Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.

Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers.

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.

Deletion mapping of N-terminal domains of surfactant protein A. The N-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion.

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 alpha-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.

Surfactant lipid peroxidation damages surfactant protein A and inhibits interactions with phospholipid vesicles.

The goal of these studies was to examine the effect of lipid peroxidation (LPO) on the function of surfactant protein A (SP-A). First, the optimal dialysis conditions for quantitative removal of EDTA and redoxactive metals from reagents were established. Surfactant phospholipids were incubated with free radical generators in the absence or presence of the SP-A or with BSA as a control. We found that SP-A inhibited copper-initiated LPO to an extent similar to BSA (P < 0.05). Exposure of SP-A to LPO was associated with an increase in the level of SP-A-associated carbonyl moieties and a marked reduction in SP-A-mediated aggregation of liposomes. LPO initiated by an azo-compound also resulted in enhanced protein oxidation and markedly inhibited SP-A-mediated liposome aggregation. The kinetics of aggregation of auto-oxidized and nonoxidized liposomes by nonoxidized SP-A was similar, suggesting that SP-A has similar affinities for oxidized and nonoxidized lipids. Oxidative inactivation of SP-A did not occur upon direct incubation of the protein with malondialdehyde alone. We conclude that exposure of SP-A to LPO results in oxidative modification and functional inactivation of SP-A by phospholipid radicals.

The Cys6 intermolecular disulfide bond and the collagen-like region of rat SP-A play critical roles in interactions with alveolar type II cells and surfactant lipids.

Rat pulmonary surfactant protein A is an oligomer of 18 polypeptide chains which are associated by triple helix formation in the collagen-like domain and interchain disulfide bridges at the NH2 terminus. The roles of the intermolecular bond at Cys6 and the collagen-like domain (Gly8-Pro80) in the interactions of SP-A with phospholipids and alveolar type II cells were investigated using mutant forms of the protein. Wild type SP-A (SP-Ahyp), SP-A with the substitution Cys6 --> Ser to prevent disulfide formation (SP-Ahyp, C6S), and SP-A with the collagen-domain deleted (SP-ADeltaG8-P80) were synthesized in insect cells using recombinant baculoviruses. The SP-As were glycosylated and secreted from the invertebrate cells and the binding affinities of the wild type and mutant proteins for the mannose-Sepharose matrix used for purification were nearly identical. The SP-Ahyp and SP-ADeltaG8-P80 were at least nonameric in solution based on gel exclusion chromatography, and demonstrated extensive sulfhydryl-dependent oligomerization under nonreducing conditions. The SP-Ahyp,C6S was also oligomeric in solution and formed disulfide-dependent dimers, indicating the presence of at least one additional interchain disulfide bond. The SPADeltaG8-P80 but not the SP-Ahyp,C6S aggregated lipid vesicles at 20 degrees C and augmented the surface tension lowering effect of extracts of natural surfactant. The SP-ADeltaG8-P80 competed poorly with native SP-A for receptor occupancy on isolated alveolar type II cells and was a potent but nonspecific (concanavalin A-like) inhibitor of surfactant secretion. In contrast, the SP-Ahyp,C6S partially competed for receptor occupancy and weakly inhibited surfactant secretion in a specific manner. Neither the SP-ADeltaG8-P80 nor the SP-Ahyp,C6S supported the association of phospholipid liposomes with type II cells. We conclude that: 1) the Cys6 interchain disulfide bond of SP-A is required for aggregation of liposomes and for potent inhibition of surfactant secretion. 2) The collagen-like region is required for competition with 125I-SP-A for receptor occupancy and specific inhibition of surfactant secretion in the presence of competing sugars. 3) Both the NH2-terminal disulfide and the collagen-like region are required to enhance the association of phospholipid vesicles with type II cells.

Epitope mapping for monoclonal antibodies identifies functional domains of pulmonary surfactant protein A that interact with lipids.

Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type II cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant SP-As in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a Cys204-Cys218 (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type II cells that is caused by SP-A. From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type II cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type II cells.

Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction.

Pulmonary surfactant proteins A (SP-A) and D (SP-D) possess similar structure as members of the mammalian C-type lectin superfamily. Both proteins are composed of four characteristic domains which are: 1) an NH2-terminal domain involved in interchain disulfide formation (denoted A1 domain for SP-A or D1 for SP-D); 2) a collagenous domain (denoted A2 or D2); 3) a neck domain (denoted A3 or D3); and 4) a carbohydrate recognition domain (denoted A4 or D4). SP-A specifically binds to dipalmitoylphosphatidylcholine, the major lipid component of surfactant, and can regulate the secretion and recycling of this lipid by alveolar type II cells. SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer), and its role in alveolar lipid metabolism remains to be clarified. To understand the relationship between the structure and the function of both proteins with respect to their interaction with lipids, we expressed recombinant wild type rat SP-D (rSP-D) and chimeric molecules of SP-A and SP-D (A1A2A3D4, A1A2D3D4, and D1D2A3A4) using a baculovirus expression system, and performed lipid binding and aggregation assays. The rSP-D effectively competed with 125I-labeled native rat SP-D in a solid phase binding assay to PI and GlcCer in a manner nearly identical to native SP-D. The rSP-D also bound to PI liposomes with approximately half the affinity of native rat SP-D. Chimera A1A2D3D4 competed with iodinated SP-D in the solid phase binding assay to both PI and GlcCer. This chimera did not bind to dipalmitoylphosphatidylcholine (DPPC) liposomes or induce their aggregation. Chimera A1A2A3D4 did not bind solid phase PI or GlcCer but was equivalent to rSP-D in binding to PI liposomes. This chimera exhibited weak binding to DPPC but failed to aggregate DPPC liposomes. Chimera D1D2A3A4 failed to bind PI and GlcCer and bound weakly to DPPC liposomes but was quite effective at inducing aggregation of DPPC liposomes. These findings demonstrate that the D3 plus D4 domains of SP-D play a role in lipid binding and that the D4 domain is essential for PI binding. Furthermore, the A3 domain of SP-A cannot account for all the lipid binding activity of this protein. In addition, the results implicate the A4 domain of SP-A as an important structural domain in lipid aggregation phenomena.

Surfactant protein A amino acids Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells.

Pulmonary surfactant protein A (SP-A) is a mammalian lectin that regulates the uptake and secretion of surfactant by alveolar type II cells and is an important component of surfactant complexes. The domains of SP-A which mediate these functions have not been fully mapped. The binding of SP-A to its high affinity receptor on alveolar type II cells is thought to be dependent on a carbohydrate recognition domain (CRD), while the interaction with lipids has been attributed to the hydrophobic neck region of the molecule. To explore the role of the CRD in the interactions of SP-A with type II cells and lipids, we introduced mutations into the cDNA to encode for the substitutions Glu195-->Gln and Arg197-->Asp (SP-Ahyp,Gln195,Asp197) and expressed the mutant protein in insect (Sf9) cells using recombinant baculoviruses. Similar mutations introduced into mannose-binding protein A have been shown to switch the carbohydrate binding specificity from mannose > galactose to the converse. Wild type SP-A produced in Sf9 cells does not contain hydroxyproline (SP-Ahyp), but like rat SP-A it binds to carbohydrate affinity columns, lipids, and the SP-A receptor and is a potent inhibitor of the secretion of surfactant from type II cells (IC50 = 0.5-1.0 micrograms/ml). The SP-Ahyp,Gln195,Asp197 also bound to affinity matrices of galactose-Sepharose and mannose-Sepharose but the indicated mutations rendered the binding at least 100 times more susceptible than SP-Ahyp to competition by free galactose. The SP-Ahyp,Gln195,Asp197 did not compete with rat SP-A for occupancy of its high affinity receptor on type II cells and the mutant protein was 25-50-fold less potent as an inhibitor of the secretion of surfactant from type II cells (IC50 = 26.0 micrograms/ml). Unlike SP-Ahyp, the inhibition of secretion of surfactant by SP-Ahyp,Gln195,Asp197 was reversed by 0.25 M alpha-methylmannoside or galactose. In addition, the SP-Ahyp,Gln195,Asp197 bound avidly to phospholipid but did not aggregate vesicles or augment the uptake of phospholipid into type II cells. We conclude that the binding of SP-A to its receptor and the inhibition of surfactant secretion are critically dependent on the carbohydrate binding specificity of the CRD. Furthermore, phospholipid aggregation and augmentation of phospholipid uptake into type II cells are mediated by the COOH-terminal region of SP-A by a mechanism that is distinct from phospholipid binding.

Alanine mutagenesis of surfactant protein A reveals that lipid binding and pH-dependent liposome aggregation are mediated by the carbohydrate recognition domain.

The carbohydrate recognition domain (CRD) of surfactant protein A (SP-A) is critical for the modulation of surfactant secretion from isolated type II cells and for the Ca(2+)-dependent aggregation of surfactant liposomes, but the domains of SP-A that mediate lipid binding have not been precisely mapped. To determine the role of the CRD in lipid interactions and other functions, the conserved amino acids of the putative Ca2+ and carbohydrate binding site (Glu195, Glu202, Asn214, Asp215) were substituted with alanine. The wild-type recombinant protein, SP-Ahyp, and mutant SP-As SP-Ahyp,E195A, SP-Ahyp,E202A, SP-Ahyp,N214A, and SP-Ahyp,D215A, were expressed in insect cells using baculovirus vectors and compared functionally. The Ca(2+)-dependent binding and aggregation of liposomes at pH 7.0 by SP-Ahyp,N214A were comparable to SP-Ahyp, but these activities were blocked in SP-Ahyp,E195A, SP-Ahyp,E202A, and SP-Ahyp,D215A. In contrast, the SP-Ahyp,D215A but not the other mutant proteins induced the Ca(2+)-independent aggregation of phospholipid liposomes at pH 4.0. The mutant recombinant proteins did not compete with 125I-labeled rat SP-A for high-affinity receptor occupancy on isolated type II cells and were much less potent than SP-Ahyp as regulators of surfactant secretion and uptake from type II cells. We conclude that (1) lipid binding and pH-dependent liposome aggregation are mediated by the CRD of SP-A, (2) distinct but overlapping domains within the CRD are required for pH- and Ca(2+)-dependent liposome aggregation, and (3) conserved acidic and polar residues of the carbohydrate binding site of SP-A are essential for interactions with type II cells.

Mutational analysis of Arg197 of rat surfactant protein A. His197 creates specific lipid uptake defects.

In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes.

The longer isoform and Cys-1 disulfide bridge of rat surfactant protein A are not essential for phospholipid and type II cell interactions.

Rat SP-A is a heterooligomer of two closely related isoforms, that requires interchain disulfide linkage for several functions including SP-A-mediated phospholipid vesicle aggregation and modulation of surfactant secretion and uptake by isolated alveolar type II cells. While the Cys6 disulfide bond of rat SP-A is known to be critical for function, the importance of the second interchain disulfide linkage within the N-terminal Isoleucine-3-Lysine-Cysteine-1 (IKC) sequence of the alternatively processed isoform is not clear. To examine the role of the Cys-1-dependent multimerization in SP-A function, we disrupted the Cys-1 disulfide bond by deletion of the IKC sequence (SP-Ahyp, DeltaIKC) or the substitution Cys-1 to Ser (SP-Ahyp,C-1S) in mutant recombinant proteins produced in insect cells. N-terminal sequence analyses revealed that the mutations influenced signal peptidase cleavage specificity, resulting in an increase in the abundance of the longer isoform of SP-Ahyp,C-1S and in N-terminal truncation of a fraction of the SP-Ahyp,DeltaIKC polypeptides at Gly8. On nonreducing SDS-PAGE analysis, both mutant proteins migrated as monomers and dimers but not the higher multimers that are characteristic of the wild-type recombinant protein (SP-Ahyp). Cross-linking analyses demonstrated that the association between trimeric SP-A subunits remained intact despite disruption of the Cys-1 bond. The SP-Ahyp,C-1S eluted in the same volume as SP-Ahyp from the gel sizing column with an apparent mass of 440 kDa, indicative of association of at least 9-10 monomers. The phospholipid binding and aggregation activities of the SP-Ahyp,C-1S were approximately 75% and 55% of the SP-Ahyp, respectively, but the SP-Ahyp,DeltaIKC was functionally comparable to SP-Ahyp. Similarly, both mutant proteins regulated the secretion and uptake of surfactant from isolated type II cells, but the SP-Ahyp,DeltaIKC was somewhat more potent than the SP-Ahyp,C-1S. Competitive binding to the SP-A receptor on type II cells was reduced by both Cys-1 mutations. We conclude that neither Cys-1-dependent multimerization nor the longer SP-A isoform is absolutely required for oligomeric association of trimeric SP-A subunits, SP-A/phospholipid interactions, or the regulation of surfactant secretion or uptake from type II cells by rat SP-A.

Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension.

Surfactant protein A (SP-A) is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation and/or stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates.