Title:	The effect of site-specific N-linked glycosylation on the protein dynamics of tissue-nonspecific alkaline phosphatase
DNr:	LiU-compute-2025-13
Project Type:	LiU Compute
Principal Investigator:	Per Magnusson <per.magnusson@regionostergotland.se>
Affiliation:	Linköpings universitet
Duration:	2025-04-04 – 2026-01-01
Classification:	10307
Keywords:

Abstract

Tissue-nonspecific alkaline phosphatase (TNALP) is a membrane-bound enzyme expressed in various tissues. TNALP is expressed by bone-forming osteoblasts that facilitate bone mineralization and is an important clinical biomarker and potential therapeutic target of bone-mineral disorders, such as hypophosphatasia and vascular calcification. Hypophosphatasia is associated with low serum TNALP activities and defective skeletal mineralization due to loss-of-function mutations in the TNALP gene, while serum TNALP levels are elevated in vascular calcification due to high osteoblastic activity (Haarhaus et al., 2022). Serum TNALP consists of several isoforms that have an identical protein structure but different post-translational modifications, i.e., glycosylation patterns. In a previous study, we demonstrated that all five hypothetically glycosylated asparagine residues (N140, N230, N271, N303 and N430) are fully occupied by long sugar chains, i.e., glycans (Atanasova et al., 2024). The N-glycan sites are also shown to be important for normal enzymatic activity (Komaru et al., 2016). Here we aim to investigate the role these N-glycan sites play in the presence of different TNALP isoforms. To date, the precise role of the N-glycan sites towards enzymatic activity is unknown. In this project, we envisage the use of lab-based methods in combination with molecular dynamics computer simulations to understand how the N-glycan sites contribute to protein function. Lab investigations will be done to study how these N-glycan sites affect protein expression and stability with biophysical methods, such as thermal shift assay, after substitution with non-glycosylated residues. The results from removing a single glycan site reveal that only removing N271 significantly diminished enzymatic activity. Strikingly, however, when two sites were removed, we observed that some combinations significantly reduced activity while others had no significant effect. Specifically, when the combinations N140/430, N230/303 and N230/430 were removed it did not significantly reduce enzymatic activity while other combinations, removal of N140/271 and N230/271, significantly reduced enzymatic activity. To rationale these results we envisage the use of molecular dynamics simulations to investigate the excluding one or a combination of N-glycan sites on protein function. This combined computational and experimental approach to protein structure exploration will further increase the understanding of the existence of TNALP isoforms and their potential as clinical biomarkers and therapeutic targets in hypophosphatasia and vascular calcification. Atanasova, D., et al. (2024). JBMR plus 8, ziae006. Haarhaus, M., et al. (2022). Nutrients 14, 2124. Komaru, K., et al. (2016). The FEBS Journal 283, 1168-1179.