| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Kanazawa University, Kanazawa 920-8641, Japan; and 2 Department of Ultramicrostructural Research, Institute for Frontier Medical Science, Kyoto University, Kyoto 606-8397, Japan
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Surfactant protein (SP)-C
is characterized by 
-helix structure and palmitoyl groups attached
to two cysteine residues. We examined the function of palmitoylation
and dimerization in promotion of tidal volume in immature newborn
rabbits. Reconstituted surfactants were made from a mixture of
synthetic phospholipids and porcine SP-B (basic mixture) by adding
various forms of SP-Cs: normal SP-C isolated from porcine lungs and
monomeric or dimeric forms of SP-C. These latter two were isolated from
patients with pulmonary alveolar proteinosis and were less
palmitoylated. Animals were ventilated at an inspiratory pressure of 25 cmH2O. Median tidal volumes were <2 ml/kg in nontreated
controls, 7.7 ml/kg in animals receiving the basic mixture without
SP-C, and >18 ml/kg in animals treated with reconstituted surfactants
containing 3% normal or 2% dimeric SP-C (P < 0.05 vs. basic mixture). The physiological effect of basic mixture was
not improved by monomeric SP-C. We conclude that palmitoyl groups are
important for the physiological effects of SP-C and that the dimeric
form also improves physiological effects.
circular dichroism; palmitoylation; pulmonary alveolar proteinosis; pulmonary surfactant; surface tension
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
PULMONARY SURFACTANT IS
PRIMARILY composed of phospholipids and surfactant proteins (SPs)
(17) and reduces surface tension of the air-liquid
interface by forming a surface film (28, 31). Hydrophobic
SPs (SP-B and SP-C) are important for facilitating surface adsorption
of phospholipid molecules and for promoting tidal volume (17,
20). The regular form of SP-C is known to be monomeric, with a
hydrophobic valine-rich -helix at the carboxy terminal
(15-17). The amino terminal part of regular SP-C is
hydrophilic, but in most species the two cysteine residues are attached
to the palmitoyl groups via thioester bonds with an overall
stoichiometry ratio of close to 1:1 between the cysteine residues and
the palmitoyl groups (6, 16). However, the relationship
between molecular configuration and the physiological function are not
yet completely understood.
Two types of abnormal SP-Cs are known to accumulate in the lungs of patients with pulmonary alveolar proteinosis (PAP). One type is monomeric but poor in palmitoyl groups, and the other type is dimeric and also deficient in palmitoyl groups (32, 34, 39). Information on the relationship between the configuration and function of SP-C may be obtained from analyses of these abnormal SP-Cs. Numerous investigations on surfactants consisting of synthetic lipids and SPs or the analogs have recently been conducted to develop artificial surfactant for therapeutic use (8, 27). The techniques used in these investigations can be applied to analysis of SP-C function. In the present study, several reconstituted surfactants (RSs), consisting of synthetic phospholipids, normal SP-B, and abnormal SP-Cs from PAP patients, were administered to surfactant-deficient immature newborn rabbits. Tidal volumes of the animals were then measured under pressure-controlled ventilation to evaluate the physiological function of abnormal SP-Cs.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Preparation of MNS, Normal SP-B, and Normal SP-C
Bronchoalveolar lavage (BAL) fluid from recently slaughtered pigs was centrifuged (150 g for 10 min) to remove cell debris, and the supernatant was further centrifuged (2,000 g for 1 h, 4°C). Modified natural surfactant (MNS) consisting of 98.0% phospholipids, 0.9% other lipids, and 1.1% hydrophobic SPs (0.37% SP-B and 0.72% SP-C) was obtained from the pellet by extraction with chloroform-methanol solution (2:1 vol/vol), by washing with 0.5% saline, and by acetone precipitation. More precise chemical compositions and isolation methods for MNS are presented elsewhere (20, 21).MNS was dissolved again in chloroform-methanol (1:1 vol/vol) containing 0.1 N hydrochloric acid at 5%. From this solution, we could separate two types of protein fractions by using Sephadex LH-60 column chromatography (Pharmacia Biotechnology, Uppsala, Sweden) (7) with the aid of ultraviolet (280 nm) absorbance using a spectrophotometer (U-2000, Hitachi, Tokyo, Japan). With further assessments described below, the protein first eluted from the column was used in the present study as normal SP-B (nSP-B), with the protein eluted second representing normal SP-C (nSP-C).
Each protein fraction was further assessed by use of tricine SDS-PAGE under the nonreducing condition and also under the reducing condition with 2-mercaptoethanol (30). Presence of SP-B was determined by Western blotting with biotinylated anti-SP-B antibody (8B5E) (33) and by amino terminal sequence analysis using a gas-phase protein sequencer (PPSQ-10, Shimazu, Kyoto, Japan) after blotting on a polyvinylidene difluoride membrane (Sequi-Blot, 0.2 µm, Bio-Rad Laboratories, Richmond, CA) according to the method of Towbin et al. (38). The relative amount of protein on tricine SDS-PAGE was determined by use of a lane and spot analyzer (AE-6920, ATTO, Tokyo, Japan). The concentration of proteins was determined according the micro-Kjeldahl method (37).
Preparation of Abnormal SP-Cs
BAL fluid was obtained from therapeutic lung lavage of two PAP patients admitted to Kanazawa University Hospital. The chloroform-soluble fraction was obtained in the same manner as used for the preparation of MNS. This fraction was further separated into pulmonary alveolar proteinosis SP-B (SP-Bpap) and dimeric (dSP-Cpap) and monomeric forms of SP-C (mSP-Cpap) under the same methods used for isolation of nSP-C (32, 34). Purity and molecular weight were determined according to the methods described above. Amount of palmitoyl groups in nSP-C and abnormal SP-Cs were quantified using gas chromatography after hydrolysis with 0.1 mol/l of potassium hydroxide (6) and by methylation with boron fluoride-methanol (25).Preparation of RSs
First, a synthetic phospholipid mixture (SPL) was prepared by mixing synthetic dipalmitoylphosphatidylcholine (Sigma Chemical, St. Louis, MO), synthetic dioleolylphosphatidylcholine (Sigma Chemical), and egg yolk phosphatidylglycerol (Sigma Chemical) at a weight ratio of 6:2:2 in chloroform-methanol (95:5) solution (36). The basic mixture was prepared by adding normal SP-B to SPL at a concentration of 0.7% (21). For RSs of the nSP-C series, nSP-C was added to the basic mixture at concentrations of 1, 2, or 3% (by weight). For the RSs of the mSP-Cpap series, mSP-Cpap was added to the basic mixture at concentrations of 1, 2, or 3%. For RSs of the dSP-Cpap series, dSP-Cpap was added to the basic mixture at concentrations of 1 or 2%. An RS containing dSP-Cpap at 3% could not be prepared because of limited supplies of the protein.After evaporation of the organic solvent by blowing nitrogen, all test
materials, i.e., MNS, SPL, basic mixture, and all types of RSs, were
suspended in normal saline by repeatedly drawing them into and
expelling them from a syringe, followed by incubation in an ultrasonic
bath (Branson 3200, Yamato, Tokyo, Japan) for 3 min. The pH values of
these suspensions of test material were corrected to 5.5-6.5,
similar to that of MNS suspension, with 0.1 N sodium hydroxide
(21). Final concentration of the test material was
adjusted at 50 mg/ml. Suspensions were stored at 
20°C.
We assessed surface activity of SPL, the basic mixture, RS containing
nSP-C at 1%, SPL supplemented with nSP-C at 3% only (without SP-B),
and MNS by using a pulsating-bubble apparatus (PBS; Electronetics,
Buffalo, NY) (9). For this purpose, the suspension
containing each test material at a concentration of 10 mg/ml was placed
in a sample chamber kept at 37°C. An air bubble communicating with
the ambient air was created in the suspension and then pulsated between
radii of 0.40 and 0.55 mm at a speed of 40 cycles/min. After 5 min of
pulsation, surface tension at maximum and minimum bubble sizes (
max,
min) were recorded.
Secondary Structure of Various SP-Cs
The circular dichroism (CD) spectra were obtained using a spectropolarimeter (J-710, JASCO, Tokyo, Japan) in a 0.2- mm cell at 25°C. Various SP-Cs (75 µg) were mixed with SPL (750 µg). These mixtures were dried and suspended in 1 ml of phosphate buffer (50 mmol/l, pH 6.0) (18). Two series of 10 scans from 240 to 200 nm were averaged, and protein-free SPL spectra were subtracted to yield the protein spectra. CD spectra were expressed as mean residue ellipticity calculated by protein concentration and estimation of the mean molecular weight of amino acid residues as 115. These spectra were analyzed for secondary structure with the aid of a neural network computer program (k2d, kindly distributed by M. A. Andrade through the World Wide Web; http://www.embl-heidelberg.de/~andrade/k2d.html) comprising a database of weights and a recall program for determining-helix and 
-sheet structure based on these weights (1,
12).
Animal Experiment 1
Validation of MNS and immature newborn rabbit model. Twenty immature newborn rabbits were delivered by hysterectomy from three pregnant does at a gestational age at between 26 days 22 h and 27 days 5 h (term = 31 days). Animals were tracheotomized under anesthesia with intraperitoneal pentobarbital sodium (0.5 mg) and randomly assigned to MNS (n = 10) or nontreated (n = 10) groups. MNS group animals were administered 100 µl of MNS suspension (50 mg/ml) via tracheal cannula before taking their first breath, whereas control group animals were administered nothing. Animals were then transferred to a system of multiple-body plethysmographs kept at 37°C (20). After all littermates were prepared, animals were relaxed with intraperitoneal pancuronium bromide (0.02 mg) and subjected in parallel to pressure-controlled ventilation. The respirator unit (Servo 900B, Siemens-Elema, Solna, Sweden) delivered 100% oxygen at 40 breaths/min with a 50% inspiration time.
The peak inspiratory pressure (PIP) was first raised to 30 cmH2O for 1 min to facilitate distribution of administered materials, then lowered to 25 cmH2O for 15 min, and then to 20 cmH2O and 15 cmH2O for 5 min each. Finally, PIP was again increased to 25 cmH2O for 5 min. No end-expiratory pressure was applied to the ventilatory circuit. Individual tidal volumes were recorded at the end of each 5-min interval by using a pneumotachograph system described elsewhere (20). Electrocardiograms were recorded immediately after tidal volume measurements, and animals showing QRS complexes at a frequency of over 100 beats/min were considered survivors. After the animals were killed by an overdose of pentobarbital, the abdomen was opened to inspect the diaphragm for evidence of pneumothorax. Animals with pneumothorax were excluded from statistical analyses.Animal Experiment 2: Function of nSP-C, mSP-Cpap, and dSP-Cpap
nSP-C series. Thirty-one immature newborn rabbits were delivered from five pregnant does and randomly assigned to groups receiving four types of RSs containing nSP-C at concentration of 0% (basic mixture, n = 7), 1% (n = 7), 2% (n = 10), or 3% (n = 7). Animals were administered 100 µl of each one of the RS suspensions (50 mg/ml) via the tracheal cannula and were ventilated in the same manner as in animal experiment 1. Tidal volumes at a PIP of 25 cmH2O were measured four times, and the last value (30 min after start of the ventilation) was used for comparisons. Animals with pneumothorax were excluded from statistical analyses.
mSP-Cpap series. Thirty-one immature newborn rabbits were delivered from five pregnant does and randomly assigned to groups receiving RSs (50 mg/ml) containing mSP-Cpap at concentrations of 0% (basic mixture, n = 7), 1% (n = 7), 2% (n = 10), or 3% (n = 7). The experiment was performed in a manner identical to the nSP-C series.
dSP-Spap series. Twenty-four immature newborn rabbits were delivered from four pregnant does and randomly assigned to groups receiving RSs (50 mg/ml) containing dSP-Cpap at concentration of 0% (basic mixture, n = 7), 1% (n = 7), or 2% (n = 10). The experiment was performed in a manner identical to the nSP-C series.
Statistical Analysis
Data for tidal volume and surface tension were expressed as median and range (parentheses). Intergroup differences were examined by using Kruskal-Wallis test followed by Mann-Whitney's test with Bonferroni correction. Data for body weight are given as means ± SD, and differences were assessed by using analysis of variance followed by Scheffé's method. Differences in survival rate and incidence of pneumothorax were assessed by using Fisher's exact test. Levels of P < 0.05 were considered statistically significant.| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Characterization of Hydrophobic SPs
Tricine SDS-PAGE of the various SP-Cs is shown in Fig. 1A. Under the nonreducing condition, the molecular weight of nSP-C, dSP-Cpap, and mSP-Cpap were 4.9 kDa, 8.5 kDa, and 4.2 kDa, respectively. Under the reducing condition, the dSP-Cpap demonstrated a prominent staining band at 4.4 kDa and a faint one at 8.5 kDa, indicating the dimeric nature of dSP-Cpap. Tricine SDS-PAGE of nSP-B and SP-Bpap is shown in Fig. 1B. Under the nonreducing condition, nSP-B (lane 1) revealed a major band at 21-22 kDa and a faint one at 16 kDa. Under the reducing condition, the major band migrated to 11 kDa, but the faint band remained at 16 kDa. A similar band was also found in the MNS prepared after isolation by sucrose-gradient centrifugation according to Frosolono et al. (10) (data not shown). The amino terminal sequence of the major band at 21-22 kDa in the nonreducing condition represented Phe-Pro-Ile-Pro-Leu-Pro-Phe-X-Trp-Leu- (X was supposed to be "Cys"), which agreed with the result of the previous report (7). Densitometry indicated that the purity of the nSP-B was 84-85%. Two major bands were demonstrated for SP-Bpap, one at 21 kDa and the other at 30 kDa. Under the reducing condition, the major bands migrated to 10 kDa with a faint band at 16 kDa. Western blotting using anti-SP-B antibody is shown in Fig. 1C. The major bands of nSP-B (21 kDa) and SP-Bpap (21-30 kDa) demonstrated the binding to anti-SP-B antibody. Their faint bands at 16 kDa did not bind with antibody. A faint binding at 10 kDa was found in the blot of dSP-Cpap, indicating the contamination with SP-B monomer. On the basis of densitometry, the contamination was calculated to 1.5%.
|
Weight ratio of SP-Bpap, dSP-Cpap, and mSP-Cpap obtained from BAL fluid of PAP patients was 27:23:50. Relative contents of palmitoyl groups in dSP-Cpap and mSP-Cpap were 42 and 38% of that contained in the identical weight of nSP-C, respectively.
Pulsating-Bubble Measurement
Findings of dynamic surface tensions are shown in Table 1. Bothmin and max of the basic
mixture composed of SPL and nSP-B were significantly lower than those
of SPL, indicating that nSP-B was functional. Similar findings were
obtained on SPL supplemented with nSP-C only, of which min was,
however, significantly higher than that of MNS. Neither nSP-B nor nSP-C
alone could constantly lower min values of SPL to <5 mN/m. The RS
consisting of SPL and both nSP-B and nSP-C demonstrated the same min
and max values as MNS.
|
Secondary Structure of Various SP-Cs
CD spectra of nSP-C demonstrated minima at 208 and 222 nm (Fig. 2), a result characteristic of-helix
(11). The spectra of mSP-Cpap revealed smaller minima at
these wavelengths than nSP-C. The minima of dSP-Cpap were somewhat
smaller than those of mSP-Cpap (Fig. 2). The k2d program indicated that
the -helical content of the nSP-C was larger than those of mSP-Cpap
or dSP-Cpap (Table 2).
|
|
Animal Experiment 1
Two of 10 animals died in the nontreated group, whereas all animals in the MNS group survived until the end of experiment. No pneumothorax was observed in the nontreated group, whereas 2 of the 10 animals in the MNS group were excluded because of pneumothorax. Body weight of all animals was 33.6 ± 3.4 g, without significant differences between the MNS and nontreated groups. As shown in Fig. 3, median tidal volume of the nontreated group were <2 ml/kg in four measurements at a PIP of 25 cmH2O, whereas the values of the MNS group was >24 ml/kg (P < 0.01 vs. nontreated group).
|
Animal Experiment 2
nSP-C series.
Body weight of all animals was 33.6 ± 3.5 g, without
significant differences among the four groups. All animals survived
until the end of the experiment, and no cases of pneumothorax were
observed. Changes in tidal volume are shown in Fig.
4. Tidal volumes with the basic mixture
were larger than those seen in the nontreated group of animal
experiment 1 at all PIPs (P < 0.01). At a PIP of
25 cmH2O, tidal volumes of animals receiving the RSs
containing nSP-C at 2 or 3% were significantly larger than those
receiving basic mixture (nSP-C at 0%). At PIPs of 15 and 20 cmH2O, tidal volumes of animals administered RS containing
SP-C at 3% were significantly larger than those receiving basic
mixture.
|
mSP-Cpap series.
Body weight of all animals was 30.4 ± 4.0 g, without
significant differences among the four groups. All animals survived
until the end of the experiment, but one animal receiving the RS
containing mSP-Cpap at 2% was excluded because of pneumothorax.
Changes in tidal volumes are shown in Fig.
5. At a PIP of 25 cmH2O,
median tidal volumes of all groups were only ~10 ml/kg.
|
dSP-Cpap series.
Body weight of all animals was 30.2 ± 4.7 g, without
significant differences among the three groups. All animals survived until the end of the experiment, and no cases of pneumothorax were
observed. Changes in tidal volumes are shown in Fig.
6. At a PIP of 25 cmH2O,
median tidal volume of the animals receiving the RS containing 2%
dSP-Cpap was nearly twice that of those receiving basic mixture
(P < 0.05).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
In animal experiment 1, tidal volumes of the MNS group were significantly larger than those of the nontreated group. Although difference in tidal volumes might have been exaggerated by relationships between alveolar opening pressures and PIPs, the same pressure-controlled ventilation used in the present study has been used previously to evaluate the physiological activity of surfactant preparations (7, 20, 21). Sucrose-gradient centrifugation, which has been used to isolate "complete" surfactant (10, 20, 21), was omitted from the present study. Lipid composition of MNS, therefore, may differ from that of complete pulmonary surfactant. Another difference between complete surfactant and MNS is the absence of SP-A and SP-D in MNS given that extraction with organic solvents results in removal of hydrophilic proteins (17, 20). However, we have found that this MNS promotes tidal volumes in surfactant-deficient immature newborn rabbits to a similar extent as complete surfactant (21). From these perspectives, we believe that the physiological activity of surfactant preparations can be assessed using the protocols of the present study and that MNS is a reasonable source for hydrophobic SPs.
An RS consisting of the same phospholipids as used in the present study
and porcine SP-B (0.7%) and porcine SP-C (1.4%) could improve
blood-gas findings in rats with acute lung injury in our previous
experiment (36). Although the reconstituted materials without SP-B exhibit some surface activity (21, 26), SP-B is believed to represent an indispensable component to optimize the
surfactant activity (21, 27). In the present study, the pulsating-bubble measurements revealed that surface tension of SPL was
significantly decreased by adding nSP-B or nSP-C. However, surface
tension did not decrease to the same level as MNS by either of the two
SPs alone. Apparent molecular size, amino terminal sequence, and
findings under Western blotting of nSP-B in the present study were
consistent with those previously reported for regular porcine SP-B
(7, 17, 33). In the present experiment, the basic mixture,
i.e., mixture of SPL and nSP-B (0.7%), increased the tidal volume of
immature newborn rabbits from <2 ml/kg to ~7.7 ml/kg. This only
modest improvement may be because the SP-B concentration was lower than
that used in a previous study (2.0%) (7). The
concentration of SP-B used in our experiment may have been suboptimal,
so that a larger tidal volume could be obtained by using a higher
concentration. It was reported that addition of SP-B at a concentration
of 2.0% reduced the min of an artificial surfactant, including
phospholipids and an analog of SP-C, to nearly the same value as that
of another surfactant preparation isolated from porcine lungs, but
decreasing the SP-B concentration from 2.0 to 0.5% did not change the
min significantly (27). Our results using a basic
mixture containing SP-B at a concentration of 0.7% would, therefore,
be valid as a yardstick for discriminating the physiological effects of
various SP-Cs.
A min value similar to that of MNS was obtained when nSP-C was added
at 1.0% to the basic mixture. Moreover, tidal volumes of the immature
rabbits increased with the concentration of nSP-C. Regular porcine SP-C
comprises 35 amino acid residues, and the residues 9-34 form an
-helix structure (19). Our findings, that nSP-C had a
molecular size of 4.9 kDa and an -helix content of 59%, do not
contradict previously reported results for regular SP-C (4,
17). Given these considerations, tidal volumes of immature
rabbits receiving the RSs containing nSP-C can be used as standards to
evaluate the function of various SP-Cs.
In the mSP-Cpap series, however, tidal volume barely improved with the
increment in concentration. This result strongly suggests that mSP-Cpap
lacks the function. Our previous experiment demonstrated that the amino
terminal sequence of mSP-Cpap and dSP-Cpap coincided with that of human
SP-C (34). The -helix part allows the anchorage of SP-C
in the phospholipid bilayer and monolayer (3) and is believed to be essential for surface activity (16). CD
spectra indicated that the -helix content in mSP-Cpap was 41%,
about two-thirds of nSP-C. The possibility therefore exists that
inefficiency of mSP-Cpap results from shortened -helix structure.
However, Qanbar et al. (29) found with the captive-bubble
technique that RS containing palmitoylated SP-C required less
compression to reach low surface tension than those with chemically
depalmitoylated SP-C. Gustafsson et al. (13) also have
shown that RS composed of palmitoylated SP-C analog forms a stable
surface film at the air-water interface but that composed of
nonpalmitoylated analog does not. In the present experiment, palmitoyl
content in mSP-Cpap was only 38% of that in nSP-C. As the cause of
functional inefficiency of mSP-Cpap, therefore, we must also consider a
deficiency in palmitoyl groups.
Interestingly, the RSs containing dSP-Cpap increased the tidal volume
of immature newborn rabbits, whereas RSs with mSP-Cpap did not. It
seems unlikely that contamination by SP-Bpap in the dSP-Cpap fraction
improved the tidal volume, because the concentration of SP-Bpap
corresponded to only 1.5% of 2% SPL, i.e., 0.03% of SPL. Baatz et
al. (2) reported that dimeric SP-C from bovine lung
predominantly consisted of -sheets. In contrast, Creuwels et al.
(5) reported that the dimeric SP-C was helical. Both studies demonstrated that dimeric SP-C improved surface activity of
synthetic phospholipid mixtures. In the present experiment, the
-helical content of mSP-Cpap was similar to that of dSP-Cpap, although both values were lower than that of nSP-C. The palmitoyl content of mSP-Cpap was also similar to that of dSP-Cpap. The functional efficiencies, however, were clearly different in the present
study. Data from the present study indicate that surfactant function is
influenced not only by the -helical content and degree of
palmitoylation of SP-C but also by the presence of dimeric SP-C.
Surfactant forms surface film at the air-liquid interface, which is
thought to be consisting of a phospholipid monolayer at the most
superficial part and bilayers in the underlying part (22, 28,
31). Some researchers have speculated that palmitoylation of
SP-C may link the monolayer to a neighboring bilayer and link two
bilayers together by anchoring palmitoyl groups and the -helix to
different layers, because the palmitoylated SP-C analog demonstrated improved adsorption of surface-associated phospholipid layers to the
air-liquid interface during surface expansion (13). Wang et al. (40) reported that regular SP-C facilitated the
adsorption of both synthetic phospholipids and phospholipids isolated
from natural surfactant better than depalmitoylated SP-C. The surface adsorption rate of surfactant molecules is an important factor in
physiological activity (20, 31). Nonionic polymers such as
dextran and polyethylene glycol are known to improve the surface adsorption rate of surfactant preparations, and presumably these polymers pull neighboring phospholipid layers together by removing water molecules (23, 24). Taking all these possibilities
into consideration, we believe that each of two -helix structures in
the dSP-Cpap molecule anchored to different lipid layers and linked
them together, improving surface adsorption and physiological function
despite the reduced content of palmitoyl groups.
The helical structure of regular SP-C is reportedly able to unfold and
form aggregates with -sheets, leading to formation of amyloid
fibrils (14, 35). Such fibrils have been found in BAL
fluid from a PAP patient (14). In the present experiment, neither polymeric forms nor aggregates of SP-C with higher molecular weights could be found on the tricine SDS-PAGE, probably because only
the chloroform-soluble fraction was used for isolation of abnormal
SP-Cs. The palmitoyl content of mSP-Cpap was found to be 38% of that
of nSP-C. The mSP-Cpap fraction could thus consist of either a largely
monopalmitoylated species or a mixture of nonpalmitoylated and
dipalmitoylated species. The respective concentrations of these three
species were not determined. Nevertheless, results of the animal
experiment indicated the importance of the palmitoyl content of nSP-C.
We used SPL for the lipid component of RSs, with synthetic surfactants
in mind. We may therefore need to examine whether the same results
could be obtained by using the phospholipids isolated from natural
surfactant (40). Although many areas remain to be
examined, we conclude that monomeric and less-palmitoylated SP-C
derived from PAP patients lacks the ability to enhance tidal volume of
immature newborn rabbits. Palmitoylation is important for the function
of monomeric SP-C. Dimeric SP-C appearing in PAP patients, however,
shows physiological function despite a deficiency in palmitoyl groups.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Professor Masako Nagai (Department of Biochemistry, Kanazawa University Faculty of Medicine, Kanazawa, Japan) for assistance with circular dichroism measurements.
| |
FOOTNOTES |
|---|
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (project nos. 10470315 and 12671457).
Address for reprint requests and other correspondence: K. Tashiro, Dept. of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Kanazawa Univ., Takara-machi 13-1, Kanazawa 920-8641, Japan (E-mail address: tashirk{at}anesth.m.kanazawa-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 25, 2002;10.1152/japplphysiol.00059.2001
Received 25 January 2001; accepted in final form 17 October 2002.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Andrade, MA,
Chacón P,
Merelo JJ,
and
Morán F.
Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network.
Protein Eng
6:
383-390,
1993.
2.
Baatz, JE,
Smyth KL,
Whitsett JA,
Baxter C,
and
Absolom DR.
Structure and functions of a dimeric form of surfactant protein SP-C: a Fourier transform infrared and surfactometry study.
Chem Phys Lipids
63:
91-104,
1992.
3.
Clercx, A,
Vandenbussche G,
Curstedt T,
Johansson J,
Jörnvall H,
and
Ruysschaert JM.
Structural and functional importance of the C-terminal part of the pulmonary surfactant polypeptide SP-C.
Eur J Biochem
229:
465-472,
1995.
4.
Creuwels, LAJM,
Demel RA,
van Golde LMG,
Benson BJ,
and
Haagsman HP.
Effect of acylation on structure and function of surfactant protein C at the air-liquid interface.
J Biol Chem
268:
26752-26758,
1993.
5.
Creuwels, LAJM,
Demel RA,
van Golde LMG,
and
Haagsman HP.
Characterization of a dimeric canine form of surfactant protein C (SP-C).
Biochim Biophys Acta
1254:
326-332,
1995.
6.
Curstedt, T,
Johansson J,
Persson P,
Eklund A,
Robertson B,
Löwenadler B,
and
Jörnvall H.
Hydrophobic surfactant-associated polypeptides: SP-C is a lipopeptide with two palmitoylated cysteine residues, whereas SP-B lacks covalently linked fatty acyl groups.
Proc Natl Acad Sci USA
87:
2985-2989,
1990.
7.
Curstedt, T,
Jörnvall H,
Robertson B,
Bergman T,
and
Berggren P.
Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant: characterization and biophysical activity.
Eur J Biochem
168:
255-262,
1987.
8.
Davis, AJ,
Jobe AH,
Häfner D,
and
Ikegami M.
Lung function in premature lambs and rabbits treated with a recombinant SP-C surfactant.
Am J Respir Crit Care Med
157:
553-559,
1998.
9.
Enhorning, G.
Pulsating bubble technique for evaluating pulmonary surfactant.
J Appl Physiol
43:
198-203,
1977.
10.
Frosolono, MF,
Charms BL,
Pawlowski R,
and
Slivka S.
Isolation, characterization, and surface chemistry of a surface-active fraction from dog lung.
J Lipid Res
11:
439-457,
1970.
11.
Greenfield, N,
and
Fasman GD.
Computed circular dichroism spectra for the evaluation of protein conformation.
Biochemistry
8:
4108-4116,
1969.
12.
Greenfield, NJ.
Methods to estimate the conformation of proteins and polypeptides from circular dichroism data.
Anal Biochem
235:
1-10,
1996.
13.
Gustafsson, M,
Palmblad M,
Curstedt T,
Johansson J,
and
Schürch S.
Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability.
Biochim Biophys Acta
1466:
169-178,
2000.
14.
Gustafsson, M,
Thyberg J,
Näslund J,
Eliasson E,
and
Johansson J.
Amyloid fibril formation by pulmonary surfactant protein C.
FEBS Lett
464:
138-142,
1999.
15.
Johansson, J.
Structure and properties of surfactant protein C.
Biochim Biophys Acta
1408:
161-172,
1998.
16.
Johansson, J,
and
Curstedt T.
Molecular structures and interactions of pulmonary surfactant components.
Eur J Biochem
244:
675-693,
1997.
17.
Johansson, J,
Curstedt T,
and
Robertson B.
The proteins of the surfactant system.
Eur Respir J
7:
372-391,
1994.
18.
Johansson, J,
Nilsson G,
Strömberg R,
Robertson B,
Jörnvall H,
and
Curstedt T.
Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C.
Biochem J
307:
535-541,
1995.
19.
Johansson, J,
Szyperski T,
Curstedt T,
and
Wüthrich K.
The NMR structure of the pulmonary surfactant-associated polypeptide SP-C in an apolar solvent contains a valyl-rich alpha-helix.
Biochemistry
33:
6015-6023,
1994.
20.
Kobayashi, T,
Li WZ,
Tashiro K,
Takahashi R,
Waseda Y,
Yamamoto K,
and
Suzuki Y.
Disparity between tidal and static volumes of immature lungs treated with reconstituted surfactants.
J Appl Physiol
80:
62-68,
1996.
21.
Kobayashi, T,
Tashiro K,
Yamamoto K,
Nitta S,
Ohmura S,
and
Suzuki Y.
Effects of surfactant proteins SP-B and SP-C on dynamic and static mechanics of immature lungs.
J Appl Physiol
83:
1849-1856,
1997.
22.
Kramer, A,
Wintergalen A,
Sieber M,
Galla HJ,
Amrein M,
and
Guckenberger R.
Distribution of the surfactant-associated protein C within a lung surfactant model film investigated by near-field optical microscopy.
Biophys J
78:
458-465,
2000.
23.
MacDonald, RI.
Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose.
Biochemistry
24:
4058-4066,
1985.
24.
Meyuhas, D,
Nir S,
and
Lichtenberg D.
Aggregation of phospholipid vesicles by water-soluble polymers.
Biophys J
71:
2601-2612,
1996.
25.
Morrison, WR,
and
Smith LM.
Preparation of fatty acid methylesters and dimethylacetals from lipids with boron fluoride-methanol.
J Lipid Res
5:
600-608,
1964.
26.
Nilsson, G,
Gustafsson M,
Vandenbussche G,
Veldhuizen E,
Griffiths WJ,
Sjövall J,
Haagsman HP,
Ruysschaert JM,
Robertson B,
Curstedt T,
and
Johansson J.
Synthetic peptide-containing surfactants: evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution.
Eur J Biochem
255:
116-124,
1998.
27.
Palmblad, M,
Johansson J,
Robertson B,
and
Curstedt T.
Biophysical activity of an artificial surfactant containing an analogue of surfactant protein (SP)-C and native SP-B.
Biochem J
339:
381-386,
1999.
28.
Pérez-Gil, J,
and
Keough KMW
Interfacial properties of surfactant proteins.
Biochim Biophys Acta
1408:
203-217,
1998.
29.
Qanbar, R,
Cheng S,
Possmayer F,
and
Schürch S.
Role of the palmitoylation of surfactant-associated protein C in surfactant film formation and stability.
Am J Physiol Lung Cell Mol Physiol
271:
L572-L580,
1996.
30.
Schäger, H,
and
von Jagow G.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
Anal Biochem
166:
368-379,
1987.
31.
Schürch, S,
Green FHY,
and
Bachofen H.
Formation and structure of surface films: captive bubble surfactometry.
Biochim Biophys Acta
1408:
180-202,
1998.
32.
Shen, HQ,
Duan CX,
Li ZY,
and
Suzuki Y.
Effects of proteinosis surfactant proteins on the viability of rat alveolar macrophages.
Am J Respir Crit Care Med
156:
1679-1687,
1997.
33.
Suzuki, Y,
Kogishi K,
Fujita Y,
Kina T,
and
Nishikawa S.
A monoclonal antibody to the 15,000 dalton protein associated with porcine pulmonary surfactant.
Exp Lung Res
11:
61-73,
1986.
34.
Suzuki, Y,
Shen HQ,
Sato A,
and
Nagai S.
Analysis of fused-membrane structures in bronchoalveolar lavage fluid from patients with alveolar proteinosis.
Am J Respir Cell Mol Biol
12:
238-249,
1995.
35.
Szyperski, T,
Vandenbussche G,
Curstedt T,
Ruysschaert JM,
Wüthrich K,
and
Johansson J.
Pulmonary surfactant-associated polypeptide C in a mixed organic solvent transforms from a monomeric -helical state into insoluble -sheet aggregates.
Protein Sci
7:
2533-2540,
1998.
36.
Tashiro, K,
Nishizuka K,
Matsumoto Y,
Ohta K,
Suzuki Y,
and
Kobayashi T.
Modified natural and synthetically reconstituted surfactant therapies for acute lung injury caused by endotoxin in rats.
Acta Anaesthesiol Scand
43:
821-828,
1999.
37.
Thompson, JF,
and
Morrison GR.
Determination of organic nitrogen: control of variables in the use of Nessler's reagent.
Anal Chem
23:
1153-1157,
1951.
38.
Towbin, H,
Staehelin T,
and
Gordon J.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354,
1979.
39.
Voss, T,
Schäfer KP,
Nielsen PF,
Schäfer A,
Maier C,
Hannappel E,
Maassen J,
Landis B,
Klemm K,
and
Przybylski M.
Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.
Biochim Biophys Acta
1138:
261-267,
1992.
40.
Wang, Z,
Gurel O,
Baatz JE,
and
Notter RH.
Acylation of pulmonary surfactant protein-C is required for its optimal surface active interactions with phospholipids.
J Biol Chem
271:
19104-19109,
1996.
This article has been cited by other articles:
|
|
 |
|
  E. Gastiasoro-Cuesta, F. J. Alvarez-Diaz, C. Rey-Santano, A. Arnaiz-Renedo, B. Loureiro-Gonzalez, and A. Valls-i-Soler Acute and Sustained Effects of Lucinactant Versus Poractant-{alpha} on Pulmonary Gas Exchange and Mechanics in Premature Lambs With Respiratory Distress Syndrome Pediatrics, February 1, 2006; 117(2): 295 - 303. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
)
and SP-Cs derived from pulmonary alveolar proteinosis lung in monomeric
(mSP-Cpap, 
) and dimeric forms (dSP-Cpap,
).
, Not given any material (nontreated group,
n = 10); 
, treated with modified
natural surfactant (MNS group, n = 8). Values are
medians (ranges). *P < 0.05 vs. nontreated group. Some
ranges are hidden by symbols.
