Three circulating human bone
alkaline phosphatase (BALP)
isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth
isoform (B1x) has been discovered in patients with
chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP
isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant
isoforms of the tissue-nonspecific ALP (TNALP)
isozyme, i.e., TNALP-flag, used extensively for mutation analysis of
hypophosphatasia mutations and sALP-FcD(10), a chimeric
enzyme recently used as therapeutic
drug in a mouse model of
infantile hypophosphatasia. The BALP
isoforms were prepared from human
osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-
nitrophenylphosphate (
pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic
pyrophosphate (PP(i)),
pyridoxal 5'-phosphate (PLP), and
phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by
lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP
isoforms. The BALP
isoforms differed in their
lectin binding properties and dose-dependent
lectin precipitation, which also demonstrated differences between native and denatured BALP
isoforms. The observed differences in
lectin specificity were attributed to N-linked
carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP
isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP
isoforms or for the recombinant TNALP
isoforms. The TNALP-flag and the sALP-FcD(10)
isoforms faithfully mimic the
biological properties of the human BALP
isoforms in vivo validating the use of these recombinant
enzymes in studies aimed at dissecting the pathophysiology and treating
hypophosphatasia.