The mutation (g.1286T>C) of the pulmonary surfactant-associated protein C gene (SFTPC) leads to the I73T substitution in the precursor protein (pro–SP–C) and results in interstitial lung disease with the histological pattern of non-specific interstitial pneumonia and pulmonary alveolar proteinosis. Central for the disease is the abnormal processing of the SP–C pro–protein to mature SP–C; however little is known about the nature of intermediates and processing products. We report here the application of high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the characterization of processing intermediates of hydrophobic pulmonary surfactant proteins SP–B and SP–C in intra-alveolar surfactant material of a patient with I73T mutation. SP–C and SP–B processing forms were separated from broncho-alveolar lavage fluid using chloroform/methanol extraction and sodium dodecyl sulfate polyacrylamide gel electrophoreis, detected by Western blot and identified by electrospray- and matrix-assisted laser desorption/ionization-FT-ICR mass spectrometry. The mass spectrometric and immuno-analytical results show the intra-alveolar accumulation of an aberrant C-terminal SP–C processing products in which the mature SP–C protein part is missing, and aberrant processing intermediates of SP–B.
WeaverT.E.ConkrightJ.J., “Function of surfactant proteins B and C”, Annu. Rev. Physiol.63, 555 (2001). doi: 10.1146/annurev.physiol.63.1.555
2.
GrieseM., “Pulmonary surfactant in health and human lung diseases: State of the art”, Eur. Respir. J.13, 1455 (1999). doi: 10.1183/09031936.99.13614779
3.
HawgoodS., “Surfactant protein B: Structure and function”, Biol. Neonate.85, 285 (2004). doi: 10.1159/000078169
4.
RyanM.A.QiX.SerranoA.G.IkegamiM.Perez-GilJ.JohanssonJ.WeaverT.E., “Mapping and analysis of the lytic and fusogenic domains of surfactant protein B”, Biochemistry44, 861 (2005). doi: 10.1021/bi0485575
5.
GonzalesL.W.AngampalliS.GuttentagS.H.BeersM.F.FeinsteinS.I.MatlapudiA.BallardP.L., “Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic”, Pediatr. Pathol. Mol. Med.20, 387 (2001). doi: 10.1080/152279501750399375
6.
SolarinK.O.BallardP.L.GuttentagS.H.LomaxC.A.BeersM.F., “Expression and glucocorticoid regulation of surfactant protein C in human fetal lung”, Pediatr. Res.42, 356 (1997). doi: 10.1203/00006450-199709000-00017
7.
BeersM.F., “Inhibition of cellular processing of surfactant protein C by drugs affecting intracellular pH gradients”, J. Biol. Chem.271, 14361 (1996).
8.
BeersM.F.KimC.Y.DodiaC.FisherA.B., “Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies”, J. Biol. Chem.269, 20318 (1994).
9.
BeersM.F.LomaxC., “Synthesis and processing of hydrophobic surfactant protein C by isolated rat type II cells”, Am. J. Physiol.269, L744–53 (1995).
10.
BeersM.F.LomaxC.A.RussoS.J., “Synthetic processing of surfactant protein C by alevolar epithelial cells. The COOH terminus of proSP-C is required for post-translational targeting and proteolysis”, J. Biol. Chem.273, 15287 (1998). doi: 10.1074/jbc.273.24.15287
11.
RussoS.J.WangW.LomaxC.A.BeersM.F., “Structural requirements for intracellular targeting of SP-C proprotein”, Am. J. Physiol.277, L1034 (1999).
12.
VorbrokerD.K.DeyC.WeaverT.E.WhitsettJ.A., “Surfactant protein C precursor is palmitoylated and associates with subcellular membranes”, Biochim. Biophys. Acta1105, 161 (1992). doi: 10.1016/0005-2736(92)90175-L
13.
BraschF.BirzeleJ.OchsM.GuttentagS.H.SchochO.D.BoehlerA.BeersM.F.MullerK.M.HawgoodS.JohnenG., “Surfactant proteins in pulmonary alveolar proteinosis in adults”, Eur. Respir. J.24, 426 (2004). doi: 10.1183/09031936.04.00076403
14.
WhitsettJ.A.WeaverT.E., “Hydrophobic surfactant proteins in lung function and disease”, N. Engl. J. Med.347, 2141 (2002). doi: 10.1056/NEJMra022387
15.
BeersM.F.MulugetaS., “Surfactant protein C biosynthesis and its emerging role in conformational lung disease”, Annu. Rev. Physiol.67, 663 (2005). doi: 10.1146/annurev.physiol.67.040403.101937
16.
BraschF.JohnenG.Winn-BraschA.GuttentagS.H.SchmiedlA.KappN.SuzukiY.MullerK.M.RichterJ.HawgoodS.OchsM., “Surfactant protein B in type II pneumocytes and intra-alveolar surfactant forms of human lungs”, Am. J. Respir. Cell Mol. Biol.30, 449 (2004). doi: 10.1165/rcmb.2003-0262OC
17.
KorimilliA.GonzalesL.W.GuttentagS.H., “Intracellular localization of processing events in human surfactant protein B biosynthesis”, J. Biol. Chem.275, 8672 (2000). doi: 10.1074/jbc.275.12.8672
18.
GuttentagS.H.BeersM.F.BielerB.M.BallardP.L., “Surfactant protein B processing in human fetal lung”, Am. J. Physiol.275, L559 (1998).
19.
JohnsonA.L.BraidottiP.PietraG.G.RussoS.J.KaboreA.WangW.J.BeersM.F., “Post-translational processing of surfactant protein-C proprotein: Targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments”, Am. J. Respir. Cell Mol. Biol.24, 253 (2001).
20.
Mayer-FliggeP.VolzJ.KrugerU.SturmE.GernandtW.SchaferK.P.PrzybylskiM., “Synthesis and structural characterization of human-identical lung surfactant SP-C protein”, J. Pept. Sci.4, 355 (1998). doi: 10.1002/(SICI)1099-1387(199808)4:5<355::AID-PSC153>3.0.CO;2-3
21.
VossT.SchaferK.P.NielsenP.F.SchaferA.MaierC.HannappelE.MaassenJ.LandisB.KlemmK.PrzybylskiM., “Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung”, Biochim. Biophys. Acta1138, 261 (1992).
22.
RosenD.M.WaltzD.A., “Hydroxychloroquine and surfactant protein C deficiency”, N. Engl. J. Med.352, 207 (2005). doi: 10.1056/NEJM200501133520223
23.
TredanoM.GrieseM.BraschF.SchumacherS.de BlicJ.MarqueS.HoudayerC.ElionJ.CoudercR.BahuauM., “Mutation of SFTPC in infantile pulmonary alveolar proteinosis with or without fibrosing lung disease”, Am. J. Med. Genet. A126, 18 (2004). doi: 10.1002/ajmg.a.20670
24.
ThomasA.Q.LaneK.PhillipsJ.IIIPrinceM.MarkinC.SpeerM.SchwartzD.A.GaddipatiR.MarneyA.JohnsonJ.RobertsR.HainesJ.StahlmanM.LoydJ.E., “Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred”, Am. J. Respir. Crit. Care Med.165, 1322 (2002). doi: 10.1164/rccm.200112-123OC
25.
NogeeL.M.DunbarA.E.IIIWertS.E.AskinF.HamvasA.WhitsettJ.A., “A mutation in the surfactant protein C gene associated with familial interstitial lung disease”, N. Engl. J. Med.344, 573 (2001). doi: 10.1056/NEJM200102223440805
26.
BraschF.GrieseM.TredanoM.JohnenG.OchsM.RiegerC.MulugetaS.MullerK.M.BahuauM.BeersM.F., “Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene”, Eur. Respir. J.24, 30 (2004). doi: 10.1183/09031936.04.00000104
27.
BaiY.GaletskiyD.DamocE.PaschenC.LiuZ.GrieseM.LiuS.PrzybylskiM., “High resolution mass spectrometric alveolar proteomics: Identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients”, Proteomics4, 2300 (2004). doi: 10.1002/pmic.200400855
28.
BaiY.GaletskiyD.DamocE.RipperJ.WoischnikM.GrieseM.LiuZ.LiuS.PrzybylskiM., “Lung alveolar proteomics of bronchoalveolar lavage from a pulmonary alveolar proteinosis patient using high-resolution FT-ICR mass spectrometry”, Anal. Bioanal. Chem.379, 1075 (2007). doi: 10.1007/s00216-007-1403-z
29.
BradfordM.M., “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding”, Anal. Biochem.72, 248 (1976). doi: 10.1016/0003-2697(76)90527-3
30.
NeuhoffV.AroldN.TaubeD.EhrhardtW., “Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250”, Electrophoresis9, 255 (1988). doi: 10.1002/elps.1150090603
31.
BaykutG.JertzR.WittM., “Matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry with pulsed in-source collision gas and in-source ion accumulation”, Rapid Commun. Mass Spectrom.14, 1238 (2000). doi:10.1002/1097-0231(20000730)14:14<1238::AID-RCM17>3.0.CO;2-H
32.
SenkoM.W.SpeirJ.P.McLaffertyF.W., “Collisional activation of large multiply charged ions using Fourier transform mass spectrometry”, Anal. Chem.66, 2801 (1994). doi: 10.1021/ac00090a003
33.
JimenezC.R.HuangL.QiuY.BurlingameA.L., “Searching sequence databases over the internet: Protein identification using MS-tag”, Curr. Protoc. Protein Sci.16, 6 (2001).
34.
WarrR.G.HawgoodS.BuckleyD.I.CrispT.M.SchillingJ.BensonB.J.BallardP.L.ClementsJ.A.WhiteR.T., “Low molecular weight human pulmonary surfactant protein (SP5): Isolation, characterization, and cDNA and amino acid sequences”, Proc. Natl. Acad. Sci. USA84, 7915 (1987). doi: 10.1073/pnas.84.22.7915
35.
HatzisD.DeiterG.deMelloD.E.FlorosJ., “Human surfactant protein-C: Genetic homogeneity and expression in RDS; comparison with other species”, Exp. Lung Res.20, 57 (1994). doi: 10.3109/01902149409064373
36.
GlasserS.W.KorfhagenT.R.PermeC.M.Pilot-MatiasT.J.KisterS.E.WhitsettJ.A., “Two SP–C genes encoding human pulmonary surfactant proteolipid”, J. Biol. Chem.263, 10326 (1988).
37.
LahtiM.MarttilaR.HallmanM., “Surfactant protein C gene variation in the Finnish population—association with perinatal respiratory disease”, Eur. J. Hum. Genet.12, 312 (2004). doi: 10.1038/sj.ejhg.5201137
38.
DamocE.YouhnovskiN.CrettazD.TissotJ.D.PrzybylskiM., “High resolution proteome analysis of cryoglobulins using Fourier transform-ion cyclotron resonance mass spectrometry”, Proteomics3, 1425 (2003). doi: 10.1002/pmic.200300482
39.
JohanssonH.NordlingK.WeaverT.E.JohanssonJ., “The Brichos domain-containing C-terminal part of pro-surfactant protein C binds to an unfolded poly-val transmembrane segment”, J. Biol. Chem.281, 21032 (2006). doi: 10.1074/jbc.M603001200
40.
WeaverT.E., “Synthesis, processing and secretion of surfactant proteins B and C”, Biochim. Biophys. Acta1408, 173 (1998).
41.
BraschF.OchsM.KahneT.GuttentagS.Schauer-VukasinovicV.DerrickM.JohnenG.KappN.MullerK.M.RichterJ.GillerT.HawgoodS.BuhlingF., “Involvement of napsin A in the C- and N-terminal processing of surfactant protein B in type-II pneumocytes of the human lung”, J. Biol. Chem.278, 49006 (2003). doi: 10.1074/jbc.M306844200
42.
UenoT.LinderS.NaC.L.RiceW.R.JohanssonJ.WeaverT.E., “Processing of pulmonary surfactant protein B by napsin and cathepsin H”, J. Biol. Chem.279, 16178 (2004). doi: 10.1074/jbc.M312029200
