Expression of the vasopressin and oxytocin genes in human hypothalami

Abstract

Poly(A)+ RNA isolated from post‐mortem human hypothalami has been used to characterize the polyprotein precursors to vasopressin and oxytocin. Translation in a cell‐free system and subsequent immunoprecipitation with antibodies raised against either vasopressin or neurophysin identified a product of M r 19000 (prepro‐vasopressin). A second less intense product of M r 16500 was tentatively identified as prepro‐oxytocin. A cDNA library derived from the human hypothalamic poly(A)+ RNA was screened for vasopressin and oxytocin‐encoding cDNA using heterologous probes; clones encoding the two precursors were identified and found to be organized as their rat and bovine counterparts. Northern blot analysis shows that the mRNAs for the two prepro‐hormones consist of ~ 840 (AVP) and ~ 700 (OT) nucleotides.

Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus.

“…Consistent with results of cell-free translation of poly(A )+ RNA from human hypothalami (20) and bovine hypothalami (25), translation of wild-type and mutant VP mRNAs in the absence of microsomes each yielded preproVP, a 1 9-kD product that was immunoprecipitable with anti-rat neurophysin antibodies (Fig. 6).…”

“…7, middle panel). This pattern is consistent with the amino acid sequence of a mixture of proVP and preproVP (2,6,20). When the more abundant 23-kD band was independently sequenced by carefully cutting the upper portion of the doublet from the PVDF blot, the peak of [3H]Phe at cycle 3 was significantly reduced (Fig.…”

“…7, top panel). This pattern ofrelease of [3H] Phe is consistent with the NH2-terminal sequence of proVP arising from cleavage of preproVP by signal peptidase at the normal site after Ala-19 (2,6,20). The increase in molecular weight of proVP (2 kD) upon translation in the presence of microsomes is most likely the result of the removal of the 19-residue signal peptide (2 kD) and the addition of core glycosylation at the single Asn-linked glycosylation site ofpreproVP as demonstrated previously with bovine proVP (25).…”

“…Although the wild-type VP cDNA was synthesized from mRNAs ofa VP-producing tumor, its nucleotide sequence was identical to the human VP cDNA sequence (20) and that deduced from the previous reports of human VP genomic sequence (2, 6). To synthesize a cDNA encoding the mutant form of preproVP, a mutagenic oligonucleotide (GCCTTC-TCCTCCACGTGCTACTTCC; nt 267 -> 291) was used.…”

NGFFFamide and echinotocin: structurally unrelated myoactive neuropeptides derived from neurophysin-containing precursors in sea urchins

“…The conserved cysteine residues in the neurophysin-like domains are underlined and numbered 1-14. The precursors of vasopressin/oxytocin-like peptides from other species include precursors of human vasopressin (Mohr et al, 1985), human oxytocin (Mohr et al, 1985), vasotocin from the lamprey Lethenteron japonicum (Suzuki et al, 1995), an oxytocin-like peptide (SOP) from the urochordate Styela plicata (Ukena et al, 2008), Lys-conopressin from the mollusc Lymnaea stagnalis (Van Kesteren et al, 1992), cephalotocin from the mollusc Octopus vulgaris (Reich, 1992), annetocin from the annelid Eisenia foetida (Oumi et al, 1994) and inotocin from the arthropod (insect) Tribolium castaneum (Aikins et al, 2008;Stafflinger et al, 2008). Also included is a precursor from the nematode Caenorhabditis elegans (GenBank: NP_001033548, GI:86564869) that has not been reported previously in the literature; this precursor contains an unusual putative vasopressin/oxytocin-like peptide comprising just eight residues (CFLNSCPY), which we have named 'nematocin'.…”