F UNCTION OF S URFACTANT P ROTEINS B AND C

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January 20, 2001 16:51 Annual Reviews AR114-22

Annu. Rev. Physiol. 2001. 63:555–78Copyright c© 2001 by Annual Reviews. All rights reserved

FUNCTIONS OF SURFACTANT PROTEINS B AND C

Timothy E Weaver and Juliana Johnson ConkrightDivision of Pulmonary Biology, Children’s Hospital Medical Center,Cincinnati, Ohio 45229-3039; e-mail: [email protected]

Key Words Type II epithelial cell, lung development, transgenic mice, respiratorydistress syndrome, structure-function

■ Abstract SP-B is the only surfactant-associated protein absolutely required forpostnatal lung function and survival. Complete deficiency of SP-B in mice and hu-mans results in lethal, neonatal respiratory distress syndrome and is characterized bya virtual absence of lung compliance, highly disorganized lamellar bodies, and greatlydiminished levels of SP-C mature peptide; in contrast, lung structure and function inSP-C null mice is normal. This review attempts to integrate recent findings in humansand transgenic mice with the results of in vitro studies to provide a better understandingof the functions of SP-B and SP-C and the structural basis for their actions.

INTRODUCTION

The maintenance of alveolar structure during cyclical changes in lung volume iscritical for normal respiration. The inherent tendency of the alveolus to collapse atend expiration is due, in large part, to high surface tension generated by an aqueouslayer lining the alveolar epithelium. Alveolar stability is achieved by maintenanceof a phospholipid-rich film (pulmonary surfactant) at the air-liquid interface thatreduces surface tension to very low levels as alveolar surface area decreases. Inthe absence of surfactant, the collapse of multiple alveoli rapidly progresses tosevere respiratory distress, a condition marked by accumulation of characteristicintra-alveolar hyaline membranes, increased alveolo-capillary permeability andthe need for ventilatory support. Human infants with surfactant insufficiency, usu-ally the result of premature birth, respond very favorably to treatment with sur-factant preparations based on the composition of native surfactant. The minimalessential elements of a therapeutic surfactant preparation include specific phos-pholipids, e.g. dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol(PG), and one of the hydrophobic surfactant peptides, surfactant protein (SP) Bor SP-C. The importance of the peptide component of pulmonary surfactant isunderscored by the observation that inherited deficiency of SP-B inevitably re-sults in lethal respiratory distress syndrome (RDS) (1). This review integratesthe findings of key studies, performed in vitro or in vivo, to provide a better

0066-4278/01/0315-0555$14.00 555

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556 WEAVER ¥ CONKRIGHT

understanding of the functions of SP-B and SP-C and the structural basis for theiractions.

STRUCTURE OF SP-B AND SP-C

The goal of this section is to summarize current understanding of SP-B and SP-Cstructure in order to provide a basis for subsequent discussions of structure/functionrelationships. For a more comprehensive treatise of SP-B and SP-C structure, thereader is directed to several recent reviews of the subject (2–4).

Isolation of SP-B and SP-C

Low-speed centrifugation of bronchoalveolar lavage fluid results in isolation ofsurfactant with excellent surface tension–reducing properties. Surfactant isolatedin this manner consists predominantly of lipid (90% by weight) with 0.5–1% eachof SP-B and SP-C (2). Because of their hydrophobicity, SP-B and SP-C co-extractwith lipids into the organic solvent phase following chloroform/methanol extrac-tion of surfactant. Although this procedure results in virtually complete separationof SP-B and SP-C from the other protein components of surfactant, separationfrom each other and from phospholipids is more challenging. Chromatographyof the acidified organic solvent extracts of surfactant on Sephadex LH-60 (5) orreverse-phase HPLC on a C18 column (6) results in elution of SP-B first, consis-tent with its overall more polar amino acid composition, followed by SP-C and,finally, phospholipids.

Structure of SP-B

The consensus sequence for SP-B, derived by alignment of the amino acid se-quences from nine different species (Figure 1A), indicates that the mature lipid-associated peptide is 79 residues in length and contains 52% hydrophobic aminoacids (Ala, Ile, Leu, Phe, Trp, Val), and a substantial number of basic aminoacids, resulting in a net charge of+5. Six of seven cysteine residues are abso-lutely conserved, and this feature identifies SP-B as a member of the saposin-like (SAPLIP) family of peptides (7, 8). Other SAPLIP family members includesaposins A, B, C, and D, NK-lysin, and the pore-forming peptide ofEntamoebahistolytica. SAPLIP domains have also been identified in acid sphingomyelinase,acyloxyacyl hydrolase, and plant aspartic proteinases (9); in addition, the propep-tides that flank the mature peptide in the SP-B proprotein each contain a SAPLIPdomain (Figure 1A). The cysteine residues of the SP-B mature peptide form in-tramolecular sulfhydryl bridges such that the innermost two residues (cysteines35 and 46) form a bridge that stabilizes a hairpin loop, the outermost two cys-teines (residues 8 and 77) form another bridge, and the two remaining cysteines(residues 11 and 71) form a disulfide bond between the inner and outer bridges(10, 11) (Figure 1B). This pattern of disulfide bonding has also been confirmed for

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FUNCTION OF SP-B AND SP-C 557

NK-lysin (12) and saposins B and C (13) and likely contributes to the remark-able thermal stability of these peptides. The three-dimensional NMR structure forNK-lysin, the only SAPLIP family member for which such data are available, sug-gests the presence of five amphipathic helices (14). The helical content of SP-B issimilar to NK-lysin (approximately 45%), and given the constraints imposed bythree disulfide bridges, it is highly likely that the three-dimensional structure ofSP-B is also similar to NK-lysin (15, 16).

All SAPLIPs interact with lipids; however, unlike other SAPLIPs, which areonly transiently lipid associated, the unique hydrophobic character of the 79 aminoacid SP-B peptide ensures that it is always associated with surfactant phospho-lipids. Although the orientation of SP-B within a lipid membrane is not clear,most evidence to date supports a model in which the polar faces of the amphi-pathic helices interact with phospholipid headgroups, and the highly conservedpositively charged amino acids interact specifically with the anionic phospholipidphosphatidylglycerol (17, 18), which constitutes approximately 10% of surfactant.This model favors location of SP-B at the surface of the membrane such that thenonpolar faces of the helices interact only with the most superficial aspect of theacyl chains.

A second unique characteristic of SP-B is the formation of oligomers, predom-inantly homodimers in most species. Dimerization occurs through formation of anintersubunit disulfide bridge involving cysteine 48, which is not present in otherSAPLIPs (11, 19). The dimer structure may be further stabilized by formationof intersubunit hydrogen bonds/ion pairs between the strictly conserved residuesGlu 51 and Arg 52 (20). It is currently unclear if both subunits are always locatedin the same lipid plane. The potential for SP-B units to reside in separate, adjacentmembranes has important implications for packaging of phospholipids in lamellarbodies and surface film dynamics.

Addition of SP-B to liposomes leads to membrane binding, destabilization, andfusion (lipid mixing) (18, 21–26). The ability of SP-B to alter membrane structuremay play a critical role in the packaging of surfactant phospholipids for storage inlamellar bodies and in the transition from storage form to the biologically activesurface film. These putative in vivo functions of SP-B (discussed below) should beinterpreted with caution as the in vitro properties of SP-B are strongly influencedby a number of variables including the method of SP-B isolation, the size andphospholipid composition of liposomes, the presence of SP-A and divalent cations,pH, and the manner in which SP-B is added to liposomes (3, 26, 27).

Structure of SP-C

Analysis of the SP-C consensus sequence, derived by alignment of amino acidsequences from 10 different species (Figure 2), indicates that the mature SP-Cpeptide is a 35–amino acid peptide composed of 69% hydrophobic amino acids(Ala, Ile, Leu, Phe, Trp, Val). The overall hydrophobicity of the peptide is in-creased by palmitoylation of cysteines at positions 5 and 6 in most species (canine

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and mink SP-C contain a single palmitoylated cysteine at position 6). A smallproportion of SP-C (less than 5%) is also palmitoylated on lysine 11 (6). Non-palmitoylated or deacylated forms of SP-C can form sulfhydryl-dependent dimersand amyloid fibrils (28–30); therefore, variable palmitoylation (non-, mono-, di-and tripalmitoylated isoforms) in combination with N-terminal truncation leads toconsiderable microheterogeneity of SP-C (4). The functional consequences of thisheterogeneity are not known.

One of the most remarkable features of SP-C is an extremely hydrophobic do-main (residues 13–28) in which valine is the predominant amino acid (69%), thusaccounting for one of the original names for this peptide, surfactant proteolipidpolyvaline (31). The NMR structure of porcine SP-C is consistent with a trans-membrane peptide in which residues 9–34 form a rigidα helix (32). This structurecan span a fluid DPPC bilayer such that the polyvaline domain is perfectly ac-commodated in the interior acyl chain region of the membrane (Figure 3). Thehelical nature of the membrane-spanning domain is promoted by the palmitatemoieties on the extramembrane N-terminal region of SP-C, which may interactwith an adjacent membrane or, more likely, with the same bilayer in which thepeptide resides (33). The transmembrane orientation of SP-C is further stabilizedby invariant Lys-Arg residues at positions 11 and 12 that interact with phospholipidheadgroups (34).

As like SP-B, SP-C has been shown to alter membrane structure in vitro. Therelative mobility of the C-terminal region of SP-C may play a role in disrupting thepacking of acyl chains in the bilayer leading to membrane destabilization (35, 36);however, unlike SP-B, SP-C does not appear to promote lipid mixing (fusion)(18, 25). The potential of SP-C to destabilize membranes and the putative roleof this property in promoting formation of a biologically active surface film arediscussed below.

SURFACTANT HOMEOSTASIS

SP-B and SP-C are associated with several different forms of surfactant (Fig-ure 3). The major intracellular form of surfactant consists of lipid bilayers thatare tightly packed into concentric layers (lamellae) within large (1–2µm dia-meter) storage granules (lamellar bodies) of the Type II alveolar epithelial cell.Exocytosis results in release of the lamellar body contents (large aggregate sur-factant) into the aqueous lining layer of the alveolus. Subsequent transition fromstorage form to functional surface film involves unraveling of the lamellae andadsorption of lipids to form an interfacial (liquid-air) film or insertion of lipidsinto an existing surface film. Turnover of the surface film results in the formationof vesicular forms of surfactant (small aggregate surfactant) that are internalizedinto catabolic pathways in Type II cells and macrophages or directed to a recyclingpathway in Type II cells. The putative roles of SP-B and SP-C in promoting transi-tion from one surfactant form to another are discussed in the succeeding sections.

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INTRACELLULAR FUNCTIONS OF SP-B AND SP-C

The Role(s) of SP-B in Surfactant Biosynthesis

The mature SP-B peptide is encoded by exons 6 and 7 of a single gene (Sftp-3)located on chromosome 2 in the human (37, 138) and chromosome 6 in the mouse(39). Translation of the human SP-B mRNA results in synthesis of a larger pre-cursor protein in which the mature SP-B peptide (residues 201–279) is flankedby an N-terminal prepropeptide of 200 amino acids and a C-terminal peptide of102 amino acids (Figure 1A). Both flanking peptides contain saposin-like do-mains that are readily identified by alignment of six conserved cysteine residues(69, 72, 100, 112, 137, and 143 in the human N-terminal propeptide; 299, 302, 325,335, 360, and 366 in the C-terminal peptide) with other members of the SAPLIPfamily (7, 40). Although the precise boundaries of the saposin-like domains havenot been established, it is apparent that these domains differ significantly fromthe mature peptide in that they are much less hydrophobic, carry a net negativecharge, and do not include an additional cysteine residue (comparable to Cys48in the mature peptide) capable of mediating sulfhydryl-dependent dimerization.Possible functions for the saposin-like domains are discussed below.

Processing of the SP-B preproprotein to its mature peptide occurs during transitthrough the secretory pathway. Entry of SP-B into the secretory pathway is me-diated by a typical N-terminal signal peptide, predicted to be 24 amino acids inlength (Signalp network server, Center for Biological Sequence Analysis; Lyngby,Denmark), which is cleaved upon translation of the proprotein into the endoplas-mic reticulum (41). The first 27 residues of the preproprotein have been shown toencode a functional signal peptide, but the exact cleavage site has not been exper-imentally determined (42). At least one point mutation in the sequence encodingthe signal peptide of human SP-B has been associated with lethal RDS (Table 1).

Transit of SP-B out of the endoplasmic reticulum is dependent on the N-terminalpropeptide, which likely facilitates folding and/or sequestration of the hydropho-bic mature peptide (42); in contrast, the C-terminal peptide is not required forintracellular trafficking of SP-B in transfected cultured cells or in transgenic mice(43, 44). Paradoxically, mutations in the saposin-like domain of the C-terminalpeptide have been associated with lethal RDS (Table 1). It is likely that thesemutations lead to misfolding and trapping of the proprotein in the endoplasmicreticulum, thereby preventing processing of the proprotein to its mature peptide inthe distal secretory pathway.

Trafficking of SP-B to the distal arm of the regulated secretory pathway(i.e. the lamellar body) requires both the N-terminal propeptide and the maturepeptide (44). Although it is not known if the proprotein is membrane bound duringtransit in the secretory pathway, it is tempting to speculate that the saposin-likedomain in the propeptide may play an important role in this sorting event, as hasbeen suggested for the saposin-like domain of the plant aspartyl protease, phytepsin(45). Mutations in the saposin-like domain in the N-terminal propeptide have beenassociated with RDS (Table 1).

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Cleavage of the N-terminal propeptide and the C-terminal peptide occurs in thelumen of the multivesicular body, prior to or during fusion of the multivesicularbody with the lamellar body (46). Processing of the proprotein to the mature peptideis detectable as early as day 17 in fetal mouse lung (47). The identity and numberof proteases involved in proprotein processing is not known, although a cathepsin-D-like protease has been implicated in cleavage of the propeptide (48). Consistentwith this hypothesis, a novel aspartyl protease, Napsin A, was recently identifiedand shown to be highly expressed in Type II epithelial cells (49–51); however,direct evidence for involvement of Napsin A in SP-B proprotein processing iscurrently lacking.

The fate of the cleaved N- and C-terminal peptides is not clear, and it is notknown if they are further processed to generate smaller peptides, consisting solelyof the saposin-like domain. The possibility that the released C-terminal peptidemay have a function independent of the proprotein is supported by a recent studyin transgenic mice (52). A human SP-B construct lacking the entire 102 amino acidC-terminal domain (SP-B1c) was able to restore lung function in SP-B (−/−) miceand completely reverse the neonatal lethal phenotype; however, lamellar bodieswere greatly enlarged in approximately 20% of Type II cells, and the intracellularsurfactant pool size was significantly increased. One possible interpretation of thisoutcome is that the C-terminal peptide plays a role in the turnover of surfactantlipids, similar to the well-characterized role of the saposins in facilitating lysosomalhydrolysis of glycolipids. It should be noted, however, that enlarged lamellar bodiesin Type II cells are common to a number of disorders including Chediak HigashiSyndrome (53), SP-D deficiency (54), LPS instillation (55), and certain lymphomas(56). The hypothesis that the C-terminal peptide modulates lamellar body size bypromoting hydrolysis of lipids in the surfactant recycling/catabolic pathway isintriguing, but has not been directly tested.

The phenotype of SP-B-deficient human infants (57) and SP-B knockout mice(58) has provided valuable insight into the role of SP-B in the surfactant biosyn-thetic pathway. Type II cells in SP-B (−/−) mice contain well-developed mul-tivesicular bodies but lack lamellar bodies with typical concentric membranes.Unlike cells from wild-type and SP-B (+/−) mice, Type II cells of SP-B (−/−)mice are characterized by inclusions containing numerous small vesicles and elec-tron dense masses. Multivesicular bodies fuse with the vesicular inclusions, andthe contents of these organelles were detected in the airway consistent with theirsecretion (47). Taken together, these data suggest that the vesicular inclusions inSP-B (−/−) mice are disorganized lamellar bodies in which the absence of SP-Bresults in failure to package surfactant phospholipids into concentric lamellae.

One of the characteristic features of the mature SP-B peptide is the formation ofhomodimers through cysteine 48 (10, 19). The proprotein does not form sulfhydryl-dependent oligomers, which suggests that dimer formation occurs following cleav-age of the peptides flanking the mature peptide. The functional significance of SP-Bhomodimerization is currently not clear. Expression of an SP-B construct in whichcysteine 48 (cysteine 248 in the proprotein) was mutated to serine completely

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reversed the neonatal lethality and disorganized lamellar body phenotype in SP-B(−/−) mice (19). This outcome suggests that sulfhydryl-dependent dimerizationof SP-B is not required for packaging of surfactant phospholipids in lamellar bod-ies or for formation and function of an interfacial film in the alveolus; however,this study cannot exclude the possibility that SP-B oligomers form through ionicor hydrophobic interactions (20).

A model that takes into account the SP-B (−/−) phenotype and the in vitroproperties of SP-B is proposed in Figure 4. In this model, the increasingly acidicmilieu of the maturing multivesicular body leads to activation of specific pro-teases and cleavage of the N-terminal propeptide and C-terminal peptide of theSP-B proprotein. Processing of the proprotein in the aqueous environment of themultivesicular body lumen results in rapid dimer formation and association of thehydrophobic mature peptide with the internal vesicles of this organelle. SP-B medi-ated lysis and fusion of internal vesicles leads to the reorganization of membranesinto lamellae, a process that continues following fusion of the multivesicular bodywith the lamellar body. This model predicts that SP-B will be associated withonly one side of a bilayer membrane, although the functional implications of thisasymmetry are not known.

The Role(s) of SP-C in Surfactant Biosynthesis

The mature SP-C peptide is encoded by exon 2 of a single gene (Sftp-2) on chromo-some 8 in the human (31, 59) and chromosome 14 in the mouse (39). Translationof the human SP-C mRNA results in synthesis of a proprotein of 197 aminoacids, or 191 amino acids following alternative splicing of the primary transcript(31, 60, 61). The organization of the proprotein is similar to SP-B in that the maturepeptide (residues 24–58) is flanked by an N-terminal propeptide and a C-terminalpeptide. However, unlike SP-B, SP-C is an integral membrane protein in whichthe hydrophobicα-helix of the mature peptide serves as both signal peptide andmembrane-spanning domain. The proprotein has been identified as a Type II (62)or Type III (63) integral membrane protein; however, since palmitoylation andpositively charged residues (K+R+) usually occur on the cytoplasmic side of themembrane (64, 65), SP-C is most likely a Type II integral membrane in whichthe N-terminal propeptide resides in the cytosol and the C-terminal peptide in thelumen of the endoplasmic reticulum.

The N-terminal propeptide and/or C-terminal peptide of the proprotein likelyplay an important role in trafficking of SP-C to the distal regulated secretorypathway. Post-Golgi sorting of integral membrane proteins is generally accepted tobe dependent upon information encoded in the cytosolic tail of the protein (66, 67).Assuming a Type II membrane orientation, the cytosolic domain of SP-C wouldconsist of the 23 amino acid propeptide and the first 10 residues of the maturepeptide (Figure 2). This region lacks a canonical di-leucine or tyrosine-basedmotif involved in adaptor protein (AP)-associated trafficking of integral membraneproteins to lysosomal or endosomal compartments, suggesting involvement of

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a novel sorting motif. This prediction is difficult to reconcile with the resultsof deletion studies suggesting that the C-terminal peptide plays a critical rolein intracellular trafficking of the proprotein (68, 69). The interpretation of thelatter studies is significantly complicated by the use of cells lacking a typicalregulated secretory pathway. It is possible that C-terminal deletions/mutationslead to misfolding and trapping of the proprotein in the endoplasmic reticulum;alternatively, but less likely, the proprotein may exist in a Type III membraneorientation such that the C-terminal peptide resides in the cytoplasm.

Trafficking of proteins through the secretory pathway involves the formationof transport vesicles in which integral membrane proteins are located in the lim-iting membrane of the vesicle. In the regulated secretory pathway, these vesiclesultimately fuse with larger storage granules, which are released in response to anextracellular stimulus. Exocytosis of regulated or constitutive secretory vesicles re-sults in the fusion of the limiting membrane of the secretory vesicle with the plasmamembrane, thereby leading to localization of integral membrane proteins on thecell surface. The transferrin receptor is an example of a palmitoylated transmem-brane protein with a Type II membrane orientation that follows the constitutivesecretory pathway to the plasma membrane. SP-C differs from the transferrin re-ceptor in that it is secreted into the alveolus, along with the other components ofsurfactant, rather than retained at the cell surface; therefore, at some point alongthe secretory pathway, SP-C must be relocated from the limiting membrane of thevesicle to its lumen. SP-C proprotein has been detected in the limiting membraneand internal vesicles of the multivesicular body, suggesting that this compartmentplays a critical role in the secretion of SP-C (63). Multivesicular bodies (endo-somes) in the endocytic pathway have previously been shown to be important forsegregation of membrane receptors destined for recycling to the cell surface fromreceptors marked for degradation. Receptors internalized into the lumen of themultivesicular body are degraded following fusion of the multivesicular body withthe lysosome (70). There is growing evidence that in addition to the endocyticpathway, multivesicular bodies may also play an important role in the secretorypathway; for example, multivesicular bodies represent a developmental stage inthe maturation ofα-granules in platelets (71). In alveolar Type II epithelial cells,multivesicular bodies can fuse with the lamellar body, which results in the deliveryof internal vesicles containing SP-C to the lumen of the lamellar body (47, 72).

The formation of internal vesicles in the multivesicular body is generally ac-cepted to be the result of inward vesiculation of the limiting membrane (Figure 5).The composition of the internal vesicle membranes is different from that of the lim-iting membrane of the multivesicular body, consistent with selective internalizationof both protein and lipid components (73, 74). Apart from facilitating secretion ofSP-C, this process may be important in refining the membrane environment inwhich SP-C is located. The lipid composition of endoplasmic reticulum and Golgimembranes through which SP-C traffics is very different from pulmonary surfac-tant, indicating that the membrane environment of SP-C changes during surfactantbiosynthesis (75, 76).

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The mechanism underlying selective internalization of membrane proteins hasnot been extensively studied. In yeast, fablp [a phosphatidylinositol(3)P-5-kinase]was shown to be essential for sorting of membrane proteins into the lumen of thevacuole, suggesting an important role for polyphosphoinositides in the formationof internal vesicles (77). The interaction of endosomal COP-1 and other proteinpartners with the cytosolic tail of specific integral membrane proteins in the lim-iting membrane of the multivesicular body may also be important for selectiveinternalization of membrane proteins (78, 79). Figure 5 proposes a model in whichthe N-terminal propeptide encodes a signal(s) for the internalization of SP-C fromthe limiting membrane of the multivesicular body.

Low levels of the mature SP-C peptide are detected on day 17 of gestation inthe mouse and day 27 of gestation in the rabbit (47, 80). Processing of the SP-Cproprotein to its mature peptide increases dramatically on day 18 or 29 in themouse and rabbit, respectively. As for the SP-B precursor, proteolytic processingof the SP-C proprotein likely occurs in the multivesicular body (63). Stepwise pro-cessing of the C-terminal peptide results in the generation of multiple processingintermediates (63, 81). The identity and number of proteases is not known, andit is not clear if there is any overlap in the proteases involved in processing ofthe SP-B and SP-C proproteins. A model of SP-C processing based on a Type IImembrane orientation of the proprotein is proposed in Figure 5. Proteases locatedin the lumen of the multivesicular body have free access to the SP-B proprotein andthe C-terminal peptide of the SP-C proprotein; the N-terminal propeptide of SP-Cis not accessible to proteases because it is located in the cytosol prior to inwardvesiculation and inside the internal vesicle following internalization. Processingof the SP-B proprotein leads to lysis and fusion of internal vesicle membranes bythe released mature peptide. SP-B-mediated vesicle lysis exposes the N-terminalpropeptide to proteases in the lumen of the multivesicular body, resulting in cleav-age of the propeptide. This model accounts for the incomplete processing of SP-Cproprotein in SP-B-deficient infants and SP-B (−/−) mice (1, 58, 82, 83). In theabsence of SP-B, levels of mature SP-C peptide are very low, and an incompletelyprocessed form of SP-C accumulates in the airway. This form of SP-C contains theN-terminal propeptide (identified by Western blotting), and its size is very closeto the predicted size of the proprotein lacking the entire C-terminal peptide (82).However, the identity of the incompletely processed SP-C proprotein has not yetbeen verified by amino acid sequence analysis.

The results of a previous study in transgenic mice suggested that the C-terminalpeptide of the SP-B proprotein was also important for SP-C processing (52). Thesemice (SP-B1c transgenic mice) expressed a truncated SP-B proprotein (lacking theentire C-terminal peptide) in the SP-B (−/−) background. A small amount of in-completely processed SP-C proprotein was detected in SP-C1c mice, suggestinga link between incomplete SP-C proprotein processing and the absence of theC-terminal peptide of SP-B. More recent studies have shown that in some trans-genic lines, the SP-B transgene is expressed in most, but not all, Type II epithelialcells, leading to small amounts of incompletely processed SP-C but normal levels

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of mature SP-C peptide in bronchoalveolar lavage fluid. Taken together, these re-sults support a role for the mature SP-B peptide, but not the C-terminal peptide,in processing of the SP-C proprotein.

EXTRACELLULAR FUNCTIONS OF SP-B AND SP-C

The Role(s) of SP-B and SP-C in Surface Film Formation

Numerous studies have characterized the effects of SP-B and SP-C on the forma-tion and properties of model surface films using the Langmuir-Wilhelmy surfacebalance, pulsating bubble surfactometer, and most recently, the captive bubble sur-factometer. The purpose of this section is not to provide a comprehensive reviewof these studies, but rather to re-examine some basic concepts regarding surfac-tant film formation and turnover in light of recent descriptions of SP-B and SP-Cknockout mice.

The alveolar lining layer consists of an aqueous subphase (hypophase) anda phospholipid-rich interfacial film. Several forms of surfactant are detected inthe hypophase, including the newly secreted contents of lamellar bodies, tubularmyelin, and loose membrane arrays. Initial formation of the surface film duringadaptation to air breathing requires rapid adsorption to and spreading of one or moreof these surfactant forms at the air-liquid interface. Despite intensive investigation,our understanding of the mechanisms leading to surface film formation and themolecular composition and structure of the interfacial film is still rudimentary.

The results of extensive in vitro studies suggest that in addition to rapid ad-sorption and spreading, a model surfactant should require only minimal surfacearea compression to achieve near zero surface tension, resist collapse when com-pressed, and respread rapidly following film compression. It is generally agreedthat in order to achieve the requisite low-surface tension during compression, thesurface film must be highly enriched in DPPC. Given the heterogeneous composi-tion of surfactant in the hypophase, it has been suggested that the interfacial film ismodified by selective incorporation of DPPC during film expansion and/or exclu-sion (squeeze-out) of non-DPPC components during film compression (84). Theprocesses involved in formation, refining, and maintenance of this surface film invitro are strongly influenced by SP-B and SP-C.

SP-B and SP-C independently accelerate film formation by enhancing theadsorption and spreading of surfactant phospholipids at an air-liquid interface. Inaddition to facilitating formation of an initial surfactant film during the first breath,SP-B and SP-C may play an important role in maintaining a functional interfacialfilm by recruiting phospholipids into the expanding film, promoting respreadingof a compressed film, and modulating film composition during dynamic compres-sion and expansion (85, 86). Although SP-B and SP-C are not equivalent in eachof these in vitro properties, surfactant preparations containing either SP-B or SP-Ccan restore lung function in surfactant-deficient animals, suggesting extensive or

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complete overlap in their ability to promote and maintain a functional surfacefilm (87–90). Consistent with this hypothesis, lung function, surfactant compo-sition, and surfactant pool sizes in SP-C (−/−) mice are indistinguishable fromwild-type littermates (91), indicating that SP-B alone is sufficient for surfactantfunction; however, rigorous testing of the functional redundancy hypothesis willrequire generation of transgenic animals that are deficient in SP-B and maintainnormal levels of mature SP-C peptide.

The structural basis for and the mechanism(s) whereby SP-B and SP-C modulateformation and function of the surface film remain unclear. Synthetic peptidescorresponding to the N and C termini of SP-B, which include helix 1 or helices 3and 4, were shown to approximate results obtained with native SP-B in vitro andin vivo (92–95). It is not known whether the fusogenic or lytic properties of SP-Bare important for the surface activities of these peptides or for native SP-B. Similarsynthetic peptide studies for SP-C indicate that the membrane-spanning domainis critical for biophysical activity and that the helical structure rather than theactual amino acid sequence seems to be more important for activity (33, 96–98).SP-B synthetic peptides localize to the surface of the membrane, whereas SP-Cpeptides adopt a transmembrane configuration, suggesting that SP-B and SP-Cwork through different mechanisms to achieve similar results; alternatively, it ispossible that the membranolytic activity of SP-B and SP-C are important for theirinterfacial activities because the peptides show overlap in this property in vitro.

The manner in which SP-B and SP-C associate with the surface film, tradi-tionally considered to be a monolayer, is not clear. Given the transmembraneorientation of SP-C, insertion of this integral membrane protein into a monolayerwould require SP-C to leave the membrane bilayer during film expansion and adopta new orientation in the monolayer [tilted instead of parallel to the phospholipidacyl chains (99)] in order to accommodate the highly hydrophobicα-helix. Subse-quent compression of the surface film would presumably require SP-C to leave themonolayer and reinsert into a lipid bilayer. A more appealing model is proposed bySchurch et al (100) who suggest that the surface film is a multi-layered structure inwhich membrane bilayers are associated with the interfacial film to form a surfacereservoir. SP-C could remain in a bilayer membrane within the surface reservoiror, perhaps, exist in a bilayer at the interface. Electron microscopic evidence fora multi-layered surface film has been reported (100, 101), although this conceptcontinues to be debated (102).

One further consequence of the integral membrane configuration of SP-Cwarrants some consideration. SP-C synthesis results in membrane asymmetry(i.e. assuming a Type II orientation, the N terminus of SP-C will always be associ-ated with the cytosolic side of the membrane) that is likely critical for intracellulartrafficking; furthermore, the model proposed in Figure 5 suggests that SP-B will al-ways be associated with the same side of a bilayer membrane (i.e. the side oppositethe N terminus of SP-C). These observations raise several questions. If SP-B andSP-C are asymmetrically distributed in surfactant bilayers, are surfactant phos-pholipids also asymmetrically distributed? Is membrane asymmetry maintained

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in the extracellular hypophase? What impact does protein and/or phospholipidasymmetry have on surface film formation and function?

The Role(s) of SP-B and SP-C in Surfactant Metabolism

Density gradient centrifugation of bronchoalveolar lavage results in isolation ofa large aggregate fraction (heavy/ultra heavy subtypes) consisting primarily oflamellar bodies, tubular myelin, and loose membrane arrays, and a small aggregatefraction (light subtype) consisting of small vesicles (103–105). Large aggregatesurfactant contains SP-B and SP-C, and is highly surface active in vitro and invivo; in contrast, the small aggregate fraction lacks SP-B and SP-C and has verypoor surface activity. Small aggregate surfactant is generally considered to be acatabolic form resulting from squeeze out during surface film compression. Thisform of surfactant is internalized more efficiently than large aggregate surfactant bylung epithelial cells in culture (106); furthermore, lipid uptake in vitro is enhancedby SP-C and, to a lesser degree, by SP-B (107). However, the interpretation ofthese results is not straight forward because large aggregate fraction appears to betaken up more rapidly than small aggregate in vivo (106); also, alveolar surfactantpool size was unaltered in SP-C (−/−) mice. [As of this writing, it is not known ifsurfactant aggregate composition and clearance kinetics are altered in SP-C (−/−)mice.] The potential for functional redundancy may make it difficult to determineif SP-C and/or SP-B play a critical role in modulating surfactant pool size byfacilitating phospholipid clearance.

A large proportion of alveolar surfactant is taken up by the Type II epithelialcell and enters a recycling pathway (105). The idea that the protein-depleted smallaggregate fraction represents the major catabolic form of surfactant lipids impliesphysical separation of SP-B and SP-C from surfactant lipids, a concept that isparticularly difficult to accept for an integral membrane protein like SP-C; fur-thermore, because SP-B and SP-C can also enter the recycling pathway and aresecreted with surfactant phospholipids, peptide/lipid re-association would have tooccur prior to or following internalization. Given the hydrophobicity of SP-B andSP-C, a more likely explanation is that the peptides remain lipid associated andco-sedimented with large aggregate surfactant or, perhaps, constitute a catabolicform with a distinct buoyant density. Overall, the metabolic form and mechanism(i.e. receptor or non-receptor mediated) (108–110) whereby SP-B and SP-C aretaken up by Type II epithelial cells is poorly understood.

Following internalization, SP-B is detected in various elements of the endocyticpathway, including multivesicular bodies (109). Multivesicular bodies are likelyinvolved in both the surfactant recycling (111, 112) and biosynthetic (46, 63, 113)pathways. Although there is currently no direct evidence for colocalization ofnewly synthesized and recycled surfactant components to the same multivesicularbody or population of multivesicular bodies, it is reasonable to speculate that thiscompartment plays a key role in surfactant homeostasis by integrating the biosyn-thetic, recycling, and catabolic pathways in Type II epithelial cells (Figure 3).

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SP-B AND SP-C DEFICIENCY

The complete absence of SP-B results in severe, neonatal respiratory distress syn-drome at term, followed by progressive respiratory failure and, inevitably, death(57, 114). Partial or transient SP-B deficiency has also been associated with chroniclung disease, and heterozygote susceptibility has been demonstrated for transgenicmice exposed to hyperoxia (115–119); however, a recent study of human patientsfailed to identify a clinical phenotype in individuals with a single functional SP-Ballele (120). The 121ins2 mutation (insertion of 2 bases into codon 121, leadingto a frame shift and early termination of translation) is present on both alleles inapproximately two thirds of SP-B-deficient patients and occurs with an allele fre-quency of 1 per 1000–3000 individuals (121). Other mutations of the SP-B geneleading to lung disease occur in combination with the 121ins2 mutation (Table 1).Prenatal diagnosis of SP-B deficiency can be made by immunologic detection ofincompletely processed SP-C and the absence of SP-B in amniotic fluid, althoughat least in one case, SP-C processing was not affected (82, 122).

SP-B deficiency in human infants has been associated with alveolar epithelialcell desquamation, congenital alveolar proteinosis, absence of tubular myelin, ab-normal distribution of SP-A and SP-C, accumulation of incompletely processedSP-C, abnormal surfactant activity, and altered levels of phosphatidylglycerol insurfactant (123–127). Some of these features are common to SP-B (−/−) mice,in particular the effects on tubular myelin formation, SP-C processing, and lungfunction (47, 58, 128); for technical reasons, surfactant composition has not beeninvestigated in newborn SP-B (−/−) mice. Other features of the SP-B-deficientphenotype appear to be unique to the human, most likely because of the geneticvariability and/or intensive postnatal care. Treatment of affected infants with ex-ogenous surfactant may produce a transient response, consistent with an ongoingrequirement for SP-B in the airspace (129). This largely ineffective therapeutic ap-proach may be limited by an additional intracellular requirement for the propeptideof SP-B. It is also possible that SP-B recycling may be compromised in these pa-tients; furthermore, the accumulation of incompletely processed SP-C or otherfactors in the hypophase may lead to inhibition of surface film formation andfunction. To date, none of these hypotheses has been experimentally tested.

In contrast to the SP-B knockout mouse, SP-C (−/−) mice process SP-B ap-propriately and lamellar body ultrastructure appears normal (91). One possibleinterpretation of this unexpected outcome is that SP-B compensates for loss ofSP-C in SP-C (−/−) mice, whereas SP-C compensation does not occur in SP-B(−/−) mice because generation of the mature SP-C peptide is dependent uponSP-B expression. This hypothesis will be difficult to test in vivo because it willrequire a strategy to restore SP-C processing in the absence of SP-B expression.Although complete loss of SP-C protein in the knockout mouse did not result in al-tered lung development or function, at least two mutations in the human SP-C genethat give rise to an altered proprotein have been linked to development of intersti-tial lung disease (130). These mutations were identified in unrelated patients and

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were present on only one allele, suggesting either a dominant-negative effect or again of function not normally associated with SP-C. One mutation (P30L) resultedin substitution of leucine for proline at position 7 in the mature peptide (position30 in the proprotein). This mutation may perturb the intracellular trafficking of theproprotein. The second mutation (c.460+ 1G> A) occurred in a donor splice siteresulting in deletion of exon 4, which encodes 37 amino acids in the C-terminalpeptide. As suggested earlier, C-terminal deletions may lead to misfolding and trap-ping of the proprotein in the endoplasmic reticulum. Intracellular accumulationof misfolded proteins has been causally linked to a number of diseases includingα-1-antitrypsin deficiency and cystic fibrosis (131, 132). Accumulation of dea-cylated SP-C has also been linked to pulmonary alveolar proteinosis, although acause-effect relationship has not been established (29, 30, 133).

SUMMARY

Although SP-C imparts important surface properties to surfactant phospholipidmixtures, the function of this peptide in vivo remains unclear. Association ofaltered SP-C structure with chronic lung disease may be of significant clinicalimportance, but a direct cause-effect relationship has not been established. SP-Bis clearly essential for surfactant homeostasis, but the molecular mechanisms un-derlying SP-B actions are not well understood. The results of very preliminarystudies suggest that SP-B may also have functions that are not directly related tosurfactant homeostasis (134), including antiflammatory properties (135) and pro-tection against oxygen-induced lung injury (117). Overall, despite considerablerecent progress in characterizing the structure and functions of SP-B and SP-C,many questions remain unanswered.

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January 31, 2001 18:29 Annual Reviews AR114-COLOR

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Figure 1 Alignment of SP-B proprotein. (A) The consensus (majority) sequence wasdetermined by alignment of SP-B proprotein from nine species. (Only mature peptidesequence is available for bovine and porcine SP-B.) Strictly conserved cysteine residues(red boxes) identify SP-B as a member of the SAPLIP family, which contains saposin-like domains in the N-terminal propeptide (blue bar), mature peptide (yellow bar), andC-terminal peptide (blue bar). Cysteine 48 in the mature peptide allows SP-B to formhomodimers (blue box). (B) The intramolecular sulfhydryl bridge formed between cysteines35 and 46 stabilizes the hairpin loop formed by the antiparallel arrangement of helices 2and 3. Additional intramolecular sulfhdryl bridges are formed between cysteines 8 and 77and cysteines 11 and 71.

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P1: FQP


Fig

ure

2A

lignm

ento

fSP

-Cpr

opro

tein

.The

cons

ensu

s(m

ajor

ity)

sequ

ence

was

dete

rmin

edby

alig

nmen

tof

SP-C

prop

rote

infr

omte

nsp

ecie

s.(O

nly

mat

ure

pept

ide

sequ

ence

isav

aila

ble

for

bovi

ne,

cani

ne,

and

porc

ine

SP-C

.)T

heSP

-Cpr

opro

tein

cont

ains

asi

ngle

mem

bran

e-sp

anni

ngdo

mai

n(b

lack

box)

.T

heN

-ter

min

alan

dC

-ter

min

alpe

ptid

es(b

lue

bars

)ar

epr

oteo

lytic

ally

clea

ved

toge

nera

teth

e35

amin

oac

id,

mat

ure,

tran

smem

bran

epe

ptid

e(y

ello

wba

r).P

alm

itoyl

atio

nof

cyst

eine

resi

dues

(red

box)

occu

rsin

alls

peci

es.

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Fig

ure

3Su

rfac

tant

hom

eost

asis

.(1)

Lam

ella

rbo

dy.(

2)L

arge

aggr

egat

esu

rfac

tant

inal

veol

arhy

poph

ase.

(3)

Surf

ace

film

.(4)

Smal

lagg

rega

tesu

rfac

tant

.(5)

Surf

acta

ntca

tabo

licpa

thw

ay.(

6)M

ultiv

esic

ular

body

.(7)

Rec

yclin

g/bi

osyn

thet

icpa

thw

ay.G

reen

arro

wsr

epre

sent

anab

olic

path

way

san

dbl

uear

row

sca

tabo

licpa

thw

ays.

Red

pept

ide

isSP

-C;b

lue

pept

ide

isSP

-B.

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Figure 4 Processing of the SP-B proprotein. SP-B is proteolytically processed by removalof the N-terminal propeptide followed by cleavage of the C-terminal peptide (black bars)releasing the 79 amino acid mature peptide (blue cylinder). SP-B mature peptide formshomodimers, which associate with the inner vesicles of the multivesicular body leading tovesicle lysis and fusion and, ultimately, reorganization of internal membranes.

Figure 5 Processing and secretion of SP-C. SP-C proprotein (red helix flanked by blacklines) interacts with unidentified cytosolic proteins (green), which selectively sort SP-Cfrom the limiting membrane to the lumen of the multivesicular body. Inward vesiculation ofthe limiting membrane results in localization of SP-C proprotein on the inner vesicles. TheC-terminal peptide of SP-C is accessible to proteases in the lumen of the multivesicular bodyand is cleaved in a step-wise fashion. Subsequent cleavage of the N-terminal propeptide ofSP-C is dependent upon the lytic activity of SP-B. Fusion of the multivesicular body withthe lamellar body results in secretion of SP-C.

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Annual Review of Physiology Volume 63, 2001

CONTENTSMedicine or Physiology: My Personal Mix, Alexander Leaf M.D 113C NMR of Intermediary Metabolism: Implications for Systemic Physiology, Robert G Shulman, Douglas L Rothman 15

Gastrin, CCK, Signaling, and Cancer, Enrique Rozengurt, John H Walsh 49

Intracellular Signaling Mechanisms Activated by Cholecystokinin Regulating Synthesis and Secretion of Digestive Enzymes in Pancreatic Acinar Cells, John A Williams

77

Specific Ca2+ Signaling Evoked by Cholecystokinin and Acetylcholine: The Roles of NAADP, cADPR, and IP3, Jose Manuel Cancela 99

The Gastrins: Their Production and Biological Activities, G J Dockray, A Varro, R Dimaline, T Wang 119

Control of Growth by the Somatropic Axis: Growth Hormone and the Insulin-Like Growth Factors Have Related and Independent Roles, Andrew A. Butler, Derek Le Roith

141

Role of Estrogen Receptor Beta in Estrogen Action, Katarina Pettersson, Jan-Åke Gustafsson 165

StAR Protein and the Regulation of Steroid Hormone Biosynthesis, Douglas M Stocco 193

The Guanylyl Cyclase Family at Y2K, BJ Wedel, DL Garbers 215MOLECULAR DIVERSITY OF PACEMAKER ION CHANNELS, U Benjamin Kaupp, Reinhard Seifert 235

Cellular Mechanisms of Oxygen Sensing, José López-Barneo, Ricardo Pardal, Patricia Ortega-Sáenz 259

Evolution and Physiological Roles of Phosphagen Systems, W Ross Ellington 289

Antifreeze and Ice Nucleator Proteins in Terrestrial Arthropods, John G Duman 327

Antifreeze Proteins of Teleost Fishes, Garth L Fletcher, Choy L Hew, Peter L Davies 359

Cytoplasmic Signaling Pathways that Regulate Cardiac Hypertrophy, Jeffery D Molkentin, Gerald W Dorn II 391

Somatic Gene Therapy in the Cardiovascular System, I Baumgartner, JM Isner 427

Genetic Assembly of the Heart: Implications for Congenital Heart Disease, Deepak Srivastava 451

Molecular Regulation of Lung Development, Wellington V Cardoso 471The Pulmonary Collectins and Surfactant Metabolism, Samuel Hawgood, Francis R Poulain 495

Surfactant Proteins A and D and Pulmonary Host Defense, Erika Crouch, Jo Rae Wright 521

Function of Surfactant Proteins B and C, Timothy E Weaver, Juliana Johnson Conkright 555

G Protein-Coupled Prostanoid Receptors and the Kidney, Matthew D Breyer, Richard M Breyer 579

Nephrogenic Diabetes Insipidus, Jean-Pierre Morello, Daniel G Bichet 607

Chloride Channels in the Loop of Henle, W Brian Reeves, Christopher J Winters, Thomas E Andreoli 631

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Molecular Analysis of Mammalian Circadian Rhythms, Steven M Reppert, David R Weaver 647

Circadian Photoperception, Paul F Devlin, Steve A Kay 677Endogenous Timekeepers in Photosynthetic Organisms, Carl Hirschie Johnson 695

Molecular Components of the Circadian System in Drosophila, Julie A Williams, Amita Sehgal 729

Genetic and Molecular Analysis of Circadian Rhythms in Neurospora, Jennifer J Loros, Jay C Dunlap 757

Dynamic Signaling Between Astrocytes and Neurons, Alfonso Araque, Giorgio Carmignoto, Philip G Haydon 795

On the Cellular and Network Bases of Epileptic Seizures, David A McCormick, Diego Contreras 815

Maintaining the Stability of Neural Function: A Homeostatic Hypothesis, Graeme W Davis, Ilya Bezprozvanny 847

Resurgence of Sodium Channel Research, Alan L Goldin 871

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Documents

F UNCTION OF S URFACTANT P ROTEINS B AND C