Proteins SP-D Research Articles

Surfactant-associated proteins: functions and structural variation

Pulmonary surfactant is a barrier material of the lungs and has a dual role: firstly, as a true surfactant, lowering the surface tension; and secondly, participating in innate immune defence of the lung and possibly other mucosal surfaces. Surfactant is composed of approximately 90% lipids and 10% proteins. There are four surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, SP-C and SP-D. Although the sequences and post-translational modifications of SP-B and SP-C are quite conserved between mammalian species, variations exist. The hydrophilic surfactant proteins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins, known as collectins, consisting of oligomers of trimeric subunits. In view of the different roles of surfactant proteins, studies determining the structure-function relationships of surfactant proteins across the animal kingdom will be very interesting. Such studies may reveal structural elements of the proteins required for surface film dynamics as well as those required for innate immune defence. Since SP-A and SP-D are also present in extrapulmonary tissues, the hydrophobic surfactant proteins SP-B and SP-C may be the most appropriate indicators for the evolutionary origin of surfactant. SP-B is essential for air-breathing in mammals and is therefore largely conserved. Yet, because of its unique structure and its localization in the lung but not in extrapulmonary tissues, SP-C may be the most important indicator for the evolutionary origin of surfactant.

The roles of surfactant proteins A and D in innate immunity.

Research over the last decade on the surfactant proteins SP-A and SP-D suggests roles beyond surfactant lipid homeostasis, involving their participation in innate immune defence. SP-A and SP-D bind and agglutinate an impressive array of non-self structures, ranging from bacteria and fungi to allergens and environmental inorganic substrates. Complementing binding. SP-A and SP-D initiate and enhance immune cell ingestion and killing of targets. Recently, some exciting developments have extended and clarified their contributions to innate immunity. Knockout mice for SP-A and SP-D have been developed. The SP-A knockout confirms that SP-A plays a key role in defence against lung pathogens and reveals the underlying defense mechanisms that require SP-A. These surfactant proteins have also been shown to have important roles in modulating the immune response, instructing, yet quenching, the immune reactions in the lung. The crystal structure of SP-D plus functional studies with recombinantly altered forms of SP-A and SP-D has begun to characterise the structural motifs responsible for mediating their immune functions. Linkage and polymorphism analysis is explaining the role these genes may play in lung diseases and infection.

Collectins and pulmonary innate immunity.

The surfactant-associated proteins SP-A and SP-D are members of a family of host defense lectins, designated collectins. There is increasing evidence that these pulmonary, epithelial-derived proteins are important components of the innate immune response to microbial challenge and participate in other aspects of immune and inflammatory regulation within the lung. Both proteins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms, and to certain organic particles, such as pollens. SP-A and SP-D have the capacity to modulate leukocyte function and, in some circumstances; to opsonize and enhance the killing of microorganisms. The biologic activity of cell wall components, such as Gram-negative bacterial polysaccharides, or viral glycoproteins, such as the hemagglutinin of influenza viruses, may be altered by interactions with collectins. In addition, complementary or cooperative interactions between SP-A, SP-D and other host defense lectins could contribute to the efficiency of this defense system. Collectins could play particularly important roles in settings of inadequate or impaired specific immunity, and acquired alterations in the levels of active collectins within the airspaces and distal airways may increase susceptibility to infection.

Genes of Surfactant Proteins SP-A, SP-B and SP-D are Expressed in Eustachian Tube

Genes of Surfactant Proteins SP-A, SP-B and SP-D are Expressed in Eustachian Tube

A novel method of purifying lung surfactant proteins A and D from the lung lavage of alveolar proteinosis patients and from pooled amniotic fluid

A simple procedure has been developed for the purification of the surfactant proteins SP-A and SP-D from lung lavage of patients with alveolar proteinosis. The SP-D is purified by fractionation of the supernatant obtained after spinning the lavage at 10 000× g for 40 min, while the bulk of the SP-A is purified by fractionation of the pellet. The supernatant is applied to a maltosyl–agarose column and the bound SP-D is specifically eluted using MnCl 2. The pellet is solubilised in 6 M urea and, following renaturation, the solubilised proteins are applied to maltosyl–agarose and SP-A eluted using a gradient of EDTA. Both SP-A and SP-D are further purified by gel-filtration on Superose-6. This procedure has also been used to prepare successfully human SP-A and SP-D from amniotic fluid and may be generally applicable to the isolation of these surfactant proteins from lung washings obtained from other species.

Functional Roles of the Lung Surfactant Proteins SP-A and SP-D in Innate Immunity

The surfactant proteins SP-A and SP-D are known to play important roles in innate immunity in the lungs in the protection against a wide variety of potential pathogens, which include viruses, bacteria and fungi. Recognition of the pathogens is, in most cases, mediated by the Ca +2-dependent binding of the C-type lectin domains of SP-A, or SP-D, to carbohydrate structures on the surface of the microorganisms. This can cause agglutination of the pathogens and also possibly their enhanced killing and clearance by phagocytic cells which carry receptors for SP-A or SP-D. Both SP-A and SP-D are found in a number of other sites in the body, in various secretions, and therefore may play a wider protective role than was initially envisaged.

Collectins and pulmonary host defense.

The surfactant-associated proteins SP-A and SP-D are members of a family of collagenous host defense lectins, designated collectins. There is increasing evidence that these pulmonary epithelial-derived proteins are important components of the innate immune response to microbial challenge, and that they participate in other aspects of immune and inflammatory regulation within the lung. The collectins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms and certain other organic particles in vitro. Although binding may facilitate microbial clearance through aggregation or other direct effects on the organism, SP-A and SP-D also have the capacity to modulate leukocyte function and, in some circumstances, to enhance their killing of microorganisms. The biologic activity of cell wall components, such as gram-negative bacterial polysaccharides, may be altered by interactions with collectins. Complementary or cooperative interactions between SP-A and SP-D could contribute to the efficiency of this defense system. Collectins may play particularly important roles in settings of inadequate or impaired specific immunity. Acquired or genetic alterations in the levels of active proteins within the airspaces and distal airways may increase susceptibility to infection.

Surfactant Protein and CC-10 Expression in Acute Lung Injury and in Response to Keratinocyte Growth Factor

The alveolar epithelium is a critical element in response to acute lung injury. Recently, the surfactant proteins SP-A and SP-D have been found to be elevated in the serum of patients with pulmonary fibrosis and diffuse type II cell hyperplasia. It is possible that the expression of these two proteins is regulated in part by inflammatory cytokines.

Lipid analysis and surfactant-associated protein expression in lung adenocarcinoma cells from pleural effusion.

Primary lung adenocarcinomas originate from the progenitor cells of peripheral airway cells. Alveolar type II cells and Clara cells are the major progenitor cells of peripheral airway cells. Alveolar type II cells produce a lipid-protein complex called surfactant, which contains surfactant proteins SP-A, SP-B, SP-C and SP-D. Phosphatidylcholine (PC) and phosphatidylglycerol (PG) are believed to be essential for the surfactant function. Clara cells also express SP-A, SP-B and SP-D but not SP-C. In this study we examined the properties of the cancer cells isolated from the pleural effusion of a patient with primary lung adenocarcinoma by analyzing lipids, proteins and mRNAs. The cancer cells, designated as LC117 cells, were isolated from the pleural effusion of a patient with primary lung adenocarcinoma. The percent distributions of [14C]-acetate incorporated into PC and PG in the cancer cells were 55.7 and 1.1%, respectively. The disaturated species in total PC was 46.2%. Immunoblotting analysis using anti-SP-D monoclonal antibody revealed that the pleural effusion from a patient with lung adenocarcinoma contained SP-D. We determined the concentrations of SP-A and SP-D by enzyme-linked immunosorbent assay. The pleural effusions from this patient and the media incubated with cancer cells exhibited significant levels of SP-D as well as SP-A. Reverse transcriptase-polymerase chain reaction demonstrated that the tumor cells expressed mRNAs for SP-C as well as the other surfactant proteins. The results demonstrate that tumor cells from lung adenocarcinoma express all of surfactant-associated proteins, indicating that LC117 cells originate from alveolar type II cells. This study indicates that the combination of analyses of lipids, proteins and mRNAs in the cancer cells isolated from pleural effusion is useful to understand the property of lung adenocarcinoma.

Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells

Cryptococcus neoformans is an opportunistic pathogen in AIDS patients causing disseminated disease and lethal meningitis after inhalation of acapsular or sparsely encapsulated yeast cells. In this study we have investigated whether a recently described family of primitive opsonins, termed collectins, contribute to innate resistance against C. neoformans. The pulmonary surfactant proteins SP-A and SP-D as well as the serum collectins mannose-binding protein and CL-43 bound in a calcium-dependent manner to acapsular C. neoformans in vitro. Binding was concentration dependent and abolished by competition with defined mono- and oligosaccharides. In contrast, no binding of the collectins was observed with the encapsulated form of the yeast. Furthermore, binding of purified collectin SP-D, but not SP-A, mannose-binding protein, or CL-43, led to a concentration-dependent agglutination of acapsular C. neoformans. These data indicate that collectins recognize carbohydrate structures in the cell wall of an initial infectious form of C. neoformans and may play a role in early antifungal defenses in the lung.

Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro

Pulmonary surfactant is synthesized and secreted into the airspaces by the alveolar type II cell. After it is secreted, surfactant undergoes a series of poorly understood transformations resulting in formation of a surface tension-reducing surface at the air-liquid interface. The by-products of the surface film and/or other products of surfactant metabolism are eventually cleared from the alveolar space. Both the alveolar type II cell and the macrophage are thought to be involved in surfactant clearance and have been shown to internalize surfactant lipid in vitro. The goal of the current investigation was to characterize further and to quantitate the role of the macrophage in surfactant clearance by investigating the uptake and metabolism of surfactant lipids and surfactant protein A (SP-A) by macrophages in vitro. SP-A enhanced the uptake of lipids by macrophages in a time-, temperature-, and concentration-dependent manner. In contrast, neither of the collagen-like proteins SP-D or C1q enhanced the uptake. Phosphatidylcholine was rapidly degraded by macrophages and the degradation occurred both in the presence and absence of SP-A. In addition, macrophages degrade SP-A by a process that is time- and temperature-dependent. These results and calculations of uptake and degradation rates suggest that macrophages may contribute significantly to the process of surfactant clearance.

Potential role of surfactant proteins A and D in innate lung defense against pathogens.

The physiological role of pulmonary surfactant is probably not limited to conferring mechanical stability to the alveoli. Increasing evidence suggests that surfactant components, in particular the hydrophilic surfactant proteins SP-A and SP-D, play potentially important roles in host defense mechanisms. Both SP-A and SP-D are collagenous C-type lectins (collectins) that are structurally and, perhaps, functionally related to collectins in the circulation. As will be discussed in this review, evidence is accumulating that the alveolar collectins SP-A and SP-D could be important components of a first-line defense system against infiltrating pathogenic micro-organisms and viruses.

A different combination of transcription factors modulates the expression of the human transferrin promoter in liver and Sertoli cells.

We have previously identified the functional regions involved in the regulation of human transferrin (Tf) gene expression in the liver and in Sertoli cells of the testis. Here, we show that a different cellular distribution of transcription factors, interacting with the same proximal promoter regions (PRI and PRII), modulates cell type-specific transcription. In the liver, hepatocyte nuclear factor 4 (HNF-4) and the chicken ovalbumin upstream promoter transcription factor (COUP-TF) act at the PRI site, while CCAAT/enhancer-binding proteins (C/EBPs) act at the PRII site. In the testis, distinct combinations of Sertoli proteins SP-A and SP-D and COUP-TF bind to the PRI site, while SP-alpha and SP-beta bind to the PRII site. Cotransfection experiments in Hep3B cells revealed that mostly HNF-4, C/EBP-alpha, C/EBP-delta, and, to a lesser extent, COUP-TF stimulated transcription driven by the -125/+39 region. In Sertoli cells, HNF-4 and COUP-TF appeared to repress, while the C/EBP factors were able to stimulate transcription driven by the -100/+39 region. However, the specific activating combination remains to be defined among the Sertoli proteins. In the non-Tf-expressing HeLa cells, the Tf promoter could be activated by C/EBP-delta. Our data revealed functional antagonism between HNF-4 and COUP-TF, binding to PRI, as well as cross-coupling interactions between HNF-4 and C/EBP, binding to adjacent sites. Thus, cell type-specific DNA-protein interactions, together with protein-protein interactions, may explain the transcriptional regulation of the Tf gene in different cell types.

Binding specificity of lung surfactant protein SP-D for glucosylceramide

The specificities of the binding of lung surfactant protein SP-D to glycolipids were examined using 125I-labeled SP-D as a probe. When the binding study was performed on TLC plates, SP-D bound exclusively to GlcCer, whereas it failed to bind to GalCer, G M1, G M2, asialo-G M1, asialo-G M2, sulfatide, Forssman antigen, ceramide dihexoside, ceramide trihexoside, globoside, paragloboside or ceramide. Excess native SP-D competed with 125I-SP-D for the binding to GlcCer. Antibody to rat SP-D inhibited 125I-SP-D binding to GlcCer. Ca 2+ was absolutely required for the binding of SP-D to GlcCer; Mg 2+ failed to substitute for Ca 2+. SP-D bound to ceramide monohexoside in glycolipids isolated from rat lung and bronchoalveolar lavage fluids of rats.

Dietary regimen and performance of high intensity ergometer exercise.

The influence of preceding diet (mixed, MD; carbohydrate CD; protein PD) on performance during high intensity endurance cycling was examined in six middle distance runners. Subjects undertook cycle ergometer exercise at a workload equivalent to 80% VO2 max until exhaustion following each of the three dietary regimens. Dietary analyses were performed using a computerised evaluation technique and cardiorespiratory, blood glucose and lactate responses to exercise were measured along with exercise time to exhaustion. Significant differences in carbohydrate and protein intakes were noted between respective diets as well as significantly higher total energy intake in MD (P less than 0.05). A significant relationship was observed between carbohydrate intake and exercise time to exhaustion (r = 0.59, P less than 0.05). No significant differences were noted in cardiorespiratory measures or blood glucose response after exhaustive exercise between the three dietary regimens but peak blood lactate concentration was lower following PD (P less than 0.05). Total time to exhaustion was significantly higher on CD (1070.0 +/- 106.7 s) than on PD (642.5 +/- 84.3 s, P less than 0.01). Performance time on MD (895.7 +/- 84.3 s) did not differ significantly for performance time on either CD or PD. It was concluded that dietary manipulation significantly improves exercise time to exhaustion during short term, high intensity cycling.