Site-specific analysis and functional characterization of N-linked glycosylation for β-Klotho protein

β-Klotho (KLB), a type I transmembrane protein, serves as an obligate co-receptor determining the tissue-specific actions of endocrine fibroblast growth factors (FGFs). Despite accumulative evidence suggesting the occurrence of N-glycosylation in the KLB protein, the precise N-glycosites, glycoforms, and the impacts of N-glycosylation on the expression and function of the KLB protein remain unexplored. Employing a mass spectrometry-based approach, a total of 12 N-glycosites displaying heterogeneous site occupancy and glycoforms were identified within the extracellular region of the recombinant human KLB protein. Molecular simulation revealed negligible impact of these N-glycans on the overall structure of the KLB protein. However, both pharmacological inhibition of N-glycosylation and mutagenesis targeting N-glycosites reduced mature KLB protein content without impacting KLB mRNA synthesis in cells, underscoring the critical role of N-glycosylation in maintaining the stability of the KLB protein. Further studies revealed that the underglycosylated KLB mutant underwent rapid degradation via both lysosomal and proteasomal pathways and was unable to be efficiently trafficked to the plasma membrane, leading to impaired FGF21 signaling transduction. Collectively, multisite N-glycosylation is essential for the stability and cell surface localization of the KLB protein, representing a novel modulatory mechanism of endocrine FGF signaling.