Disulfide bonds in the SAPA domain of the pulmonary surfactant protein B precursor

The disulfide bonds formed in the SAPA domain of a recombinant version of the NH₂-terminal propeptide (SP-B_N) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through sequential digestion of SP-B_N with GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cys³²-Cys⁴⁹ and Cys⁴⁰-Cys⁵⁵, and we propose a disulfide connectivity pattern of 1-3 and 2-4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z ∼ 2136 and ∼ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptides ²⁴AWTTSSLACAQGPE³⁷ and ⁴⁵QALQCR⁵⁰ linked by Cys³²-Cys⁴⁹ and ³⁸FWCQSLE⁴⁴ and ⁵¹ALGHCLQE⁵⁸ linked by Cys⁴⁰-Cys⁵⁵ respectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1-3/2-4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-B_N propeptide, the MS analysis of both digests identified the bond Cys¹⁰⁰-Cys¹¹², numbered 7-8, which is coincident with the bond position in the kringle motif. SIGNIFICANCE: The SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.