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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.

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.

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 bioactivity in 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.

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.

The accompanying paper [Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L302-L310, 1991] showed that in the radiation pneumonitis model of adult respiratory distress syndrome (ARDS) there was an excess of the proximate, higher buoyant density subtypes of alveolar surfactant, and a decrease in the light buoyant density form. Because the surfactant subtypes normally evolve from the former to the latter a delay in the alveolar metabolism of surfactant could explain this disproportion. Three possible mechanisms of a delay in surfactant metabolism in radiation pneumonitis were explored using an in vitro model of surfactant subtype metabolism called "cycling". The first was that the surfactant of mice with radiation pneumonitis was intrinsically less capable of conversion to the light subtype. It was found, however, that the proximate forms of surfactant of mice with radiation pneumonitis were as capable of generating light subtype as those of control mice. The second was that there was a deficit in the serine protease activity, called "convertase", that mediates the conversion. But it was found that lungs of mice with radiation pneumonitis released convertase activity to the same extent as control lungs. The third was that an inhibitor of convertase activity was present in the alveoli. It was found that the alveolar lavage fluid of mice with radiation pneumonitis inhibited the conversion of exogenous surfactant by exogenous convertase. Moreover, it contained an 18-fold excess of antiprotease activity. The present data are interpreted as suggesting that an inhibitor in the alveolar space is responsible for the delay in surfactant subtype metabolism in radiation pneumonitis, resulting in the disproportion of surfactant subtypes in radiation pneumonitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary surfactant as lavaged from the alveoli exists in at least three structural subtypes, lamellar body-like, tubular myelin and vesicular forms that can be separated on the basis of their buoyant densities. Previous studies have suggested that surfactant is secreted in the lamellar body form and metabolized through the other subtypes in sequence. This metabolic sequence can be reproduced in vitro by cyclic expansion and contraction ('cycling') of the surface area of nascent surfactant at 38 degrees C. Cycling of nascent secretion, which is predominantly of lamellar body-like buoyant density, rapidly converted it to the buoyant density of tubular myelin and then to that of the vesicular subtype. We examined the role of proteinases in the conversion of nascent surfactant subtypes in vitro. Addition of metallo-, cysteine- and acid-proteinase inhibitors to the cycling mix did not inhibit the conversion of tubular myelin to vesicular subtype. However, a variety of serine proteinase inhibitors inhibited the formation of vesicular subtype. Their inhibitory effect was dose-related and most marked for alpha 1-antitrypsin where a concentration equal to that found in the alveolar fluid lining layer resulted in 50% inhibition of the generation of light subtype, suggesting physiological relevance. The enzyme(s) responsible for promoting the generation of light subtype was sedimentable and therefore presumably in particulate form. By differential centrifugation of lung secretions it was separable from alveolar macrophages and partially separable from surfactant itself. It has not been identified, nor has its substrate. We conclude that in vitro cycling provides a model for the study of alveolar surfactant metabolism and that the conversion of tubular myelin to vesicular forms of surfactant requires serine proteinase activity.