Nigel P. Birch

Differential glycosylation of N‐POMC1–77 regulates the production of γ3‐MSH by purified pro‐opiomelanocortin converting enzyme A possible mechanism for tissue‐specific processing

The amino terminus of bovine pro‐opiomelanocortin (N‐POMC1–77) is partially processed in the intermediate lobe of the pituitary to N‐POMC1–49 and lys‐γ3 ‐melanotropin. Two pools of N‐POMC1–77 were isolated which were differentially glycosylated at threonine45, while N‐POMC1–49 isolated from bovine intermediate lobe extracts existed in a non‐glycosylated form. This suggested that differential O‐linked glycosylation of N‐POMC1–77 may regulate cleavage at the Arg49‐Lys50 processing site. We tested this hypothesis by incubating N‐POMC1–77 glycoforms with purified pro‐opiomelanocortin converting enzyme. Only non‐O‐glycosylated N‐POMC1–77 and O‐glycosylated N‐POMC1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys‐γ3 ‐melanotropin, identified by high‐performance liquid chromatography. These data provide the first functional evidence to support a role for differential O‐linked glycosylation in the regulation of the processing of the N‐terminus of bovine POMC.

Coordinate regulation of mRNA levels of pro-opiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in rat pituitary intermediate lobe.

Pro-opiomelanocortin is a multivalent hormone precursor which is processed at pairs of basic residues in a tissue-specific manner to release biologically active peptides. We have examined the message levels of three candidate pro-opiomelanocortin processing enzymes in the intermediate lobe of the rat pituitary following treatment with a dopamine receptor agonist and antagonist which are known to regulate pro-opiomelanocortin mRNA levels. Message levels for PC2 and PC3 but not furin were coordinately regulated with pro-opiomelanocortin transcripts supporting a role for PC2 and PC3 in the maturation of the pro-opiomelanocortin precursor in the rat pituitary intermediate lobe.

Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis

Carboxypeptidase H is an exopeptidase which is highly specific for C-terminal basic amino acids and thought to play a role in neuropeptide biosynthesis. The distribution of carboxypeptidase H mRNA was examined in adult rat brain using in situ hybridization histochemistry. Enzyme transcripts were detected in all major brain areas. Very high levels of carboxypeptidase H mRNA were found in the hippocampus associated with pyramidal cells. Other brain regions showed varied levels of labelling, ranging from high levels in the magnocellular cells of hypothalamic nuclei to very low levels in the striatum. The appearance of enzyme transcripts throughout brain supports a role for this enzyme in the biosynthesis of many neuropeptides. The expression of transcripts in certain ventricular ependymal cells identified them as a new potential peptidergic cell type. The variations in levels of expression of carboxypeptidase H mRNA may reflect differences in peptidergic activity in different neuronal systems.

Regulated Secretion of Pro‐Opiomelanocortin Converting Enzyme and an Aminopeptidase B‐Like Enzyme from Dispersed Bovine Intermediate Lobe Pituitary Cells

Abstract: Coordinate secretion of two prohormone/proneuropeptide processing enzymes [pro‐opiomelanocortin converting enzyme (PCE) and an aminopeptidase B‐like enzyme (APBE)] and α‐melanotropin (α‐MSH) from bovine intermediate lobe pituitary cells was studied. Stimulation of secretion with 8‐bromo‐cyclic AMP produced significant increases in levels of immunoreactive α‐MSH, PCE, and APBE. Treatment of cells with the dopaminergic agonist 2‐bromo‐α‐ergocryptine resulted in significant decreases in secretion of α‐MSH, PCE, and APBE. In neither case were there significant changes in levels of cytosolic lactic dehydrogenase or lysosomal β‐glucuronidase in the medium. The secreted PCE activity was shown to process frog and mouse pro‐opiomelanocortin primarily to 23,000‐Mr corticotropin (ACTH), 13,000‐Mr ACTH, β‐lipotropin, a β‐endorphin‐like peptide, and β‐endorphin, products comparable to those synthesized by the mouse and frog intermediate lobe in situ. The secreted enzymatic activity had a pH optimum between 4.0 and 5.0, was strongly inhibited by pepstatin A, and had an inhibitor profile similar to the purified bovine intermediate lobe PCE. The secreted APBE activity cleaved Arg0‐[Met]‐enkephalin to [Met]‐enkephalin and had a pH optimum and inhibitor profile similar to that previously reported for an activity from purified secretory vesicle fractions of bovine intermediate and neural lobes. The coordinate regulated secretion of α‐MSH and enzyme activities (PCE and APBE) strongly indicates their colocalization in the same secretory vesicle compartment within the cell. The characteristics of the two enzymes secreted in the medium paralleled those seen in the tissue and further support their role in pro‐opiomelanocortin processing in vivo.

Distribution and regulation of the candidate prohormone processing enzymes SPC2 and SPC3 in adult rat brain.

A number of candidate mammalian prohormone processing enzymes related to the yeast Kex2 endoprotease have been cloned and demonstrated to cleave several prohormone precursors at single, pairs and tetra basic amino acid processing sites. We have mapped the distribution of the mRNAs encoding two of these endoproteases in adult rat brain. SPC3 message levels showed a more restricted distribution and generally lower levels than SPC2 transcripts. The highest levels of SPC2 mRNA were found in the pyramidal cells of the hippocampus, several thalamic nuclei, the habenula and selected nuclei in the hypothalamus. SPC3 mRNA was most abundant in dentate gyrus granule cells, the habenula and selected hypothalamic nuclei. In the hypothalamus overlapping and unique distributions of the two transcripts were seen in the paraventricular nucleus with SPC3 mRNA predominantly expressed in lateral magnocellular cells. Both SPC2 and SPC3 mRNA were upregulated in the paraventricular and supraoptic hypothalamic nuclei following chronic salt loading. Combined immunocytochemistry/in situ hybridization histochemistry demonstrated that SPC2 and SPC3 transcripts were both expressed in the vasopressinergic subpopulation of magnocellular neurons in the supraoptic nucleus. SPC3 mRNA, but not SPC2 transcripts, also colocalized with immunoreactive vasopressin-associated neurophysin in the suprachiasmatic nucleus. These results remain consistent with roles for SPC2 and SPC3 in the biosynthesis of neuropeptides and for a specific role for SPC3 in the processing of provasopressin. Increased levels of SPC2 and SPC3 transcripts following a chronic osmotic stimulus suggests these proteases are coregulated with prohormone substrates and may be useful as an indicator of peptidergic activity.

Pro-opiomelanocortin and pro-vasopressin converting enzyme in pituitary secretory vesicles

Peptide hormones are synthesized from larger precursors by cleavages at paired basic residues. We have isolated a pro-hormone converting enzyme from bovine neural and intermediate lobe secretory vesicles that cleaves pro-vasopressin and pro-opiomelanocortin at Lys-Arg residues to yield vasopressin, and adrenocorticotropin/endorphin-related peptides, respectively. The enzyme from both lobes is an aspartyl protease of approximately 70,000 Da, is a glycoprotein and has an optimum pH range of 4.0-5.0. Present within the same secretory vesicles is an aminopeptidase B-like enzyme which is a metalloprotease that is inhibited by Co2+ and Zn2+. This enzyme may play a role in trimming off the N-terminal extended basic residues from peptides liberated by the pro-hormone converting enzyme.

Homology cloning of aspartic proteases from an endocrine cell line using the polymerase chain reaction

Oligonucleotides directed towards the active site regions of aspartic proteases were used as primers for the polymerase chain reaction to identify a unique sequence (asppcr1) from the AtT-20 anterior pituitary corticotrope cell line. Asppcr1 showed the greatest similarity (85% identity) to human cathepsin E [(1989) J. Biol. Chem. 264, 16748-16753]. Northern blot analysis of AtT-20 RNA revealed a single 1.9 kB message. Nuclease protection experiments indicated that asppcr1 mRNA was present in pancreas, spleen, testis and liver at low levels and undetectable in heart and brain. This contrasted with the lysosomal aspartic protease, cathepsin D whose mRNA showed a broader tissue distribution. The restricted message distribution of asppcr1 supports a more specific role for this aspartic protease in aspect(s) of cellular physiology.

Rat mitochondrial coupling factor 6: molecular cloning of a cDNA encoding the imported precursor

A cDNA clone encoding the precursor to the rat mitochondrial protein coupling factor 6 (F6) has been isolated and sequenced. The deduced amino acid sequence of the rat precursor protein shows 78% and 74% identity with the human and bovine F6 pre-proteins, respectively.