Abstract
Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed. TRAP 5a and 5b protein levels were evaluated as osteoclast differentiation/activity markers in serum and in culture, and their correlation to the resorption marker CTX-I was examined. Serum TRAP 5a and 5b concentrations in healthy men were 4.4 ± 0.6 ng/ml and 1.3 ± 0.2 ng/ml, respectively, and they correlated moderately to each other suggesting that their secretion is coupled under healthy conditions. A correlation was also observed between serum TRAP 5a and 5b with CTX-I, suggesting that both TRAP isoforms associate with osteoclast number. During osteoclast differentiation on plastic/bone, predominantly 5b increased in media/lysate from M-CSF/RANKL-stimulated CD14+ PBMCs. However, substantial levels of 5a were detected at later stages suggesting that both isoforms are secreted from differentiating OCs. More TRAP 5b was released on bone indicating a connection to osteoclast resorptive activity, and a peak in TRAP 5b/5a-ratio coincided with rapid CTX-I release. At the end of the culture period of M-CSF + RANKL-stimulated CD14+ PBMCs, there was a correlation between the secretion of TRAP 5a and 5b proteins with CTX-I. The correlation of not only 5b but also 5a with collagen degradation, both in serum and osteoclast cultures indicates that a considerable proportion of the TRAP 5a originates from osteoclasts and may reflect a hitherto undisclosed regulatory mechanism during bone resorption and bone remodeling.
Highlights
IntroductionTRAP has two isoforms in human sera, 5a and 5b
Tartrate-resistant acid phosphatase type 5 (TRAP; ACP5, EC 3.1.3.2) has been linked to osteoclast (OC) biology for decades, both with respect to the physiological function [1] and as a biomarker for bone resorption correlating to OC number [2].TRAP has two isoforms in human sera, 5a and 5b
This carry-over of TRAP 5a would lead to falsely high TRAP 5b protein levels, since the monoclonal antibodies (mAbs) 25.44 does not discriminate between the isoforms
Summary
TRAP has two isoforms in human sera, 5a and 5b. TRAP is translated as a monomeric protein, referred to as TRAP 5a of ~ 35 kDa in which a peptide loop (aa 165–177 in P13686, UniProt) interacts with the active site to prevent phosphatase activity [3]. Proteolytic cleavage in the loop region [3] results in the dimeric TRAP 5b (16 + 23 kDa) [3] with augmented phosphatase activity. TRAP 5a has been regarded as a pro-form, it does have growth factor activity [4]. TRAP 5b has been implied to regulate OC migration by dephosphorylation of osteopontin (OPN) [5], and inactivation of the human TRAP gene (Acp5) leads to hyperphosphorylation of OPN [6]
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