Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2

Abstract

The bioactivity of neuropeptides can be regulated by a variety of post-translational modifications, including proteolytic processing. Here, gene-targeted mice producing defective prohormone convertase 2 (PC2) were used to examine the post-translational processing of two neuroendocrine prohormones, pro-opiomelanocortin (POMC) and pro-orphanin FQ (pOFQ)/nociceptin (N), in the brain. Reversed-phase HPLC and gel-exclusion chromatography were combined with specific radioimmunoassays to analyze the processing patterns of these two prohormones in the hypothalamus and the amygdala. In the case of POMC, the lack of PC2 activity completely prevented carboxy-shortening of beta-endorphins and greatly diminished conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin. Although conversion of beta-lipotropin to beta-endorphin decreased, the lack of PC2 activity caused an increase in beta-lipotropin and beta-endorphin levels in the mutant animals, but no increases in POMC or biosynthetic intermediates were seen. The extent of OFQ/N production was significantly lower in PC2-deficient mice and there was an accumulation of relatively large amounts of pOFQ/N and biosynthetic intermediates. These results demonstrate that PC2 is directly involved in the biogenesis of two brain neuropeptides in vivo and suggest that the specific prohormone and cellular context influences neuropeptide processing by PCs.

Fig. 1.

A, C-terminal proteolytic processing of pre-POMC (pPOMC). A schematic representation of the β-LPH–β-endorphin processing pathway in the rodent hypothalamus. B, A schematic representation of ppOFQ/N processing in the rodent hypothalamus. Predicted and potential cleavages by PC1and PC2 based on pituitary POMC processing are indicated. The processing patterns shown are thought to occur in mice expressing WT PC2. K, Lysine;R, arginine. Asterisks denote potential paired basic cleavages in OFQ/N. Arrows denote PC cleavage sites.

Fig. 2.

β-LPH domain processing in WT and PC2 mutant mouse hypothalamic extracts. RP-HPLC fractions were assayed for β-endorphin and β-MSH immunoreactivity. The dashed lineindicates the acetonitrile gradient. A, WT β-MSH and mutant animals. B, Mutant PC2. Results shown are representative of four separate determinations.

Fig. 3.

Sephadex G-50 gel-exclusion chromatography of WT and mutant PC2 hypothalamic extracts. The fractions were assayed for β-endorphin immunoreactivity. The arrow indicates the elution position of ¹²⁵I-acetyl-β-endorphin 1–27.V₀, Exclusion volume;V_t, total column volume. Theinset shows the percentage of conversion of β-LPH to β-endorphin in a WT compared with a PC2 mutant mouse hypothalamus. The percentage of conversion is the ratio of the immunoreactivity eluting 3–3.5 kDa β-endorphin to the total β-endorphin immunoreactivity. Several RP-HPLC analyses were used for this calculation. n = 3; p = 0.0063; unpaired t test using StatView (Abacus Concepts, Calabasas, CA).

Fig. 4.

RP-HPLC fractionation of WT and PC2-deficient mouse hypothalamic extracts after gel exclusion, as in Figure 3. The pooled extracts of β-endorphin-sized material were fractionated on the gradient indicated, and the fractions were assayed for β-endorphin immunoreactivity. The arrows indicate the elution times of authentic β-endorphin peptides. The dashed line indicates the acetonitrile gradient. Similar results were seen in two experiments. Ac, Acetyl.

Fig. 5.

RP-HPLC fractionation of (WT) and PC2 mutant mouse hypothalamic extracts (one mouse hypothalamic equivalent). Extracts from WT (A), PC2-deficient (B), and heterozygous (C) mice were fractionated using a linear gradient of 8–36% acetonitrile (dashed line). The fractions were assayed for OFQ/N immunoreactivity. The arrow indicates the elution position of synthetic OFQ/N. The profile shown is representative of four separate determinations.

Fig. 6.

Sephadex G-50 chromatography of WT and PC2 mutant mouse hypothalamic extracts. Gel filtration was performed as described in Materials and Methods. Portions of the fractions were vacuum dried and assayed for OFQ/N immunoreactivity. ¹²⁵I-OFQ/N elutes at fraction 55. Cytochrome C (molecular mass, 22.5 kDa) elutes at fraction 33–34. V₀, Exclusion volume; V_t, total column volume. Conversion of total OFQ/N immunoreactivity to mature peptide in WT and PC2 mutant mouse hypothalamus is shown in the inset. The percentage of conversion is the ratio of the immunoreactivity co-eluting with synthetic OFQ/N at 31 min to the total OFQ/N immunoreactivity. We used RP-HPLC analyses for this calculation.n = 3; p = 0.002; unpairedt test using StatView.

Fig. 7.

RP-HPLC fractionation of WT and PC2 mutant amygdala extracts. Extracts from WT animals (open circles) and PC2 mutant animals (filled circles) were fractionated using a linear gradient of 8–36% acetonitrile (dashed line). The fractions were assayed for OFQ/N immunoreactivity. The arrow indicates the elution time of synthetic OFQ/N. The profile shown is representative of four separate determinations.

Fig. 8.

Fractionation of pOFQ/N C-terminal (c-term)-containing peptides in WT and PC-2-deficient mice. Extracts were fractionated as described in Figure 5, and the fractions were assayed for OFQ/N and C-terminal peptide immunoreactivity. The elution positions of synthetic peptides are indicated by the arrows. The dashed lineindicates the percentage of acetonitrile. A, Heterozygote;B, PC null.