Quantitative proteomic identification of host factors involved in the Salmonella typhimurium infection cycle.

To identify host factors involved in Salmonella replication, SILAC-based quantitative proteomics was used to investigate the interactions of Salmonella typhimurium with the secretory pathway in human epithelial cells. Protein profiles of Golgi-enriched fractions isolated from S. typhimurium-infected cells were compared with those of mock-infected cells, revealing significant depletion or enrichment of 105 proteins. Proteins annotated to play a role in membrane traffic were overrepresented among the depleted proteins whereas proteins annotated to the cytoskeleton showed a diverse behavior with some proteins being enriched, others being depleted from the Golgi fraction upon Salmonella infection. To study the functional relevance of identified proteins in the Salmonella infection cycle, small interfering RNA (siRNA) experiments were performed. siRNA-mediated depletion of a selection of affected proteins identified five host factors involved in Salmonella infection. Depletion of peroxiredoxin-6 (PRDX6), isoform ß-4c of integrin ß-4 (ITGB4), isoform 1 of protein lap2 (erbin interacting protein; ERBB2IP), stomatin (STOM) or TBC domain containing protein 10b (TBC1D10B) resulted in increased Salmonella replication. Surprisingly, in addition to the effect on Salmonella replication, depletion of STOM or ITGB4 resulted in a dispersal of intracellular Salmonella microcolonies. It can be concluded that by using SILAC-based quantitative proteomics we were able to identify novel host cell proteins involved in the complex interplay between Salmonella and epithelial cells.

Identification of host factors involved in coronavirus replication by quantitative proteomics analysis.

In this study, we applied a quantitative proteomic approach, based on SILAC, to investigate the interactions of coronaviruses with the secretory pathway of the host cell, with the aim to identify host factors involved in coronavirus replication. Comparison of the protein profiles of Golgi-enriched fractions of cells that were either mock infected or infected with mouse hepatitis virus revealed the significant depletion or enrichment of 116 proteins. Although ribosomal/nucleic acid binding proteins were enriched in the Golgi-fractions of mouse hepatitis virus-infected cells, proteins annotated to localize to several organelles of the secretory pathway were overrepresented among the proteins that were depleted from these fractions upon infection. We hypothesized that proteins, of which the abundance or distribution is affected by infection, are likely to be involved in the virus life cycle. Indeed, depletion of a small subset of the affected proteins by using small interfering RNAs identified several host factors involved in coronavirus infection. Transfection of small interfering RNAs targeting either C11orf59 or Golgi apparatus glycoprotein 1 resulted in increased virus replication, whereas depletion of vesicle-trafficking protein vesicle-trafficking protein sec22b enhanced the release of infectious progeny virus. Overexpression of these proteins, on the other hand, had a negative effect on virus replication. Overall, our study shows that the SILAC approach is a suitable tool to study host-pathogen interactions and to identify host proteins involved in virus replication.

Lipids in host-pathogen interactions: pathogens exploit the complexity of the host cell lipidome.

Lipids were long believed to have a structural role in biomembranes and a role in energy storage utilizing cellular lipid droplets and plasma lipoproteins. Research over the last decades has identified an additional role of lipids in cellular signaling, membrane microdomain organization and dynamics, and membrane trafficking. These properties make lipids an attractive target for pathogens to modulate host cell processes in order to allow their survival and replication. In this review we will summarize the often ingenious strategies of pathogens to modify the lipid homeostasis of host cells, allowing them to divert cellular processes. To this end pathogens take full advantage of the complexity of the lipidome. The examples are categorized in generalized and emerging principles describing the involvement of lipids in host-pathogen interactions. Several pathogens are described that simultaneously induce multiple changes in the host cell signaling and trafficking mechanisms. Elucidation of these pathogen-induced changes may have important implications for drug development. The emergence of high-throughput lipidomic techniques will allow the description of changes of the host cell lipidome at the level of individual molecular lipid species and the identification of lipid biomarkers.

Surfactant protein D/anti-Fc receptor bifunctional proteins as a tool to enhance host defence.

BACKGROUND: Drug-resistant pathogens are an increasing threat, particularly for hospitalised patients. In search of a new approach in pathogen targeting, we developed bifunctional proteins that combine broad spectrum pathogen recognition with efficient targeting to phagocytes. Pathogen recognition is provided by a recombinant fragment of surfactant protein D (rfSP-D) while targeting to phagocytic cells is accomplished by coupling rfSP-D to F(ab') fragments directed against Fcalpha receptor I (FcalphaRI) or Fcgamma receptor I (FcgammaRI). FcalphaRI and FcgammaRI are expressed on myeloid cells, and induce rapid internalisation of particles after crosslinking. OBJECTIVE/METHODS: In this review we discuss the roles of SP-D and Fc receptors in host defence as a rationale for rfSP-D/anti-FcR bifunctional proteins. Furthermore we summarise the available data on rfSP-D/anti-FcR proteins as well as opportunities and considerations for future use of such bifunctional proteins. RESULTS/CONCLUSION: rfSP-D/anti-FcR bifunctional proteins could be of great value for the treatment of a variety of infectious diseases. The focus in the near future should be on proof-of-principle by testing the bifunctional proteins in different mouse models of infectious disease.

Monocyte CD64 or CD89 targeting by surfactant protein D/anti-Fc receptor mediates bacterial uptake.

We recently showed that a chimeric protein, consisting of a recombinant fragment of human surfactant protein D (rfSP-D) coupled to a Fab' fragment directed against the human Fcalpha receptor (CD89), effectively targets pathogens recognized by SP-D to human neutrophils. The present study evaluates the effectiveness of chimeric rfSP-D/anti-Fc receptor proteins targeting Escherichia coli to CD89 or to the Fcgamma receptor I (CD64) on monocytes. Both chimeric rfSP-D/anti-Fc receptor proteins increased internalization of E. coli by the human promonocytic cell line U937, but only after induction of monocytic differentiation, despite the fact that the expression levels of CD64 and CD89 on undifferentiated cells were at least as high as on differentiated cells. The two chimeric rfSP-D/anti-Fc receptor proteins did not enhance each other's effect on E. coli uptake. Targeting to differentiated U937 cells was inhibited by blocking the interaction either between the rfSP-D part of the chimeric molecule and E. coli, or between the anti-Fc receptor Fab' fragment and the Fc receptor on the U937 cell. In conclusion, both CD64 and CD89 on U937 cells prove to be suitable for targeting by rfSP-D/anti-Fc receptor proteins. However, in addition to mere Fc receptor expression, effective targeting requires monocytic differentiation.

Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus.

Oxidants and neutrophils contribute to lung injury during influenza A virus (IAV) infection. Surfactant protein (SP)-D plays a pivotal role in restricting IAV replication and inflammation in the first several days after infection. Despite its potent anti-inflammatory effects in vivo, preincubation of IAV with SP-D in vitro strongly increases neutrophil respiratory burst responses to the virus. Several factors are shown to modify this apparent proinflammatory effect of SP-D. Although multimeric forms of SP-D show dose-dependent augmentation of respiratory burst responses, trimeric, single-arm forms either show no effect or inhibit these responses. Furthermore, if neutrophils are preincubated with multimeric SP-D before IAV is added, oxidant responses to the virus are significantly reduced. The ability of SP-D to increase neutrophil uptake of IAV can be dissociated from enhancement of oxidant responses. Finally, several other innate immune proteins that bind to SP-D and/or IAV (i.e., SP-A, lung glycoprotein-340 or mucin) significantly reduce the ability of SP-D to promote neutrophil oxidant response. As a result, the net effect of bronchoalveolar lavage fluids is to increase neutrophil uptake of IAV while reducing the respiratory burst response to virus.

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.

Effective targeting of pathogens to neutrophils via chimeric surfactant protein D/anti-CD89 protein.

Targeting of specific pathogens to FcRs on immune effector cells by using bispecific Abs was reported to result in effective killing of the pathogens, both in vitro and in vivo. Instead of targeting a specific pathogen to an FcR, we assessed whether a broad spectrum of pathogens can be targeted to an FcR using surfactant protein D (SP-D). SP-D is a collectin that binds a great variety of pathogens via its carbohydrate recognition domain. A recombinant trimeric fragment of SP-D (rfSP-D), consisting of the carbohydrate recognition domain and neck domain of human SP-D, was chemically cross-linked to the Fab' of an Ab directed against the human Fc alpha RI (CD89). In vitro, the chimeric rfSP-D/anti-CD89 protein enhanced uptake of Escherichia coli, Candida albicans, and influenza A virus by human neutrophils. Blocking of the interaction between rfSP-D/anti-CD89 and either the pathogen or CD89 abolished its stimulatory effect on pathogen uptake by neutrophils. In addition, rfSP-D/anti-CD89 stimulated killing of E. coli and C. albicans by neutrophils and enhanced neutrophil activation by influenza A virus. In conclusion, rfSP-D/anti-CD89 effectively targeted three structurally unrelated pathogens to neutrophils. (Col)lectin-based chimeric proteins may thus offer promise for therapy of infectious disease.

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.

Rab3D and actin reveal distinct lamellar body subpopulations in alveolar epithelial type II cells.

Rab3D is a small GTP-binding protein associated with secretory vesicles in various exocrine and endocrine cells, where it has been implicated in regulated exocytosis. Data obtained previously in pancreas have suggested that rab3D is involved in the coating of secretory granules with filamentous actin. In the present study we employed Western blot analysis, immunofluorescence, and immunoelectron microscopy to examine the distribution of rab3D in rat lung. Rab3D immunoreactivity was detected in bronchiolar Clara cells and alveolar epithelial type II (AET-II) cells. In both cell types, rab3D displayed preferential localization to secretory vesicles that were identified using specific antibodies against Clara Cell Secretory Protein and p180 lamellar body protein, respectively. Interestingly, rab3D was associated with only 24% of the lamellar bodies in AET-II cells. Rab3D-positive lamellar bodies were typically in close proximity of the apical plasma membrane, where exocytosis occurs. Another subpopulation of lamellar bodies, constituting only 2%, was not only rab3D-positive but could also be labeled with the filamentous-actin probe phalloidin. A third subpopulation, constituting 9%, displayed actin coating without rab3D staining. We propose that these three lamellar body subpopulations represent consecutive intermediates along the regulated exocytotic pathway, implying that rab3D release and actin coating are intimately linked processes.

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.

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.

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.

Involvement of cathepsin H in the processing of the hydrophobic surfactant-associated protein C in type II pneumocytes.

Surfactant protein C (SP-C) is synthesized by type II pneumocytes as a 21-kD propeptide (proSP-C) which is proteolytically processed to a 4.2-kD dipalmitoylated protein. To characterize the processing of proSP-C and the role of the cysteine protease cathepsin H, we studied the localization of proSP-C and cathepsin H in human as well as proSP-C in rat lungs, the enzymatic cathepsin H activity in isolated rat lamellar bodies, and the cleavage of human proSP-C by purified cathepsin H. Using antisera directed against the N-terminal E(11)-R(23) (NPROSP-C(11-23)), the C-terminal G(162)-G(174) domain (CPROSP-C(162-174)) of proSP-C, and against cathepsin H, immunogold labeling identified all three in electron-dense multivesicular bodies, but only NPROSP-C(11-23) and cathepsin H in composite as well as lamellar bodies of type II pneumocytes. Immuno double-labeling further distinguished electron-dense vesicles containing cathepsin H or electron light vesicles/multivesicular bodies containing proSP-C. Isolated lamellar bodies contained enzymatically active cathepsin H, a 6-kD proSP-C processing intermediate detected only by NPROSP-C(11-23), and mature SP-C. Using enzyme activities comparable to those in isolated lamellar bodies, purified cathepsin H generated a partially N-terminal processed proSP-C intermediate in vitro. In conclusion, our results indicate that after the fusion of electron-dense vesicles containing cathepsin H and electron-light vesicles or multivesicular bodies containing proSP-C, cathepsin H is involved in the first N-terminal processing step of proSP-C in electron-dense multivesicular bodies of type II pneumocytes.

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.

SP-A-enriched surfactant for treatment of rat lung transplants with SP-A deficiency after storage and reperfusion.

BACKGROUND: The function of pulmonary surfactant is affected by lung transplantation, contributing to impaired lung transplant function. A decreased amount of surfactant protein-A (SP-A) after reperfusion is believed to contribute to the impaired surfactant function. Surfactant treatment has been shown to improve lung transplant function, but the effect is variable. We investigated whether SP-A enrichment of surfactant improved the efficacy of surfactant treatment in lung transplantation. METHODS: Left and right lungs of Lewis rats, inflated with 50% O2, were stored for 20 hr at 8 degrees C. Surfactant in bronchoalveolar lavage fluid from right lungs was investigated after storage (n=6). Left lungs were transplanted into syngeneic recipients and treated with SP-A-deficient surfactant (n=6) or SP-A-enriched surfactant (n=6) just before reperfusion. Air was instilled into untreated lung transplants (n=6). Sham operated (n=4) and normal (n=8) animals served as controls. Lung function was measured during 1 hr of reperfusion; surfactant components in bronchoalveolar lavage fluid were measured after reperfusion. RESULTS: After storage the amount of SP-A decreased by 27%, whereas surfactant phospholipids changed minimally. After reperfusion a further decrease of SP-A was paralleled by profound changes in surfactant phospholipids. Lung transplant function, however, remained relatively good. After instillation of SP-A-enriched surfactant, PO2 values were reached that approximated sham control PO2 values, whereas after SP-A-deficient surfactant treatment, the PO2 values did not improve. CONCLUSION: Enrichment of surfactant with SP-A for treatment of lung transplants improves the efficacy of surfactant treatment.

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.

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.