Post-transcriptional regulation of Surfactant Protein B expression

Abstract

Though a minor constituent by weight, surfactant protein B (SP-B) plays a major role in surfactant function. It is the unique structure of SP-B that promotes permeabilization, cross-linking, mixing, and fusion of phospholipids, facilitating the proper structure and function of pulmonary surfactant as well as contributing to the formation of lamellar bodies. SP-B production is a complex process within alveolar type 2 cells, and is under hormonal and developmental control. Understanding the post-translational events in the maturation of SP-B may provide new insight into the process of lamellar body formation and into the pathophysiology of pulmonary disorders associated with surfactant abnormalities.

Alveolar patency is critical to gas exchange in the lung. To support the gas exchange function of the lung, the alveolar walls exhibit a conservation of cellularity ¹. The epithelial surfaces, lined by thin, expansive type I epithelial cells and surfactant-producing type II cells, are separated by minimal connective tissue, providing little structural support to the alveolus. Instead, inflated distal airspaces support each other by their interconnectedness. Alveolar lining fluid is essential to protect the alveolar epithelium from dehydration during the respiratory cycle, and fetal lung fluid plays an important role in lung growth and development. However, even a thin fluid layer within the alveolus presents a surface tension problem in the distal lung. The surface tension of the alveolar lining fluid or of fetal lung fluid is a force sufficient to overcome the tensile strength of the alveolar walls, thereby promoting alveolar collapse and opposing inflation.

Together, the phospholipids, neutral lipids and proteins of pulmonary surfactant at the alveolar air-liquid interface to reduce surface tension ². The amphipathic nature of phospholipids in pulmonary surfactant mitigate the surface tension at the air-liquid interface ³. Though a minor constituent by weight, surfactant protein B (SP-B) plays a major role in surfactant function. SP-B is a small, hydrophobic protein that commonly exists as a homodimer ^{4, 5}. The 79 amino acid monomer consists of 5 alpha helices that play important roles in the fusion and lysis of phospholipid mono/bilayers ⁶. Although synthetic peptides based on helices 1 and 2 exhibit biophysical activity, it is likely that optimal function is achieved by the complete SP-B structure. It is the unique structure of SP-B that promotes permeabilization, cross-linking, mixing, and fusion of phospholipids. These “fusogenic” properties of SP-B contribute to phospholipid adsorption at the air-liquid interface and reduction in alveolar surface tension.

Absence or deficiency of SP-B, due to developmental, genetic, or acquired disease processes, is associated with high surface tension in the alveolar space, resulting in atelectasis even in the presence of sufficient alveolar phospholipid levels ⁷. In animal models, selective inactivation of SP-B from instilled monoclonal antibodies ⁸, in response to acute lung injury ⁹, or upon conditional inactivation of SP-B gene expression in transgenic mice ¹⁰ results in poor surface activity and loss of alveolar stability. The observation that correcting phospholipid content of surfactant obtained from human patients with ARDS ex vivo without correcting SP-B levels did not restore surfactant function highlights the importance of SP-B ¹¹. However, the significance of SP-B fusogenic properties is not limited to alveolar surface tension. SP-B deficiency, in vivo or in vitro, is associated with abnormal type 2 cell ultrastructure due to the abnormal development of lamellar bodies, the lysosome-related structures that store and release surfactant, within type 2 cells ^12–16.

Lamellar bodies arise in part from transformation of multivesicular bodies within alveolar type 2 cells ¹⁷. Multivesicular bodies are membrane-limited organelles with small internal membranous vesicles formed by invagination of the limiting membrane. The predominance of MVB and absence of lamellar bodies in SP-B deficiency suggest that SP-B promotes the lysis of these small internal vesicles, which then become the first phospholipid layers of the lamellar body. It is unclear whether administration of surfactant preparations containing SP-B to patients or animals with SP-B deficiency can correct this poorly understood intracellular process of lamellar body genesis.

SP-B production is a complex process within alveolar type 2 cells, and is under hormonal and developmental control. Understanding the post-translational events in the maturation of SP-B may provide new insight into the process of lamellar body formation and into the pathophysiology of pulmonary disorders associated with surfactant abnormalities. Mature SP-B is the result of extensive modification and proteolysis of a 381 amino acid proSP-B precursor. This large proprotein is folded, glycosylated, and transferred through the secretory pathway of the type 2 cell, during which several proteolytic events occur sequentially to release the bioactive, mature protein. The discussion that follows will highlight post-translational events in SP-B processing and trafficking, emphasizing in the conclusion that the regulation of these events is as important in contributing to the expression of SP-B as transcriptional events in gene expression.

PROTEIN FOLDING

ProSP-B shares structural homology with the saposin-like protein family, SAPLIP ¹⁸. Like the saposins, proSP-B exhibits the regular repeat of 6 cysteines that contributes to the arrangement of 3 intramolecular disulfide bonds. ProSP-B contains three SAPLIP domains, with mature SP-B protein residing in the second domain ¹⁹. The importance of intramolecular disulfide bonds was established when the SP-B deficient phenotype in mice could not be rescued when one of these bonds (between Cys235 and Cys246 within mature SP-B) was disrupted ²⁰. Abnormally folded proSP-B accumulated in type 2 cells and did not undergo further proteolytic processing, leading to SP-B deficiency

GLYCOSYLATION

Human proSP-B has 2 sites for glycosylation, at Asn¹²⁹ and Asn³¹¹, within the amino-and carboxyl-terminal saposin-like domains; neither is within the mature SP-B protein sequence. Altered proSP-B glycosylation has been implicated in human disease. A polymorphism of the aminoterminal glycosylation consensus sequence, Thr131Ile, confers glycosylation at Asn¹²⁹. The Thr/Thr genotype associated with ARDS, more strongly when ARDS was due to direct (pneumonia) rather than indirect (trauma) lung injury ²¹. The Thr/Thr genotype has also been associated with infant RDS, and in a family with congenital alveolar proteinosis ²². The Thr/Thr proSP-B protein is expressed as a glycosylated proSP-B while the Ile/Ile proSP-B is unglycosylated in lung tissue ²³. It is as yet unclear what mechanism could explain increased susceptibility to surfactant deficiency or dysfunction from altered glycosylation of proSP-B.

PROTEOLYSIS

There are multiple proteolytic cleavage events in the metabolism of proSP-B to the mature SP-B monomer. PreproSP-B contains a signal peptide that is cleaved within the endoplasmic reticulum resulting in proSP-B. Three enzymes have been identified to participate in proSP-B proteolysis: Napsin A, Cathepsin H, and Pepsinogen C. Like prosaposin proteolysis ²⁴, the 3 saposin-like domains of proSP-B are separated sequentially through the action of endopeptidases, with bulk separation of the aminoterminus followed by bulk cleavage of the carboxylterminus ²⁵. These bulk cleavages of proSP-B leave small residual peptides on the amino- and carboxyltermini of mature SP-B, which are then removed through exopeptidase activity. Although the amino- and carboxylterminal sequences are removed in bulk, it is unclear whether they function independently in the type 2 cell or in the alveolar space.

Napsin A, or kidney-derived aspartyl protease, was first described as a product of renal tubular cells ²⁶. Characterization of protein expression led to the observation that this protease was also expressed in the lung, specifically in alveolar type 2 cells ²⁷. Napsin A can remove the aminoterminus of proSP-B in vitro, although there is some discrepancy as to whether Napsin A can remove the carboxylterminus of proSP-B ²⁸²⁸, ²⁹. The aminoterminal Napsin A cleavage site in proSP-B is between L¹⁷⁸ and P¹⁷⁹, leaving 21 amino acids attached to mature SP-B. More importantly, RNAi experiments showed that interference with Napsin A production resulted in decreased production of mature SP-B ²⁹.

Pepsinogen C is another aspartyl protease found in alveolar type 2 cells ³⁰, and the evidence suggests that it participates in bulk removal of the carboxylterminus of proSP-B 31. The possibility that two separate aspartyl proteases could be involved in proSP-B proteolysis has presented challenges to discriminate between the actions of Napsin A and Pepsinogen C. In vitro proteolysis using purified Pepsinogen C and recombinant human proSP-B demonstrated two specific proteolytic sites: between Lys¹⁹⁶-Ser¹⁹⁷ of the aminoterminus of proSP-B, and between Met³⁰²-Ser³⁰³ of the carboxylterminus. Effective silencing of Pepsinogen C expression using RNAi during in vitro differentiation of type 2 cells decreased the production of mature SP-B, further reinforcing its role in proSP-B processing.

The first protease to be associated with proSP-B processing, Cathepsin H, is likely responsible for the exopeptidase trimming of both the N- and C-termini prior to release of the mature SP-B protein ^{12, 29}. Cathepsin H is a cysteine protease for which there are several inhibitors of high specificity ³². Inhibition of Cathepsin H action, either by protease inhibitors or RNAi, also results in decreased production of mature SP-B ^{12, 29, 33}.

TRAFFICKING

Like many secreted proteins, proSP-B begins in the lumen of the endoplasmic reticulum. Glycosylation studies have shown that proSP-B exits the endoplasmic reticulum and moves to the Golgi intact. Modification of carbohydrates in the cisGolgi by mannosidases renders them resistant to carbohydrate removal by endoglycosidase H. We have observed that some proSP-B is endoglycosidase H-sensitive and some endo H-resistant ³⁴. The 25 kDa intermediate of proSP-B processing is similarly endo H-resistant. Since carbohydrate modification occurs in the cisGolgi, this also suggests that the removal of the aminoterminus of proSP-B resulting in the 25 kDa intermediate protein occurs beyond the cisGolgi.

Inhibitors of subcellular trafficking have further elucidated the path of proSP-B through the alveolar type 2 cell. Brefeldin A results in the collapse of proximal Golgi compartments toward the ER, effectively blocking movement of proteins beyond the cisGolgi. Alveolar type 2 cells treated with Brefeldin A do not process proSP-B to smaller forms, further evidence that proteolytic steps occur beyond the cisGolgi ³⁴. Monensin, a carboxylic ionophore that induces progressive swelling of the Golgi, blocks movement of proteins beyond the Golgi ³⁵. Alveolar type 2 cells treated with monensin produced proSP-B, 25 kDa intermediate and 9-12 kDa intermediates of proSP-B, but no mature SP-B. Thus, bulk separation of the amino- and carboxyl-termini proSP-B occur within Golgi compartments after maturation of carbohydrates of proSP-B, leaving the exopeptidase cleavages by Cathepsin H as post-Golgi events.

Whether proSP-B is chaperoned through the secretory pathway toward the lamellar body is unclear. ProSP-B has no transmembrane domain that would allow for interactions with traditional cytoplasmic chaperones. However, the tight membrane association of mature SP-B suggests that proSP-B and intermediates may be able to associate with transmembrane proteins in the limiting membrane of the secretory pathway. Regardless of mechanism, it is clear that after leaving the transGolgi, proSP-B intermediates enter the multivesicular body where proteolytic processing is completed, since inhibition of Cathepsin H action interferes with lamellar body formation and leads to increased numbers of MVB ³².

DIMERIZATION

Unlike other SAPLIP family members, the second domain of proSP-B contains an extra cysteine (Cys²⁴⁸ of proSP-B which is Cys⁴⁸ of mature SP-B) that contributes to SP-B homodimer formation. The physiologic importance of dimerization was challenged when mutation of Cys²⁴⁸ was shown to enable rescue of the SP-B deficient phenotype in transgenic mice ³⁶. However, in vitro analysis of monomeric SP-B indicated that monomers have poor activity at low concentrations. At high concentration, monomers associated into non-covalent dimers that functioned more normally. Furthermore, mice expressing monomeric SP-B appear to have altered pulmonary function studies despite being born alive with well-formed lamellar bodies. Together, these findings indicate that dimerization is necessary for the optimal function of alveolar SP-B.

REGULATION OF SP-B EXPRESSION VIA POST-TRANSLATIONAL EVENTS

The complex post-translational process of SP-B production serves more than one function. First, it allows for SP-B to become fully active in the right place within type 2 cells. As suggested previously ⁷, the fusogenic properties of mature SP-B appear to be important for the transformation of small vesicles in multivesicular bodies into the plate-like structures of lamellar bodies, although this has not been confirmed experimentally. Activation of lytic properties of SP-B elsewhere in the type 2 cell would likely be detrimental to the membranes surrounding subcellular organelles. Second, complex post-translational processing allows for SP-B production at the right time. Regulation of protease expression, for example, may allow for fine-tuning the timing of SP-B expression. SP-B RNA can be detected in developing human lung as early as 12 weeks gestation ^{37, 38} and proSP-B is found in second trimester lung ²⁵. However, mature SP-B protein is not readily detectable until the third trimester of human gestation despite SP-B RNA levels that are near 50% of adult lung. By comparison, Pepsinogen C is not detectable in second trimester human fetal lung, at the RNA or protein level ³⁰. The regulation of protease expression may restrict mature SP-B production to the third trimester. Thus, complex post-translational processing of proSP-B allows for SP-B expression in the right place at the right time in the developing lung, contributing a burst of surfactant production in anticipation of the transition to air breathing at birth. The critical role of SP-B in the biogenesis of lamellar bodies and in the proper function of alveolar surfactant suggests that alterations in post-translational events, due to genetic polymorphisms, pathologic states, or drug interactions, may have adverse effects on SP-B expression that contribute to lung disease.