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Gomez Gil, L. (2009). The interaction between cholesterol and surfactant protein-c in lung surfactant (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté des sciences appliquées – Chimie, Bruxelles.

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D 03639

p6| O 3

Université Libre de Bruxelles Faculté des Sciences

Structure et Fonction des Membranes Biologiques



Ph.D. Thesis Leticia Gômez Gil

Director: Erik Goormaghtigh Co-director: Jésus Pérez Gil

Brussels, 2009




Captive Bubble Surfactometry






Lamellar bodies


Néonatal Respiratoty Dsitress Syndrome


Polyacrylamide Gel Electrophoresis












Respiratory Distress Syndrome


Sodium Dodecilsulfate


Surfactant Protein B


Surfactant Protein C


Phase-Transition Température




1.1. Lungs: Structure and Function... 2

1.2. Surface Tension and Pulmonary Surfactant...2

1.3. Pulmonary Surfactant: Composition... 3

1.4. Surfactant Protein B... 4

1.5. Surfactant Protein C... 5

1.6. Cholestérol and Surfactant Protein C... 7

1.7. References...9


3. CHAPTER I: Pulmonary Surfactant Protein SP-C Reduces The Deleterious Effects Of Cholestérol On The Activity Of Surfactant Films Under Physiologically Relevant Compression-Expansion Dynamics...14

Submitted to BiophysicalJoumal, 04/09. MS ID#: BIOPHYSJ/2009/159921

Abstract... 15

1. Introduction... 15

2. Materials and Methods...17

3. Results... 19

4. Discussion...33

5. References...38

4. CHAPTER II: Cholestérol Modulâtes The Exposure And Orientation Of Pulmonary Surfactant Protein SP-C In Model Surfactant Membranes... 41

Biochimica et Biophysica Acta. 2009, May 21 (Epub). PMID 19464999.

Abstract... 42

1. Introduction...42

2. Materials and Methods... 44

3. Results... 47


4. Discussion. 56

5. Référencés...61

5. CHAPTER III: Cloning And Production Of Single-Cysteine Mutants Of Recombinant SP-C And Their Characterization By EPR... 65

Abstract... 66

1. Introduction... 66

2. Materials and Methods... 67

3. Results... 70

4. Discussion... 74

5. Référencés... 77



1. Introduction


1. INTRODUCTION 1.1. Lungs: Structure and Function

Lungs are the essential respiration organs for most air-breathing animais. They transport oxygai from the atmosphère into the bloodstream and release carbon dioxide from the bloodstream into the atmosphère. Human beings hâve two lungs, divided into lobes, located within the chest cavity, on either side of the heart (Figure 1). Atmospheric air enters through the trachea, which divides into two bronchi. Each bronchus branches successively into bronchioles and alveoli. The average adult's lungs contain about 600 million of these tiny thin-walled air sacs where the exchange of gases is accomplished. Alveoli are surrounded by a dense network of blood vessels, or capillaries, which connects to the heart. Oxygen passes through the walls of the alveoli into the capillaries and is then carried back to the heart via the pulmonary veins. At the same time, carbon dioxide is removed from the blood through the same process of diffusion.

Figure I. The structure of lungs and their location within the human body. A. Air entCTS through the trachea, which divides into bronchi and bronchioles, and finally reaches the alveoli.

B. A detailed view of the bronchioles and alveoli. Taken from [1].

Breathing is controlled by the brain, which saises changes in gas concentrations, and driven by the diaphragm. When we breathe in, this group of muscles between the chest and the abdomen contracts, forcing the lungs to inflate with the air. When we breathe out, the diaphragm relaxes and the lungs deflate.

Lungs can process 15,000 liters of air each day under resting conditions, which can be up to 50,000 liters/day under hi^ly demanding exercise . This constant exposure to the extemal environment makes lungs vulnérable to a range of illnesses, particularly to those resulting from bacterial or viral infection or offiCT environmental insults.

1.2. Surface Tension and Pulmonary Surfactant

Molécules in a liquid State expérience strong intomolecular attractive forces. At the air- liquid interface, liquid molécules are not attracted as intensely by molécules in the neighbouring


medium, the air, and are pulled inwards by other molécules deeper Inside the liquid. The molécules will squeeze together until a minimum surface area is reached. The surfece tension, measured as force per unit of length, quantifies the energy required to expand the surfece area.

Water has a surface tension of 72 mN/m at 37°C, but this surfece tension can be modulated by the presence of different solutés. Surfactants, or surfece-active agents, are defined as molécules that lower surface tension to a minimum, either at a liquid-gas or a liquid-liquid interface. The internai side of alveoli, covered by a thin layer of aqueous fluid, is coated by a complex mixture of lipids and proteins, known as pulmonary surfactant, which reduces surface tension at the alveolar air-liquid interface of lungs, avoiding alveolar collapse at the end of expiration and fecilitating the work of breathing (reviewed in [2]). The three main requirements for an efficient lung surfactant would thus be:

a. Rapid adsorption into the air-liquid interface.

b. Réduction of the surfece tension to values close to 0 mN/m upon compression of the lungs, during exhalation.

c. Efficient re-spreading during expansion, upon inspiration.

Although the phospholipid fi’action within lung surfactant, as explained below, is essentially responsible for the maximal surfece tension réduction upon compression, the presence of proteins, particularly SP-B and SP-C, is absolutely required for interfacial adsorption, film stability and re-spreading capacities [2, 3]. Pulmonary surfectant is synthesized in Type 13 cells, which rqjresent about 3% of the total alveolar surfece. The other 97% of alveolar epithelium is composed by Type I cells, forming a thin layCT whidi is very permeable to gases. Once it is synthesized, surfectant adsorbs rapidly and eflficiently at the liquid-air interface in the inner side of the alveoli.

1.3. Pulmonary surfactant: composition

Pulmonary surfactant, in most mammals, is composed of about 90% lipids, mainly phospholipids, and 8-10% proteins. About 80% of surfectant by mass is composed of phosphatidylcholine (PC), about half of which is dipalmitoylphosphati^lcholine (DPPC) [4].

The acidic phospholipids phosphatidylglycerol (PG) and phosphatidylinositol (PI) account for 8-15% of the total surfectant phospholipid pool. Cholestérol is the major neutral lipid component, accounting for up to 8-10% of surfectant by weight, or as much as 20%

cholesterol/phospholipid molar ratio. The phospholipid fraction within lung surfectant, and particularly DPPC, is essentially responsible for the maximal surfece tension réduction upon compression. The melting température of DPPC is 41°C, which is higher than that of most phospholipids in animal cell membranes. At the physiological température of 37®C the acyl


chains of DPPC are still in a gel State and they are rigid aiough to sustain large pressures at the end of expiration, stabilizing the lung at those compressed States.

Overall, the main fimction of unsaturated phospholipids is to reduce the melting point of the surfactant membranes so they are liquid and thus dynamic at physiological températures.

Moreover, these lipids increase the spreadibility of the material, whidi is largely reduced in pure DPPC membranes [5, 6]. Saturated and unsaturated lipids are organized in surfectant membranes in such a way that surfectant exhibits simultaneously the dynamic properties of fluid membrane régions enriched in unsaturated species and the mechanical stability provided by segregated DPPC-enriched domains [7].

Surfactant proteins represent about 6-8% of the total surfactant wei^t. There are four surfactant proteins, the hydrophilic SP-A and SP-D, and the hydrophobie SP-B and SP-C (reviewed in [2]). SP-A and SP-D account for about 5 and 0.5% of the surfectant dry weight, respectively. Both of them belong to the collectin fàmily of proteins, although SP-A is strongly associated with surfactant phospholipids and hydrophobie proteins and SP-D is not. Their rôle in lung surfactant is related mostly with innate host defense mechanisms [8], contributing to maintain the stérile condition of the respiratory surfece. Neither of them is directly related to the formation of interfàcial films or the réduction of surface tension at the alveoli. SP-A or SP-D knock-out mice are more susceptible to infections than wild-type animais, but they don't sufiFer fi-om any apparent respiratory dysfimetion [9,10].

The hydrophobie proteins in surfactant, SP-B (dimer, ~ 18 KDa) and SP-C (~ 4.2 KDa), are synthesized as large precursors that imdergo post-translational processing. Both proteins play critical rôles in formation and stabilization of pulmonary surfactant films [11] and are absolutely required for interfecial adsorption, film stability and re-spreading capacities [2].

1.4. Surfactant Protein B

Surfactant Protein B is synthesized in pneumocytes as a 381 residue precursor, proSP-B, which is processed along its transit through the trans-Golgi network and the multivesicular bodies until being assembled into surfectant membranes in the lamellar bodies. The final processed form is a dimer of approximately 18 KDa and 158 residues. The monomers are bound together through an intermolecular disulfide link in cysteine 48 and each of them contains three additional intramolecular disulfide bridges in positions 8-77, 11-71 and 35-46, respectively. SP- B has been classified, by sequence homology, as one of the members of the saposin-like femily of proteins [12]. Although the three-dimensional structure of SP-B is still unknown, the common topology model for these proteins suggests the presence of four-helices, some of them amphipathic, which are lined in SP-B by residues 8-22, 27-38,42-50 and 67-74 [13].


Figure 2. Model for the structure of pulmonary surfactant protein SP-B. A. Model of the structure of SP-B according to the saposin-like folding. B. Potential disposition of SP-B associated with surfectant bilayers and monolayers. Taken from [2].

Although its molecular mechanism is still unknown, it is clear that SP-B plays a critical rôle in adult lung function. Deficiency of SP-B in humans and in SP-B knock-out mice causes léthal respiratory distress [14-16]. The tmiporary loss of SP-B in adult mice also results in respiratory fàilure, regardless of the presence of SP-C and an appropriate lipid composition [17], The presence of SP-B is also required for the synthesis and packing of pulmonary surfectant [18-20] and for the processing of SP-C. Indeed, the absence of SP-B in transgenic mice leads to a deficiency of mature SP-C in alveolar airspaces [11, 14, 21], caused by an incomplète Processing of the SP-C precursor protein.

1.5. Surfactant Protein C

SP-C has been considered as a relatively modem protein in évolution, as it was only detected in mammals, until very recently; the expression of SP-C has now been reported in amphibian lungs as well [22]. The protein is therefore présent in ail types of alveolar lungs, but not in the tubular airspaces of birds, indicating that SP-C mi^t be specifically related with the physical stabilization of sacular gas-exchangjng organs. It is an int^ral membrane protein which consista of 35 residues, with a hydrophobie transmonbrane domain and a 10-12 amino acid N-terminus extramembrane segment (Figure 3A). The sequence of the protein is highly conserved through different species, and, in most of them, there are two palmitoyl groups esterifying Cys at positions 5 and 6 (reviewed in [23]) . Human SP-C is synthesized in the endoplasmic réticulum (ER) as a propeptide of 197 amino acids and approximately 21 KDa [24]. The mature protein is obtained after cleavage of the N- and C-terminal ends. The protein goes through the Golgi apparatus, where palmitoyl chains are attached, and moves toward the multivesicular bodies (MVB) to finally be stored at the lamellar bodies (LB). As mentioned in section 1.4, the processing of SP-C is dépendent on the presence of the precursor of SP-B, proSPB [14,21].


The three-dimensional structure of SP-C has been d^ainined by NMR in chloroform/methanol [23]. The transmembrane domain consists of a highly structured a-helix, whose length matdies that of a fluid DPPC bilayer (37 Â), while the Nt segmait is unstructured in this solvent.

Unlike SP-B, deficiency of SP-C in knock-out mi ce (SP-C-/-) is not léthal, but induces pneumonitis and other chronic respiratory diseases at short or long term after birth, depending on genetic background [25, 26]. The protein promotes formation of interfacial phospholipid films [27-30], possibly through perturbation of surfectant bilayers and films by the N-terminus segment, which is the most dynamic part of the molécule. Recent studies using synthetic Nt peptides hâve shown the affinity of this segment toward the interfaces of both phospholipid membranes and monolayers [31]. This partitioning is observed regardless of the palmitoylation degree of the protein and it can afifect lipid packing and membrane permeability, which could be exhibited towards either the same phospholipid membrane where the a-helix is inserted or towards a second bilayer. However, non-palmitoylated SP-C is not able to remain associated with phospholipid films when these are hi^y compressed, i.e. when the lowest surface tensions are reached at the end of expiration [32]. The suggested rôle of the protein within surfactant is illustrated in the cartoon of Figure 3. The palmitoylated N-terminal segment would maintain the association of membrane-based surfectant réservoirs with the interfacial film at the end of ejq^iration, therefore fecilitating the reinsertion of surface active molécules fi'om the réservoirs during re-expansion [33-36].

Fig. 3. A model for the structure and activity of pulmonary surfactant protein SP-C. A.

The structure of the protein as determined in organic solvents. The palmitoylated N-taminal segment of the protein would promote phospholipid adsorption and re-insertion (B) and maintain the association of surfactant réservoirs with the interfacial film at the end of expiration (C). Taken from [2].

SP-C shows a complex oligomérization behavior and a transition to p-amyloid-like fibril structures which are not yet flilly understood. According to a model published by Johansson et al. [37], monomeric a-helical SP-C would aggregate through a non- helical Intermediate into


higher- order structures, ultimately fibrils. The presence of the palmitoyl groups stabilizes the a- helix and thus prevents the formation of such fibrils. The formation of fibrils is not limited to in vitro situations, but also occurs in association with pulmonary alveolar proteinosis (PAP) [38].

Recent research by the same group has proved the anti-amyloid effect of the C-taminal domain of proSP-C (CTC), which could hâve an active rôle in protecting the transmanbrane part of SP-C fi'om aggregation. CTC can also interact with the transmembrane sèment of other proteins, such as the P-amyloid associated with Alzheimer’s disease and medin [39].

The existence of an SP-C spécifie a-helical dimer at low to neutral pH has also been suggested by Mass Spectometry and diemical CTOss-linking data combined with CD spectra [40]. Ccxnputational doddng of two SP-C helices reveals the dimerization potential of a SP-C helix-helix interface that resembles that of glycophorin A and is mediated by an AxxxG motif similar to the experimaitally determined GxxxG pattern of glycophorin (Figure 4).

Dimerization may therefore be important for the fimetion of this protein in surfactant.

Monolayer Water

Nt Nt

Figure 4. Suggested SP-C dimers in a phospholipid multilamellar structures. The C- terminus of SP-C would be located in the hydrophobie core of multilamellar surfactant arrays and stabilized by the dimerization. Modified fi'om [40].

1.6 Cholestérol and Surfactant Protein C

As stated in section 1.3, cholestérol is the major neutral lipid component, accounting for iç» to 8-10% of surfectant by weight, or as much as 20% cholesterol/phospholipid molar ratio.

However, its functicmal rôle within lung surfectant has long been debated and remains unknown. Due to its molecular structure, cholestérol contributes to diflferent flinctions such as stabilizing and fiuidizing lipid mono- and bilayCTS [34]. It has been proposed that the hydroxylic group of cholestérol interacts with the headgroups of the lipids and prevents SP-C fi'om aggregating, leading to a better miscibility of the protein into lipid/protein complexes [41]. It has also been proved that cholestérol maintains the native structure of pulmonary surfectant membranes, promoting the coexistence of two distinct micrometer-sized fluid phases (fluid


ordered and fluid disordered-like) at physiologjcal températures [7]. The hydrophobie proteins SP-B and SP-C are located exclusively in the fluid disordered-like phase, and it has been suggested that this latéral structure sustained by lung surfactant under physiological conditions provides particular structural and dynamic properties for its medianical fonction. The presence of cholestérol also introduces an intrinsically dynamic component to the behavior in ternis of order, mobility and latCTal diffusion of surfactant phospholipids in the different surfectant membrane phases [42],

With respect to the structure of pulmonary surfactant into^facial films, cholestérol has been also shown to introduce a particularly dynamic contribution. Native surfactant films or films formed fi-om the whole surfectant hydrophobie fiaction, including cholestérol, exhibit complex compression-driven latéral transitions, including ségrégation and remixing of phases, whose molecular nature is not understood but that are profoundly altered when cholestérol is removed [43-45].

Recent studies suggest that physiological levels of cholestérol, up to 10% by weight with respect to phospholipids, may play important rôles in modulating surfectant function [46- 48]. The baieficial function of physiological diolesterol levels within surfectant has been clearly established for heterothermic animais, such as dunnarts or bats. In these animais, cholestérol concentration varies rapidly in response to body température changes [49].

CholestCTol is also involved in lung stabilization in some deep-diving mammals [50]. However, several studies hâve reported that the ability of different surfectant models to reach minimum surface tension during different compression-expansion régimes is markedly impaired in the presence of cholestérol [51-55]. Furthermore, the impaired surfece activity of surfectant obtalned fi’om patients of ARDS has been in part attributed to an elevated contmt of neutral lipids, mainly diolesterol [56]. Thus, cholestérol has traditionally been removed fi-om most clinically used surfectants and it is not added to artifidal surfectants.

The existence of SP-C/cholesterol complexes in membranes has been suggested in the past [34, 41], althou^ direct évidences are lacking, as well as a model including the potential implication of SP-C/cholesterol interactions in surfectant function. It also seems that concomitant changes in the concentration of cholestérol and SP-C occur in lung surfectant of heterothermic animais shifting between a warm-active and a torpid State, such as dunnarts or bats [57], suggesting potential connected rôles of these two molécules in surfectant. Recent experiments indicate that the presence of SP-C shifts phase diagrams of some lipid model membranes towards régimes corresponding to lower cholestérol contents, which would be consistent with SP-C directly complexing cholestérol [58].



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The rôle of cholestérol within lung surfectant fùnction remains unknown. So does the possible interaction of the former with hydrophobie protein SP-C. The main goal of the présent study was to gain fnrther knowledge on the occurrence and potential significance of interactions between cholestérol and SP-C through the study of the structure, disposition and dynamics of SP-C in model surfectant membrane environments containing different concentrations of cholestérol.

Herein, we report the thermodynamic characterization of model membranes containing cholestérol and SP-C and a fimctional study of these membranes by Captive Bubble Surfectometry (Chapter I). We also report the analysis of the effect of cholestérol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes (Chapter II) and the cloning and expression of single-cysteine recombinant SP-Cs for their fùrther study by ESR (Chapter EŒ). The results are discussed in each chapter, in ternis of a combined rôle of both components in lung surfectant and the possible modulation of the effect of cholestérol in surfectant by SP-C.


Chapter I.

Pulmonary Surfactant Protein SP-C Counteracts The Deleterious Effects Of Cholestérol On The Activity Of

Surfactant Films Under Physiologically Relevant

Compression-Expansion Dynamics



The presence of cholestérol is critical in defining a dynamic latéral structure in pulmonary surfactant membranes, including the ségrégation of fluid-ordered and fluid-disordered phases.

However, an excess of cholestérol has been associated with impalred surface actlvity both in surfactant models and in surfectant from injured lungs. It has also been reported that surfectant protein SP-C interacts with diolesterol in lipid/protein interfecial films. In the présent stucfy, we hâve analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholestérol and on the ability of lipid/protein complexes containing surfactant proteins and cholestérol to form and re-spread interfecial films capable of producing very low surface tensions upon répétitive compression-expansion cycling.

SP-C modulâtes the effect of cholestérol to reduce the enthalpy associated with the gel-to- liquid-crystalline melting transition in dipalmitoylphosphatidylcholine (DPPC) bilayers, as analyzed by differential scanning calorimetry. Incorporation of 1% or 2% SP-C (protein/phospholipid by weight) promotes almost instantaneous adsorption of suspensions of DPPC/palmitoyloleoylphospatidylcholine(POPC)/palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15, w/w/w) into the air-liquid interface of a captive bubble, both in the absence and in the presence of cholestérol. However, cholestérol significantly impaired the ability of SP-C- containing films to achieve and sustain very low surface tensions in bubbles subjected to compression-expansion cycling. Cholestérol also impaired substantially the ability of DPPC/POPC/POPG films containing 1% surfactant protein SP-B to mimic the interfacial behaviour of native surfectant films, which are characterized by very low minimum surface tensions with only limited area change during compression and practically no compression- e}q)ansion hystérésis. However, simultaneous presence of 2% SP-C practically restores compression-expansion dynamics of cholestérol- and SP-B-containing films to the efficient behaviour shown in the absence of cholestérol, suggesting that coopération between the two proteins is required for lipid-protein films containing cholestérol to achieve optimal performance under physiologically relevant compression-expansion dynamics.


Pulmonary surfectant is a complex mixture of lipids and proteins whose main fùnction is to reduce the surface tension at the alveolar air-liquid interfece of lungs in order to avoid alveolar collapse at the end of ejq)iration and to fecilitate the work of breathing. The lack, deficiency or inactivation of this essential material is the cause of severe respiratory disorders, sometimes léthal, such as the Néonatal Respiratory Distress Syndrome (NRDS) [59] or the pulmonary dysfunction associated with the Acute Respiratory Distress Syndrome (ARDS) in cases of lung injury [60].


Pulmonaiy surfactant, in most mammals, is composée! of about 90% lipids, mainly phospholipids, and 8-10% proteins. About 80% of surfectant by mass is composed of phosphatidylcholine (PC), about half of which is dipalmitoylphosphatidylcholine (DPPC) [4].

The acidic phospholipids phosphatidylglycerol (PG) and phosphatidylinositol (PI) account for 8-15% of the total surfectant phospholipid pool. Cholestérol is the major neutral lipid component, accounting for up to 8-10% of surfactant by weight, or as much as 20%

cholesterol/phospholipid molar ratio. The phospholipid fraction within lung surfactant, and particularly DPPC, is essaitially responsible for the maximal surfece tension réduction upon compression. However, the presence of proteins, particularly SP-B and SP-C, is absolutely required for interfacial adsorption, film stability and re-spreading capacities [2, 3].

SP-B (dimer, ~ 18 KDa) and SP-C (~ 4.2 KDa), are very small hydrophobie polypeptides, which resuit from the proteolytic processing of larger precursors along the exocytic pathway of pulmonary surfectant in type II pneumocytes. Each of then accounts for no more than 1-1.5% of total surfectant weight but plays critical rôles in formation and stabilization of pulmonary surfactant films [11]. Thus, SP-B deficiency in humans [15] and in SP-B knock-out mice [14, 16] causes léthal respiratory failure at birth. The temporary loss of SP-B in adult mice also results in severe surfactant dysflmction and respiratory feilure in spite of maintenance of SP-C and surfectant lipid composition [17], confirming a critical rôle of SP-B in adult lung function.

The second hydrophobie protein, SP-C, is not absolutely required for survival.

However, targeted délétion of the SP-C gene (SP-C^') in mice leads to the development of severe progressive pneumonitis within a shorter or longer period of time after birth, depending on the genetic background [25, 26]. SP-C is an intégral membrane protein, which consists of a hydrophobie transmembrane hélix and a 10-12 amino acid extramembrane segment, which includes 2 palmitoylated Cys at positions 5 and 6 in most species, located at the N-terminus of the mature peptide. Expa-imental data suggest that SP-C, principally via its palmitoylated N- terminal segment, would be required to maintain the association of surfectant complexes (the réservoir) ^vith the interfecial film at the most compressed States, those reached at the end of ejqjiration [2]. The SP-C-promoted attachment would then fecilitate the reinsertion of surface active molécules from the réservoirs, with the probable critical participation of SP-B, during re­

expansion [2, 33-36].

Some authors hâve suggested in the past the existence of SP-C/cholesterol complexes in membranes [34, 41], although direct évidences are laddng as well as a model including the f)otential implication of SP-C/cholesterol interactions in surfactant fimetion. Some modulating effects hâve been proposed for cholestérol on the general properties of surfactant layers, mainly through the stabilization and fluidization of lipid mono- and bilayers [34]. The hydroxylic group


of cholestérol interacts with the headgroups of the phospholipids and could contribute to prevent SP-C from aggregating, leading to a better miscibility of the protein into monolayers [41]. On the other hand, it seems that concomitant changes in the concentration of cholestérol and SP-C occur in surfactant of heterothermic animais, sudi as dunnarts or bats [57], which shift between a warm-active and a torpid State, suggesting potential connected rôles of these two molécules in surfactant.

The main goal of the présent study is thus to gain fiirther knowledge on the occurrence and potential significance of interactions between cholestérol and SP-C. Herein, we report the thermodynamic characterization of model membranes containing cholestérol and SP-C and a fiinctional study of these membranes. The results are discussed in terms of a combined rôle of both components in lung surfactant and the possible modulation of the effect of cholestérol in surfactant by SP-C.


Materials. l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 [phospho-rac-( 1 - glycerol)] (POPG) and cholestérol were from Avant! Polar Lipids (Alabaster, AL). Chloroform and methanol solvents, HPLC grade, were from Scharlau (Barcelona, Spain). The palmitoylated N-terminal peptide from SP-C (LRIPCpaimCpaimPVNLKRL) was synthesized by Dr. David Andreu, from Pompeu Fabra University, as previously described [32]. Native surfactant was purified from porcine lung lavage as previously described [61]. Surfactant proteins SP-B and SP-C were isolated from minced porcine lungs as described elsewhere [62]. Quantitation of purified proteins was achieved by acidic hydrolysis and amino acid analyses.

Reconstitution of lipid and lipid/protein samples. The standard lipid mixture used in this study to reconstitute pulmonary surfactant model membranes contained DPPC/POPC/POPG (50/25/15, w/w/w). This mixture simulâtes the proportion of saturated/unsaturated and zwitterionic/anionic phospholipids in surfectant. The mixture was modified by adding different proportions of diolesterol and surfactant proteins and peptides, described in each set of ejqjeriments. Multilamellar suspensions were prepared by mixing the appropriate amount of protein and lipids in chloroform/methanol 2:1; samples were dried ovemight under vacuum and resuspended in the desired final volume of 5 mM Tris/HCl buffer (pH 7) containing 150 mM NaCl, with incubation for Ih at 51°C with occasional vortexing.

Differential scanning calorimetry. To investigate the gel to liquid-crystalline phase transition of DPPC in the presence or absence of SP-C and/or cholestffol, excess beat capacity (Cp) was measured at constant pressure in a Microcal VP-DSC differential scanning microcalorimeto' (Northampton, MA). Ail DSC experiments were performed using DPPC or


DPPC/SP-C multilamellar suspensions (50:1 or 10:1, w/w) at a final phospholipid concentration of 0.5 mg/ml. The vesicles contained 0, 3, 5, 10 or 20% cholestérol to total phospholipid (w/w).

Ail the samples were degassed before calorimetric measurements. At least six buffer versus buffer scans were performed to obtain a reproducible baseline. After the reference measurements, the sample cell containing 0.6 mL of the desired lipid or lipid/protein suspension and the reference cell filled with the same volume of buffer solution were heated fi’om 25 to 85

°C at a heating rate of 0.5 “C/min. An average of 12 scans were obtained fi'om each experiment.

The calorimetric data were analyzed using MicroCal ORIGIN software (MicroCal Software Inc.). Ail the thermograms were obtained fi'om the calorimetric profiles after baseline correction and concentration normalization. The molar enthalpy of the phase transition, AH, was obtained fi'om the area under the main DSC peak and the concentration of lipids in each sample. The température of the transition, Tm, was here reported as the température at which Cp is maximum.

Captive bubble surfactometry. The surface activity of the model surfectant mixtures was determined using a fidly computer-controlled Captive Bubble Surfactometer (CBS), built for us by Prof Sam Schürch and Dr. Michael Schoel, from University of Calgary. This device has been described in detail elsewhere [63, 64] and allows a reliable and reproducible estimation of the surface taision (y) of surfactant films subjected to compression-expansion dynamics. We hâve used a modification of the procedure as described elsewhere [65]. The chamber of CBS was filled with 1.5 mL of a solution 5 mM Tris, 150 mM NaCl, pH 7.0. Sucrose (10% w/w) was added to the buffer to increase its density so that the model surfectant suspensions would float and remain in contact with the bubble upon Injection. The presence of sucrose does not affect surface activity of surfactant [65]. The température was maintained between 36.8 and 37.1 °C.

To start each experiment, a small air bubble (approx. 50 pL) was introduced into the chamber and allowed to fioat up to the diamber's concave agarose ceiling. Then, ~250 ni of lipid or lipid/protein surfectant suspension (10 mg/ml) were deposited directly at the air-bufifer interface of the bubble by means of a thin transparent capillary. This allowed a precisely defined volume of surfectant to be dqjosited under visual control. The bubble was imaged by a video caméra (Pulnix TM 7 CN) and recorded for later analysis. A 5 min period followed the introduction of the surfectant model suspensions into the diamber during which the bubble was not manipulated and the change in y was monitored to follow film formation. The chamber was then sealed and the bubble was rapidly (1 s) expanded to a volume of 0.15 ml. Five min after the bubble was expanded (to permit for post-expansion adsorption and film équilibration) quasi- static cycling commenced. The bubble size was first reduced and then enlarged in several subséquent steps, each including a change in volume and 5 s of delay. Compression was stopped when minimum surfece tension was obtained, as seen when the bubble height no longer


decreases upon volume réduction, but only the diameter. Along these quasi-static compression- expansion isothams the film was allowed to relax, in contrast to the isotherms obtained in the dynamic cycling mode. There was an intercycle delay of 0.5 min between each of the four quasi-static cycles and a further 2 min delay between the quasi-static and the dynamic cycles. In the latter, the bubble size was continuously varied for 20 cycles at a rate of 20 cycles/min.

Bubble volume and area and surfece tension were calculated fi'om examination of the shape of the bubble in the video recordings, using an axisymmetric model as previously described [66].

At least three independent experiments wa-e performed for each surfectant model suspension, using at least two different batches of purified protein, with qualitatively ccanparable results.

Figures represent illustrative example experiments of each surfectant model.


We hâve investigated the thamotropic liquid-crystalline to gel transition of DPPC bilayers in the absence or presence of SP-C and cholestérol in order to détermine the nature of the interaction between SP-C and lipids in the model membranes fi'om a thermodynamic point of view. Figure 1 shows the efifect of the presence of increasing proportions of cholestérol on the thermotropic behaviour of DPPC membranes, either in the absence or presence of SP-C 2%

OT 10% (protein to lipid, w/w), équivalent to 1:273 and 1:55 protein to lipid molar ratios, respectively.


2% 10%

Figure 1. Thermotropic behaviour of DPPC bilayers in the presence of Cholestérol and SP-C. Diflferential Scanning Calorimetry thermograms of DPPC multilamellar suspensions hâve been obtained in the absence (left) or in the presence of 2% (center) ot 10%

(right) protein-to-phospholipid by weigjit of porcine SP-C, containing the indicated proportions of cholestérol (w/w with respect to phospholipid).


The phase transition of pure DPPC bilayers is abrupt, as demonstrated by the peak sharpness. The effects of a graduai increase of the concentration of cholestérol on the thermotropic behaviour of the DPPC membranes, include the disappearance of the Lp-Pp pretransition peak, which occurs at approximately 35 “C for pure DPPC, the broadening of the main transition, which exours at 41 “C, and a remarkable réduction of the transition enthalpy, revealed by a decrease in the area under the main calorimetric peak. In the presence of SP-C the broadening of the transition is even more noticeable, even though the effects of both manbrane components are not additive, as shown in Figure 2 and Table 1.

Table 1 summarizes the parameters that define the main phase transition of the assessed lipid and lipid/protein samples as determined experimentally by DSC. As described above, increasing concentrations of cholestérol produced a progressive decrease in the main transition enthalphy, with slight effects on the Tm at which the transition exxurs, either in the absence or in the presence of SP-C. The combined efifect of cholestérol and the protein requires further analysis.


(% w/w) AH (Kcal/mol) Tm fC)

DPPC . 7.1 ±0.06 41.4

3 6.5 ±0.13 40.7

5 3.9 ±0.13 43.7

10 2.1 ±0.05 40.3

20 0.56 ±0.15 41.4

DPPC/SP-C 2% w/w - 6.4 ±0.5 40.2

3 5.1 ±0.09 39.1

5 3.9 ±0.36 39.8

10 2.02 ± 0.6 41.3

20 0.89 ±0.1 41

DPPC/SP-C 10% w/w - 4.1 ±0.05 41.2

3 3.2 ± 0.03 41.5

5 2.8 ±0.6 41.6

10 2.5 ±0.7 41.0

20 0.6 ±0.3 41.0

Table 1. Thermcxlynamic parameters of the main calorimetric transition of DPPC bilayers in the absence or presence of cholestérol and SP-C.

Figure 2A plots the enthalpy of the phospholipid phase transition, calculated pw mol of DPPC, as a fùnction of the DPPC/cholesterol molar ratio, in the presence and absence of SP-C 2% or 10% (protein to phospholipid, w/w). The presence of diolesterol within the DPPC membranes reduced progressively the enthalpy associated with transition in a linear fashion. In the presence of SP-C, the interaction followed the same linear trend seen at high proportions of cholestérol. However, at lower concentrations of cholestool, the presence of the sterol does not seem to significantly reduce the enthalpy associated with phase transition in the lipid/protein


membrane complexes. The amount of cholestérol above which the calorimetric enthalpy of the lipid complexes is again reduced dépends on the proportion of SP-C présent. Cholestérol proportions higher than 10% (mol/mol) with respect to phospholipid are required to reduce the calorimetric enthalpy in membranes containing 2% SP-C, while only proportions of cholestérol hi^er than 20% reduce substantially the enthalpy of membranes containing 10% SP-C. The concentration of cholestérol needed to significantly affect the thermodynamics of the System, likely through formation of phospholipid/cholesterol-enriched manbrane riions, is thus higher in the presence of a higher concentration of protein, which may indicate a direct interaction between these two membrane components.

SP-CDPPC (nmol/mmol)

Figure 2. Dependence of the phase transition enthalpy of DPPC bilayers on the cholestérol content, in the absence or presence of SP-C. A) The enthalpy associated with the main phase transition of DPPÇ bilayers, determined from the area under the main calorimetric peak in thermograms of figure 1, has been plotted against the DPPC:cholestCTol molar ratio, in the absence (diamonds) or presence of SP-C 2% (squares) or 10% (triangles) by weight with respect to phospholipids. B) Contour plot of values of équivalent enthalpy determined from the calorimethric behaviour of DPPC bilayers containing variable amounts of cholestérol and/or SP- C. Lines connect combined proportions of cholestérol and SP-C that produce the similar enthalpy value indicated in each line, once incorporated into DPPC suspensions.

The additivity of the effects of cholestérol and SP-C has been ftuther analyzed in the contour plot that represents the cholestérol and DPPC proportions producing équivalent enthalpy values, once included in DPPC membranes (Fig. 2B). Lines in this plot should be straight diagonal Unes connecting the two axes if the combined action of cholestérol and SP-C is merely additive. On the other hand, if the combined effect is not additive, eg. one of the components buffers the effect of the other, the Unes should be parallel to one of the axes. Figure 2 B illustrâtes how the presence of increasing amounts of cholestérol and DPPC produces non- additive effects, consistent with partial counteraction between SP-C and cholestérol with respect to their mutual effect on the transition enthalpy within DPPC membranes.


In order to characterize a possible modulation of the surfectant fonction by the interaction between cholesto-ol and SP-C, the activity of model surfectant membranes containing different concentrations of cholestérol and SP-C was evaluated in a captive bubble surfactometer (CBS). As a reference, Figure 3 shows the behavior of native pulmonary surfactant, as purified from porcine lungs, when applied onto the captive bubble under conditions mimicking exposure of alveolar inteifeces to thin surfectant-enriched aqueous layers [49]. Injection of 250 nL of concentrated surfectant at the surface of a 50 pL air bubble formed in a dense sucrose-containing subphase led to the formation of a thin water layer maintaining a higji surfectant concentration in contact with the air-water bubble interface. Under these conditions, native surfectant adsorbed to the interfece extraordinarily fest (Fig. 3A), producing equilibrium surface tensions close to 22 mN/m within the first minute. Compression of such adsorbed surfectant films under quasi-static conditions produced suifece tensions lower than 1 mN/m with only 20% of area réduction, while suifece tension hardly increased above 30 mN/m upon bubble expansion (Fig 3B). Furthermore, the compression and expansirai moieties of the cycling isothams showed almost no hystérésis, indicative of the extraordinary stability of the native surfactant film during cycling dynamics. This ranarkable behavior is even more pronounced when the films are subjected to rapid, physiological-like compression-expansion dynamics, at 20 cycles/minute (Fig 3C). The ability of surfectant to reach and sustain very low surface tensions with only limited area change during compression, the relatively low maximal surface tensions upon expansion, and the little hystérésis, are considered essential requirements of a functional lung surfectant. The compression-expansion isotherms of Figure 3 could therefore be considered représentative of the biophysical behavior expected of an eflficient pulmonary surfectant.

0.4 Relative Su rfece Area

Figure 3. Functional behaviour of native porcine surfactant in a captive bubble surfactometer. A) Time course for initial (•) and post-expansion (A) adsorption of native surfactant (250 nL, 10 mg/mL phospholipid) deposited at the captive bubble interface. B).

Cycles number !(♦), 2(b), 3(A) and 4(X) are represented. C) Compression-expansion isothams under dynamic cycling. Cycles number !(♦), 10(b), and 20(A) are represented.


Figure 4 shows the time course for the réduction of surface tension upon adsorption of model membranes consisting of the mixture DPPC/POPC/POPG (50/25/15, w/w/w), in the absence or in the presence of cholestérol 5 or 10% (w/w) and SP-C 1% or 2% (protein to phospholipid, w/w). Surfece tension decay is associated with film formation during adsorption of lipid or lipid/protein manbranes, eitha" after the initial spreading on the bubble interface or due to fürther adsorption upon bubble expansion. In the absence of protein, adsorption of pure lipid samples was limited, producing decays of surface tension to values around 45 mN/m, and furtha" impaired in the presence of cholestérol. Expansion of the film produced with these samples resulted in almost no fürther change in surface tension, likely because no fiirtho- lipid could be incorporated into the interfece. In contrast, ail the samples containing SP-C showed an initial sudden drop of surfece tension to values of ~ 25-30 mN/m, reflecting the rapid spreading and adsorption of the sample fi’om the point of contact along the whole interface, and continuing with a slower fürther drop of surface torsion to values of ~ 22 mN/m likely due to subséquent rearrangements within the lipid film. The minimum surface tensions reached were lowest in the absence of cholesto-ol and in the presence of the highest concentration of protein. The presence of cholestérol increased the minimal values of surface tension reached in the two proportions of sterol tested althou^ the observed différence was not significant in the case of SP-C 2%. The introduction of the higher concentrations of cholestérol, restored» at least partially, the adsorption to very low surfece tensions observed in the absence of cholestérol, possibly due to a direct eflfect of the sterol on the membrane fluidity or the ségrégation of membrane phases [7].

Film expansion, in the presence of SP-C, led to the adsorption of additional surfectant material associated with the surfece. In most cases, such post-expansion adsorption did not produce surfece tensions as low as those reached aftw initial adsorption, reflecting the limited amount of material associated with the interfece. The minimum surfece taisions observed post- eiqiansion in the presence of SP-C 2% were higher than those observed in the presence of SP-C 1%. In both cases, lower decreases in surfece tension occurred at cholestérol 5%, reflecting a lower efficiency in the insertion of new material from réservoirs. However, increasing the concentration of cholestérol fi-om 5 to 10% produced a slight improvement in the surface tension réduction.


InKiai Adsorption Post Expansion Acteorption

Figure 4. Effect of SP-C and cholestérol on the interfacial adsorption of phospholipids at a bubble air-liquid interface. In left panels, surfece tension versus time adsorption isotherms hâve been obtained after spreading 0.5 pL of a suspension 10 mg/mL of the mixture DPPC/POPC/POPG (50:25:15:1, w/w/w), in the absence (upper panels) or in the presence of SP-C 1 or 2% (w/w) (central and Iowctpanels, respectively), at the surface of a 50 pL air bubble formed into a subphase of 5 mM Tris, 150 mM NaCl, pH 7.0 containing sucrose (10% w/w). At the right panels, interfacial adsorption leading to a decrease in surface tension was monitored upon expansion of the bubble from 0.05 to a volume of 0.15 mL. The samples assayed contained 0 (•), 5 (o) or 10% (V) cholestérol to phospholipid by weight.

Figures 5 and 6 respectively, show the quasi-static and dynamic compression-expansion isotherms of films formed from suspensions of DPPC/POPC/POPG (50/25/15, w/w/w)


containing SP-C 1 or 2%, in the absence or in the presence of 5 or 10% cholestérol. In the presence of 1% SP-C, the filins were not able to reach low enough minimum surface tensions under quasi-static compression, either in the presence or in the absence of cholestérol, and the presence of increasing proportions of cholestérol produced progressively higher minimal tensions. Ail the isotherms presented conspicuous plateaus at around 22-23 mN/m, which hâve been interpreted as due to compression-driven three-dimensional transitions.


0% 5% 10%


Reiaiive Surface Area

Figure 5. EfTect of SP-C and cholestérol on the quasi-static compression-expansion isotherms of phospholipid films in a captive bubhie surfactometer. Quasi static compression-expansion isotherms of films adsorbed fi'om DPPC/POPC/POPG (50:25:15, w/w/w) suspaisions containing 1% (upper panels) or 2% (lower panels) SP-C and 0 (left), 5 (central) or 10% (right) cholestérol to phospholipid by weight. Plotted are isotherms obtained fi-om the 1“ (♦), 2"‘‘ (■), 3^“* (A) and 4* (X) sequential compression and expansion cycles.

It has been proposed that some films undergoing quasi-static compression may require such transitions to adiieve a configuration able to withstand low surface tensions without collapse (13). In the absence of diolesterol and in the presence of 1% SP-C, quasi-static


compression of DPPC/POPC/POPG films seems to proceed partially through such type of film transition, allowing progression to minimum surface tensions on the order of 13-15 mN/m, still hi^er than those required to properly stabilize the interface. Maximal surface tensions, reached at the end of expansion, were also high, suggesting that excluded material is not re-spreading into the interfece efficiently. Inclusion of progressively higher proportions of cholestérol impairs fiirther the ability of the films to progress towards very low surfece tensions, presumably because compressed films are not rigid aiough to sustain them. Interestingly, increasing proportions of cholestérol also reduce the maximal surface tensions achieved along the different quasi-static compression-expansion cycles, indicating that those relatively fluid compressed States can at least re-spread rapidly. In the absence of cholestérol and in the presence of SP-C 2%, the films had optimal surfece properties in ternis of their ability to reach close to zéro surfece tension upon compression. However, in the presence of cholestool 5 or 10%, the films were not able to reach surface taisions lowa than 5 and 20 mN/m, respectively, unda quasi-static compression, indicating again a delaerious eflfect of cholestaol.

When subjected to dynamic compression-expansion cycling, ail the films containing 1%

or 2% SP-C wae able to reach very low tensions, either in the absaice or presence of cholestérol (Figure 6). Howeva, the changes in relative surface area that wae required for such a low tension values to be readied wae considaable (see Table 2), and showed large compression-expansion hystaesis, reflecting low stabillty of compressed phases. Inaeasing even fiirther the concoitration of SP-C, up to 3 or 5% by weight with respect to phospholipids, did not re-establish the original behaviour of these films in the absence of cholestérol (Figure SI).



Cholesteiol (wiM

5% 10%

Figure 6. Efifect of SP-C and cholestérol on the dynamic compression-expansion isotherms of phospholipid films in a captive bubble surfectometCT. Dynamic compression- ejqjansion isotherms, obtained at 20 cycles/min, of films adsorbed fi'om DPPC/POPC/POPG (50:25:15, w/w/w) suspensions containing 1% (upper panels) or 2% (lower panels) SP-C and 0 (left), 5 (central) or 10% (right) cholestérol to phospholipid by weight. Plotted are isotherms obtained fi’om the Ist O, lOth (■), and 20th (A) sequential compression and expansion cycles.


Q-Stat, cycle 4 Dynamic, cycle 20


(% w/w) Omin (mN/m) (mN/m) . %compression* O min (mN/m) Om«(mN/m) %compression

1%SP-C - 16 ±0.5 67 ± 1.4 80 ± 0.9 7 ±0.4 72 ± 0.7 70 ± 2.1

5 20 ± 1.2 61 ± 0.8 71 ± 1 20 ± 0.8 75 ± 1.4 64 ± 1.6

10 21 ±2 35 ± 1.1 71 ± 1.3 6 ±0.7 73 ± 0.6 57 ± 2

2% SP-C - 2 ±0.3 57 ± 2.3 54 ± 0.4 3 ±0.4 69 ±0.9 58 ±0.7

5 9 ±0.6 58 ± 1 60 ± 0.5 7 ±0.5 66 ± 1 62 ± 0.9

10 18± 1.3 61 ± 1.4 78± 1.1 4 ±0.8 68 ±2 74 ± 1.4

1%SP-B - 2 ±0.2 25 ±0.7 22 ± 0.8 2 ±0.3 34 ± 1 18 ±0.7

5 8 ±0.4 64 ± 0.5 58 ±0.9 8 ±0.6 72 ± 0.8 51 ±0.6

1% SP-B + 2% SP-C 5 3 ±0.2 31 ±0.4 28 ±0.5 3 ±0.3 42 ± 0.7 23 ± 0.4

2.5 mol Nt SPC + 1% SP-B 5 22 ± 1.8 71 ±1.3 73 ±1.2 16± 1.1 73 ±2 79 ± 0.8

Table 2. Parameter defining the surface behaviour of DPPC/POPC/POPG films, in the absence or presence of variable amounts

of cholestérol and surfactant proteins SP-B, SP-C or an N-terminal synthetic SP-C peptide, upon quasi-static or dynamic compression-

expansion cycling in the captive bubble surfactometer.


Ouail Staitic Dynamic

Relative Suifac« Aiea

Figure SI. Quasi-static (left panels) and dynamic (right panels) compression-expansion isotherms of DPPC/POPC/POPG (50:25:15, w/w/w) films in the presence of 5% cholestérol to phospholipid ratio by wei^t and 3% (upper panels) or 5% (Iowctpanels) SP-C (protein to lipid, w/w). Quasi-static cycles 1®' (♦), 2"‘‘ (■), 3^“* (A) and 4* (X) and dynamic cycles 1®‘ (♦), lO"’ (■) and 20"’ ( A ) are represented.

The déficient surface properties of films containing SP-C as the only protein component and proportions of unsaturated lipids similar to those in natural surfactant membranes but higher than those included in most clinical surfectants, were typical of films lacking the main hydrophobie surlàctant protein, SP-B. For this reason, we repeated our experiments in the presence of 1% SP-B purified fi’om porcine lungs. Membranes containing SP-B or both SP-B and SP-C wa-e fimctionally tested in the CBS in the absence or presence of cholestérol 5%.

Figure 7 shows the time course of interfecial film formation fi'om SP-B-containing membranes, either during the initial or the post expansion adsorption. In the absence of cholestérol, SP-B promoted a very rapid adsorption of lipids into the interface, associated with a rapid decrease of surface tension to equilibrium values of around 22-23 mN/m, independently of the presence or not of SP-C. Introduction of cholestérol impaired significantly the ability of lipid-protein complexes containing SP-B to rapidly achieve low surfece tensions. This was particularly évident during post-expansion adsorption, where DPPC/POPC/POPG/Chol/SP-B samples did



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