Surfactant protein D binding to terminal alpha1-3-linked fucose residues and to Schistosoma mansoni.

Pulmonary surfactant protein (SP)-D is an important component of the innate immune system of the lung, which is thought to function by binding to specific carbohydrates on the surface of viruses and unicellular pathogens. SP-D has been shown to have a relatively high affinity for the monosaccharides mannose, glucose, and fucose. However, there is limited information on SP-D binding to complex carbohydrate structures, and binding of SP-D to fucose in the context of an oligosaccharide has not yet been investigated. In this study, we used surface plasmon resonance spectroscopy to examine the potential of SP-D to bind to various synthetic fucosylated oligosaccharides, and identified Fucalpha1-3GalNAc and Fucalpha1-3GlcNAc elements as strong ligands. These types of fucosylated glycoconjugates are presented at the surface of Schistosoma mansoni, a parasitic worm that, during development, transiently resides in the lung. In line with the findings by surface plasmon resonance, we found that SP-D can bind to larval stages of S. mansoni, demonstrating for the first time that SP-D interacts with multicellular lung pathogens.

Inhibition of phosphatidylcholine synthesis is not the primary pathway in hexadecylphosphocholine-induced apoptosis.

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.

Collectins: players of the innate immune system.

Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. They bind preferentially to monosaccharide units of the mannose type, which present two vicinal hydroxyl groups in an equatorial position. High-affinity interactions between collectins and microorganisms depend, on the one hand, on the high density of the carbohydrate ligands on the microbial surface, and on the other, on the degree of oligomerization of the collectin. Apart from binding to microorganisms, the collectins can interact with receptors on host cells. Binding of collectins to microorganisms may facilitate microbial clearance through aggregation, complement activation, opsonization and activation of phagocytosis, and inhibition of microbial growth. In addition, the collectins can modulate inflammatory and allergic responses, affect apoptotic cell clearance and modulate the adaptive immune system.

Interactions of influenza A virus with sialic acids present on porcine surfactant protein D.

Pigs can be infected with both human and avian influenza A virus (IAV) strains and are therefore considered to be important intermediates in the emergence of new IAV strains due to mixing of viral genes derived from human, avian, or porcine influenza viruses. These reassortant strains may have potential to cause pandemic influenza outbreaks in humans. The innate immune response against IAV plays a significant role in containment of IAV in the airways. We studied the interactions of IAV with porcine surfactant protein D (pSP-D), an important component of this first line defense system. Hemagglutination inhibition analysis shows that the distinct interactions of pSP-D with IAV mediated by the N-linked carbohydrate moiety in the carbohydrate recognition domain of pSP-D depend on the terminal sialic acids (SAs) present on this carbohydrate. Analysis by both lectin staining and by cleavage with linkage-specific sialidases shows that the carbohydrate of pSP-D is exclusively sialylated with alpha(2,6)-linked SAs, in contrast to surfactant protein A, which contains both alpha(2,3)- and alpha(2,6)-linked SAs on its N-linked carbohydrate. Enzymatic modification of the SA-linkages present on pSP-D demonstrates that the type of SA-linkage is important for its hemagglutination-inhibitory activity, and correlates with receptor-binding specificity of the IAV strains. The SAs present on pSP-D appear especially important for interactions with poorly glycosylated IAV strains. It remains to be elucidated to what extent the unique sialylation profile of pSP-D is involved in host range control of IAV in pigs, and whether it facilitates adaptation of avian or human IAV strains that can contribute to the production of reassortant strains in pigs.

Porcine pulmonary collectins show distinct interactions with influenza A viruses: role of the N-linked oligosaccharides in the carbohydrate recognition domain.

Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant strains; this may result in the appearance of new pandemic IAV strains in humans. We have studied the role of the porcine lung collectins surfactant proteins D and A (pSP-D and pSP-A), two important components of the innate immune response against IAV. Hemagglutination inhibition assays revealed that both pSP-D and pSP-A display substantially greater inhibitory activity against IAV strains isolated from human, swine, and horse, than lung collectins from other animal species. The more potent activity of pSP-D results from interactions mediated by the asparagine-linked oligosaccharide located in the carbohydrate recognition domain of pSP-D, which is absent in SP-Ds from other species characterized to date. Presence of this sialylated oligosaccharide moiety enhances the anti-influenza activity of pSP-D, as demonstrated by assays of viral aggregation, inhibition of infectivity, and neutrophil response to IAV. The greater hemagglutination inhibitory activity of pSP-A is due to porcine-specific structural features of the conserved asparagine-linked oligosaccharide in the carbohydrate recognition domain of SP-A. A more efficient lung collectin-mediated immune response against IAV in pigs may play a role in providing conditions by which pigs can act as "mixing vessel" hosts that can lead to the production of reassortant, pandemic strains of IAV.

The transmembrane domain of surfactant protein C precursor determines the morphology of the induced membrane compartment in CHO cells.

Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.

Inhibition of phosphatidylcholine synthesis induces expression of the endoplasmic reticulum stress and apoptosis-related protein CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153).

Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40 degrees C resulted in PC depletion via an inhibition of CT within 24 h. Early apoptotic features appeared in several cells around 30 h, and most cells were apoptotic within 48 h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16 h, to a maximum at 24 h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40 degrees C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40 degrees C. Furthermore, heat-shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress-related protein CHOP, without raising a general ER stress response.

Effects of cholesterol on surface activity and surface topography of spread surfactant films.

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.

Palmitoylation and processing of the lipopeptide surfactant protein C.

Pulmonary surfactant, a mixture of lipids and proteins, reduces the surface tension at the air-water interface of the lung alveoli by forming a surface active film. This way, it prevents alveoli from collapsing and facilitates the work of breathing. Surfactant protein C (SP-C) plays an important role in this surfactant function. SP-C is expressed as a proprotein (proSP-C), which becomes posttranslationally modified with palmitate and undergoes several rounds of proteolytical cleavage. This results in the formation of mature SP-C, which is stored in the lamellar bodies (LB) and finally secreted into the alveolar space. Recently, new insights into the sorting, processing and palmitoylation of proSP-C have been obtained by mutagenesis studies. Moreover, reports on the association of development of lung disease with SP-C deficiency have led to new insights into the importance of SP-C for proper surfactant homeostasis. In addition, new information has become available on the role of the palmitoyl chains of SP-C in surface activity. This review summarizes these recent developments in the processing and function of SP-C, with particular emphasis on the signals for and role of palmitoylation of SP-C.

Porcine surfactant protein D is N-glycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers.

Surfactant protein D (SP-D) belongs to a subgroup of mammalian collagenous Ca(2+)-dependent lectins known as the collectins. It is thought to play a significant role in the innate immune response against microorganisms within the lungs and at other mucosal surfaces. This report documents the isolation and characterization of SP-D purified from porcine lung lavage using mannan affinity chromatography and gel filtration. Ultrastructural analysis shows both dodecameric and higher order oligomeric complexes of SP-D. The molecular mass of monomeric porcine SP-D (50 kD) is larger than that of SP-D from humans (43 kD). The difference in mass is due to the presence of an Asparagine-linked glycosylation in the carbohydrate recognition domain of porcine SP-D, which is absent in SP-D of other species investigated so far. Analysis of this carbohydrate moiety indicates that it is a highly heterogeneous, complex type oligosaccharide which is sialylated. The heterogeneity of oligosaccharide sialylation results in the existence of many differently charged porcine SP-D isoforms. The removal of the carbohydrate moiety reduces the inhibitory effect of porcine SP-D on influenza A virus haemagglutination. Therefore, the carbohydrate moiety may influence interactions with pathogens.

Multilayer formation upon compression of surfactant monolayers depends on protein concentration as well as lipid composition. An atomic force microscopy study.

The determinants for the formation of multilayers upon compression of surfactant monolayers were investigated by compressing films, beyond the squeeze-out plateau, to a surface tension of 22 millinewtons/m. Atomic force microscopy was used to visualize the topography of lipid films containing varying amounts of native surfactant protein B (SP-B). These films were compared with films containing synthetic peptides based on the N terminus of human SP-B: monomeric mSP-B-(1-25) or dimeric dSP-B-(1-25). The formation of typical hexagonal network structures as well as the height of protrusions were shown to depend on the concentration of SP-B. Protrusions of bilayer height were formed from physiologically relevant concentrations of 0.2-0.4 mol % (4.5-8.5 wt %) SP-B upwards. Much higher concentrations of SP-B-(1-25) peptides were needed to obtain network structures, and protrusion heights were not equal to those found for films with native SP-B. A striking observation was that while protrusions formed in films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DPPG) (80/20) had single bilayer thickness, those formed in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (80/20) had various heights of multilayers, whereas those seen in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/DPPG (60/20/20) were mainly of bilayer height. For the first time direct observations by atomic force microscopy show (i) that a certain minimal concentration of SP-B is required for the formation of layered protrusions upon film compression, (ii) that protrusion height depends on whether the phospholipids contain an unsaturated fatty acyl chain, and (iii) that protrusion height also depends on whether the unsaturated acyl chain is present in phosphatidylcholine or in phosphatidylglycerol.

Differential effect of brefeldin A on the palmitoylation of surfactant protein C proprotein mutants.

The surfactant protein C precursor (proSP-C) is palmitoylated on two cysteines adjacent to its transmembrane domain. We showed previously that palmitoylation of proSP-C occurs in a postendoplasmic reticulum compartment and is not affected by the Golgi-disturbing agent brefeldin A (BFA). In contrast, the investigations presented here showed that BFA almost completely abolished palmitoylation of proSP-C mutants that contained alterations in the region between the palmitoylated cysteines and the transmembrane domain, including a Pro 30 to Leu mutant associated with interstitial lung disease. This differential effect of BFA was not caused by differences in the palmitoylation kinetics between wild-type proSP-C and the mutants and was not mimicked by nocodazole and monensin. However, differences between the mutants and wild-type proSP-C in the relative degree of processing suggest that BFA may unmask a difference in routing. This would imply that the amino acids just N-terminal of the transmembrane domain may be important for a proper sorting of proSP-C.

Structural requirements for palmitoylation of surfactant protein C precursor.

Pulmonary surfactant protein C (SP-C) propeptide (proSP-C) is a type II transmembrane protein that is palmitoylated on two cysteines adjacent to its transmembrane domain. To study the structural requirements for palmitoylation of proSP-C, His-tagged human proSP-C and mutant forms were expressed in Chinese hamster ovary cells and analysed by metabolic labelling with [3H]palmitate. Mutations were made in the amino acid sequence representing mature SP-C, as deletion of the N- and C-terminal propeptide parts showed that this sequence by itself could already be palmitoylated. Substitution of the transmembrane domain by an artificial transmembrane domain had no effect on palmitoylation. However, an inverse correlation was found between palmitoylation of proSP-C and the number of amino acids present between the cysteines and the transmembrane domain. Moreover, substitution by alanines of amino acids localized on the N-terminal side of the cysteines had drastic effects on palmitoylation, probably as a result of the removal of hydrophobic amino acids. These data, together with the observation that substitution by alanines of the amino acids localized between the cysteines and the transmembrane domain had no effect on palmitoylation, suggest that the palmitoylation of proSP-C depends not on specific sequence motifs, but more on the probability that the cysteine is in the vicinity of the membrane surface. This is probably determined not only by the number of amino acids between the cysteines and the transmembrane domain, but also by the hydrophobic interaction of the N-terminus with the membrane. This may also be the case for the palmitoylation of other transmembrane proteins.