N-linked glycosylation plays an essential role in the stability and function of tissue-nonspecific alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNALP) is a membrane-anchored glycoprotein with five N-linked glycosylation sites (N140, N230, N271, N303, N430) that is crucial for bone mineralization. TNALP is released into the bloodstream, serving as a biomarker for bone and mineral disorders. This study explores the role of N-linked glycosylation in the secretion, enzymatic activity, stability, and structure of TNALP. To eliminate the N-linked glycosylation site specifically, a soluble TNALP expression construct was created with the following substitution mutations N140Q, N230Q, N271Q, N303Q and N430D, and expressed in mouse osteoblasts. The effect of glycosylation was also studied by computational modeling (molecular dynamics simulations and the GlycoSHIELD tool). We observed that substituting glycosylation sites reduced TNALP secretion, particularly in the double-site mutations N140Q/N271Q and N230Q/N271Q, due to increased cellular retention. Mutations comprising site N271 (N271Q, N140Q/N271Q, N271Q/N303Q and N271Q/N430D) significantly impaired the enzymatic activity. The computational modeling indicated that N-glycans can stabilize regions of the protein, including the Ca²⁺-binding domain. Further, interactions between N-glycans can compensate for specific double-site glycan losses. Protein thermal stability analysis showed that, compared to WT, N271Q/N430D and N303Q/N430D had increased stability at 56°C. TNALP isoform analysis revealed no differences in isoform patterns for mutations with retained enzymatic activity. The study suggests that N-linked glycosylation, particularly the presence of glycans at N271, is vital for TNALP stability, secretion, and enzymatic function, offering insights into the structural and functional properties of TNALP.