Impact of N-linked glycans on the dual short fibulin/LTBP-4 axes regulating elastogenesis

Elastic fibers are key extracellular components, providing elasticity to blood vessels, lungs, skin, and bladder. Elastic fiber formation requires the accessory proteins fibulin-4, fibulin-5, and the long and short isoforms of the latent TGFβ binding protein-4 (LTBP-4L/S). We established two molecular axes, LTBP-4L/fibulin-4 and LTBP-4S/fibulin-5, defined similar and distinct functions, and determined the role of N-linked glycans in this context. Glycoproteomic analysis identified the specific N-linked glycans in these proteins. Biophysical analyses revealed that the N-linked glycans of LTBP-4L, but not fibulin-4, were critical for fibulin-4-mediated conformational extension of LTBP-4L, impacting its function and assembly. Biochemical and recombinant removal of N-linked glycans from fibulin-4 enhanced its interaction with tropoelastin and elastic fiber formation, indicating an inhibitory role for these N-glycans. Fibulin-5 strongly interacted with and robustly induced a conformational extension of LTBP-4S, leading to enhanced binding to fibronectin, increased LTBP-4S deposition, and doubling of elastic fiber formation. Loss of N-linked glycans from fibulin-5, but not LTBP-4S, reduced their interaction by about 10-fold and abolished the ability of fibulin-5 to extend LTBP-4S conformationally. The presence of fibulin-5-extended LTBP-4S did not trigger tropoelastin aggregation in an in vitro assembly assay but boosted elastic fiber-like assembly massively when fibulin-4 and LTBP-4L were additionally present, suggesting synergistic effects. N-linked glycans in fibulin-5 were essential in this process. The study uncovers novel mechanisms that regulate elastic fiber formation, including overlapping and distinct roles of the LTBP-4L/fibulin-4 and the LTBP-4S/fibulin-5 axes and the importance of N-linked glycans of each of these proteins.