Surfactant Convertase Research Articles

HDL, heart disease, and the lung

HDL, heart disease, and the lung

Contribution of HMSE-1 to surfactant conversion under acute inflammatory conditions

A reduced content of biophysically active large surfactant aggregates is a common finding in acute inflammatory lung disease. Cyclic surface area changes and a carboxylesterase activity (surfactant convertase) are thought to mediate this subtype conversion. However, data concerning regulation of surfactant convertase are scarce. We therefore investigated the expression and activity of lung surfactant convertase and HMSE-1, a potential macrophage-derived human convertase, under normal and acute inflammatory conditions.

Liver carboxylesterase cleaves surfactant protein (SP-) B and promotes surfactant subtype conversion

Conversion of the biophysically active large surfactant aggregate subtype of alveolar surfactant into the less surface active small surfactant aggregates occurs in vitro and in vivo, possibly in dependency of a carboxylesterase, entitled surfactant convertase. The substrate has yet not been safely identified. Utilizing the in vitro cycling assay we investigated conversion of an organic rabbit lavage extract reconstituted with SP-A. Porcine liver carboxylesterase, which is closely related to surfactant convertase, induced subtype conversion to a similar degree as compared with native lavage fluid containing endogenous convertase. In addition, we asked for cleavage products of SP-B and identified a ∼12 kDa band upon cycling with liver carboxylesterase, having the same N-terminus as mature SP-B. A band of same molecular weight was found in native lavage fluid after in vitro conversion mediated by the endogenous convertase. We conclude that SP-B plays a pivotal role during subtype conversion and represents the substrate for surfactant convertase.

Inhalation of α1-Protease Inhibitor in Cystic Fibrosis does not affect Surfactant Convertase and Surface Activity

The inhalation of α1-protease inhibitor (α1-PI) was assessed in a pilot study to restore the protease-antiprotease balance in the lungs of cystic fibrosis (CF) patients. In addition, the effect of this treatment on the surface active properties of lung surfactant and the metabolic conversion of aggregate forms was studied. Eight young adults with CF inhaled 100 mg of α1-PI twice daily over 8 weeks and bronchoalveolar lavages (BAL) were obtained before and 12 h after the last inhalation. Large aggregate (LA) forms of surfactant were isolated from the in vivo material by ultracentrifugation and their conversion into small aggregates (SA) was assessed by an in vitro surface area cycling assay. Although α1-PI partially restored the protease–anti-protease imbalance and reduced BAL protein content, no effects were noted on the impaired minimal surface tension and on the in vivo and in vitro conversion of LA to SA. Antiserum against the specific carboxyl esterase ES-2, previously identified in mice and rats as the putative surfactant convertase, did not detect a protein of the appropriate size in CF BAL. Whereas short-term inhalation of α1-PI was beneficial for the proteolytic aspects of CF lung injury, this appeared not to be the case for surfactant conversion and surface activity.

Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions.

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222-230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L404-L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.

Molecular cloning, characterization, and differential expression pattern of mouse lung surfactant convertase.

We recently reported the purification and partial amino acid sequence of "surfactant convertase," a 72-kDa glycoprotein involved in the extracellular metabolism of lung surfactant (S. Krishnasamy, N. J. Gross, A. L. Teng, R. M. Schultz, and R. Dhand. Biochem. Biophys. Res. Commun. 235: 180-184, 1997). We report here the isolation of a cDNA clone encoding putative convertase from a mouse lung cDNA library. The cDNA spans a 1,836-bp sequence, with an open reading frame encoding 536 amino acid residues in the mature protein and an 18-amino acid signal peptide at the NH2 terminus. The deduced amino acid sequence matches the four partial amino acid sequences (68 residues) that were previously obtained from the purified protein. The deduced amino acid sequence contains an 18-amino acid residue signal peptide, a serine active site consensus sequence, a histidine consensus sequence, five potential N-linked glycosylation sites, and a COOH-terminal secretory-type sequence His-Thr-Glu-His-Lys. Primer-extension analysis revealed that transcription starts 29 nucleotides upstream from the start codon. Northern blot analysis of RNA isolated from various mouse organs showed that convertase is expressed in lung, kidney, and liver as a 1,800-nucleotide-long transcript. The nucleotide and amino acid sequences of putative convertase are 98% homologous with mouse liver carboxylesterase. It thus may be the first member of the carboxylesterase family (EC 3.1.1.1) to be expressed in lung parenchyma and the first with a known physiological function.

Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant.

Surfactant convertase is required for conversion of heavy density (H) natural surfactant to light density (L) subtype during cycling in vitro, a technique that reproduces surfactant metabolism. To study mechanisms of H to L conversion, we prepared liposomes of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), or the phospholipids (PL) in combination with either surfactant protein A (SP-A), surfactant protein B (SP-B), or both SP-A and SP-B. Phospholipids alone showed time-dependent conversion from heavy to light subtype on cycling in the absence of convertase, which was decreased by adding SP-B, but not SP-A, to phospholipids (p < 0.01 for PL+SP-B, or PL+SP-A+SP-B vs. PL, or PL+SP-A). The ultrastructure, surface activity, buoyant density, and L subtype generation on cycling PL+SP-A+SP-B with partially purified convertase or with phospholipase D were similar to those of natural TM. In conclusion, a reconstituted surfactant mimics the behavior of natural surfactant on cycling, and reveals that interaction of SP-B with phospholipids decreases L subtype generation. In addition, esterase/ phospholipase D activity is required for conversion of heavy to light subtype on cycling.

Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase.

In the alveolar lumen, pulmonary surfactant converts from the contents of secreted lamellar bodies to tubular myelin to apoprotein-depleted vesicles during respiration. Using an in vitro system, researchers have reported that the conversion of tubular myelin to vesicles is blocked by inhibitors of serine hydrolase activity and have tentatively ascribed "convertase" activity to a diisopropyl fluorophosphate (DFP)-binding protein in mouse bronchoalveolar lavage (BAL). We purified and sequenced the homologous enzyme from rat BAL fluid. Amino acid sequence from the amino terminus and an internal cyanogen bromide peptide of the purified rat DFP-binding protein perfectly match the sequence of the carboxylesterase ES-2. Although ES-2 was initially cloned from liver, we found a 1.8-kilobase mRNA for ES-2 in decreasing relative abundance in rat liver, kidney, and lung but not in heart or spleen. Although further studies are required to establish the identity between "convertase" and ES-2 or a homologous member of the carboxylesterase family, our results raise the possibility that a protein with esterase/lipase activity plays a role in extracellular surfactant metabolism.

Surfactant convertase action is not essential for surfactant film formation.

A serine-active enzyme, "surfactant convertase", is required for the conversion of surfactant from the tubular myelin (TM) form to the small vesicular (SV) form. This transformation involves at least two steps, the conversion of TM to a surface-active film at the air-fluid interface and the reorientation of the film into the surface-inactive SV form; we asked if convertase was required for the first of these steps. Rat and mouse TMs were pretreated with diisopropyl fluorophosphate (DFP) to inactivate endogenous convertase activity or with vehicle and then were analyzed for their ability to lower surface tension in vitro as an index of the conversion of TM to a surface film. DFP pretreatment did not alter the ability of TM preparations to lower surface tension, as assessed by pulsating bubble, and it did not affect the behavior of TM in a surface balance. In an experiment designed to test the ability of TM to feed a surface film to exhaustion, TMs that had been pretreated with DFP or vehicle performed similarly. These experiments show that convertase activity is not required for the conversion of TM to a monolayer and suggest, instead, that convertase acts at a post surface film stage.

Lung “Surfactant Convertase” Is a Member of the Carboxylesterase Family

The extracellular conversion of lung surfactant from tubular myelin to the small vesicular form has previously been shown to require a serine-active enzyme called “surfactant convertase.” In the present study, a 72kD serine-active enzyme previously identified in mouse lung alveolar lavage and having convertase activity was partially sequenced. Sixty-eight residues obtained from amino acid sequencing of this protein show that it is a new member of the mouse carboxylesterase family (EC 3.1.1.1). The 72kD lung protein also has esterase activity. A commercial esterase of the same family was able to reproduce surfactant convertase bioactivityin vitro,unlike several serine proteinases previously tested. We conclude that surfactant convertase is a carboxylesterase which mediates a biochemical step in the extracellular metabolism of surfactant.

Extracellular metabolism of pulmonary surfactant: the role of a new serine protease.

Lung surfactant, an organized complex of lipids and specific apoproteins that is responsible for maintaining alveolar stability, is a product of alveolar epi­ thelial type II cells in which it is packaged for secretion in the form of lamellar bodies (LB) (41, 43, 44). Following its release from type II cells into the alveolar fluid-lining layer, surfactant undergoes a number of structural and metabolic changes. Ultimately, some of the extracellular surfactant is taken back into type II cells for recycling, and the remainder is probably removed by alveolar clearance mechanisms. The structural transformation that surfac­ tant undergoes after its secretion from the type II cell includes at least one step requiring the action of a recently discovered enzyme. We review the evidence for enzymatic activity in this sequence, what is known of its role and actions, as well as some of the properties of the putative enzyme, which we call surfactant convertase.

The role of alpha 1-antitrypsin in the control of extracellular surfactant metabolism

Alveolar surfactant exists in several structural forms that are believed to be secular products of the secreted form, lamellar bodies. The conversion of tubular myelin to the small vesicular form appears to require the action of a novel serine protease, surfactant convertase. As the in vitro activity of this enzyme is quite sensitive to inhibition by the serine protease inhibitor alpha 1-antitrypsin, a normal constituent of the alveolar fluid lining layer, we explored the possibility that the alveolar level of alpha 1-antitrypsin might affect the rate of subtype conversion in vivo. When the alveolar alpha 1-antitrypsin level was augmented by tracheal instillation of exogenous alpha 1-antitrypsin, the newly synthesized surfactant phospholipids of spontaneously breathing mice accumulated in the heavy subtype, and virtually none was found in the light subtype form up to 72 h later. An in vivo turnover study suggested that when the alveolar alpha 1-antitrypsin level was raised by the same means, the flux of surfactant through the light (small vesicular) compartment was virtually shut off, despite the availability of abundant amounts of its precursor, heavy subtype (tubular myelin). Finally, mouse lungs that were lavaged to remove resident surfactant and mechanically ventilated for 1-5 h released surfactant that progressed through heavy and light subtype compartments as in vivo. But in mice whose lung alpha 1-antitrypsin might affect the metabolism of alveolar surfactant in addition to its well-known role in lung defense.

Requirements for extracellular metabolism of pulmonary surfactant: tentative identification of serine protease.

Pulmonary alveolar surfactant is secreted by the alveolar epithelium in the form of lamellar bodylike structures that evolve sequentially into tubular myelin and vesicular forms that can be separated by centrifugation. Using an in vitro procedure by which the extracellular metabolism of pulmonary surfactant can be mimicked, namely cyclic variation in surface area, we previously reported that serine protease activity, which we called "convertase," was required for the conversion of tubular myelin to the vesicular form. In the present studies we explored the biochemical requirements of this activity and sought the enzyme in alveolar products. Convertase activity has unusual requirements; in addition to being dependent on repetitive variations in surface area (cycling), it requires the presence of a high g fraction of lung secretions that is heat stable and not inhibitable by diisopropyl fluorophosphate (DFP) or alpha 1-antitrypsin, both typical serine protease inhibitors. The enzyme does not require calcium ions and has a pH optimum of 7.4. Convertase appears to be a component of surfactant itself because the ability of purified surfactant to convert to the vesicular form on cycling is impaired by pretreating it with DFP. A protein of Mr 75,000 that reacts with DFP and is heat sensitive was found in alveolar lavage, lamellar body preparations, and lung homogenate. It copurifies with lung surfactant in sucrose gradients. A similar DFP-reactive protein was observed in stable human neoplastic peripheral airway cell lines that express type II properties, suggesting that it may be a product of type II cells. We tentatively conclude that surfactant convertase is a 75,000 serine protease that is closely associated with surfactant phospholipid and that may be a product of alveolar type II cells.