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• W2019562364 abstract "The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80(domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1–2), and 1, 2, and 3 (TM1–2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1–2 (at Cys−1) and TM2 proteins (at Cys−1 and Cys6), and to a lesser extent in TM1 (at Cys−1), but not in TM1–2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1–2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1–2, and TM2 but not the TM1–2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess α-methylmannoside and therefore nonspecific. TM1 and TM1–2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1–2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities. The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80(domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1–2), and 1, 2, and 3 (TM1–2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1–2 (at Cys−1) and TM2 proteins (at Cys−1 and Cys6), and to a lesser extent in TM1 (at Cys−1), but not in TM1–2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1–2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1–2, and TM2 but not the TM1–2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess α-methylmannoside and therefore nonspecific. TM1 and TM1–2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1–2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities. Pulmonary surfactant lines the distal airspaces and stabilizes the gas exchanging alveolar units of the lung by reducing surface tension (1Goerke J. Clements J.A. Handbook of Physiology. 3. American Physiological Society, Bethesda1986: 247-261Google Scholar). Surfactant is composed of phospholipids and proteins that are organized as lattice-like arrays (e.g. tubular myelin) and vesicular aggregates within the alveolar lining fluid, and as a film at the alveolar air-liquid interface. Surfactant protein A (SP-A) 1The abbreviations used are: SP-A, surfactant protein A; TM, truncated mutant; CRD, carbohydrate recognition domain; IKC, isoleucine-lysine-cysteine; BSA, bovine serum albumin; DSG, disuccinimidyl glutarate; DPPC, dipalmitoylphosphatidylcholine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PAGE, polyacrylamide gel electrophoresis. is a hydrophilic, Ca2+-dependent phospholipid-binding protein that is secreted into the airspace by alveolar type II cells (2King R.J. Klass D.J. Gikas E.G. Clements J.A. Am. J. Physiol. 1973; 224: 788-795Crossref PubMed Scopus (193) Google Scholar). The function of SP-A is not fully understood, but reports that SP-A binds to a high affinity receptor on the surface of alveolar type II cells (3Wright J.R. Borchelt J.D. Hawgood S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5410-5414Crossref PubMed Scopus (102) Google Scholar, 4Kuroki Y. Mason R.J. Voelker D.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5566-5570Crossref PubMed Scopus (166) Google Scholar), is required for the formation and/or stability of tubular myelin (5Suzuki Y. Fujita Y. Kogishi K. Am. Rev. Respir. Dis. 1989; 140: 75-81Crossref PubMed Scopus (277) Google Scholar, 6Korfhagen T.R. Bruno M.D. Ross G.F. Huelsman K.M. Ikegami M. Jobe A.H. Wert S.E. Stripp B.R. Morris R.E. Glasser S.W. Bachurski C.J. Iwamoto H.S. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9594-9599Crossref PubMed Scopus (376) Google Scholar) and large surfactant aggregates (7Veldhuizen R.A. Yao L.J. Hearn S.A. Possmayer F. Lewis J.F. Biochem. J. 1996; 313: 835-840Crossref PubMed Scopus (56) Google Scholar), and protects surfactant surface activity in the presence of foreign protein contamination (8Cockshutt A.M. Weitz J. Possmayer F. Biochemistry. 1990; 29: 8424-8429Crossref PubMed Scopus (254) Google Scholar) suggest one or more roles in surfactant function. The notion that SP-A is also a pulmonary host defense protein is supported by membership in the structurally homologous collectin family of antibody-independent opsonins (including mannose-binding protein A) (9Drickamer K. Dordal M.S. Reynolds L. J. Biol. Chem. 1986; 261: 6878-6887Abstract Full Text PDF PubMed Google Scholar,10Hoppe H.J. Reid K.B. Protein Sci. 1994; 3: 1143-1158Crossref PubMed Scopus (191) Google Scholar), reports that SP-A binds to, aggregates, and opsonizes multiple microorganisms (for review see Ref. 11McCormack F.X. Whitsett J.A. Ezekowitz A. Reid K.B.M. Sastry K. Collectins and Innate Immunity. R. G. Landes, Austin, TX1996: 9-50Google Scholar), and the finding that the SP-A gene-targeted mouse is susceptible to infection with group B streptococcus (12LeVine A.M. Bruno M.D. Huelsman K.M. Ross G.F. Whitsett J.A. Korfhagen T.R. J. Immunol. 1997; 158: 4336-4340PubMed Google Scholar). Rat SP-A is a large oligomer composed of 18 very similar subunits that are distinguished by variable glycosylation at one or bothN-linked carbohydrate attachment sites (13Whitsett J.A. Ross G. Weaver T. Rice W. Dion C. Hull W. J. Biol. Chem. 1985; 260: 15273-15279Abstract Full Text PDF PubMed Google Scholar, 14McCormack F.X. Calvert H.M. Watson P. Smith D.L. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 5833-5841Abstract Full Text PDF PubMed Google Scholar) and microheterogeneity in the N-terminal amino acid sequences. The latter most probably arises by alternative site cleavage by signal peptidase, which results in a slightly elongated variant containing an Ile-Lys-Cys (IKC) sequence upstream of the predicted N terminus of Asn1(15Elhalwagi B.M. Damodarasamy M. McCormack F.X. Biochemistry. 1997; 36: 7018-7025Crossref PubMed Scopus (33) Google Scholar). The primary structure of the SP-A subunits are otherwise identical, composed of several discrete domains including the following (16White R.T. Damm D. Miller J. Spratt K. Schilling J. Hawgood S. Benson B. Cordell B. Nature. 1985; 317: 361-363Crossref PubMed Scopus (297) Google Scholar, 17Ross G.F. Notter R.H. Meuth J. Whitsett J.A. J. Biol. Chem. 1986; 261: 14283-14291Abstract Full Text PDF PubMed Google Scholar): 1) a short N-terminal segment, 2) a collagen-like sequence of Gly-X-Y repeats (where X is any amino acid and Y is often Pro or hydroxyproline) containing a midpoint interruption (Gly44), 3) a hydrophobic neck domain, and 4) a carbohydrate recognition domain (CRD). Trimeric association of monomeric subunits occurs by the folding of the collagen-like domains into triple helices (18King R.J. Simon D. Horowitz P.M. Biochim. Biophys. Acta. 1989; 1001: 294-301Crossref PubMed Scopus (64) Google Scholar). The role of the neck domain in the oligomeric assembly of SP-A has not been reported, but in related collectins, the formation of α-helical coiled coil bundles in this region is critical for trimerization (19Hoppe H.J. Barlow P.N. Reid K.B. FEBS Lett. 1994; 344: 191-195Crossref PubMed Scopus (113) Google Scholar). Fully assembled SP-A is a hexamer of trimers that are laterally associated through the N-terminal segment and the first half of the collagen-like domain and stabilized by inter- and intratrimeric interchain disulfide bonds at Cys−1 and Cys6 (18King R.J. Simon D. Horowitz P.M. Biochim. Biophys. Acta. 1989; 1001: 294-301Crossref PubMed Scopus (64) Google Scholar, 20Voss T. Eistetter H. Schafer K.P. Engel J. J. Mol. Biol. 1988; 201: 219-227Crossref PubMed Scopus (206) Google Scholar). The domains of SP-A that interact with type II cells and surfactant phospholipids have been mapped by site-directed mutagenesis. The CRD of the molecule contains binding sites for carbohydrate, the major surface glycoprotein of the pulmonary pathogen Pneumocystis carinii, the high affinity SP-A receptor on type II cells, and surfactant phospholipids, especially dipalmitoylphosphatidylcholine (DPPC) (21McCormack F.X. Stewart J.J. Voelker D.R. Damodarasamy M.D. Biochemistry. 1997; 36: 13963-13971Crossref PubMed Scopus (38) Google Scholar, 22McCormack F.X. Festa A.L. Andrews R.P. Linke M. Walzer P.D. Biochemistry. 1997; 36: 8092-8099Crossref PubMed Scopus (68) Google Scholar, 23Kuroki Y. McCormack F.X. Ogasawara Y. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 29793-29800Abstract Full Text PDF PubMed Google Scholar, 24Ogasawara Y. McCormack F.X. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 29785-29792Abstract Full Text PDF PubMed Google Scholar). We have recently reported that the N-terminal domains that contribute to oligomeric assembly, including the interchain disulfide bond at Cys6 and the collagen-like region, are essential for SP-A function (25McCormack F.X. Pattanajitvilai S. Stewart J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The collagen-like region was required for competitive binding to the SP-A receptor and the specific inhibition of surfactant secretion from type II cells. Disruption of the Cys6 interchain disulfide bond, by substitution of serine for cysteine, blocked the aggregation of liposomes by SP-A and reduced the inhibitory effect of SP-A on surfactant secretion from isolated alveolar type II cells. Recent analyses indicate that the N-terminal IKC sequence, and specifically the interchain disulfide-forming cysteine at Cys−1, is not essential for the interactions of SP-A with type II cell or surfactant phospholipids (26Zhang M. Damodarasamy M. Elhalwagi B.M. McCormack F.X. Biochemistry. 1998; 37: 16481-16488Crossref PubMed Scopus (11) Google Scholar). In the current study, we used truncated mutant forms of SP-A to examine the role of the N-terminal segment and defined regions of the collagen-like domain in oligomeric assembly, phospholipid binding and aggregation, and SP-A receptor-mediated functions. The synthesis of the mutant recombinant proteins TM1, TM2–3, and TM1–2-3 has been previously reported (15Elhalwagi B.M. Damodarasamy M. McCormack F.X. Biochemistry. 1997; 36: 7018-7025Crossref PubMed Scopus (33) Google Scholar). The TM2 protein was generated by similar methods. Briefly, the TM2 mutant cDNA encoding the deletion of amino acids Gly8-Gly44 of rat SP-A was produced using mutagenic oligonucleotides by overlapping extension polymerase chain reaction amplification of a 1.6-kilobase pair rat SP-A cDNA (27Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar, 28Sano K. Fisher J. Mason R.J. Kuroki Y. Schilling J. Benson B. Voelker D. Biochem. Biophys. Res. Commun. 1987; 144: 367-374Crossref PubMed Scopus (81) Google Scholar). The TM2 cDNA was ligated into the EcoRI site of PVL 1392 (Invitrogen), and the correct orientation was confirmed using KpnI. Nucleotide sequencing of the Gly8-Gly44 coding region of the TM2 cDNA confirmed the intended deletion and the absence of spurious mutations (29Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463Crossref PubMed Scopus (52769) Google Scholar). A recombinant baculovirus containing the TM2 cDNA for SP-A was produced by homologous recombination in Spodoptera frugiperda (Sf-9) cells following contransfection with linear viral DNA and the PVL 1392/TM2 construct (BaculoGold, PharMingen). Fresh monolayers of 107 Trichoplusia ni(T. ni) cells were infected with plaque-purified recombinant virus at a multiplicity of infection of 10 and then incubated with serum-free media (IPL-41) supplemented with 0.4 mm ascorbic acid and antibiotics for 72 h. Recombinant SP-A was purified from the culture media by adsorption to mannose-Sepharose 6B columns in the presence of 1 mm Ca2+ and elution with 2 mm EDTA (30Fornstedt N. Porath J. FEBS Lett. 1975; 57: 187-191Crossref PubMed Scopus (213) Google Scholar). The purified recombinant SP-A was dialyzed against 5 mm Tris (pH 7.4) and stored at −0 °C. Surfactant was isolated by bronchoalveolar lavage of silica-pretreated Sprague-Dawley rats (31Dethloff L.A. Gilmore L.B. Brody A.R. Hook G.E.R. Biochem. J. 1986; 233: 111-118Crossref PubMed Scopus (77) Google Scholar) and floated on NaBr gradients (32Hawgood S. Benson B. Hamilton R.J. Biochemistry. 1985; 24: 184-190Crossref PubMed Scopus (258) Google Scholar, 33Kuroki Y. Mason R.J. Voelker D.R. J. Biol. Chem. 1988; 263: 3388-3394Abstract Full Text PDF PubMed Google Scholar). SP-A was isolated and purified from the surfactant pellicle by delipidation, mannose-Sepharose affinity chromatography (30Fornstedt N. Porath J. FEBS Lett. 1975; 57: 187-191Crossref PubMed Scopus (213) Google Scholar), and gel permeation chromatography with Bio-Gel A-5m. The SP-A content of tissue culture media containing recombinant SP-A was determined with a rabbit polyclonal IgG against rat SP-A using a sandwich enzyme-linked immunosorbent assay (34McCormack F.X. Fisher J.H. Suwabe A. Smith D.L. Shannon J.M. Voelker D.R. Biochim. Biophys. Acta. 1990; 1087: 190-198Crossref PubMed Scopus (14) Google Scholar). The lower limit of sensitivity of the assay was 0.20 ng/ml, and the linear range extended from 0.16 to 10.0 ng/ml. Routine protein concentrations were determined with the bicinchoninic protein assay kit (BCA) (Pierce) using bovine serum albumin as a standard. The effects of mutations on the oligomeric structure of the recombinant proteins were assessed by chemical cross-linking. Variant SP-As (10 μg) were incubated with the 5 mm disuccinimidyl glutarate (DSG, Pierce) in 20 mm HEPES containing 0.1 mm EGTA, 0.15m NaCl, and 100 mm KCl at room temperature for 30 min. The reaction was stopped by addition of reducing sample buffer, and the proteins were size-fractionated on 8–16% gradient SDS-PAGE gels and stained with Coomassie Blue. Experiments to assess the effect of N-terminal deletions on the SP-A-mediated inhibition of surfactant secretion were performed as described previously (14McCormack F.X. Calvert H.M. Watson P. Smith D.L. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 5833-5841Abstract Full Text PDF PubMed Google Scholar). Briefly, alveolar type II cells were isolated from male Sprague-Dawley rats by tissue dissociation with elastase and purification on metrizamide gradients (35Dobbs L.G. Mason R.J. J. Clin. Invest. 1979; 63: 378-387Crossref PubMed Scopus (279) Google Scholar). The type II cells were seeded into tissue culture flasks and incubated overnight in [3H]choline (0.5 μCi/ml) containing Dulbecco's modified Eagle's medium and 10% fetal calf serum (D10) at 37 °C in a 10% CO2 atmosphere. After washing, SP-A variants were tested for their ability to antagonize 12-O-tetradecanoylphorbol-13-acetate (10−7m)-stimulated surfactant secretion by coincubation with the monolayer for 3 h. In some experiments, 0.125 mα-methylmannoside was added simultaneously with the 12-O-tetradecanoylphorbol-13-acetate and SP-A to determine if the SP-A effect was reversible with excess monosaccharides. Secretion was measured using [3H]phosphatidylcholine (PC) as a marker for surfactant and expressed as percent secretion (radioactivity in the media/radioactivity in the cells + media). A whole cell competitive binding assay was performed to determine the ability of various recombinant forms of SP-A to compete with 125I-rat SP-A for receptor occupancy on the surface of isolated type II cells (36Kuroki Y. Mason R.J. Voelker D.R. J. Biol. Chem. 1988; 263: 17596-17602Abstract Full Text PDF PubMed Google Scholar). Following isolation, 2 × 106 type II cells/35-mm dish were incubated overnight in D10 at 37 °C in a 10% CO2 atmosphere. The following morning, the nonadherent cells were removed by washing the monolayers three times at 4 °C with 10 ml of Dulbecco's modified Eagle's medium containing 1 mg/ml BSA. The monolayers were then incubated with 1 μg/ml 125I-rat SP-A and various recombinant SP-As in D10 for 3 h at 37 °C in a 10% CO2 atmosphere. After washing three times on ice with buffer containing 50 mm Tris (pH 7.4), 100 mmNaCl, 2 mm CaCl2, and 1 mg/ml BSA, the cells were solubilized in 0.1 n NaOH, and radioactivity was quantified in a gamma radiation counter. Liposome binding and aggregation experiments were performed using lipids purchased from Avanti Polar Lipids, as described previously (14McCormack F.X. Calvert H.M. Watson P. Smith D.L. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 5833-5841Abstract Full Text PDF PubMed Google Scholar). Unilamellar vesicles were produced by probe sonication of lipid mixtures composed of DPPC:egg PC:phosphatidylglycerol (PG), 9:3:2, and equilibrated with SP-As (lipid:protein ratio 20:1 by weight) in 50 mm Tris, 150 mm NaCl buffer (buffer A) at 20 °C. Aggregation was determined by measuring light scattering (A 400 nm) at 1-min intervals after the addition of 5 mm Ca2+ (final). For lipid binding, multilamellar liposomes produced by vigorous vortexing of the same lipid mixture were incubated with 10 μg/ml SP-A (lipid:protein ratio 50:1) in buffer A containing 2.0% BSA and 5 mmCa2+. Following incubation for 1 h at room temperature, the reaction mixture was centrifuged at 14,000 ×g av for 10 min and washed once, and the SP-A content of the pellet and pooled supernatant fractions were determined by enzyme-linked immunosorbent assay. Percent binding was defined as SP-Apellet/(SP-Apellet + supernatant) × 100. Control experiments in which liposomes or Ca2+ were individually deleted were also performed. Uptake of phospholipid liposomes by type II cells was performed according to the method of Wright et al. (37Wright J.R. Wager R.E. Hawgood S. Dobbs L. Clements J.A. J. Biol. Chem. 1987; 262: 2888-2894Abstract Full Text PDF PubMed Google Scholar) with minor modifications (38McCormack F.X. Kuroki Y. Stewart J.J. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 29801-29807Abstract Full Text PDF PubMed Google Scholar). Freshly isolated alveolar type II cells (1 × 106/tube) were incubated with unilamellar liposomes (100 μg/ml) composed of [3H]DPPC (1600 cpm/nmol):egg PC:PG, 7:2:1, and SP-A variants in 0.5 ml of Dulbecco's modified Eagle's medium, 10 mm HEPES (pH 7.4) for 1 h at 37 °C. The media and cells were separated by centrifugation at 160 × g for 5 min at 4 °C, and the cells were washed three times in ice-cold phosphate-buffered saline containing 1 mg/ml BSA. An additional volume of 0.5 ml of phosphate-buffered saline was added to each tube, and the cells and media were transferred to separate liquid scintillation vials and counted. Percent uptake was calculated according to the equation: [3H]DPPCcells/([3H]DPPCcells+ 3[H]DPPCmedia) × 100. The structures of the mutant recombinant proteins used in this study are shown in Fig.1. Recombinant SP-A that is overproduced in insect cells has functional characteristics that are comparable to the natural protein, despite incomplete hydroxylation of prolines in the collagen-like region (designated “hyp” for hydroxyproline-deficient) (14McCormack F.X. Calvert H.M. Watson P. Smith D.L. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 5833-5841Abstract Full Text PDF PubMed Google Scholar). For the purposes of this study we have designated the domains of SP-A from N to C terminus as the follows: I-K-C domain, domain 1 (N-terminal segment), domain 2 (N-terminal half of the collagen-like domain), domain 3 (C-terminal half of the collagen-like domain), neck, and CRD. The synthesis and physical characterization of the mutant recombinant SP-As containing telescoping deletions from Asn1 through the end of the N-terminal segment (TM1 or ΔAsn1-Ala7), the midpoint of the collagen-like region (TM1–2 or ΔAsn1-Gly44), and end of the collagen region (TM1–2-3 or ΔAsn1-Pro80) have been previously reported (15Elhalwagi B.M. Damodarasamy M. McCormack F.X. Biochemistry. 1997; 36: 7018-7025Crossref PubMed Scopus (33) Google Scholar). For this study, an additional mutant SP-A containing a nested deletion of the proximal collagen-like region (TM2 or ΔGly8-Gly44) was produced using similar methods. The TM2 migrated slightly more rapidly than the SP-Ahyp on SDS-PAGE gels under reducing conditions, consistent with the deletion of 37 amino acids (Fig.2). Like the SP-Ahyp, the TM2 formed disulfide-linked multimers at least as large as tetramers under non-reducing conditions. This migration profile is consistent with interchain disulfide bond formation at both sulfhydryls which are available for interchain linkage, at Cys−1 and Cys6. We have previously reported that disulfide bond formation at Cys−1 in TM1 and TM1–2 results in 17 and 51% dimers, respectively, based on densitometry of nonreducing SDS-PAGE gels. SP-Ahyp, by comparison, is 60% dimers and higher oligomers (15Elhalwagi B.M. Damodarasamy M. McCormack F.X. Biochemistry. 1997; 36: 7018-7025Crossref PubMed Scopus (33) Google Scholar). The TM1–2-3 is monomeric due to the near-absence of the Cys−1-containing tripeptide sequence at the N terminus (<3% of polypeptide chains), presumably because the mutations introduced lead to exclusive signal peptidase cleavage site at the Cys−1-Ala81 bond rather than the alternative site cleavage that occurs in SP-Ahyp, TM1, and TM1–2 (15Elhalwagi B.M. Damodarasamy M. McCormack F.X. Biochemistry. 1997; 36: 7018-7025Crossref PubMed Scopus (33) Google Scholar). The preservation of partial and near wild type levels of Cys−1-dependent disulfide cross-linking at the N terminus of TM1 and TM1–2, respectively, allows for evaluation of the functional consequences of the peptide sequence deletions that are distinct from those that are due to loss of covalent interchain linkage. Noncovalent interactions that also contribute to the assembly of the SP-A oligomer were analyzed by DSG cross-linking, and the results are shown in Fig. 3. Treatment of the SP-Ahyp with DSG followed by size fractionation on reducing SDS-PAGE resulted in the appearance of a series of 8–9 distinct bands, indicating that the same number of polypeptide chains were closely associated in the soluble protein, most likely as three trimers. Previously reported gel filtration chromatography data are also consistent with nonameric assembly of SP-Ahyp (25McCormack F.X. Pattanajitvilai S. Stewart J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Cross-linking analyses further revealed that the TM1–2-3 was a noncovalent trimer, demonstrating that collagen-like region and interchain disulfide bond formation are not required for triple-stranded folding of SP-A during synthesis or to maintain the association of three chains in the fully processed protein. The TM1 and TM1–2 did not form detectable oligomers greater than trimers, but the TM2 was at least hexameric. These results indicate that N-terminal interchain disulfide linkage is not sufficient for the association of trimers and that either the N-terminal segment itself or two interchain bridge-forming cysteines are required for the interaction. We have recently reported that individual disruption of Cys6 or Cys−1 by substitution with Ser did not prevent the assembly of large SP-A oligomers, based on cross-linking analyses 2F. X. McCormack, unpublished data. and gel exclusion chromatography (25McCormack F.X. Pattanajitvilai S. Stewart J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Zhang M. Damodarasamy M. Elhalwagi B.M. McCormack F.X. Biochemistry. 1998; 37: 16481-16488Crossref PubMed Scopus (11) Google Scholar). Taken together, these data suggest that the neck + CRD are sufficient for trimerization of SP-A, and that the N-terminal segment plays an important role in the association of trimeric subunits in solution that is not solely attributable to disulfide bond formation.Figure 2Electrophoretic analysis of TM2 SP-A.Rat SP-A, wild type recombinant SP-A (SPAhyp), and TM2 SP-As were subjected to 8–16% SDS-PAGE under reducing and nonreducing conditions and stained with Coomassie Brilliant Blue. hyp, hydroxyproline-deficient.View Large Image Figure ViewerDownload (PPT)Figure 3Cross-linking analysis of oligomeric association of polypeptide chains of TM SP-As. The wild type recombinant SP-A (SP-Ahyp), TM1, TM1–2, TM1–2-3, or TM2 were incubated with the cross-linking reagent DSG for 30 min at room temperature and then size-fractionated on 8–16% reducing SDS-PAGE gels and stained with Coomassie Brilliant Blue. hyp, hydroxyproline-deficient.View Large Image Figure ViewerDownload (PPT) The effects of N-terminal deletions on the direct binding of TM SP-As to multilamellar liposomes was assessed by determining the percent of added protein that cosedimented with the phospholipid pellet upon centrifugation, as described previously (14McCormack F.X. Calvert H.M. Watson P. Smith D.L. Mason R.J. Voelker D.R. J. Biol. Chem. 1994; 269: 5833-5841Abstract Full Text PDF PubMed Google Scholar). Specific binding was defined by subtracting Ca2+-independent sedimentation of SP-A (i.e. in the presence of EDTA) from total Ca2+-dependent binding. As shown in Fig.4, 53.8 ± 2.2% of SP-Ahyp specifically bound to the liposomes, compared with 32.2 ± 2.5% of TM1 and 43.8 ± 1.6% of TM1–2. The TM2 protein exhibited wild type levels of phospholipid binding activity (54.5 ± 2.6%), but binding by TM1–2-3 was negligible (2.1 ± 1.3%). We have previously reported that the binding of TM2–3 to lipid is about half that of SP-Ahyp (25McCormack F.X. Pattanajitvilai S. Stewart J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These data indicate that domain 2 of SP-A does not contribute to lipid binding and that domain 3 is required for wild type levels of lipid binding activity. The finding that TM1–2 bound to lipid approximately 80% as effectively as SP-Ahyp demonstrates that N-terminal disulfide linkage can functionally replace the N-terminal segment with respect to lipid binding. We have previously determined that the Cys6 interchain disulfide bond but not the collagen-like domain of rat SP-A is required for aggregation of phospholipid liposomes (25McCormack F.X. Pattanajitvilai S. Stewart J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The role of the N-terminal segment in aggregation was examined by incubation of truncated mutant forms of SP-A with unilamellar liposomes in the presence of Ca2+ and measurement of light scattering. As sh" @default.
• W2019562364 created "2016-06-24" @default.
• W2019562364 creator A5046611314 @default.
• W2019562364 creator A5080061052 @default.
• W2019562364 creator A5086486920 @default.
• W2019562364 date "1999-01-01" @default.
• W2019562364 modified "2023-09-27" @default.
• W2019562364 title "Deletion Mapping of N-terminal Domains of Surfactant Protein A" @default.
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