Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: Implications for PTHrP peptideproduction in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteoly tic processing to generate PTHrP-related peptide products that possess regu latory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically activ e peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multib asic, dibasic, and monobasic residues. These basic residues are predicted t o serve as proteolytic cleavage sites for converting the PTHrP precursor in to active peptide products. The coexpression of the prohormone processing e nzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cel ls, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study , PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MA LDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites t o generate biologically active PTHrP-related peptides that correspond to th e NH2-terminal domain (residues 1-37) that possesses PTH-like and growth re gulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that mod ulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage si tes. Overall, these results demonstrate the ability of PTP to cleave the PT HrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreacti vity in PTHrP-containing lung cancer cells suggests PTP as a candidate proc essing enzyme for the PTHrP precursor. (C) 2001 Academic Press.