Synthetic pre-proparathyroid hormone leader sequence inhibits cell-free processing of placental, parathyroid, and pituitary prehormones.

Abstract

A chemically synthesized peptide consisting of the NHAerminal precursor-specific leader, or signal, sequence of pre-proparathyroid hormone was used as a molecular probe to investigate the role of the leader sequence in translocation across membranes of polypeptides destined for secretion.When added to a reticulocyte cell-free translation system supplemented with canine pancreatic microsomes that process precursor proteins to their mature -forms, this peptide inhibited in a specific manner the processing of four separate prehormones (bovine pre-proparathyroid hormone, bovine pregrowth hormone, bovine preprolactin, and human preplacental lactogen) to their mature forms.Processing was defined as (a) a reduction in the size of a translation product to ,that of the authentic hormone or prohormone and ( b ) demonstration of resistance of the processed product to limited proteolysis.These criteria are indicative of cleavages of leader sequences from the nascent precursors and the sequestration of the processed products within the microsomes.Several tests supported the specificity of the inhibition by the synthetic precursor-specific peptide.The inhibitory effect of the synthetic peptide was not due to nonspecific disruption of the integrity of the microsomal membranes through detergent-like effects, inasmuch as processed translation products were not released from within microsomes when the peptide was added after the translation reactions were completed.No inhibition of processing was seen when another synthetic peptide consisting of an internal fragment of parathyroid hormone structurally similar in length and hydrophobicity to the precursor-specific peptide was used; inhibition of processing appeared to reside specifically in the leader sequence.These results suggest that microsomal membranes contain specific, saturable recognition elements that function in the membrane-related binding, translocation, and/or cleavage of nascent polypeptides destined for secretion.This synthetic biologically functional peptide of pre-proparathyroid hormone should be useful in the further delineation of the mechanisms involved in the binding of prehormones to, and their translocation across, microsomal membranes, as well as in the identification and isolation of presumed precursor-specific receptors in these membranes.