Scientists crack code on hidden bone disease marker, could transform diagnostics
Researchers have identified structural differences in a key blood test used to diagnose bone disorders, revealing why the same marker produces vastly different results across patients. The discovery could lead to more accurate tests for kidney disease complications and rare genetic bone conditions—opening new markets for diagnostic companies and improving treatment decisions for millions of patients.
Originaltitel: Exploring the glycosylation of tissue-nonspecific alkaline phosphatase : A biomarker in bone and mineral disorders
Tissue-nonspecific alkaline phosphatase (TNALP), mainly from bone and liver, is a serum biomarker of skeletal disease. Although the bone and liver TNALP isoforms share an identical protein structure, differences in their post-translational glycosylation alter protein structure and function, adding complexity to TNALP as a biomarker. Osteoblasts produce four bone alkaline phosphatase (BALP) isoforms, essential for normal bone and mineral metabolism. TNALP activity is reduced in the genetic disorder hypophosphatasia (HPP), whereas unexplained elevations occur in benign transient hyperphosphatasemia (BTH). In chronic kidney disease, elevated BALP levels may provide prognostic insight into bone turnover and cardiovascular disease. However, limited understanding of TNALP glycosylation and insufficient differentiation between liver ALP and BALP underscore the need for more sensitive and specific clinical assays. The aim of this thesis is to characterize TNALP glycosylation and evaluate its influence on BALP as a biomarker. Structural differences in glycosylation patterns across cell types and TNALP isoforms were examined with novel structural methods and established BALP detection techniques. Paper 1 applies glycoproteomics to define glycan structures and site occupancy in human TNALP. Paper 2 investigates the functional role of N-glycan sites in enzymatic activity, folding, and stability of TNALP, using site-directed mutagenesis and molecular dynamics simulations. Paper 3 expands the glycoproteomic profile with HPLC, gel electrophoresis, and analyses of TNALP expressed in multiple cell types, asfotase alfa (recombinant ALP for HPP treatment), the B2 BALP isoform, and TNALP in an overexpression mouse model. Paper 4 examines BALP status, TNALP isoform profile and glycosylation patterns in children with BTH. The findings indicate that TNALP has five fully glycosylated sites with high heterogeneity in glycosylation, core fucosylation and sialylation between sites and cell sources. Glycan interactions with the protein are essential for normal folding and function. Increasing evidence in terminal sialylation variations might explain the differences between the TNALP isoforms and aid in developing new isoform-specific assays. However, more studies are needed to confirm these structural differences.