From the
Pulmonary surfactant protein C (SP-C) is a small, extremely
hydrophobic peptide with a highly conservative primary structure. The
protein is characterized by two adjacent palmitoylated cysteine
residues, two positively charged residues (one arginine residue and one
lysine residue) in the N-terminal region, and a long hydrophobic
stretch. SP-C enhances the adsorption of phospholipids into an
air-water interface. To determine the importance of the positively
charged residues, we carried out experiments with natural porcine SP-C
and modified porcine SP-C (SP-C
Pulmonary surfactant, essential for breathing, is a complex
mixture of lipids and proteins. It is present at the air-liquid
interface of the lung, and its main function is to stabilize the lung
by reducing the surface tension. Surfactant consists of 90% lipids and
of 8-10% specific proteins(1, 2) . Four
surfactant-associated proteins have been described, of which two are
hydrophobic. These hydrophobic proteins, surfactant protein (SP)
SP-B is
essential for the reduction of surface tension in the lung. A recent
case report presented clinical evidence that deficiency of this protein
is life threatening(4) . If the activity of SP-B was blocked by
monoclonal antibodies, an acute inflammatory reaction was seen,
together with a decrease in lung-thorax compliance(5) . Evidence
has been presented that the activity of SP-B is enhanced by the
hydrophilic surfactant protein A(6, 7) .
SP-C, a
protein of 35 amino acid residues, is very hydrophobic due to a high
content of Val, Ile, and Leu (60% of sequence). The hydrophobic amino
acid residues form a long contiguous stretch, reaching from residue 13
to residue 28. The primary structure is highly conservative. In
monolayers(8) , as well as in bilayer
systems(9, 10, 11) , the secondary structure is
mainly
To
elucidate the importance of the positively charged amino acids for the
function or structure of the SP-C, we used the reaction of
phenylglyoxal with arginine residues(22, 23) . When
proteins are treated with a large excess of phenylglyoxal for a long
period, the
The proteins were separated from the lipids by Sephadex LH-60
chromatography after butanol extraction of pulmonary surfactant. The
fractions were analyzed by Tricine/SDS-polyacrylamide gel
electrophoresis under non-reducing conditions and colored by silver
staining. The SP-C fractions were pooled and, after modification,
analyzed on a reverse phase C
In this study, we investigated the role of the positively
charged residues in the SP-C protein. It has been proposed that the
positive charges play a role in the determination of the transmembrane
orientation(31) . Two positive charges, one lysine residue and
one arginine residue, are found on positions 11 and 12, respectively,
in a variety of species(14) . In this study, the positive
charges in the SP-C molecule were neutralized by a modification of the
methods described by Takahashi(22) . With this method, all
arginine is changed (23) into a sufficiently stable non-charged
derivative of arginine. As phenylglyoxal is somewhat less selective for
arginine than is e.g. butanedione, also the lysine residue
will be (partly) modified(32) . Amino acid analysis and HPLC
analysis proved that the majority of the arginine residues and part of
the lysine residues were modified. We show by analysis on a reverse
phase C
The insertion rate
of phospholipids from DPPC/PG (7:3, w/w) SUV into a monolayer of the
same composition containing natural porcine SP-C was calcium and
protein dependent. If negatively charged phospholipids were absent in
the monolayer, the insertion rate of lipids catalyzed by natural
porcine SP-C in the presence of calcium was much higher, due to an
increased association of vesicles with the monolayer. This causes
smaller distances, and as vesicle monolayer interactions are partially
determined by electrostatic forces, Van der Waals and hydration forces
will become more important(33) . With the use of
As the pK of the guanido group of arginine
is about 12.5 (32), the positive charge of the porcine SP-C can be
altered if the pH of the subphase is changed in Wilhelmy plate
experiments. The highest insertion rate of lipids from negatively
charged vesicles was found if a subphase was used with a pH of 7.0.
With increasing pH, the insertion rate decreased.
The reduced
insertion had to be the result of a reduced function of the SP-C after
changing the positive charges of the protein. Insertion of
phospholipids was possible, even at a pH of 11.0. This insertion was
induced by SP-C and started after the addition of calcium ions. This
indicated that there was no denaturation of SP-C. The expected increase
in
It has been
shown earlier that SP-C did not catalyze mixing of lipids of negatively
charged lipid vesicles(30) . If 300 nmol of non-charged vesicles
containing SP-C were mixed with pyrene-PC-labeled negatively charged
vesicles, SP-C-induced lipid mixing was possible(15) . In the
present paper, SP-C was present in negatively charged or neutral
vesicles. When 15 nmol of these were mixed with 300 nmol of negatively
charged vesicles, the relatively small quantity of SP-C was not able to
induce lipid mixing. The modified protein, however, was able to induce
lipid mixing, even in the absence of calcium. The mechanism by which
this process is possible is not yet understood.
In all experiments
after the addition of calcium, a decrease in excimer/monomer ratio was
observed. This indicates that the positive charge of Ca
In conclusion, the positive charges of the
pulmonary surfactant protein C are important both for structure and
function of SP-C. The positive charges are responsible for binding of
the PG-containing small unilamellar vesicles to the monolayer, a
process that precedes the insertion of phospholipids into the
monolayer.
Samples were collected at a surface pressure of
30 mN/m. Average numbers (± S.D.) derived from spectra of two or
three different monolayer preparations are given as percentages of
total structure.
) in which the positive
charges had been blocked by phenylglyoxal. Circular dichroism
experiments showed that SP-C
had an increased content of
-helix. Natural SP-C, but not SP-C
, catalyzed
insertion of phospholipids into a monolayer at the air-water interface.
This reduced insertion was due to a strong reduction of binding of
phospholipid vesicles to the monolayer. The insertion catalyzed by the
natural porcine SP-C was decreased by an increased pH of the subphase.
In contrast to natural SP-C, SP-C
induced lipid mixing
between phospholipid vesicles. The extent of lipid mixing was a
function of the SP-C content. We conclude that the positively charged
residues of SP-C are important for the binding of phospholipid vesicles
to the monolayer, a process that precedes the insertion of
phospholipids into the monolayer.
(
)B (SP-B) and surfactant protein C (SP-C), make up for
13% of the surfactant-specific proteins(3) .
-helical. Two palmitoylated cysteine residues can be found
on positions 5 and 6(12, 13) , with the exception of
canine SP-C, which has only one cysteine residue(14) . The exact
function of the acylation is not clear yet, but it has been
demonstrated that acylation of SP-C alters structure and physical
properties of the protein(15) . A dimeric form of SP-C has been
demonstrated, which exhibits surface tension-lowering properties
differing from those of monomeric SP-C(16, 17) . SP-C
from all known species has two positively charged residues on positions
11 and 12 (a lysine residue and an arginine residue,
respectively)(14) , of which the function remains to be
elucidated. SP-C, when present in phospholipid vesicles (18, 19) or monolayers(15, 20) , was able
to catalyze the insertion of phospholipids into a monolayer or to
induce the lipid mixing between neutral phospholipid vesicles and
pyrene-PC-labeled vesicles(15) . It also altered the
thermodynamic properties of membranes(11, 21) .
-amino group of the lysine residue may be modified
too, thereby neutralizing all positive charges of SP-C. The reaction is
very effective, and the formed derivative is sufficiently stable,
especially under mildly acidic conditions(22) . We used the
Wilhelmy plate method and circular dichroism and fluorescence
experiments to study the role of the positively charged residues of
SP-C. The experiments show that these residues are important to bind
vesicles prior to the insertion of phospholipids from the vesicles into
the monolayer.
Materials
SP-C was isolated from
porcine lung lavage. Fresh porcine lungs were lavaged three to five
times with a solution of 154 mM NaCl. The proteins were
isolated by the method of Hawgood and co-workers(24) .
Extraction of pulmonary surfactant was done with n-butanol(25) , which was later removed by rotary
evaporation. The residue was dissolved in chloroform/methanol/0.1 M HCl (1:1:0.05, v/v/v), and the solution was centrifuged to remove
the insoluble particles. Isolation and separation of SP-B and SP-C from
the lipids was done by Sephadex LH-60 chromatography. The proteins were
stored in a mixture of chloroform/methanol (1:1, v/v) at -20
°C. Fluorescamine was used to determine the concentration of the
proteins(26) . The content of arginine and lysine amino acid
residues was determined by amino acid analysis. Phospholipid phosphorus
was measured with the method of Bartlett(27) . L--Dipalmitoylphosphatidylcholine (DPPC) was bought from
Avanti Polar Lipids, Inc. (Alabaster, AL). L-
-Phosphatidyl-DL-glycerol and L-
-phosphatidylcholine were obtained from Sigma. L-
-Phosphatidylcholine 1-palmitoyl-2-(1-pyrenedecanoyl
(pyrene-PC) was purchased from Molecular Probes.
Modification of SP-C
To modify SP-C, 300
µg of porcine SP-C was dried under nitrogen and redissolved in 50
µl of a mixture of N-ethylmorpholine (final concentration
0.2 M) in water and n-butanol (1:9, v/v), pH 8.0.
After sonification for 15 s in a water bath sonicator, 50 µl of a
6% phenylglyoxal hydrate solution dissolved in the same buffer was
added, and the sample was vortexed. After 60 h (25 °C), the buffer
was replaced by 200 µl of chloroform/methanol/0.1 M HCl
(1:1:0.05, v/v/v). The reagents were removed from the protein by
passage of the reaction mixture through a Sephadex G-25 column. The
modified protein was stored under mildly acidic conditions to prevent
the decomposition of the derivative(22) .
SDS-Polyacrylamide Gel
Electrophoresis
Electrophoresis of the protein was
performed by one-dimensional Tricine/SDS-polyacrylamide gel
electrophoresis(28) . Staining of the protein was done with
silver stain (Bio-Rad).
HPLC
SP-C was analyzed on a reverse phase
C column as described earlier(13, 15) .
Monolayer Studies
Lipids (DPPC/PG, 7:3
(w/w)), dissolved in chloroform/methanol (1:1, v/v), were dried under a
continuous stream of nitrogen at room temperature. The remaining lipid
film was hydrated in 25 mM Hepes (pH 7.0) or 25 mM Tris (pH 7, 9, or 11) and vortexed for 1 min. Sonication with a
Branson B12 sonifier with a 0.5-inch flat-top disrupter tip for 5
20 s (10-s intervals) at 30 watts and at a temperature of 55
°C resulted in small unilamellar vesicles (SUV). The vesicles were
kept at 37 °C and used the same day at which they were prepared.
Measurements were done by the Wilhelmy plate method, using a Cahn 2000
electrobalance, in a temperature-controlled box at 37 °C. For the
experiments with a constant surface area, a 5.5-ml Teflon trough was
filled with subphase (Hepes buffer or Tris buffer) and stirred
continuously. To determine the amount of lipid associated with the
monolayer, a trace amount of [
C]DPPC was added
to the lipid vesicles. For these measurements, a 20-ml Teflon trough
(5.3
5.7 cm) was used. Lipids (DPPC/PG or DPPC/PC, 7:3 (w/w))
were mixed with SP-C in various amounts (lipid to protein ratio 5:1 or
10:1 (w/w)). The mixture was spread on the subphase to a surface
pressure of 20.0 ± 0.1 mN/m. SUV (end concentration, 10-20
nmol/ml) made of DPPC/PG (7:3, w/w) were injected through an injection
hole into the subphase. Lipid insertion from SUV took place without
further addition or was initiated after the injection of CaCl
(final concentration of the subphase, 3 mM) through the
injection hole. The surface pressure was recorded for maximally 45 min.
The surface radioactivity was detected using a gas-type detector. After
the experiment was ended and a sample of the subphase was taken, the
subphase was flushed for 10 min with 25 mM Hepes containing 10
mM EGTA (pH 7.0), with a flow of 10 ml/min. The radioactivity
that was not bound to the monolayer was removed; the surface pressure
was unaffected by this procedure. Afterward, the monolayers were
collected. The interface radioactivity was corrected for the
radioactivity of collected subphase(20, 29) .
CD Measurements
Secondary structure of
SP-C was analyzed by use of a CD meter as described before(15) .
CD Spectra Analysis
CD spectra were
analyzed with the protein secondary structure program obtained from the
Japan Spectroscopic Co., Ltd (Tokyo). A r.m.s. error was obtained and
gave an indication of the validity of the prediction of the secondary
structure. The lower the value, the more reliable the prediction. If
the r.m.s. value was higher than 10, the result was not considered as
reliable.
Lipid-mixing Experiments
The method used
was a modification of the method previously described(30) . All
experiments were done at 37 °C. SUV (15 nmol of lipid and
DPPC/PG/pyrene-PC or DPPC/PC/pyrene-PC, 63:27:10 (w/w/w)), containing
various amounts of protein, were mixed with SUV (300 nmol of lipid,
DPPC/PG, 7:3 (w/w)). Hepes (25 mM, pH 7.0) with EGTA (0.2
mM) was used as a buffer. CaCl was added to a
final concentration of 3 mM. Fluorescence measurements were
done on a Perkin-Elmer luminescence spectrometer (LS50), linked to a
personal computer, under continuous stirring. Fluorescence emission
spectra were recorded (excitation wavelength, 343 nm; emission
wavelength, 360-550 nm) immediately after the initiation of the
experiment. The process of lipid mixing was at that time completed. The
monomer fluorescence maximum was found at 377 nm and the excimer
fluorescence maximum at 475 nm. The excimer-monomer ratio was
calculated and used to express the extent of lipid mixing.
Pulsating Bubble Measurements
Lipids
(DPPC/PG, 7:3 (w/w)) were mixed with 2% SP-C (by weight), and dried
under a stream of nitrogen. The film was stored overnight in an
exsiccator. The next day, the sample was rehydrated while shaking for
10 min in 200 µl of 0.9% NaCl with 1.5 mM CaCl at 60 °C. To ensure that all lipids were in suspension, the
glass tube was held twice in a sonicator bath for 15 s. In all cases,
before samples were taken for assay, the tubes were vortexed for a
short time. Surface tension was measured with a pulsating bubble
surfactometer (Electronetics Corp., Amherst, NY) at 37 °C. The
surface tension of a bubble with a radius of 0.4-0.55 mm was
recorded for 10 pulsations (20 pulsations/min).
column. The modified SP-C
exhibited a peak at 45 min, whereas the natural SP-C fraction came off
the column at 37 min (Fig. 1). Amino acid analysis showed that
94.7% of the arginine residues and 24.2% of the lysine residues were
modified. Circular dichroism measurements showed a difference in
secondary structure between the natural porcine SP-C and SP-C
collected from the interface (). Due to the
modification of the natural SP-C, an increase in the
-helix
content from 63.5 to 73.6% was observed. The content of random coil
decreased from 36.4 to 26.3%. There was no
-structure found in
these proteins.
Figure 1:
HPLC chromatograms for normal and
neutralized porcine SP-C.
We followed the calcium-dependent insertion of
phospholipids from SUV (10 nmol/ml) into protein-containing monolayers
(lipid to protein ratio, 5:1 (w/w)) as a function of time by monitoring
the increase in surface pressure and surface radioactivity. The maximum
surface (equilibrium) pressure was 48.0-48.5 mN/m. Natural SP-C
catalyzed the insertion of phospholipids into a negatively charged
monolayer in a protein-dependent way, which agreed with our earlier
studies (8, 15) (Fig. 2A, insertion rate
2.25 mN/m/min). If SP-C was used, the insertion rate of
phospholipids into the monolayer was strongly reduced (Fig. 2B, insertion rate 0.46 mN/m per min). In the
absence of negative charges in the monolayer, the insertion of
phospholipids into the monolayer catalyzed by the natural SP-C was
faster (Fig. 2, AversusC; insertion
rates 5.17 and 0.64 mN/m/min for SP-C and SP-C
,
respectively). In the absence of protein, no insertion of phospholipids
into the monolayer was observed (not shown). The increase in surface
radioactivity showed the same pattern as the increase in surface
pressure (Fig. 2). The subphase was flushed with a buffer
containing 10 mM EGTA to remove all vesicles that were not
bound to the monolayer. While flushing, there was no visible effect on
the surface pressure. The radioactivity in the subphase dropped to
nearly background values. The amount of radioactivity at the interface
was just slightly diminished after the flushing, indicating that
phospholipids had been inserted into the monolayer, or phospholipid
vesicles were firmly bound to the monolayer.
Figure 2:
Effect of modification of SP-C on binding
to and insertion of phospholipids into preformed SP-C-containing
monolayers. A monolayer of DPPC/PG or DPPC/PC and SP-C was spread on a
subphase of 25 mM Hepes (pH 7.0) to an initial surface
pressure of 20 mN/m. The surface pressure (uppercurve) and the surface radioactivity (lowercurve) were measured simultaneously with time. After 5
min, vesicles (SUV of DPPC/PG, containing trace amounts of
[C]DPPC; final concentration, 10 nmol of
lipid/ml) and Ca
, to a final concentration of 3
mM, were injected into the subphase. A, monolayer of
DPPC/PG and natural SP-C; B, monolayer of DPPC/PG and modified
SP-C; C, monolayer of DPPC/PC and natural SP-C; D,
monolayer of DPPC/PC and modified SP-C.
If instead of a Hepes
buffer a Tris buffer was used for the subphase (both of pH 7.0), there
was no difference in insertion rate. The calcium-dependent insertion
rate was reduced if a subphase with a pH higher than 7 was used (Fig. 3A, insertion rates of 10.98, 6.02, and 1.49
mN/m/min at pH 7.0, 9.0, and 11.0, respectively). If no negative
charges were present in the monolayer, the insertion of phospholipids
proceeded in the absence of calcium. In this case, the insertion was
also pH-dependent; if the pH was higher than 7, a lower insertion rate
was observed (1.85 and 0.10 mN/m/min at pH 9.0 and 11.0, respectively,
compared with 9.00 mN/m/min at pH 7.0). After 15 min, calcium was added
to the subphase. This enhanced the insertion rate (to 6.38 and 6.13
mN/m/min at pH 9.0 and 11.0, respectively), although the insertion rate
leveled off quickly at pH 11.0. After the injection of calcium into the
subphase, a small decrease in surface pressure was observed. The higher
the pH of the subphase, the less increase there was in surface pressure (Fig. 3B).
Figure 3:
Effect of pH on phospholipid insertion
from SUV into preformed SP-C-containing monolayers. SUV of DPPC/PG
(7:3, w/w) were injected into the subphase of 25 mM Tris (pH
7, 9, 11; 20 nmol of lipid/ml) underneath a preformed monolayer of
DPPC/PG or DPPC/PC and natural porcine SP-C (20 mN/m). Lipid to protein
ratio in the preformed monolayer is 10:1 (w/w). A, monolayer
of DPPC/PG; at time 0, Ca was injected to a final
concentration of 3 mM to start the insertion. B,
monolayer of DPPC/PC; Ca
was added after 15
min.
The surface properties of the surfactant
proteins can also be measured with a pulsating bubble
surfactometer(18) . The minimum surface tension of the pulsating
bubble immediately fell to ±5 mN/m with the natural protein.
Samples containing SP-C showed a reduction of the surface
tension to only 10 mN/m (Fig. 4).
Figure 4:
Comparison by pulsating bubble
surfactometer of the surface tension lowering properties of natural and
modified SP-C. Results are a mean of at least five measurements. The
maximum and minimum surface tensions (mean ± S.D., n 5) are indicated by open and closedsymbols, respectively.
, natural SP-C;
,
modified SP-C.
To investigate whether SP-C
was able to induce lipid mixing, the protein was incorporated in
pyrene-PC-containing vesicles. No protein-induced lipid mixing was
observed if DPPC/PG/pyrene-PC or DPPC/PC/pyrene-PC vesicles containing
natural SP-C were mixed with DPPC/PG vesicles (Fig. 5). If the
vesicles contained SP-C, protein-induced lipid mixing was
observed, both in the presence of DPPC/PG and DPPC/PC vesicles. The
extent of lipid mixing was a function of the SP-C content.
Figure 5:
Lipid mixing as a function of SP-C
concentration. Pyrene-PC-labeled vesicles of (A) DPPC/PG (30
mol% PG) or (B) DPPC/PC (30 mol% PC) containing SP-C were
mixed with DPPC/PG vesicles (30 mol% PG). The excimer/monomer ratio is
a parameter of lipid mixing. The experiment was done at 37 °C. The
average and S.D. of three experiments are shown. , vesicles
containing natural SP-C and in the absence of CaCl
;
,
vesicles containing natural SP-C and in the presence of 3 mM CaCl
;
, vesicles containing modified SP-C and in
the absence of CaCl
;
, vesicles containing modified
SP-C and in the presence of 3 mM CaCl
.
column that SP-C
was more hydrophobic
than the natural porcine SP-C. Circular dichroism measurements
demonstrated that SP-C
had an increased amount of
-helix. Both experiments are in line with the expected
neutralization of the positive charges of SP-C.
C-labeled vesicles, we showed that binding of the
negatively charged vesicles to the monolayer by the SP-C
was decreased, and consequently a reduced insertion was obtained.
There was no indication of a reduced insertion of phospholipids into
the monolayer.
-helix can possibly contribute to the insertion. At the moment
that Ca
was added, a small dip in the surface
pressure was visible (Fig. 3), which could be explained by the
condensing effect of the Ca
ions on the (already
inserted) PG in the monolayer(8) . With the pulsating bubble
surfactometer, cyclic adsorption measurements were done, which also
showed the reduced function of the neutralized SP-C.
alone is sufficient to overcome partly the repulsive forces
between the vesicles.
Table: Comparison of the secondary structures of
natural and modified SP-C at the air-water interface, as determined by
circular dichroism
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.