Biochimica el Biophvsi(,: Aria 1086( 1991) 185- ItS) ,c 1991 Elsevier .%"icncePublishers B.V. All rights reserved [MX)5-2760/91/$(13.5~)
185
BBALIP 53765
Characterization of pulmonary surfactant protein D: its copurification with lipids Yoshio Kuroki, Masanori Shiratori, Yoshinori Ogasawara, Akihiro Tsuzuki and Toyoaki Akino Department of Biochemixt~'. Sapl~m~ [~(,dit'al ('oll(~(,. Sapporo (Japan/ (Received I~ February 19gl )
Key words: Pulmonarysurfaclanl: Surfaclant prolcin D: Pl~,pholipid
Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II calls. SP-D was purified from the supernatant of rat bronchoalvcolar iavagc fluids obtained by centrifugation at 3 3 0 0 0 x g , ~ for 16 h. The contents of SP-D and SPA in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of S P A in these lavage fluids. 99.1% of SP-A was present in the 33000£ pellet, whereas 71.1% of SP-D was in the 33000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SI' A, SP-D in the purified and delipidated form failed to compete with '~l-laheled SP-A for plmsphotidylcholine binding, and to aggregate phosphlipid liposomes. The present study demonstrates that lipids are copurifled with SP-D, that SP-D and S P A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.
Introduction Pulmonary surfactant, a complex mixture of lipids tnd proteins, functions to stabilize alveoli by lowering the surface tension at the air/liquid interface [I]. One of the major protein components of surfactant (surfactant protein A, SP-A) is a glycoprotein that has monomeric molecular masses of 26-38 kDa in the rat [2]. This protein is characterized by the striking features of collagen-like sequences [3] and a carbohydrate-binding property [4]. SP-A pos~sses a variety of biological activities. SP-A interacts with surfactantlike phospholipids [5,6], specifically binds dipa;mitoylphosphatidylcholine [7], and causes phospholipid ag-
Abbreviations: SP-D, surfaclanl associated protein D: SP-A. surfacrant associated protein A: llcpcs, N-2 hydro~elhylpipcrazine-N'-2ethanesulfonic acid: ELISA. enzyme-linked immumv~rbcnl assay: TLC, thin-layer chromatography: HPLC, high performance liquid chromatography; dipalmitoylplv,)sphatidylcholineDPPC: EDTA. elhylenediaminetetraacelate. Correspondence: Y. Kuroki, [:~partmenl of Bk~chemislry. Sapporo Medical College, South-l, West-17. Chuo-ku. Sapporo (~|. Japan.
gregation [8]. This protein has al~) been sht~vn to function as a negative regulator of surfactant ph(vspholipid secretion by alveolar type II cells [9,10], to enhance the uptake of phimpholipid liposomes by the,we cells [II] and to enhance the host-defense mcchanism of rat alveolar type il cells [12]. Another hydrophilic surfactant protein (SP-D) has recently been i.5olated from the culture medium of alveolar type II ceils and from rat bronchoalveolar lavagc fluids [13.14]. SP-D is a glycoprotein that shows similarities to SP-A in its collagcnous structure and carbohydrate-binding property [ 13-15]. Similarities betwecn SP-A and SP-D suggest that SP-D may function like SP-A. The purpose of the present study was: (I) to puri!~ and characterize SP-D isolated from rat bronchoalveolar lavagc and (2) to examine interactions of SP-D with iipids in compari.qm with thirst of SP-A.
Materials and Methods Purification o f sur~actant proteins Bronchoalveolar lavage was obtained from rats given intratracheal instillation of IO mg per rat silica in .saline 4 weeks before lung lavage according to the method of Dcthloff et al. [16]. Prelreatmcnt of the rats with silica
186 increased the yield of surfactant approximately 2¢l-fold. Lungs were lavaged l0 times with 5 mM ftcpes buffer (pH 7.4)containing 150 mM NaCI and bronehoalvcolar lavage fluid~ were centrifuged at 15l) × g for Ill rain to remove cell debris. The supernatant was centrifuged at 33(XX) ×g,,~ at 4°C 16 h after the addition of CaCI, at a final concentration of 5 raM. The supcrnatant was used fi)r the isolation of SP-D. which was purified by the modified methods essentially based on those de.scribed by Persson el al. [14,15]. The supernatant was applied to an affinity column of mannose-Sepharosc 4B (Pharmacia Fine Chemicals) prepared by the method of Fornstcdt and Porath [17]. SP-D was eluted with 5 mM Tris buffer (pH 7.4) containing 150 mM NaCI and 2 mM EDTA. The cluted fraction was dialyzed against 5 mM Tris buffer (pH 7.4) containing 100 mM NaCI at 4°C. Barium sulfate powder (Katayama Chemical Corp.) was then added to the dialysate at 2 mg/ml when calculated as a volume of the lavagc fluids and the suspension was centrifuged at 10(X10x g,,, for ~1 min. The pellet obtained was washed twice with 50 mM Tris buffer (pH 7.4) containing 150 mM NaCI and 2.5 mM .sodium citrate, and then clutcd with 2(10 mM .'sodium citrate (pH 7.4). SP-D was finally purified by passing the eluate through a Scpharose 4B (Pharmacia Fine Chemicals) column c(~alently linked with monoclonal antibody [2] against rat SP-A to remove any possible contamination by SP-A, followed by dclipidation with I-butanol (98 ml of butanol per 2 ml of SP-D suspension). The pellet after ccntrifugation of bronchoalveolar lavagc fluids at 33000 x g ~ was dispersed in 5 mM Hcpes buffer (pH 7.4) containing 150 mM NaCI and 1.64 M :.~lium bromide and surfactant was isolated by the method of H a w g ( ~ ct al. [8]. SP-A was purified from the surfactant by the method of Kuroki ct al. [18]. The dclipidated surfactant was suspended in 5 mM Tris buffer (pH 7.4) and dialyzed against the same buffer. Insoluble materials were then removed by centrifugation at 13(I(MMlxg,,, for i h at 4 ° C and the supernatant was collected. SP-A was isolated from the supernatant by affinity chromatography on mannoseSepharosc 6B and further purified by gel filtration on Bio-Gel ASm (Bio-Rad). The protein content was cstiI~ated by the method of Lowry el al. [19]. S{~ium dt~lecyl sulfatc-polyacrylamidc gel clcctrophoresis was performed according to the m c t h ~ of Laemmli [~}].
Distributions of SPA and SP-D hi bronehoah'eolar Ire'age flnid Bronchoalveolar lavage fluids were obtained from normal male Sprague-Dawley rats (200-250 g) by lavaging the rat lungs five times with 5 mM Hepes buffer (pH 7.4) containing 150 mM NaCI. After the cell debris was sedimented at 15(l×g for 10 min. the supernatant was centrifuged at 33000 x g~, at 4°C. The
supernatant and pellet obtained after centrifugation fiw 16 h were collected. The ctmtents of SP-A and SP-D in these fractions were de'~ermined by enzymelinked immunosorbcnt assay (ELISA) using specific antibodies to SP-A [21] and SP-D [22].
Direct binding of I-~~I-SP-A to ph~:pholipid on thin-layer chromatogram The binding of SP-A to phosphatidylcholine was performed by the method described recently [7]. Rat SP-A was iodinated by the method of Bolton and Hunter [23]. The specific activity of the I:~I-SP-A used was 137 cpm/ng. 10 nmol of dipalmitoylphosphatidylcholine was applied to one-dimensional thin-layer chromatography on Polygram sil G (Macherry-Nagel, Germany) and developed with the solvent system of chloroform/methanol/water (70 : 30 : 5, v/v). The plates of TLC were incubated at room temperature for 1 h with 0.5 ~ g / m l of mz-~I-SP-Ain the absence or presence of 20, 50 or 100 # g / m i of unlabeled proteins in 50 mM Tris buffer (pH 7.4) containing 0.15 M NaCI, 2 mM CaCI~, and 20 mg/ml bovine serum albumin. The plates were then washed, air-dried and exposed to Fuji X-ray film at - 8 0 °C for 3 days.
Turbidity measurements Turbidity, as a measure of iiposome aggregation, was estimated by a modified method based on that described by Hawgood et al. [8]. Phospholipids used in the present study were purchased from Sigma. A phospholipid mixture (dipalmitoylphosphatidylcholine/egg phosphatidylcholine/phosphatidylglycerol, 9: 3 : 2, by weight) in chloroform/methanol (2: !, v/v) was dried under nitrogen and hydrated in 10 mM Tris buffer (pH 7.4) containing 0.15 M NaCI (1.0 mg phospholii~.u/mi) for 1 h at 45°C and then probe-sonicated three times for 30 s with a Sonifier cell disruptor (Heat system-Ultrasonics Inc.). Phospholipid liposomes were used at a concentration of 100 ~tg/ml in 10 mM Tris buffer (pH 7.4) containing 0.15 M NaCI. Proteins were added at a concentration of 20 # g / m l to the phospholipid preparations in a I-ml glass cuvette containing 0.25 mM EDTA (time 01 and the change in turbidity was measured at 4IX) nm in a Shimazu UV 350 Recording Spectrophotomcter (Shimazu-Co. Ltd.) at 37°C. 180 s later CaCI, was added at a final concentration of 5 mM and the absorbance was further monitored.
Other methods Isolated SP-D before and after the butanol extraction was analyzed by a CI reverse-phase column (0.46 × 7.5 cm) for high perforff, dnce liquid chromatography (HPLC) (Toyo Corporation) equilibrated with 0.1% trifluoroacetic acid (TFA). Bound materials were eluted with a linear gradient of 0-100% acetonitrile in 0.1% TFA at a flow rate of 0.5 ml/min.
1~7 The soluble fraction after the extraction of SP-D with I-butanol was evaporated and suspended with c h l o r o f o r m / m e t h a n o l (2: !, v / v ) . Lipids associated with SP-D were analyzed by one-dimensional thin-layer chromatography ( T L C ) on Polygram sil G (MachereyNagel) with the solvent system o f hc.xane/diethyl e t h e r / a c e t i c acid (85 : 15 : 0.5, v / v ) . P h ~ p h o l i p i d s were also analyzed by two-dimensional TLC on silica gel G plates. The solvent systems were c h l o r o f o r m / m e t h a n o l / a c e t i c acid ( 6 5 : 2 5 : ! 0 , v / v ) for the first dimension, followed in the second dimension with c h l o r o f o r m / m e t h a n o l / 8 8 % formic acid (65 : 25 : ]0, v / v ) [24]. Lipids were visualized with iodine vapor. The phospholipid content was determined by measuring phospholipid phosphorus [25].
TABLE I
Di~tnhutum, o[ SP-D and SP-A m hr~mch~uh¢'r,~arla~age fluUL~ Br~mcl~lalvcolar latagc fluids i~dalcd |tom normal ral~ were ¢cnlri|ugcd at 3311110x g~ for Ifl h and the conlcnl~ o[ 5P-l) and .%P-,% in the ,,upcrnatanl and the t~:11¢1oblairlcd v,cr: dclermincd ;l~ dc~cril'~d undi:r Malcrial~ and Mclh~A. %,'aluc~t ~ gl rlll I Jill."liic;lll~ +5.E. |tit Ihr~c diltet'cnl prcparalNm~. Value,, (% t rcpr¢,,cnl pclccntagc ill each prolcin in |raciSms. Proichl contcnt~. SP-D
~up ppt
#glial 8.45 ~, ILI~,I 2 ~ + 074
'.:
SP-A
,.up ppl
O7, + ILk;It
ft.q
q4l.lq~+ 31.~!
q~.i
7li 2x.q
Results Eiectrophoretic analysis o f purified surfactant prowins P u r i f i e d s u r f a c t a n t p r o t e i n s w c r c s u b j e c t e d to SDSp o l y a c r y l a m i d e gel electrophoresis and the results are
shown in Fig. i. SP-A appears as a triplet of molecular masses of 26-38 kDa under reducing conditions and migrated as oligomcrs under nonreducing conditions (Fig. 1, lanes B and D). Purified SP-D dclipidated with butanol migrated with a molecular mass of 43 kDa under reducing conditions and formed disulfide-bonded oligomers under nonreducing conditions (Fig. I, lanes
Mol.Wt. (kDa) 94 67
A
B
C
D
E
I
43 30
=0
l
8 Fig. I. Elcclmplmr¢lic analy.~i~*ff purified SP-A and SP-D. Proteins were analyzed u~inl 13~; p~dyacr~lamidc~el under reducing ~mdifion.~(lance.A. B and C) and mmrcduclng o~illdilkWl~.(lanes D and E) and ~tained wilh ('oorna,~,: brilliam blue. Lane~ B and D. 5P-A: lan¢~C and E, SP-D.
C and El. Purified surfactant proteins in the dclipidated forms were u ~ d for the experiments dc~:ribcd below. Distrihuliom o f SP-A attd SP-D m bnmchahueolar /alw.,e
fluids The contents of SP-A and SP-I) in [ractkms i.,~datcd from normal rat lung la~agc by ccntrifugation at 33lk1(1 x g.,, Ior Ih h were determi==cd as sh, rwn in Table I. Total content o f SP-D was approx. 12% of that of SP-A in the lavagc fluids. 99.1~ o f SP-A w&,, present in the crude surfactant pellet, whereas 71.1% (d SP-D was in the supcrnatant. 1"his result dcrm)n~tratc5 that SP-A and SP-D distribute differently in bronchalv¢olar lavagc fluids. A n a h 'si~ Of rwl~wd SP-D hy hq;h I~'rf.,man(e hqmd
chr~.~mat~eqraph) UIPL('J SP-D wa~ i ~ l a l c d from the supvrnatant obtained after the centrifugati(m i d the lavaging fluids at j3lgl(I × g j, by manno~-Scpharow.: 4B column chromatilgraphy, and analyzed by rcvcr.~-pha.v,: HPLC (Fig. 21. T w o main peak~ wcrc obtained ~ ' eluting with acetonit rile gradient in (1.1% "rFA (Fig. 2A). The.so fractions (peaks a and b) were further analyzed by elcctrophore~is under reducing conditions (Fig. 2. i n , t . lanes a and b. respectively). Electrophorctic analysis demonstrates that peak b consists o f a 43 kDa pr(~cin, whereas peak a contains no protein band. Next, isolated SP-D wa~ extracted with butamd and the butanol-insoluble mate. rial was analyzed by HPL(" (Fig. 2BI. The main peak was cluted at the .,mmc p ~ i t k m where peak b was eluted, and migrated as a 43 kDa protein band by clcctrophorctic analysis (not shown). T h c ~ d~ta indicate that butanol-soluble material, presumably lipids. appears to b¢ copurificd with SP-D i.,,olated from thc lavagc SUl~rnatant.
188 1.0- A--:--
laW. at. (i~qll
l
b
We
.....,,,I ~,..,."
6"1- 9111
/./
43- II
/.......!
30--~ ~ N ~
O.$-
~
I~
SO
20-~ ~1,-~
.i
O.S-
-SO
I
Ofz:
i/''/'i"1//
/I .... )
Tme
(min)
Fi~. 2. Analysis of isolated SP-D by high performance liquid chromatography (HPLC). SP-D was isolated from the 33000g supernatant of the bronchoalveolar lavage fluids by manm',~,c-Sepharosc 4B column chromatography and analyzed before (panel A) and after (panel B) the butanol extraction by reversc-pha~ HPLC and ra~mitored by absorbance at 215 nm as described under Materials and Methods. In panel A. the eluted fractions (peaks a and b) were further analyzed by sodium dodecyl sulfate-polyacq,'lamide gel electrophoresis (13q~) under reducing conditions (inset. lanes a and b. respectively)and stained with Coomassie brilliant blue.
AnalysisoflipidscopurifiedwithSP-D SP-D was purified from the lavaging fluids by mannose-Sepharose 4B column chromatography and adsorption with barium sulfate, followed by passage through a column covalently linked with monoclonal antibody to rat SP-A, and then further delipidated with butanol. The butanol-soluble material was then analyzed by one-dimensional thin-layer chromatography. The thin-layer chromatogram revealed that the butanol-soluble materials are lipids and consist of cholesterol, triglyceride, fatty acid. and mainly phospholipids (not shown). Next, phospholipids copurified with SP-D were analyzed by two-dimensional thin-layer chromatography and phospholipid composition was determined as shown in Table II. The main componcnt of the phospholipids was phosphatidylcholine. The phospholipid composition of lipids copurifled with SP-D does not appear to be significantly different from that of the surfactant pellet [26], The decreased percentage of phosphatidylglycerol and the increased percentage of phosphatidylinositol could be a consequence of intratracheal instillation of silica, which is consistent with a previous report [26]. 745 + 186 nmol of phospholipids
(mean + S.D., it = 4) per 1 mg of SP-D was determined. These results demonstrate that lipids are copurifled with SP-D.
Binding to phospho!ipids and lipid aggregation We have recently reported that SP-A specifically binds dipalmitoylphosphatidylcholine [7]. CopurificaTABLE II
('~.nlx~itiovt o f phospholipids copurified .'ith SP-D Indh.'idual phospholipids isolated by extraction of SP-D with lbutanol w e r e separated as described under Materials and Methods. Values (¢'i pl~sphorus) are means for two different preparations of SP-D Composition (moire;) Phosphatidylcholine Lysophosphatidyleholine Phosphatidylinositol Phosphatidylglyeerol Phosphatidylcthanolamine Phosphatidylscrinc Sphin~myelinc Others
84.8 1.0 7.3 1.6 1.4 trace 1.3 2.4
189
unlabeled proteins (l~glml)
SP-D 0
/0
50
SP-A /00
~0
50
!100
tit n of phospholipids, mainly phosphatidylcholir, e, with SP-D suggests that lipids may be associated with SP-D, and SP-D may bind to phosphatidylcholine in the same fashion as SP-A. To examine this possibility, we studied whether excess SP-D competes with n-'~I-SP-A for phosphatidylcholine binding. '-'51-SP-A binding (11.5 /zg/ml) to dipalmitoylphosphatidylcholine on thin-layer chromatogram was performed in the pre~ncc of 211, 50 and 1(1(1 # g / m l of either unlabeled SP-D or SP-A. Unlabeled SP-A inhibited L'51-SP-A binding to phospholipid in a concentration-dependent manner, whereas unlabeled SP-D failed to compete with radiolabeled SP-A (Fig. 3). SP-A alg~ aggregated phospholipid liposomes in the presence of calcium ions, but SP-D did not aggregate liposomes (Fig. 4). T h e ~ data suggest that the association of phosphoiipids with SP-D appears to be different from that with SP-A.
Discussion Fig. 3. Purified SP-D failed to compete with J-'~I-SP-A for phosplmlipid binding. Dipalmitoylphosphatidyleholine (10 nmol/lan¢) was developed with chloroform~methanol~water(7(h.'~h51 and t-'SI-SPA (0.5/1 g/ml) binding was performed in the absence (01 or presence of 20, 50 and 100 # g / m l 120, 50 and IIXD)of either purified SP-D or SP-A and autoradiographed as described under Materials and Methods.
0.2
calcium chloride (SmM)
0
C
0.1 o
0.0
I
200
, ~
400
E
11 A B D
600
lime(seconds) Fig. 4. Effect of purified SP-D on liposome aggregation. P h ~ p h o lipid liposomes (lllfi p g / m l ) containing II.Z4i mM EDTA were incubaled alone (A) or with 211 p g / m l of either SP-D t e l or SP-A (C). SP-A alone and SP-D alone without phospholipid I i p ~ m e s IB and D, respectively) were a l ~ incubated and turbidity l a b ~ r b a n c e at 4U(I nm) was measured at 3"PC. CaCI: was added 180 s later at a final concentration of 5 mM and the absorbance at 400 nm was further monitored as de~ribcd under Materials and Methods. Data are from a representative experiment of four performed.
SP-D has rcccntly becn de~ribed to be synthesized and secreted by alveolar type !1 cells, to be a glycoprotein that contains collagcnous and noncollagenous sequences and to function as a calcium-dependent carbohydrate-binding protein [13-15]. The present study demonstrates that lipids are eopurified with SP-D isolated from the ,soluble fraction of rat bnmchoalveolar lavage fluids, that the distribution of SP-D in the lavage fluids are clearly different from that of SP-A, and that SP-D does not possess the ~ m e activities for phospholipids as SP-A. An immunoas~y of SP-D developed in our laboratory [22] made it possible to compare the absolute amounts of SF-D attd SP-A. The total amount of SP-D determined in the bronchoah, colar lavage from normal rats was quite low compared with that of SP-A. Unlike SP-A. a significant amount of SP-D (more than 711% of total amount of SP-D) is in the 33(l(MIg supcrnatant of rat bronchoalveolar lavage, which is essentially consistent with the study de~ribed by Pers.~m ct al. [14] who estimated the relative amount by immunoblotting and densitomctory of silver-stained gels. It is surprising that SP-D distributes predominantly to the supcrnatant in the 331100 x g centrifugal(on. SP-D ma: be p r e ~ n t in a soluble form with a small am~mnt of I?pids, and may function in the alvcoli in a different fashion to SP-A, most of which associates with surfactant pellet. SP-D and SP-A appear to show different states in brondu~alveolar fluids. The p r e ~ n t study confirmed that a significant am~mnt of SP-D is ~duble in the bronchoalveolar lavage fluids, and a l ~ demonstrated that .some lipids are copurified with isolated SP-D in the u~luble form (the 3311011g supernatantL If a small amount of SP-A were to contaminate the preparation of SP-D in the purification steps of mannose-Sepharo~ 4B col-
190 umn chromatography followed by barium adsorption. one possibility comes up, that is. the contaminated SP-A rather than the SP-D may interact with phospholipids since SP-A binds phospholipids [5-7]. Howc~,cr. wc accomplished the purification of SP-D by passing it through a column covalently linked with monockmal antibody to rat SP-A, so that any chance of contamination by SP-A has bccn removed completcly, as shown in Fig. I. Antibody to SP-A failed to recognize any contamination of SP-A in our preparations of purified SP-D by immunoblotting analysis (not shown). There is also a possibility that surfactant lipids might be retained in the affinity column, and be eluted in the prepuce of EDTA since surfactant lipid may aggregate in the prepuce of calcium. However, we at first centrifi,ged thc bronchoalveolar lavagc fluids in the presence of 5 mM CaCI, at 33000 ×g~,, overnight. Thus, lipid aggregates caused by the addition of calcium appeared to be removed before the lavage supernatant was applied to the affinity column. Although phosphatidylchotine is the main component of ph~rspholipids copurificd with SP-D, SP-D failed to compete with labeled SP-A fi~r phosphatidylcholi~ ~ binding. SP-D also failed to aggregate phospholipid lipc~,omes. Although SP-D may bind phosphatidylcholine with a lower affinity than SP-A, or SP-D may bind another component of lipids, the present study suggests that SP-D may asg~ciate with lipids in a different fashion from SP-A. In conclusion, the present study demonstrates that lipids are copurified with SP-D, that approximately three quarters of SP-D cxists as the .soluble form, that SP-D and SP-A distribute differently in rat broncholveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fitshion as SP-A. Acknowledgements We thank Profes~,r Fumi Morita (Faculty of Science. University of Hokkaido) and Dr. Sohma (Sapporo Medical College) for the u,~ of Shimazu UV 350 Recording Spectrophotometer. This re,~arch was in part supported by Grants in Aid for Scientific Research from the Ministry of Education, Japan.
References I King, R.J. and ('lcmcnts, J.A. ( 19721 Am. J. Physiol. 223. 707-714. 2 Weaver. T.E., Ilull. W.R., Ross, G.F. and Whitr, ctt, J.A. (1985) Biochim. Biophys. Acta 827, 260-267. 3 White. R.T., Datum. D.. Miller, J.. SpraU. K., Schilling, J., tlawg~a~d. S., Bcnson. B. and Cordell. B. (1985) Nature (London) 317, 361-363. 4 Itaagsman, tI.P., Flawg~a~d, S.. Sargeant, T.. Buckley, D., While, R.T., Drickamer, K. and Bcnson. B.J. (19871 J. Biol. Chem. 262, 13877-13880. 5 King, R.J., Carmichael. C. and ilorowitz, P.M. (1983) J. Biol. Chem. 25g, 111672-11168(I, 6 Ross. G.F.. NoUer, R.H.. Meuth, J. and Whitsen. J.A. (1986)J. Biol. Chem. 261, 14283-14291. 7 Kuroki. Y. and Akino. T. (1991) J. Biol. Chem. 266. 3068-31173. 8 llawgood. S., Bcn~m. B, and tlamilton. R.J. (1985) Biochemistry 24, 184-190. 9 Dobbs. L.G.. Wright, J R . . Hawgood. S.. Gonzalez, R.. Venstrom. K. and Nellenbogen, J. (1987) Proc. Natl. Acad. Sci. USA 84. 1010-1014. 10 Rice, W., Ross, G.F.. Singleton, F.M., Dingle. S. and W h i t e n , J.A. (Its87)J. Appl. Physiol. 63. 692-698. I I Wright. J,R.. Wager. R.E.. Hawgood. S.. Dobbs, L. and CJements, J.A. (1t~871J. l~iol. Chem. 262. 2888-2894. J2 Van Iv,'aarden. F.. Welms, B.. Verhoeh, J.. Haagsman. H.P. and Van Golde, L, M.G. (19911) Am. J. Respir. Cell Mol. Biol. 2. 91-98. 13 Persson, A., Rust, K.. Chang, D.. MoxJey, M.. Longmore. W. and Crouch. E. (19881 Biochemistry 27. 8576-8584. 14 Pcrsson, A.. Chang, D., Rust. K.. Moxley. M.. Longmore, W. and Crouch, E. (19891 Biochemistry 8. 6361-6367. J5 Persson. A., Chang, D. and Crouch. E. ( 199111J. Biol. Chem. 265. 5755-57~0. 16 Dethloff. L.A., Gilrm~re. UB.. Brody. A.R. and Hook. G.E,R. (1986) BkK'hem. J. 233. 111-118. 17 Fornstedt. N. and Porath. J. (10751 FEBS Len. 57, 187-19118. 18 Kuroki. Y.. Mason. R.J. and Voelker. D.R. (19881 Pro¢. Nat. Acad. Sci. USA 85, 5566-5570. It/ I,owry, O.tt.. Rosebrough. N.J.. Farr. A.L. and Randall. R J . (19511J. Biol. Chem, ITS3.265-275. 211 Laemmli, U.K. (19701 Nature. 227, 6811-685. 21 Shimizu. tt.. Miyamura. K. and KurokL Y. (It~l) Biochim. Biophys, Acta 1081, 53-611. 22 Ogasawara. Y.. Kuroki. Y.. Shiratori. M.. Shimizu. H., Miyamura. K. and Akino. T. (It~,~l) Bit~:him. Biophys. Acla 1(183. 252-256. 23 Dohon, A.E. and Ilunter. W.M. (19731 Biocl'em, J. 133. 529-539. 24 Esh~, J.D. and Raetz. C.R.H. (198111J. Biol. Chem. 255. 447444811, 25 Bartlett. G R . (1959) J. Biol. Chem. 234, 466-468. 26 Adachi, H., Hayashi. H.. Sato. H.. Dempo. K. and Akino. T. (1989) Biochem, J. 262. 781-780.
185
BBALIP 53765
Characterization of pulmonary surfactant protein D: its copurification with lipids Yoshio Kuroki, Masanori Shiratori, Yoshinori Ogasawara, Akihiro Tsuzuki and Toyoaki Akino Department of Biochemixt~'. Sapl~m~ [~(,dit'al ('oll(~(,. Sapporo (Japan/ (Received I~ February 19gl )
Key words: Pulmonarysurfaclanl: Surfaclant prolcin D: Pl~,pholipid
Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II calls. SP-D was purified from the supernatant of rat bronchoalvcolar iavagc fluids obtained by centrifugation at 3 3 0 0 0 x g , ~ for 16 h. The contents of SP-D and SPA in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of S P A in these lavage fluids. 99.1% of SP-A was present in the 33000£ pellet, whereas 71.1% of SP-D was in the 33000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SI' A, SP-D in the purified and delipidated form failed to compete with '~l-laheled SP-A for plmsphotidylcholine binding, and to aggregate phosphlipid liposomes. The present study demonstrates that lipids are copurifled with SP-D, that SP-D and S P A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.
Introduction Pulmonary surfactant, a complex mixture of lipids tnd proteins, functions to stabilize alveoli by lowering the surface tension at the air/liquid interface [I]. One of the major protein components of surfactant (surfactant protein A, SP-A) is a glycoprotein that has monomeric molecular masses of 26-38 kDa in the rat [2]. This protein is characterized by the striking features of collagen-like sequences [3] and a carbohydrate-binding property [4]. SP-A pos~sses a variety of biological activities. SP-A interacts with surfactantlike phospholipids [5,6], specifically binds dipa;mitoylphosphatidylcholine [7], and causes phospholipid ag-
Abbreviations: SP-D, surfaclanl associated protein D: SP-A. surfacrant associated protein A: llcpcs, N-2 hydro~elhylpipcrazine-N'-2ethanesulfonic acid: ELISA. enzyme-linked immumv~rbcnl assay: TLC, thin-layer chromatography: HPLC, high performance liquid chromatography; dipalmitoylplv,)sphatidylcholineDPPC: EDTA. elhylenediaminetetraacelate. Correspondence: Y. Kuroki, [:~partmenl of Bk~chemislry. Sapporo Medical College, South-l, West-17. Chuo-ku. Sapporo (~|. Japan.
gregation [8]. This protein has al~) been sht~vn to function as a negative regulator of surfactant ph(vspholipid secretion by alveolar type II cells [9,10], to enhance the uptake of phimpholipid liposomes by the,we cells [II] and to enhance the host-defense mcchanism of rat alveolar type il cells [12]. Another hydrophilic surfactant protein (SP-D) has recently been i.5olated from the culture medium of alveolar type II ceils and from rat bronchoalveolar lavagc fluids [13.14]. SP-D is a glycoprotein that shows similarities to SP-A in its collagcnous structure and carbohydrate-binding property [ 13-15]. Similarities betwecn SP-A and SP-D suggest that SP-D may function like SP-A. The purpose of the present study was: (I) to puri!~ and characterize SP-D isolated from rat bronchoalveolar lavagc and (2) to examine interactions of SP-D with iipids in compari.qm with thirst of SP-A.
Materials and Methods Purification o f sur~actant proteins Bronchoalveolar lavage was obtained from rats given intratracheal instillation of IO mg per rat silica in .saline 4 weeks before lung lavage according to the method of Dcthloff et al. [16]. Prelreatmcnt of the rats with silica
186 increased the yield of surfactant approximately 2¢l-fold. Lungs were lavaged l0 times with 5 mM ftcpes buffer (pH 7.4)containing 150 mM NaCI and bronehoalvcolar lavage fluid~ were centrifuged at 15l) × g for Ill rain to remove cell debris. The supernatant was centrifuged at 33(XX) ×g,,~ at 4°C 16 h after the addition of CaCI, at a final concentration of 5 raM. The supcrnatant was used fi)r the isolation of SP-D. which was purified by the modified methods essentially based on those de.scribed by Persson el al. [14,15]. The supernatant was applied to an affinity column of mannose-Sepharosc 4B (Pharmacia Fine Chemicals) prepared by the method of Fornstcdt and Porath [17]. SP-D was eluted with 5 mM Tris buffer (pH 7.4) containing 150 mM NaCI and 2 mM EDTA. The cluted fraction was dialyzed against 5 mM Tris buffer (pH 7.4) containing 100 mM NaCI at 4°C. Barium sulfate powder (Katayama Chemical Corp.) was then added to the dialysate at 2 mg/ml when calculated as a volume of the lavagc fluids and the suspension was centrifuged at 10(X10x g,,, for ~1 min. The pellet obtained was washed twice with 50 mM Tris buffer (pH 7.4) containing 150 mM NaCI and 2.5 mM .sodium citrate, and then clutcd with 2(10 mM .'sodium citrate (pH 7.4). SP-D was finally purified by passing the eluate through a Scpharose 4B (Pharmacia Fine Chemicals) column c(~alently linked with monoclonal antibody [2] against rat SP-A to remove any possible contamination by SP-A, followed by dclipidation with I-butanol (98 ml of butanol per 2 ml of SP-D suspension). The pellet after ccntrifugation of bronchoalveolar lavagc fluids at 33000 x g ~ was dispersed in 5 mM Hcpes buffer (pH 7.4) containing 150 mM NaCI and 1.64 M :.~lium bromide and surfactant was isolated by the method of H a w g ( ~ ct al. [8]. SP-A was purified from the surfactant by the method of Kuroki ct al. [18]. The dclipidated surfactant was suspended in 5 mM Tris buffer (pH 7.4) and dialyzed against the same buffer. Insoluble materials were then removed by centrifugation at 13(I(MMlxg,,, for i h at 4 ° C and the supernatant was collected. SP-A was isolated from the supernatant by affinity chromatography on mannoseSepharosc 6B and further purified by gel filtration on Bio-Gel ASm (Bio-Rad). The protein content was cstiI~ated by the method of Lowry el al. [19]. S{~ium dt~lecyl sulfatc-polyacrylamidc gel clcctrophoresis was performed according to the m c t h ~ of Laemmli [~}].
Distributions of SPA and SP-D hi bronehoah'eolar Ire'age flnid Bronchoalveolar lavage fluids were obtained from normal male Sprague-Dawley rats (200-250 g) by lavaging the rat lungs five times with 5 mM Hepes buffer (pH 7.4) containing 150 mM NaCI. After the cell debris was sedimented at 15(l×g for 10 min. the supernatant was centrifuged at 33000 x g~, at 4°C. The
supernatant and pellet obtained after centrifugation fiw 16 h were collected. The ctmtents of SP-A and SP-D in these fractions were de'~ermined by enzymelinked immunosorbcnt assay (ELISA) using specific antibodies to SP-A [21] and SP-D [22].
Direct binding of I-~~I-SP-A to ph~:pholipid on thin-layer chromatogram The binding of SP-A to phosphatidylcholine was performed by the method described recently [7]. Rat SP-A was iodinated by the method of Bolton and Hunter [23]. The specific activity of the I:~I-SP-A used was 137 cpm/ng. 10 nmol of dipalmitoylphosphatidylcholine was applied to one-dimensional thin-layer chromatography on Polygram sil G (Macherry-Nagel, Germany) and developed with the solvent system of chloroform/methanol/water (70 : 30 : 5, v/v). The plates of TLC were incubated at room temperature for 1 h with 0.5 ~ g / m l of mz-~I-SP-Ain the absence or presence of 20, 50 or 100 # g / m i of unlabeled proteins in 50 mM Tris buffer (pH 7.4) containing 0.15 M NaCI, 2 mM CaCI~, and 20 mg/ml bovine serum albumin. The plates were then washed, air-dried and exposed to Fuji X-ray film at - 8 0 °C for 3 days.
Turbidity measurements Turbidity, as a measure of iiposome aggregation, was estimated by a modified method based on that described by Hawgood et al. [8]. Phospholipids used in the present study were purchased from Sigma. A phospholipid mixture (dipalmitoylphosphatidylcholine/egg phosphatidylcholine/phosphatidylglycerol, 9: 3 : 2, by weight) in chloroform/methanol (2: !, v/v) was dried under nitrogen and hydrated in 10 mM Tris buffer (pH 7.4) containing 0.15 M NaCI (1.0 mg phospholii~.u/mi) for 1 h at 45°C and then probe-sonicated three times for 30 s with a Sonifier cell disruptor (Heat system-Ultrasonics Inc.). Phospholipid liposomes were used at a concentration of 100 ~tg/ml in 10 mM Tris buffer (pH 7.4) containing 0.15 M NaCI. Proteins were added at a concentration of 20 # g / m l to the phospholipid preparations in a I-ml glass cuvette containing 0.25 mM EDTA (time 01 and the change in turbidity was measured at 4IX) nm in a Shimazu UV 350 Recording Spectrophotomcter (Shimazu-Co. Ltd.) at 37°C. 180 s later CaCI, was added at a final concentration of 5 mM and the absorbance was further monitored.
Other methods Isolated SP-D before and after the butanol extraction was analyzed by a CI reverse-phase column (0.46 × 7.5 cm) for high perforff, dnce liquid chromatography (HPLC) (Toyo Corporation) equilibrated with 0.1% trifluoroacetic acid (TFA). Bound materials were eluted with a linear gradient of 0-100% acetonitrile in 0.1% TFA at a flow rate of 0.5 ml/min.
1~7 The soluble fraction after the extraction of SP-D with I-butanol was evaporated and suspended with c h l o r o f o r m / m e t h a n o l (2: !, v / v ) . Lipids associated with SP-D were analyzed by one-dimensional thin-layer chromatography ( T L C ) on Polygram sil G (MachereyNagel) with the solvent system o f hc.xane/diethyl e t h e r / a c e t i c acid (85 : 15 : 0.5, v / v ) . P h ~ p h o l i p i d s were also analyzed by two-dimensional TLC on silica gel G plates. The solvent systems were c h l o r o f o r m / m e t h a n o l / a c e t i c acid ( 6 5 : 2 5 : ! 0 , v / v ) for the first dimension, followed in the second dimension with c h l o r o f o r m / m e t h a n o l / 8 8 % formic acid (65 : 25 : ]0, v / v ) [24]. Lipids were visualized with iodine vapor. The phospholipid content was determined by measuring phospholipid phosphorus [25].
TABLE I
Di~tnhutum, o[ SP-D and SP-A m hr~mch~uh¢'r,~arla~age fluUL~ Br~mcl~lalvcolar latagc fluids i~dalcd |tom normal ral~ were ¢cnlri|ugcd at 3311110x g~ for Ifl h and the conlcnl~ o[ 5P-l) and .%P-,% in the ,,upcrnatanl and the t~:11¢1oblairlcd v,cr: dclermincd ;l~ dc~cril'~d undi:r Malcrial~ and Mclh~A. %,'aluc~t ~ gl rlll I Jill."liic;lll~ +5.E. |tit Ihr~c diltet'cnl prcparalNm~. Value,, (% t rcpr¢,,cnl pclccntagc ill each prolcin in |raciSms. Proichl contcnt~. SP-D
~up ppt
#glial 8.45 ~, ILI~,I 2 ~ + 074
'.:
SP-A
,.up ppl
O7, + ILk;It
ft.q
q4l.lq~+ 31.~!
q~.i
7li 2x.q
Results Eiectrophoretic analysis o f purified surfactant prowins P u r i f i e d s u r f a c t a n t p r o t e i n s w c r c s u b j e c t e d to SDSp o l y a c r y l a m i d e gel electrophoresis and the results are
shown in Fig. i. SP-A appears as a triplet of molecular masses of 26-38 kDa under reducing conditions and migrated as oligomcrs under nonreducing conditions (Fig. 1, lanes B and D). Purified SP-D dclipidated with butanol migrated with a molecular mass of 43 kDa under reducing conditions and formed disulfide-bonded oligomers under nonreducing conditions (Fig. I, lanes
Mol.Wt. (kDa) 94 67
A
B
C
D
E
I
43 30
=0
l
8 Fig. I. Elcclmplmr¢lic analy.~i~*ff purified SP-A and SP-D. Proteins were analyzed u~inl 13~; p~dyacr~lamidc~el under reducing ~mdifion.~(lance.A. B and C) and mmrcduclng o~illdilkWl~.(lanes D and E) and ~tained wilh ('oorna,~,: brilliam blue. Lane~ B and D. 5P-A: lan¢~C and E, SP-D.
C and El. Purified surfactant proteins in the dclipidated forms were u ~ d for the experiments dc~:ribcd below. Distrihuliom o f SP-A attd SP-D m bnmchahueolar /alw.,e
fluids The contents of SP-A and SP-I) in [ractkms i.,~datcd from normal rat lung la~agc by ccntrifugation at 33lk1(1 x g.,, Ior Ih h were determi==cd as sh, rwn in Table I. Total content o f SP-D was approx. 12% of that of SP-A in the lavagc fluids. 99.1~ o f SP-A w&,, present in the crude surfactant pellet, whereas 71.1% (d SP-D was in the supcrnatant. 1"his result dcrm)n~tratc5 that SP-A and SP-D distribute differently in bronchalv¢olar lavagc fluids. A n a h 'si~ Of rwl~wd SP-D hy hq;h I~'rf.,man(e hqmd
chr~.~mat~eqraph) UIPL('J SP-D wa~ i ~ l a l c d from the supvrnatant obtained after the centrifugati(m i d the lavaging fluids at j3lgl(I × g j, by manno~-Scpharow.: 4B column chromatilgraphy, and analyzed by rcvcr.~-pha.v,: HPLC (Fig. 21. T w o main peak~ wcrc obtained ~ ' eluting with acetonit rile gradient in (1.1% "rFA (Fig. 2A). The.so fractions (peaks a and b) were further analyzed by elcctrophore~is under reducing conditions (Fig. 2. i n , t . lanes a and b. respectively). Electrophorctic analysis demonstrates that peak b consists o f a 43 kDa pr(~cin, whereas peak a contains no protein band. Next, isolated SP-D wa~ extracted with butamd and the butanol-insoluble mate. rial was analyzed by HPL(" (Fig. 2BI. The main peak was cluted at the .,mmc p ~ i t k m where peak b was eluted, and migrated as a 43 kDa protein band by clcctrophorctic analysis (not shown). T h c ~ d~ta indicate that butanol-soluble material, presumably lipids. appears to b¢ copurificd with SP-D i.,,olated from thc lavagc SUl~rnatant.
188 1.0- A--:--
laW. at. (i~qll
l
b
We
.....,,,I ~,..,."
6"1- 9111
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43- II
/.......!
30--~ ~ N ~
O.$-
~
I~
SO
20-~ ~1,-~
.i
O.S-
-SO
I
Ofz:
i/''/'i"1//
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Tme
(min)
Fi~. 2. Analysis of isolated SP-D by high performance liquid chromatography (HPLC). SP-D was isolated from the 33000g supernatant of the bronchoalveolar lavage fluids by manm',~,c-Sepharosc 4B column chromatography and analyzed before (panel A) and after (panel B) the butanol extraction by reversc-pha~ HPLC and ra~mitored by absorbance at 215 nm as described under Materials and Methods. In panel A. the eluted fractions (peaks a and b) were further analyzed by sodium dodecyl sulfate-polyacq,'lamide gel electrophoresis (13q~) under reducing conditions (inset. lanes a and b. respectively)and stained with Coomassie brilliant blue.
AnalysisoflipidscopurifiedwithSP-D SP-D was purified from the lavaging fluids by mannose-Sepharose 4B column chromatography and adsorption with barium sulfate, followed by passage through a column covalently linked with monoclonal antibody to rat SP-A, and then further delipidated with butanol. The butanol-soluble material was then analyzed by one-dimensional thin-layer chromatography. The thin-layer chromatogram revealed that the butanol-soluble materials are lipids and consist of cholesterol, triglyceride, fatty acid. and mainly phospholipids (not shown). Next, phospholipids copurified with SP-D were analyzed by two-dimensional thin-layer chromatography and phospholipid composition was determined as shown in Table II. The main componcnt of the phospholipids was phosphatidylcholine. The phospholipid composition of lipids copurifled with SP-D does not appear to be significantly different from that of the surfactant pellet [26], The decreased percentage of phosphatidylglycerol and the increased percentage of phosphatidylinositol could be a consequence of intratracheal instillation of silica, which is consistent with a previous report [26]. 745 + 186 nmol of phospholipids
(mean + S.D., it = 4) per 1 mg of SP-D was determined. These results demonstrate that lipids are copurifled with SP-D.
Binding to phospho!ipids and lipid aggregation We have recently reported that SP-A specifically binds dipalmitoylphosphatidylcholine [7]. CopurificaTABLE II
('~.nlx~itiovt o f phospholipids copurified .'ith SP-D Indh.'idual phospholipids isolated by extraction of SP-D with lbutanol w e r e separated as described under Materials and Methods. Values (¢'i pl~sphorus) are means for two different preparations of SP-D Composition (moire;) Phosphatidylcholine Lysophosphatidyleholine Phosphatidylinositol Phosphatidylglyeerol Phosphatidylcthanolamine Phosphatidylscrinc Sphin~myelinc Others
84.8 1.0 7.3 1.6 1.4 trace 1.3 2.4
189
unlabeled proteins (l~glml)
SP-D 0
/0
50
SP-A /00
~0
50
!100
tit n of phospholipids, mainly phosphatidylcholir, e, with SP-D suggests that lipids may be associated with SP-D, and SP-D may bind to phosphatidylcholine in the same fashion as SP-A. To examine this possibility, we studied whether excess SP-D competes with n-'~I-SP-A for phosphatidylcholine binding. '-'51-SP-A binding (11.5 /zg/ml) to dipalmitoylphosphatidylcholine on thin-layer chromatogram was performed in the pre~ncc of 211, 50 and 1(1(1 # g / m l of either unlabeled SP-D or SP-A. Unlabeled SP-A inhibited L'51-SP-A binding to phospholipid in a concentration-dependent manner, whereas unlabeled SP-D failed to compete with radiolabeled SP-A (Fig. 3). SP-A alg~ aggregated phospholipid liposomes in the presence of calcium ions, but SP-D did not aggregate liposomes (Fig. 4). T h e ~ data suggest that the association of phosphoiipids with SP-D appears to be different from that with SP-A.
Discussion Fig. 3. Purified SP-D failed to compete with J-'~I-SP-A for phosplmlipid binding. Dipalmitoylphosphatidyleholine (10 nmol/lan¢) was developed with chloroform~methanol~water(7(h.'~h51 and t-'SI-SPA (0.5/1 g/ml) binding was performed in the absence (01 or presence of 20, 50 and 100 # g / m l 120, 50 and IIXD)of either purified SP-D or SP-A and autoradiographed as described under Materials and Methods.
0.2
calcium chloride (SmM)
0
C
0.1 o
0.0
I
200
, ~
400
E
11 A B D
600
lime(seconds) Fig. 4. Effect of purified SP-D on liposome aggregation. P h ~ p h o lipid liposomes (lllfi p g / m l ) containing II.Z4i mM EDTA were incubaled alone (A) or with 211 p g / m l of either SP-D t e l or SP-A (C). SP-A alone and SP-D alone without phospholipid I i p ~ m e s IB and D, respectively) were a l ~ incubated and turbidity l a b ~ r b a n c e at 4U(I nm) was measured at 3"PC. CaCI: was added 180 s later at a final concentration of 5 mM and the absorbance at 400 nm was further monitored as de~ribcd under Materials and Methods. Data are from a representative experiment of four performed.
SP-D has rcccntly becn de~ribed to be synthesized and secreted by alveolar type !1 cells, to be a glycoprotein that contains collagcnous and noncollagenous sequences and to function as a calcium-dependent carbohydrate-binding protein [13-15]. The present study demonstrates that lipids are eopurified with SP-D isolated from the ,soluble fraction of rat bnmchoalveolar lavage fluids, that the distribution of SP-D in the lavage fluids are clearly different from that of SP-A, and that SP-D does not possess the ~ m e activities for phospholipids as SP-A. An immunoas~y of SP-D developed in our laboratory [22] made it possible to compare the absolute amounts of SF-D attd SP-A. The total amount of SP-D determined in the bronchoah, colar lavage from normal rats was quite low compared with that of SP-A. Unlike SP-A. a significant amount of SP-D (more than 711% of total amount of SP-D) is in the 33(l(MIg supcrnatant of rat bronchoalveolar lavage, which is essentially consistent with the study de~ribed by Pers.~m ct al. [14] who estimated the relative amount by immunoblotting and densitomctory of silver-stained gels. It is surprising that SP-D distributes predominantly to the supcrnatant in the 331100 x g centrifugal(on. SP-D ma: be p r e ~ n t in a soluble form with a small am~mnt of I?pids, and may function in the alvcoli in a different fashion to SP-A, most of which associates with surfactant pellet. SP-D and SP-A appear to show different states in brondu~alveolar fluids. The p r e ~ n t study confirmed that a significant am~mnt of SP-D is ~duble in the bronchoalveolar lavage fluids, and a l ~ demonstrated that .some lipids are copurified with isolated SP-D in the u~luble form (the 3311011g supernatantL If a small amount of SP-A were to contaminate the preparation of SP-D in the purification steps of mannose-Sepharo~ 4B col-
190 umn chromatography followed by barium adsorption. one possibility comes up, that is. the contaminated SP-A rather than the SP-D may interact with phospholipids since SP-A binds phospholipids [5-7]. Howc~,cr. wc accomplished the purification of SP-D by passing it through a column covalently linked with monockmal antibody to rat SP-A, so that any chance of contamination by SP-A has bccn removed completcly, as shown in Fig. I. Antibody to SP-A failed to recognize any contamination of SP-A in our preparations of purified SP-D by immunoblotting analysis (not shown). There is also a possibility that surfactant lipids might be retained in the affinity column, and be eluted in the prepuce of EDTA since surfactant lipid may aggregate in the prepuce of calcium. However, we at first centrifi,ged thc bronchoalveolar lavagc fluids in the presence of 5 mM CaCI, at 33000 ×g~,, overnight. Thus, lipid aggregates caused by the addition of calcium appeared to be removed before the lavage supernatant was applied to the affinity column. Although phosphatidylchotine is the main component of ph~rspholipids copurificd with SP-D, SP-D failed to compete with labeled SP-A fi~r phosphatidylcholi~ ~ binding. SP-D also failed to aggregate phospholipid lipc~,omes. Although SP-D may bind phosphatidylcholine with a lower affinity than SP-A, or SP-D may bind another component of lipids, the present study suggests that SP-D may asg~ciate with lipids in a different fashion from SP-A. In conclusion, the present study demonstrates that lipids are copurified with SP-D, that approximately three quarters of SP-D cxists as the .soluble form, that SP-D and SP-A distribute differently in rat broncholveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fitshion as SP-A. Acknowledgements We thank Profes~,r Fumi Morita (Faculty of Science. University of Hokkaido) and Dr. Sohma (Sapporo Medical College) for the u,~ of Shimazu UV 350 Recording Spectrophotometer. This re,~arch was in part supported by Grants in Aid for Scientific Research from the Ministry of Education, Japan.
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