Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Abstract

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A(2)) and SP-A2 (1A(0)), and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A(0)) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A(2)) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.

Figure 1

Image of monolayers of DPPC containing 17% SP-B spread over a subphase of 0.68 μg/ml of porcine SP-A (Panel A) and human SP-A (Panel B). Surface pressures at which images were obtained are given at the sides of the Figure. Three phases in the images were observed, including lipid liquid expanded (LE) phase (bright regions, circles), lipid liquid-condensed (LC) phase (small black domains, arrows), and surface clusters characteristic of SP-A and SP-B complexes (grey regions, block arrows). The morphology of the DPPC monolayers containing SP-B and human SP-A in panel B was similar to that of the lipid monolayers plus SP-B and porcine SP-A in panel A.

Figure 1

Image of monolayers of DPPC containing 17% SP-B spread over a subphase of 0.68 μg/ml of porcine SP-A (Panel A) and human SP-A (Panel B). Surface pressures at which images were obtained are given at the sides of the Figure. Three phases in the images were observed, including lipid liquid expanded (LE) phase (bright regions, circles), lipid liquid-condensed (LC) phase (small black domains, arrows), and surface clusters characteristic of SP-A and SP-B complexes (grey regions, block arrows). The morphology of the DPPC monolayers containing SP-B and human SP-A in panel B was similar to that of the lipid monolayers plus SP-B and porcine SP-A in panel A.

Figure 2

Images of films of DPPC: egg PG (8:2, mol/mol) containing 17% SP-B spread over human SP-A at 0.68 μg/ml. Surface pressures are given at the side of each image. In these images of monolayers of DPPC:eggPG plus SP-B spread on human SP-A show that the interactions between SP-A and SP-B produced the protein-rich phase (grey clusters, block arrows) in the monolayers regardless of the presence of 20 mol% unsaturated PG.

Figure 3

Images from films of DPPC with SP-B (17%) spread over the product of the 6A² of SP-A1 (0.68 μg/ml). Surface pressures are given at the side of each image. Panels A and B are from separate experiments. Both panel A and B show three characteristic features typical for the monolayers: a fine network of grey phase (block arrows) which extended throughout the LE phase, large protein-rich patches (large dark grey regions, stars), and LC phase (small black domains, arrows).

Figure 3

Images from films of DPPC with SP-B (17%) spread over the product of the 6A² of SP-A1 (0.68 μg/ml). Surface pressures are given at the side of each image. Panels A and B are from separate experiments. Both panel A and B show three characteristic features typical for the monolayers: a fine network of grey phase (block arrows) which extended throughout the LE phase, large protein-rich patches (large dark grey regions, stars), and LC phase (small black domains, arrows).

Figure 4

Images from films of DPPC/SP-B (17%) spread over the product of the 1A⁰ of SP-A2 (0.68 μg/ml). Surface pressures are given at the side of each image. Panels A and B are from separate experiments. The morphology of the films was very similar to that observed in the films containing combinations of SP-B and porcine SP-A (Figure 1A) or human SP-A (Figure 1B).

Figure 4

Images from films of DPPC/SP-B (17%) spread over the product of the 1A⁰ of SP-A2 (0.68 μg/ml). Surface pressures are given at the side of each image. Panels A and B are from separate experiments. The morphology of the films was very similar to that observed in the films containing combinations of SP-B and porcine SP-A (Figure 1A) or human SP-A (Figure 1B).

Figure 5

Images of films of DPPC/SP-B (17%) spread over a coexpressed product (1A⁰/6A²) of SP-A1 and SP-A2 (0.68 μg/ml). Surface pressures are given at the side of each image. Panels A and B are from different experiments. The morphology of the films containing SP-A1 and SP-A2 variants appears to combine features of the lipid-protein monolayers containing SP-A1 (Figure 3) and SP-A2 (Figure 4). Large likely protein-rich patches (stars), similar to those seen in the monolayers spread on SP-A1 (Figure 3), and a network of grey phase (block arrows), similar to those seen in the monolayer spread on SP-A2 (Figure 4), are observed in the films containing SP-A1 and SP-A2.

Figure 5

Images of films of DPPC/SP-B (17%) spread over a coexpressed product (1A⁰/6A²) of SP-A1 and SP-A2 (0.68 μg/ml). Surface pressures are given at the side of each image. Panels A and B are from different experiments. The morphology of the films containing SP-A1 and SP-A2 variants appears to combine features of the lipid-protein monolayers containing SP-A1 (Figure 3) and SP-A2 (Figure 4). Large likely protein-rich patches (stars), similar to those seen in the monolayers spread on SP-A1 (Figure 3), and a network of grey phase (block arrows), similar to those seen in the monolayer spread on SP-A2 (Figure 4), are observed in the films containing SP-A1 and SP-A2.