Role of N-glycosylation of surfactant protein SP-BN in lipid and SP-B interacting properties. Implications in disease

Abstract

SP-BN is an independent protein derived from the precursor of pulmonary surfactant protein B (SP-B), a critical component of the pulmonary surfactant (PS), the membrane-based system that coats the alveolar air-liquid interface and is essential for both respiratory mechanics and innate defense. In humans, a single-nucleotide polymorphism (SNP) defining hSP-BN glycosylation has been associated with propensity to certain respiratory diseases, but molecular studies in this regard are scarce. Previous studies with the murine SP-BN, nonglycosylated, have suggested a role for this protein in lipid transfer during PS biogenesis. This study focuses on the structural and functional characterization of both glycosylated and nonglycosylated human SP-BN protein variants to elucidate the impact of N-glycosylation. Recombinant proteins (hSP-BN, glycosylated, and hSP-BN-T73I, nonglycosylated) were produced in Pichia pastoris and purified to homogeneity. The structural characterization confirmed the main features of hSP-BN as a member of the SAPLIP protein family: mainly α-helical, a propensity to dimerization and a high stability. Interestingly, N-glycosylation did not significantly affect hSP-BN structure. Regarding lipid interactions, both hSP-BN variants were able to bind and perturb membranes in lipid vesicles with a PS-like composition at acidic, but not neutral pH, which is relevant given the acidification during PS biogenesis. Remarkably, N-glycosylation impaired the synergistic effect of hSP-BN and mature SP-B to promote lipid mixing/transfer activity. These results support the joint action of both proteins in PS biogenesis and, more importantly, suggest that this combined activity affected with the SNP-induced glycosylation of hSP-BN could be behind certain PS defects acquired during biogenesis causing some susceptibility to respiratory diseases.

NEW & NOTEWORTHY The impact of N-glycosylation on the structure and function of human SP-BN protein has been studied. Homogeneous production of glycosylated hSP-BN and nonglycosylated hSP-BN-T73I was achieved in Pichia pastoris. Structural characterization and lipid interaction properties at acidic pH revealed no significant differences due to glycosylation. N-glycosylation impairs the synergistic action of hSP-BN and SP-B in lipid transfer/mixing activity. N-glycosylation of hSP-BN could impair PS biogenesis, in agreement with its potential involvement in respiratory disease.