The surfactant peptide KL4 sequence is inserted with a transmembrane orientation into the endoplasmic reticulum membrane

Abstract

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic surfactant peptide KL(4), a peptide based on a C-terminal amphipathic helical region of human SP-B, efficiently mimics some functional properties of SP-B and is included in therapeutic surfactant preparations used in trials to treat respiratory distress syndrome. The membrane orientation of this peptide is controversial. We used an in vitro transcription-translation system to study the insertion of hydrophobic sequences into microsomal membranes, and showed that the KL(4) sequence integrates efficiently with a transmembrane orientation despite the presence of intermittent lysines throughout the sequence. In contrast, the precise sequence of the C-terminal SP-B amphipathic region failed to integrate, indicating a nontransmembrane orientation. Differences in the membrane insertion between KL(4) and the SP-B-inspiring sequence match predictions from calculated free energies of insertion of the two sequences into membranes.

FIGURE 1

(A) Schematic of the engineered leader peptidase (Lep) model protein. (B) The H-segment sequences examined in this study. (C) Calculated ΔG_app values for the assessed sequences using the ΔG Prediction Server (see text). (D) In vitro translation in the presence (+) or absence (−) of MMs and PK. Nonglycosylated protein bands are indicated by a white dot; single and double glycosylated proteins are indicated by one and two black dots, respectively. The protected double-glycosylated P2 fragment is indicated by an asterisk (*). (E) Left, KL₄ canonical helix structure. Center, SP-C helical region (residues 13–35) from the structure determined by NMR (Protein Data Bank: 1SPF) (15). Right, SP-B region (residues 63–78) based on the structure of Mini-B in sodium dodecylsulfate micelles (Protein Data Bank code: 2DWF). Side chains of Arg and Lys residues are shown.