Isolation of pulmonary surfactant proteins from bronchoalveolar lavage fluids

Shigeru Ariki; Chiaki Nishitani; Yoshio Kuroki

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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.

Isolation of pulmonary surfactant proteins from bronchoalveolar lavage fluids

Shigeru Ariki, Doctor of Science, Ph.D.

Sapporo Medical University

Email: pj.ca.dempas@bcskiras

Corresponding author.

Chiaki Nishitani, Doctor of Science, Ph.D.

Sapporo Medical University

Yoshio Kuroki, M.D., Ph.D.

Sapporo Medical University

Email: moc.liamg@4150yikoruk

Created: September 30, 2021; Last Revision: March 18, 2022.

Introduction

Alveolar type II cells produce and secrete a complex mixture of lipids and proteins called pulmonary surfactant, which keeps the alveoli from collapsing at the end of expiration (1,2). Surfactant enables an effortless process that occurs with a frequency of ~25,000 cycles/d in breathing (2,3). Pulmonary surfactant contains four specific proteins: SP-A, SP-B, SP-C, and SP-D (4). The hydrophobic surfactant proteins, SP-B and SP-C, affect the biophysical functions of surfactant. The hydrophilic surfactant proteins, SP-A and SP-D, belong to the C-type lectin superfamily. SP-A and SP-D along with mannose-binding lectin comprise a subgroup of the C-type lectins that possess collagen-like domains and are called collectins (5). SP-A specifically binds dipalmitoyl phosphatidylcholine that is essential for biophysical function of surfactant (6). SP-A is a potent negative regulator of surfactant phospholipid secretion and affects the regulation of recycling of dipalmitoyl phosphatidylcholine (7), whereas SP-D binds to phosphatidylinositol (8). Additionally, SP-A and SP-D have been implicated in the regulation of pulmonary host defense and inflammation (9). SP-A and SP-D directly interact with various microorganisms, including bacteria and viruses, and inhibit their growth (10). Pulmonary collectins bind to cell surface receptors, including CD14, Toll-like receptors, SIRPα, and celrectculin/CD91, and attenuate or enhance inflammation in a microbial ligand-specific manner (11,12).

Protocol

Because SP-A, SP-D, and surfactant lipids exist in the alveoli, these materials are obtained in bronchoalveolar lavage (BAL) fluids recovered by washing the lung. BAL fluids are the starting material for isolating SP-A and SP-D. After the BAL fluids are centrifuged at 85,000 ×g at 4°C overnight, SP-A is sedimented with surfactant lipids, whereas SP-D is present in the supernatant (13).

In this chapter, protocols for isolating pulmonary collectins (SP-A and SP-D) will be described.

Materials

1.: Sepharose 6B (GE Healthcare, Little Chalfont, UK)
2.: Buffer A (0.5 M sodium carbonate, pH 11.0)
3.: Buffer B (0.5 M sodium carbonate, pH 10.0)
4.: Buffer C (0.5 M sodium carbonate, pH 8.5)
5.: Divinyl sulfone (Sigma, St Louis, MO)
6.: D-Mannose solution (20% (w/v) D-mannose in buffer B)
7.: Pooled BAL fluids from rat or pooled fluids of therapeutic BAL from individuals with pulmonary alveolar proteinosis.
8.: Buffer D (10 mM of Tris-HCl buffer, pH 7.4, containing 150 mM of NaCl)
9.: Buffer E (10 mM of Tris-HCl buffer, pH 7.4)
10.: NaBr solution (1.64 M NaBr in buffer D)
11.: Butanol (Sigma, St Louis, MO)
12.: Buffer F: loading buffer for SP-A isolation (10 mM of Tris-HCl buffer, pH 7.4 containing 5 mM of CaCl₂)
13.: Buffer G: loading buffer for SP-D isolation (10 mM of Tris-HCl buffer, pH 7.4 containing 150 mM of NaCl and 5 mM of CaCl₂)
14.: Buffer H: elution buffer for SP-A isolation (10 mM of Tris-HCl buffer, pH 7.4 containing 5 mM of EDTA)
15.: Buffer I: elution buffer for SP-D isolation (10 mM of Tris-HCl buffer, pH 7.4 containing 150 mM of NaCl and 5 mM of EDTA)
16.: Sepharose 6 10/300 GL (GE Healthcare, Little Chalfont, UK)
17.: Dialysis membrane (MWCO: 14,000) (Viskase Companies Inc., Darien, IL)

Instruments

1.: Ultracentrifuge (CP60E; Himac, Ibaraki, Japan)
2.: Column for open column chromatography
3.: AKTA purifier (GE Healthcare, Little Chalfont, UK)

Methods

1.

Preparation of mannose-Sepharose

a.: Put ~80 mL of Sepharose 6B (1:1 slurry) on the glass filter.
b.: Wash the resin with 1 L of distilled water.
c.: Transfer the resin to glass bottle and suspend with 50 mL of buffer A.
d.: Add 5 mL of divinyl sulfone to the suspension.
e.: Incubate 70 min at room temperature with rocking.
f.: Put the resin on the glass filter and wash with 1 L of distilled water.
g.: Transfer the resin to glass bottle and suspend with 50 mL of D-mannose solution.
h.: Incubate overnight at room temperature with rocking.
i.: Put the resin on the glass filter and wash with 1 L of distilled water.
j.: Transfer the resin to glass bottle and suspend with 50 mL of buffer C.
k.: Add 1 mL of β-mercaptoethanol to the suspension.
l.: Incubate 2 h at room temperature with rocking.
m.: Put the resin on the glass filter and wash with 1 L of distilled water.
n.: Transfer the resin to a glass bottle and suspend with 50 mL of distilled water.
o.: Store at 4°C until use (Note 1).

2.

Protocol for isolation and purification of SP-A from BAL fluids

a.: Prepare pooled BAL fluids (starting materials) after lavaging with buffer D (Note 2).
b.: Add 0.5 M CaCl₂ into the BAL fluids to a final concentration of 5 mM.
c.: Centrifuge the pooled BAL fluids at 85,000 ×g at 4°C overnight.
d.: Separate the supernatant and the precipitate (Note 3).
e.: Suspend the precipitate with NaBr solution (Note 4).
f.: Homogenize thoroughly until a homogeneous suspension is obtained (Note 5).
g.: Centrifuge the homogenate at 60,000 ×g at 4°C for 4 h (Note 6).
h.: Collect and suspend the pellicle with buffer D (Note 4).
i.: Centrifuge the suspension at 100,000 ×g at 4°C for 1 h.
j.: Collect the precipitate, which is the surfactant fraction, and discard the supernatant.
k.: Suspend the precipitate with 1–2 mL of distilled water.
l.: Inject the suspension of the surfactant fraction into 100 mL of 1-butanol, which is strongly being mixed by a stir bar, and continue mixing at room temperature for at least 1 h (the process of delipidation of the surfactant).
m.: Centrifuge the surfactant-butanol mixture at 1,600 ×g at room temperature for 30 min.
n.: Discard the supernatant and collect the precipitate (delipidated surfactant) (Note 7).
o.: Vapor the residual butanol in the precipitate by a gentle stream of nitrogen.
p.: Suspend the delipidated surfactant with 3–4 mL of distilled water.
q.: Dialyze the delipidated surfactant against buffer E at 4°C for 2–3 d with 3–4 exchanges of the same buffer (Note 8).
r.: Centrifuge the dialysate at 150,000 ×g at 4°C for 1h.
s.: Collect the supernatant.
t.: Add 0.5 M CaCl₂ into the supernatant to a final concentration of 5 mM (Note 9).
u.: Apply the supernatant to a mannose-Sepharose column (bed volume: 3 mL) after equilibrating the column with the buffer F (loading buffer).
v.: Wash the column with buffer F until the absorbance of the eluate at 280 nm becomes <0.01.
w.: Elute the bound components with buffer H (elution buffer), monitor the absorbance at 280 nm, and collect the protein peak (Note 10) (Fig. 1).
x.: Dialyze against buffer E and store at −30°C.

3.

Protocol for isolation and purification of SP-D from BAL fluids

a.: Collect the supernatant after centrifugation of the BAL fluids at 85,000 ×g (Protocols 2[c] and [d]).
b.: Apply the supernatant to a mannose-Sepharose column (bed volume: 3 mL) after equilibrating the column with buffer G (loading buffer).
c.: Wash the column with buffer G until the absorbance of the eluate at 280 nm becomes <0.01.
d.: Elute the bound components with buffer I (elution buffer), monitor the absorbance at 280 nm, and collect the protein peak (Note 10) (Figure 1).
e.: Dialyze against buffer D and store at −30°C.

4.

Protocol for further purification of pulmonary collectins (Note 11).

a.: Apply the collected eluate (Protocol 2[w] or 3[d]) to the Superose 6 10/300 GL column on AKTA purifier, which is equilibrating with the buffer E (for SP-A purification) or buffer D (for SP-D purification).
b.: Elute the protein while monitoring the absorbance at 280 nm.
c.: Collect the first protein peak, which is the purified pulmonary collectin (Figures 2 and 3).

Notes

1.: If the resin will be stored over a week, the addition of 0.02% NaN₃ is recommended.
2.: Lavage with 10–15 mL of buffer D and repeat 5–7 times for each rat. Pool the BAL fluids from a few dozen rats and use them as starting material.
3.: Do not discard the supernatant as it contains SP-D.
4.: The volume of NaBr solution or buffer D depends on the size of the homogenizer and the centrifuge tube to be used.
5.: To avoid heat denaturation, this procedure should be performed on ice.
6.: After centrifugation, surfactant forms white membrane (referred as “pellicle” in the next procedure) on the surface of the solution.
7.: Carefully discard the supernatant as the precipitate is easy to peel off.
8.: Dialyze thoroughly until the butanol odor disappears.
9.: If the solution becomes cloudy white, use it as is.
10.: Use appropriate elution buffer. SP-A and SP-D cannot be eluted with buffers I and H, respectively.
11.: These steps should be performed when a highly purified protein is needed.

References

1.: Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to beta-adrenergic stimulation. J Clin Invest. 1979 Mar;63(3):378-87. doi: 10.1172/JCI109313. PMID: 34631. [PMC free article: PMC371964] [PubMed: 34631] [CrossRef]
2.: Goerke J, Clements JA. Handbook of Physiology-The Respiratory System III. Washington: American Physiological Society; 1986. Alveolar surface tension and lung surfactant; p. 247-261.
3.: King RJ, Clements JA. Surface active materials from dog lung. I. Method of isolation. Am J Physiol. 1972 Sep;223(3):707–14. [PubMed: 5068619] [CrossRef]
4.: Kuroki Y, Voelker DR. Pulmonary surfactant proteins. J Biol Chem. 1994 Oct 21;269(42):25943–6. [PubMed: 7929300] [CrossRef]
5.: Day AJ. The C-type carbohydrate recognition domain (CRD) superfamily. Biochem Soc Trans. 1994 Feb;22(1):83–8. [PubMed: 7515837] [CrossRef]
6.: Kuroki Y, Akino T. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine. J Biol Chem. 1991 Feb 15;266(5):3068–73. [PubMed: 1993679] [CrossRef]
7.: Kuroki Y, Mason RJ, Voelker DR. Pulmonary surfactant apoprotein A structure and modulation of surfactant secretion by rat alveolar type II cells. J Biol Chem. 1988 Mar 5;263(7):3388–94. [PubMed: 2449439] [CrossRef]
8.: Ogasawara Y, Kuroki Y, Akino T. Pulmonary surfactant protein D specifically binds to phosphatidylinositol. J Biol Chem. 1992 Oct 15;267(29):21244–9. [PubMed: 1400434] [CrossRef]
9.: Kuroki Y, Takahashi M, Nishitani C. Pulmonary collectins in innate immunity of the lung. Cell Microbiol. 2007 Aug;9(8):1871–9. [PubMed: 17490408] [CrossRef]
10.: Ariki S, Kojima T, Gasa S, Saito A, Nishitani C, Takahashi M, Shimizu T, Kurimura Y, Sawada N, Fujii N, Kuroki Y. Pulmonary collectins play distinct roles in host defense against Mycobacterium avium. J Immunol. 2011 Sep 1;187(5):2586–94. [PubMed: 21821801] [CrossRef]
11.: Sano H, Sohma H, Muta T, Nomura S, Voelker DR, Kuroki Y. Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14. J Immunol. 1999 Jul 1;163(1):387–95. [PubMed: 10384140]
12.: Yamada C, Sano H, Shimizu T, Mitsuzawa H, Nishitani C, Himi T, Kuroki Y. Surfactant protein A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response. Importance of supratrimeric oligomerization. J Biol Chem. 2006 Aug 4;281(31):21771–21780. [PubMed: 16754682] [CrossRef]
13.: Kuroki Y, Shiratori M, Ogasawara Y, Tsuzuki A, Akino T. Characterization of pulmonary surfactant protein D: its copurification with lipids. Biochim Biophys Acta. 1991 Nov 5;1086(2):185–90. [PubMed: 1932100] [CrossRef]

Footnotes

: The authors declare no competing or financial interests.

Figures

Figure 1:

Schematic diagram of typical elution pattern from mannose-Sepharose column. Both SP-A and SP-D exhibit similar elution pattern. The fractions indicated by the solid line contain pulmonary collectin.

Figure 2:

Schematic diagram of typical elution pattern from Superose 6 10/300 GL column. Both SP-A and SP-D exhibit similar elution pattern. The fractions indicated by the solid line contain pulmonary collectin. Arrowhead indicates void volume. Arrows indicate elution time of molecular mass standards; 1, thyroglobulin (669 kDa); 2, ferritin (440 kDa); 3, aldolase (158 kDa); 4, ovalbumin (43 kDa); and 5, ribonuclease A (13.7 kDa).

Figure 3:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of isolated SP-A and SP-D under reducing and nonreducing conditions. Purified proteins were separated on 10% Laemmli’s gel and stained with Coomassie Brilliant Blue. SP-A and SP-D show highly oligomerized forms under nonreducing conditions.

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Bookshelf ID: NBK593992PMID: 37590721

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