Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific
alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization.
Enzyme replacement therapy (ERT) with partially purified plasma
enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for
hypophosphatasia may require delivering functional TNSALP
enzyme to bone. We tagged the C-terminal-anchorless TNSALP
enzyme with an acidic
oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged
enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified
enzymes tagged with the acidic
oligopeptide were the same as the untagged
enzyme. In vitro affinity experiments showed the tagged
enzymes had 30-fold higher affinity for
hydroxyapatite than the untagged
enzyme.
Lectin affinity chromatography for
carbohydrate structure showed little difference among the three
enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled
enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged
enzyme retained in bone was 4-fold greater than that of the untagged
enzyme. In vitro mineralization assays with the bone marrow from a
hypophosphatasia patient using each of the three
enzymes in the presence of high concentrations of
pyrophosphate provided evidence of bone mineralization. These results show the anchorless
enzymes tagged with an acidic
oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged
enzymes in ERT for
hypophosphatasia, which should be explored.