NORMAL GENOMIC

• Zaltash S, Palmblad M, Curstedt T, Johansson J, Persson B
• Pulmonary surfactant protein B: a structural model and a functional analogue.
• Biochim Biophys Acta. 2000; 1466: 179-86
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• Surfactant proteins B and C (SP-B and SP-C), together with phospholipids, are important constituents of pulmonary surfactant and of preparations used for treatment of respiratory distress syndrome (RDS). SP-B belongs to the saposin family of homologous proteins, which include other lipid-interacting proteins, like the membranolytic NK-lysin. SP-B, in contrast to other saposins, is hydrophobic and a disulfide-linked dimer, and its mechanism of action is not known. A model of the three-dimensional structure of one SP-B subunit was generated from the structure of monomeric NK-lysin determined by nuclear magnetic resonance, and the SP-B dimer was formed by joining two subunits via the intersubunit disulfide bond Cys48-Cys48'. After energy minimization, intersubunit hydrogen bonds/ion pairs were formed between the strictly conserved residues Glu51 and Arg52, which creates a central non-polar region located in between two clusters of positively charged residues. The structural features support a function of SP-B in cross-linking of lipid membranes. Mixtures of phospholipids, an SP-C analogue and polymyxin B (which cross-links lipid vesicles but is structurally unrelated to SP-B) exhibit in vitro surface activity which is indistinguishable from that of analogous mixtures containing SP-B instead of polymyxin B. This suggests an avenue for identification of SP-B analogues that can be used in synthetic surfactants for treatment of RDS.

• Zhai Y, Saier MH Jr
• The amoebapore superfamily.
• Biochim Biophys Acta. 2000; 1469: 87-99
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• Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed.

• Cochrane CG
• Surfactant protein B and mimic peptides in the function of pulmonary surfactant.
• FEBS Lett. 1998; 430: 424425-424425

• McCormack F
• The structure and function of surfactant protein-A.
• Chest. 1997; 111: 114119-114119

• Zhekova N
• [Pulmonary surfactant--its structure and function]
• Akush Ginekol (Sofiia). 1997; 36: 32-6

• Roach PJ, Skurat AV
• Self-glucosylating initiator proteins and their role in glycogen biosynthesis.
• Prog Nucleic Acid Res Mol Biol. 1997; 57: 289-316

• Drablos F, Petersen SB
• Identification of conserved residues in family of esterase and lipase sequences.
• Methods Enzymol. 1997; 284: 28-61

• Kang JH, Lee MK, Kim KL, Hahm KS
• The relationships between biophysical activity and the secondary structure of synthetic peptides from the pulmonary surfactant protein SP-B.
• Biochem Mol Biol Int. 1996; 40: 617-27
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• Synthetic pulmonary surfactants consisting of a mixture of phospholipids with synthetic peptides based on human and bovine surfactant-associated protein SP-B were prepared. These surfactants were analyzed for their biophysical activities by Wilhemly balance experiments and for their secondary structures by circular dichroism (CD) spectroscopy. Four synthetic peptides (SP-1, SP-2, SP-3, and SP-4) combined with the phospholipid mixture displayed significant surfactant properties. The CD spectra showed that the alpha-helical propensities of the peptides in SDS micelles were related to their surfactant activities. These results suggested that the several truncated peptides originated from SP-B protein, when appropriately recombined with phospholipids, could be used as an effective synthetic surfactant for clinical use.

• Cruz A, Casals C, Perez-Gil J
• Conformational flexibility of pulmonary surfactant proteins SP-B and SP-C, studied in aqueous organic solvents.
• Biochim Biophys Acta. 1995; 1255: 68-76
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• The structure of hydrophobic pulmonary surfactant-associated proteins SP-B and SP-C have been studied in different acetonitrile (ACN)/water and trifluorethanol (TFE)/water mixtures by circular dichroism and fluorescence spectroscopy to analyze the conformational flexibility of these proteins in response to changes in solvent composition. SP-B presented a very stable conformation in all the assayed ACN/water mixtures and in TFE/water mixtures containing until 70% TFE, showing around 40% alpha-helix. When SP-B was transferred to mixtures containing more than 70% TFE, the percent of alpha-helix in SP-B increased up to 60%. The fluorescence emission spectra of SP-B in the different solvents showed that tryptophan residues are more sensitive to solvent changes than those of tyrosine, reflecting differential effects on different protein microenvironments. The effect of solvent changes on the two tryptophan populations detected by fluorescence spectra was also different. A model for the folding of SP-B dimers, dominated by intra- and intermolecular disulphide bonds, is proposed. Surfactant protein SP-C revealed a secondary structure much more sensitive to solvent composition than SP-B. It had a main alpha-helical conformation in ACN/water solvents which was up to 63% in mixtures containing more than 60% ACN. When the protein was transferred to solvents containing less than 60% ACN, its secondary structure possessed less percent of alpha-helix and an increased percent of beta-structure. On the other hand, SP-C had a main beta-sheet secondary structure in all the assayed TFE/water mixtures, with 30-40% alpha-helix and around 50% beta-structure. The strong dependence of SP-C conformation on the nature of the solvent is interpreted to arise from its high hydrophobicity and the possible occurrence of protein-protein interactions.

• Andersson M, Curstedt T, Jornvall H, Johansson J
• An amphipathic helical motif common to tumourolytic polypeptide NK-lysin and pulmonary surfactant polypeptide SP-B.
• FEBS Lett. 1995; 362: 328-32
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• The tumour-lysing and antimicrobial polypeptide NK-lysin and the pulmonary surfactant-associated polypeptide SP-B exhibit 24% residue identities (49% similarities), including six half-cystine residues in the same disulphide bonding pattern, and similar far-UV circular dichroism spectra corresponding to 45-55% alpha-helix and 20-25% beta-sheet structures. From this, we conclude that the conformations of NK-lysin and SP-B are similar. In contrast, the functional properties of the two proteins are dissimilar: SP-B does not exhibit antibacterial activity and NK-lysin does not significantly effect phospholipid spreading at an air/water interface. Saposins, which solubilize lipids and activate lysosomal hydrolases, the pore-forming amoebapores, and parts of acid sphingomyelinase and acyloxyacylhydrolase, also share 18-27% sequence identities with NK-lysin (and SP-B), including the six conserved half-cystine residues. The inclusion of NK-lysin extends the family of saposin-like polypeptides, all members of which appear to interact with lipids. Strictly conserved structural features with implications for helix topology and lipid interactions are observed.

• Henikoff S
• Comparative methods for identifying functional domains in protein sequences.
• Biotechnol Annu Rev. 1995; 1: 129-47
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• This chapter reviews the different approaches that have been applied to the problem of protein motif identification. Several methods are available for finding motifs within protein families, for clustering databases to identify family relationships and for searching databases that consist of motif representations. With the rapid expansion of sequence databases, which currently appear to represent most protein families, these methods are becoming increasingly important for interpretation of molecular sequence information.

• Motwani M, White RA, Guo N, Dowler LL, Tauber AI, Sastry KN
• Mouse surfactant protein-D. cDNA cloning, characterization, and gene localization to chromosome 14.
• J Immunol. 1995; 155: 5671-7
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• Surfactant protein-D (SP-D) is a collectin found associated with surfactant in the lung. SP-D has also been functionally characterized as an opsonin for diverse microorganisms and a chemoattractant for phagocytic cells. To determine the structure of mouse SP-D, we isolated and characterized clones from a B6/CBAF1J strain lung cDNA library using a PCR-derived genomic probe. The deduced sequence predicts a 19-amino acid signal sequence, a 25-amino acid long NH2 terminus with two cysteines, followed by an uninterrupted collagen domain with 59 Gly-X-Y repeats. Next, a short "neck" domain of 28 amino acids, with a potential to form trimeric alpha-helical coiled coil is found ending in a COOH-terminal 125-amino acid carbohydrate recognition domain. The mature mouse SP-D protein of 355 amino acids shows strong homology to rat (92% identity), human (76%), and bovine (72%) SP-D amino acid sequences. Northern blot and RT-PCR analysis revealed that the mouse SP-D gene is expressed predominantly in lung and, surprisingly, also in heart, stomach, and kidney but not in brain. In contrast, mouse surfactant protein-A (SP-A) mRNA expression was found to be restricted to lung. Human lung and stomach, but not heart or liver, were found to express SP-D mRNA, as determined by PCR. The mouse SP-D gene (Sftp4) has been localized to chromosome 14 (to a region syntenic to human chromosome 10), closely linked to the genes for other collagenous lectins, mannose-binding protein-A (MbI1), and SP-A (Sftp1).

• Johansson J, Persson P, Lowenadler B, Robertson B, Jornvall H, Curstedt T
• Canine hydrophobic surfactant polypeptide SP-C. A lipopeptide with one thioester-linked palmitoyl group.
• FEBS Lett. 1991; 281: 119-22
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• The amino acid sequence and the posttranslational modification of the hydrophobic surfactant polypeptide SP-C from canine, rabbit and bovine lungs were established by direct sequence analysis and plasma-desorption time-of-flight mass spectrometry. The results reveal that canine SP-C has only one cysteine residue which, however, is palmitoylated, like the two Cys residues in other characterized SP-C molecules. In addition, canine SP-C is N-terminally truncated, with only 34 amino acid residues in its longest form. Thus, SP-C molecules can apparently vary to some extent in the N-terminal lipid-modified part, whereas the extremely hydrophobic middle and C-terminal parts are well conserved.

• Singh G, Katyal SL, Brown WE, Kennedy AL, Wong-Chong ML, Gottron SA
• Identification, isolation, and partial characterization of a 7.5-kDa surfactant-associated protein.
• Exp Lung Res. 1991; 17: 559-67
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• Human surfactant was analyzed for proteins associated with the lipids. Surfactant was isolated from lung lavage by the salt gradient centrifugation method, and the soluble proteins binding to the lipids were recovered by extraction with a low pH buffer. Antiserum to this preparation reacted with surfactant apoprotein A and a 7.5-kDa protein. The 7.5-kDa protein was isolated from reduced and alkylated lung lavage pellet by chromatography. The N-terminal amino acid sequence of the protein indicates that it is a novel protein.

• Johansson J, Curstedt T, Robertson B, Jornvall H
• Size and structure of the hydrophobic low molecular weight surfactant-associated polypeptide.
• Biochemistry. 1988; 27: 3544-7
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• The most abundant low molecular weight protein of pulmonary surfactant has unusual properties. Its primary structure has now been determined by analysis at the protein level. The highly hydrophobic polypeptide is resistant to cleavage with proteolytic enzymes, but it was possible to generate fragments by limited cleavage with concentrated HCl or with sodium in liquid ammonia. Acid hydrolysis of the peptide required exceptional conditions for release of all residues. The N-terminus is heterogeneous, and in its longest form the primary structure consists of 35 residues. This analysis establishes that the size of the major native hydrophobic surfactant polypeptide is considerably smaller than previously proposed. Biological effects of the polypeptide recombined with phospholipids are confirmed in vitro by using a pulsating bubble system and in vivo by using premature newborn rabbits. The molecule has branched-chain amino acid residues at about two-thirds of all positions and lacks nine types of residue. The middle third is composed entirely of hydrophobic residues, and fragments from this part are sparingly soluble even in organic solvents. The hydrophobic region is preceded by a more hydrophilic, N-terminal segment. Thus, the molecule has two contrasting parts, like a detergent, which may explain its essential role in the pulmonary surfactant system.