Surfactant protein A without the interruption of Gly-X-Y repeats loses a kink of oligomeric structure and exhibits impaired phospholipid liposome aggregation ability.

Pulmonary surfactant protein A (SP-A) belongs to the collectin subgroup of the C-type lectin superfamily. SP-A oligomerizes as an octadecamer, which forms a flower bouquet-like structure. A collagen-like domain of human SP-A consists of 23 Gly-X-Y repeats with an interruption near the midpoint of this domain. This interruption causes a kink, but its role remains unknown. To define the importance of the kink region of SP-A, two mutated proteins were constructed to disrupt the interruption of Gly-X-Y repeats: SP-ADEL, which lacks the Pro47-Cys48-Pro49-Pro50 sequence at the interruption, and SP-AINS, in which two glycines were introduced to insert Gly-X-Y repeats (Gly-Pro47-Cys48-Gly-Pro49-Pro50). Electron microscopy using rotary shadowing revealed that both mutants form octadecamers that lack a bend in the collagenous domain. Electrophoretic analysis under nondenaturing conditions and gel filtration chromatography demonstrated that SP-AINS consisted of a large assembly of oligomers whereas SP-ADEL formed mainly octadecamers. Both SP-ADEL and SP-AINS mutants as well as wild-type SP-A bound to liposomes containing dipalmitoylphosphatidylcholine and galactosylceramide at equivalent levels, but the abilities of the mutants to induce phospholipid liposome aggregation were significantly less developed than that of the wild type. The mutants SP-ADEL and SP-AINS augmented liposome uptake by alveolar type II cells and inhibited secretion of phospholipids from type II cells at a level comparable to that of wild-type SP-A. These results indicate that the interruption of Gly-X-Y repeats in the SP-A molecule is critical for the formation of a flower bouquet-like octadecamer and contributes to SP-A's capacity to aggregate phospholipid liposomes.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.