Antibodies against synthetic amphipathic helical sequences of surfactant protein SP‐B detect a conformational change in the native protein

Abstract

Syntltetic peptides based on the native human sequen¢~ of surfactant protein B have I~,en used to generate polyclonal monospecific antibodies against specific segments of the native SP-B protein, Circular dichroism analysis or the synthr.tic p<ptides shows the>, have a dominant helical content in structure promotinll environments and tensiometric measurements indicate these peptides lower surface tension at air=water interfaces implyin~ that they contain amphipathic alpha helical motifs, Antibodies directed aBainst the C-terminal segment of SP.B react with the native protein in the oxidized and reduced state, Antibodies directed against the N.tem~inal sequence of SP-B react with the native protein only in the reduced stat~ suue..aiai| that this domain has a conformation dependent on disulfide bond formation.Surfactant pcptide; Amphtpathie helix: lmmunodominance

Conformation and molecular topography of the N‐terminal segment of surfactant protein B in structure‐promoting environments

“…The "blue" shift in the fluorescence maximum for SP-BI-2s is most likely due to peptide aggregation in PBS. Previous CD studies of SP-B,-,, in PBS indicated a concentration-dependent change in structure typical of peptide-self association (Fan et al, 1991). This aggregation phenomenon is further indicated by the characteristic ESR spectral broadening for SP-BT-,, in PBS (see above), which suggested a significant population of peptide aggregates in solution.…”

“…These results complement a n earlier immunologic study (Longo et al, 1992) of SP-BI-,, bound to liposomes that also provided information on the exposure of protein domains to the aqueous buffer. Antibodies raised to the amphipathic helical region encompassing SP-B residues Cys-8 to Gly-25 (Fan et al, 1991) showed that this sequence is accessible to the bulk aqueous solvent with SP-B,_,, bound to surfactant liposomes (Longo et al, 1992). In this context, it should be noted that our polarized FTIR measurements indicate that the helical axis (SP-B residues 8-25) inserts in surfactant lipids at an oblique angle with respect to the surface of the film (Table 1).…”

“…Another indication of specific interaction of SP-B with lipids is insertion of the protein into monolayers at varying surface pressures (OosterlakenDijksterhuis et al, 1991). The critical insertion pressure, an index of the degree of peptide association with lipid films and membrane systems, is very high for the amino-terminal peptide of SP-B in contrast to the C-terminal domain of the protein Fan et al, 1991). The N-terminal domain of SP-B has also been shown to mediate lipid-vesicle mixing (Longmuir et al, 1992), as does the native SP-B (OosterlakenDijksterhuis et al, 1992).…”

“…residues 1-25) helical segments near the N-terminus and C-terminus of the 78-residue molecule (Waring et al, 1989;Takahashi et al, 1990;Bruni et al, 1991). CD, FTIR, and Raman spectroscopy of native SP-B and selected amphipathic segments indicates a dominant helical conformation in structure-promoting solvents, such as TFE:water, surfactant lipid dispersions, oriented lipid films, and detergent micelle dispersions Fan et al, 1991;Vincent et al, 1991;Longo et al, 1992;OosterlakenDijksterhuis et al, 1992;Vandenbussche et al, 1992;PCrez-Gil et al, 1993). Oriented CD measurements and polarized FTIR determinations of the native protein in surfactant lipid films further indicate that the protein helical axis is oriented approximately parallel to the surface of the film (OosterlakenDijksterhuis et al, 1991;Vandenbussche et al, 1992).…”

Binding of GM1 ganglioside to a synthetic peptide derived from the lysosomal sphingolipid activator protein saposin B

“…This protein is a major component of the lung surfactant together with other lipid-binding proteins and phospholipids. It is interesting to note that a synthetic peptide (Leu4"pLeu66 of SP-B), corresponding approximately to the position of sapB-18 in saposin B, also adopts an a-helical conformation and binds lipids [23]. This similarity between SP-B and saposin B suggests that these two similar proteins may bind lipids using similar structural elements and mechanisms.…”