Biallelic mutations in ACP5, encoding
tartrate-resistant acid phosphatase (
TRACP), have recently been identified to cause the inherited immuno-osseous disorder,
spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on
TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established.
TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of
monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable
enzyme activity. All ACP5 mutant constructs were translated into intact
proteins by HEK-293 cells. The mutant
TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of
TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing
enzyme function.
TRACP exists as two
isoforms.
TRACP 5a is a less active monomeric
enzyme (35kD), with the intact loop
peptide and
TRACP 5b is proteolytically cleaved highly active
enzyme encompassing two subunits (23 kD and 16 kD) held together by
disulfide bonds. None of the
mutant proteins were proteolytically processed into
isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact
isoform 5a-like
proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K
mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted
TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive
protein. All
mutant proteins were attacked by Endo-H sensitive
glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in
TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.