Bovine NK-lysin: Copy number variation and functional diversification.

NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30-35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer's patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants.

S-palmitoylation and s-oleoylation of rabbit and pig sarcolipin.

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.

The amoebapore superfamily.

Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed.

Damage to surfactant-specific protein in acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) develops in association with many serious medical disorders. Mortality is at least 40%, and there is no specific therapy. A massive influx of activated neutrophils, which damage pulmonary vascular endothelium and alveolar epithelium, leads to alveolar oedema and pulmonary surfactant dysfunction. In-vitro studies show that neutrophil elastase can cleave surfactant-specific proteins and impair surfactant function. If this happens in vivo in ARDS, the response to surfactant therapy will be limited. METHODS: Samples of pulmonary surfactant were obtained from the lungs of 18 patients with ARDS and six healthy controls by bronchoalveolar lavage. We separated proteins in these samples according to molecular weight by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We then used western blotting with monoclonal antibody E8 to detect the major surfactant-specific protein A (SP-A). FINDINGS: By contrast with controls, 14 of 18 patients had evidence of in-vivo damage to SP-A that resembled damage caused to SP-A when it is cleaved by neutrophil elastase. Controls showed a single band of normal dimers at 66 kDa, whereas 14 of 18 patients showed multiple bands at 66 kDa, 55 kDA, and 30-36 kDa, and six showed additional bands at 36-40 kDa. INTERPRETATION: Direct damage to surfactant-specific proteins occurs in lungs of patients with ARDS, probably by proteolysis. Trials of protein-containing therapeutic surfactant are in progress in ARDS, and our results indicate that the frequent failure to maintain response may result from continuing damage to surfactant by products of activated neutrophils. A combination of surfactant and antiprotease therapy may improve therapeutic prospects.

Localization of surfactant protein synthesis in human lung by in situ hybridization.

In order to investigate the sites of synthesis of the pulmonary surfactant-associated proteins, we performed tissue in situ hybridization. We used frozen sections of human lung tissue and 35S-UTP-labeled cRNA probes to localize mRNAs for the 35 kDa surfactant-associated protein (PSP-A) and for the precursor of one of the hydrophobic, low molecular weight surfactant-associated proteins (PSP-B). We found that PSP-A mRNA is present only in the alveolar epithelial type II cells with alveolar macrophages, bronchiolar epithelium, and other cells of the interstitutium being negative. PSP-B mRNA is present in both alveolar type II cells and in some cells of the bronchiolar epithelium. Macrophages and other cells were negative. The data in this report demonstrate that: (1) type II pneumonocytes are capable of synthesizing both PSP-A and PSP-B, (2) some cells of the human bronchiolar epithelium contain PSP-B mRNA but not PSP-A, and (3) human alveolar macrophages do not synthesize either PSP-A or PSP-B.