (2005).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: