Purification and characterization of a secreted T cell beta-endorphin endopeptidase.

Abstract

Primary murine CD4+ and CD8+ T cells and the EL-4 thymoma cell line secrete a metal-dependent, thiol endopeptidase that hydrolyzes β-endorphin at essentially equivalent rates at one of either of two peptide bonds (leu17-phe18 or phe18-lys19) yielding β-endorphin1–17 (γ-endorphin) or β-endorphin1–18 and their corresponding C-terminal fragments β-endorphin18–31 and β-endorphin19–31. This endopeptidase was purified to homogeneity from EL-4 cell conditioned medium (>1,000 fold, 26% yield, a single band of ≈110 kDa via SDSPAGE). The enzyme also cleaves dynorphin A1–7 and dynorphin B1–13 but not Leuenkephalin, dynorphin A1–9, γ-endorphin, or β-endorphin18–31. The endopeptidase is expressed constitutively in EL-4 cells and upon anti-CD3 activation of CD4+ and CD8+ T cells. Based on cleavage sites identified in a number of opioid peptide substrates, it can be concluded that the enzyme exhibits a complex specificity with a preference for cleaving on the amino side of hydrophobic or basic residues of larger peptide substrates. The presence of such an extracellularly acting peptidase clearly could be important in attenuating or modifying the biological activity of opioid peptides in their role as extracellular chemical signals between cells.