Rescue of SP-B Knockout Mice with a Truncated SP-B Proprotein

Abstract

Pulmonary surfactant is a complex mixture of lipids and proteins that is synthesized exclusively by the alveolar type II epithelial cell. Surfactant is stored in large inclusions (lamellar bodies) that are secreted into the alveolar airspace by exocytosis. Newly secreted lamellar bodies unravel into a tubular network (tubular myelin) that subsequently adsorbs and spreads as a phospholipid-rich film that reduces surface tension at the air-liquid interface. Rapid adsorption and spreading of the phospholipid film are critical and require the presence of specific proteins, in particular surfactant proteins B and C (1-3).Surfactant protein B (SP-B) 1 is a hydrophobic peptide of 79 amino acids that avidly associates with surfactant phospholipids in the alveolar airspace (reviewed in Ref. 4). Homozygous mutations leading to the complete absence of SP-B in newborn human infants result in the rapid onset of respiratory distress syndrome which is refractory to mechanical ventilation and surfactant replacement (5, 6). The latter observation suggests that simple addition of mature peptide to the SP-B-deficient airway is not sufficient to restore lung function. Therefore, despite an obvious requirement for SP-B in normal lung function, the precise role(s) of SP-B in the structure, function, and metabolism of surfactant remains unclear.Human SP-B is synthesized by the type II epithelial cell as a preproprotein of 381 amino acids. The mature peptide is generated by sequential cleavage of the signal peptide (23 amino acids), the N-terminal propeptide (177 residues), and the Cterminal propeptide (102 residues) (7, 8). Propeptide cleavage occurs within the late endosome/multivesicular body which subsequently directs newly synthesized SP-B to the lamellar body (9); mature SP-B is also recycled from the alveolar airspace, via the endocytic pathway to the multivesicular body (10). Previous in vitro evidence supports the concept that trafficking of the mature peptide through the biosynthetic pathway requires the N-terminal but not the C-terminal domain of the propeptide (11,12). In the absence of both propeptide domains the mature peptide is degraded within the endoplasmic reticulum (11); however, an SP-B construct encoding both the Nterminal propeptide and the mature peptide (hSP-B ⌬c ) produces a truncated proprotein that is sorted to the multivesicular body where the propeptide is appropriately cleaved to liberate mature SP-B (12). These results clearly demonstrate that the N-terminal propeptide is required for the intracellular trafficking of SP-B; the function of the C-terminal propeptide, however, remains unknown.Lung morphogenesis and surfactant phospholipid synthesis in SP-B(Ϫ/Ϫ) mice proceed normally prior to birth (13). However, lamellar body formation is disrupted, resulting in abnormal inclusions consisting of multivesicular bodies and disorganized lamellae. Neither mature lamellar bodies nor tubular myelin were detected in the SP-B(Ϫ/Ϫ) mice. In addition to effects on lamellar body biogenesis, SP-B deficiency resulted in

Surfactant proteins in pediatric interstitial lung disease

“…SP-B, a dimer of 16 kDa, was detected easily in all cases investigated except for the single case of hereditary SP-B deficiency. Together with the finding of lacking SP-B in all other reported cases of this genetic disorder (7,17,18,(24)(25)(26) and the successful rescue of SP-B-deficient knockout mice (27), the analysis of BALF for SP-B concentration appears diagnostically helpful when pediatric DPLD potentially including this rare condition are investigated. In this context, it must be recommended to sample BAL before exogenous surfactant is applied, as the latter usually contains both SP-B and SP-C whose in vivo half-lives may be very long (28)(29)(30).…”

Prolonged Survival in Hereditary Surfactant Protein B (SP-B) Deficiency Associated with a Novel Splicing Mutation

“…Mature SP-B corresponds to the middle (34,35). Genetically engineered mice that expressed only SP-B constructs that lacked sequences encoding the proSP-B carboxy terminal domains produced mature SP-B but had perturbed proSP-C processing and abnormally large LB, suggesting a role for the carboxy terminal region in LB biosynthesis and SP-C proprotein processing (36). However, SP-C metabolism was abnormal in the children we describe despite the presence of the aberrant protein, suggesting that the carboxy terminal region of the abnormal peptide was nonfunctional or that the reduced level of mature SP-B protein was also important in altered proSP-C processing.…”

Increased and Prolonged Pulmonary Fibrosis in Surfactant Protein C-Deficient Mice Following Intratracheal Bleomycin

“…Supernatants were then analyzed for the four surfactant proteins, SP-A, SP-B, SP-C, and SP-D. Lavage content of SP-B and SP-D was measured by specific enzyme-linked immunosorbent assay (ELISA) as previously reported. [22][23][24] Western blots were performed for SP-A and the mature form of SP-C using polyclonal antibodies as previously described. 17 Densitometry was performed using Gelworks 1D Advanced V3.01 software (Ultra-Violet Products, Cambridge, UK) and the individual band density was normalized to a positive control run on each gel.…”