Dynamic processing of neuropeptides: sequential conformation shaping of neurohypophysial preprohormones during...

Neurohypophysial preprohormones are single polypeptide chains folded into 3/4 domains, namely a signal prepeptide (18/20 residues), a hormone peptide (9 residues), and a propeptide neurophysin-copeptin (93/134 residues). Neuro-hormone and neurophysin contain 1 and 7 disulfide bridges, respectively, whose pairing depends on correct primordial folding within the endoplasmic reticulum (ER) compartment (pH 7.0) of hypothalamic magnocellular neurons. During intracellular travel in the secretory pathway from ER to secretory granules (SC), the precursor is submitted to successive processings (glycosylation, proteolysis, amidation) in distinct compartments, leading to domain separation and reshaping. In particular the hormone domain is subjected, in the SG, pH 5.5, to a 4-enzyme cascade in order to reach the bioactive conformation. We have purified SG from rat and ox neurohypophyses and characterized: 1) the processed domains (neurohormone, neurophysin, copeptin); 2) the four processing enzymes acting successively at the level of the processing sequence, namely a Lys-Arg calcium-dependent endopeptidase, a carboxypeptidase B-like enzyme, a peptidyl-glycine monooxygenase and a peptidyl-hydroxyglycine lyase (amidating enzyme). A reconstitution of the processing has been carried out in vitro using purified granular enzymes and synthetic nonactive prohormone peptides, vasopressinyl-Gly-Lys-Arg, vasotocinyl-Gly, and oxytocinyl-Gly. Vasopressin (yield 17% at pH 6.0, 30% at pH 8.0) has been identified by both coelution in high-performance liquid chromotography (HPLC) and bioactivity. In the homozygote mutant Brattleboro rats, a single nucleotide deletion in the gene entails a complete change in aminoacid sequence of neurophysin from residue 64 onwards. A misrouting in the ER or a misprocessing in the SG could occur so that neither vasopressin nor associated-neurophysin are found in the neurohypophysis, this lack determining diabetes insipidus. In addition there is a 50% decrease of the Lys-Arg-endoendopeptidase activity in the SG of the homozygote Brattleboro.