The Use of Synthetic Peptides in the Formation of Biophysically and Biologically Active Pulmonary Surfactants

Abstract

ABSTRACf. Synthetic pulmonary surfactants consisting of mixtures of phospholipids with synthetic peptides based on the amino acid sequence of human surfactant apoprotein SP-B were prepared. These surfactants were analyzed for their ability to lower surface tension on a pulsating bubble surfactometer and for their capacity to improve lung compliance and increase alveolar expansion in a fetal rabbit model of surfactant deficiency. The data demonstrate that several peptides, ranging from 17 to 45 residues in length, matching the carboxy-terminal sequence of the SP-B protein, when appropriately recombined with the phospholipids dipalmitoylphosphatidylcholine and phosphatidylglycerol (3:1), are capable of producing a synthetic surfactant with biophysical and biologicactivity approaching that of human surfactant derived from amniotic fluid. (Pediatr Res 29: 460-465, 1991) Abbreviations DPPC, dipalmitoylphosphatidylcholine lA, iodoacetic acid PG, phosphatidylglycerol PL, phospholipid SP, surfactant protein or swine, or synthetic lipids, generally in combination with SPBand SP-C (7, 10, 11, 13-15, 17-19, 21-23, 27), are being evaluated for their potential use in surfactant replacement therapies.Our previous studies (10) and those of Curstedt et al. (2), and Yu and Possmayer (19), suggested that when recombined with PL, SP-B resulted in greater surface tension lowering and biologic activity than did SP-C. For this reason, we decided to focus our attention on this protein. SP-B, also called SP-18 (4, 10), SPL(Phe) (3), PSP-B (5), and SAP-6-14 (7,16,17,23), has been shown to be a disulfide-linked dimer of two identical polypeptides of approximately 9000 D having an amino terminal sequence of phe-pro-ile-pro-leu-pro-tyr-(human) (3, 5, 10). Data based on amino acid compositions (10) and fast atom bombardment mass spectroscopy (unpublished observations) have led us to conclude that human SP-B is 80-81 residues in length. The sequence is shown in Figure I.To investigate the mechanisms by which SP-B protein augments the surfactant capacity of PL, peptides were synthesized that conform to portions of the native sequence of this protein. These peptides could be combined with PL to reconstitute the biophysical and biologic activities of natural surfactant. We report here the results of studies, using the pulsating bubble surfactometer to measure the capacity of these synthetic surfactants to lower surface tension at an air/liquid interface and the fetal rabbit model to determine their ability to increase lung compliance and alveolar expansion. MATERIALS AND METHODS Preparation ofpurified SP-B monomer, dimer, and oligomers.SP-B was isolated from human amniotic fluid surfactant ad escribed previously (10). One hundred J.Lg of SP-B, in a volume of 206 J.LL methanol, were incubated with 5.14 mg DTT (Sigma Chemical Co, St. Louis, MO) in 17 J.LL methanol, for 1 h at 37°C. Analysis of 2 J.LL on SDS-PAGE showed the SP-B to be approximately 70% reduced as noted by a shift in band position from 18000 D to approximately 9000 D. The SP-B/...

NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

“…This study examines the structure of the 16 C-terminal residues of SP-B (SP-B CTERM ) which contains the predicted C-terminal helix and several residues N-terminal to the putative helix. A similar Cterminal peptide has been shown to possess partial biological activity (32). Since determination of the structure of fulllength SP-B has long been hampered by its high degree of hydrophobicity, structural studies of the C-terminal peptide were initiated to add to our understanding of SP-B's mechanism of function.…”

Surfactant for Pediatric Acute Lung Injury

“…Two important conceptual approaches being followed to develop synthetic exogenous surfactants are: (1) Combining human sequence recombinant proteins or synthetic amphipathic peptides with synthetic biological glycerophospholipids; and (2) combining synthetic amphipathic peptides with novel synthetic lipids designed to have favorable physicochemical properties such as high surface activity and structural resistance to inflammatory phospholipases during lung injury. Surfaxin® (KL4) [87,107,123,[189][190][191][192][193][194][195] and Venticute® (recombinant SPC surfactant) [26,[196][197][198][199][200][201] are two current examples of the first of these approaches (Table 3). However, the 21 amino acid KL4 peptide is only a very rough structural analog to native SP-B, and advances in peptide molecular modeling and synthesis technology support the feasibility of preparing new SP-B peptides with significantly greater sequence and molecular folding specificity, and correspondingly higher activity ( [202][203][204] for review).…”

Lung Surfactant and Its Use in Lung Diseases

“…Пептид представляет собой амфипатическую спираль повторяющихся субъединиц из одно го лизина и четырех лейцинов [117]. Surfaxin получают путем добавления пептида, растворенного в хлороформ -метаноле (1:1, об/об), к смеси ДПФХ и ФГ, нагревания при температуре 43°C и дальнейшего высушивания в токе азота или под вакуу мом.…”