Use of human surfactant low molecular weight apoproteins in the reconstitution of surfactant biologic activity.

Abstract

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at -9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-proile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence.Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.

Surfactant for Pediatric Acute Lung Injury

“…Survanta has an SP-B content by ELISA of only 0.044% by weight relative to phospholipid, while CLSE (Infasurf®) has an SP-B content of 0.9% by weight that approaches that of lavaged whole surfactant [118]. SP-B is known to be the most active of the hydrophobic surfactant proteins in enhancing the adsorption, dynamic surface activity, and inhibition resistance of phospholipids [118,[176][177][178][179][180][181][182][183][184]. The addition of bovine SP-B or synthetic SP-B peptides to Survanta® significantly improves its surface and physiological activity towards that of Infasurf and whole surfactant [106,118,175] (Figure 5)‥ Despite its lack of SP-B, other active components such as SP-C in Survanta® allow it to have significant efficacy in some forms of ALI/ARDS as noted earlier (e.g., [130,131,133]).…”

“…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). The development of new synthetic SP-B peptides is particularly important for optimal synthetic exogenous surfactants given the greater activity of SP-B compared to SP-C in native surfactant noted earlier [118,[176][177][178][179][180][181][182][183][184]. In addition to peptide/protein components in new synthetic surfactants, it is also important to consider their lipid constituents.…”

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

“…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.…”

“…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).…”

“…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.…”