Ablation of a critical surfactant protein B intramolecular disulfide bond in transgenic mice

The 79-amino acid, mature SP-B peptide contains three intramolecular disulf ide bonds shared by all saposin-like proteins. This study tested the hypoth esis that the disulfide bond formed between cysteine residues 35 and 46 (re sidues 235 and 246 of the SP-B proprotein) is essential for proper function of SP-B, To test the role of this bridge in SP-B function in vivo, a const ruct was generated in which cysteine residues 235 and 246 of the human SP-B proprotein were mutated to serine and cloned under the control of the 3.7- kilobase hSP-C promoter (hSP-B-C235S/C246). In two transgenic mouse lines, expression of the mutant peptide in the wild-type murine SP-B background wa s invariably lethal in the neonatal period. In four additional lines, survi val was inversely related to the level of transgene expression. To test the ability of the mutant peptide to functionally replace the wild type protei n, transgenic mice were crossed into the SP-B null background. No animals t hat expressed hSP-B-C235S/C246S in the murine SP-B-/- background survived t he neonatal period. hSP-B-C235S/C246S proprotein accumulated in the endopla smic reticulum and was not processed to the mature, biologically active pep tide, The results of these studies demonstrate that the intramolecular brid ge between residues 235 and 246 is critical for intracellular trafficking o f SP-B and suggest that overexpression of mutant SP-B in the wild-type back ground may be lethal.