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, 107 (6), 1596-613

Peptidomics of Cpe(fat/fat) Mouse Brain Regions: Implications for Neuropeptide Processing

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Peptidomics of Cpe(fat/fat) Mouse Brain Regions: Implications for Neuropeptide Processing

Xin Zhang et al. J Neurochem.

Abstract

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe(fat/fat) mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe(fat/fat) mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe(fat/fat) mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe(fat/fat) mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe(fat/fat) mice; these peptide processing intermediates with C-terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.

Figures

Figure 1
Figure 1
Diagram of typical processing of peptide precursors by selective peptidases, and model of the intracellular location of these enzymes. Left: Typical peptide precursors contain multiple sites for proteolytic processing that are usually pairs of basic amino acids, with Lys-Arg (KR) and Arg-Arg (RR) the most common. These sites (as well as sites with single basic residues) are initially cleaved by an endopeptidase such as prohormone convertase 1 or 2, which cleaves to the C-terminal side of the basic residue. A carboxypeptidase is then required to remove the C-terminal basic residues from the processing intermediates. On occasion, additional post-translational processing (amidation, sulfation, and others) is required for biological activity (not shown). Right: Carboxypeptidase D (CPD) is enriched in the trans Golgi network and immature secretory vesicles, along with endopeptidases such as furin (not shown). However, CPD is retrieved from the immature vesicles to the trans Golgi network and is not detected in mature secretory vesicles. In contrast, carboxypeptidase E (CPE) is present throughout the secretory pathway but is inactive at the neutral pH of the Golgi. CPE is activated by the drop in pH that occurs during the late secretory pathway, and is maximally active in mature vesicles where the pH is typically 5–5.5.
Figure 2
Figure 2
Selected propeptides and processed peptides identified in the present study in WT mice; some of these peptides were also detected in Cpefat/fat mice, but others were only present at detectable levels in the WT mice (see Table 1). When basic amino acids are present in the cleavage sites, they are indicated above the prohormone. Solid lines indicated peptides identified in the present study. If the peptide has not previously been named, the amino acid location within the precursor is indicated (i.e. as for procholecystokinin); if the peptide has been named, the name is indicated and then shorter fragments of this peptide (if detected) are indicated by the numbering within this peptide. Abbreviations: A8, dynorphin A8; Ac, acetyl; BAM18, bovine adrenal medulla peptide of 18 residues; C-term, C-terminal; DesAc, des-acetyl; End, endorphin; HP, enkephalin heptapeptide; J-peptide; joining peptide; LE, Leu-enkephalin; MA, metorphamide; ME, Met-enkephalin; MSH, melanocyte stimulating hormone; NE, neoendorphin; and OP, enkephalin octapeptide. Note that ProSAAS and the various names of the proSAAS-derived peptides are not abbreviations.
Figure 3
Figure 3
Representative MS data showing peptides that are either increased or decreased in Cpefat/fat mice, relative to WT mice. Left panels: Extracts from WT mice were labeled with H9-TMAB and those from Cpefat/fat mice were labeled with D9-TMAB. Right panels: The labeling scheme was reversed; extracts from WT mice were labeled with D9-TMAB while those from Cpefat/fat mice were labeled with H9-TMAB, as described in Experimental Procedures and also shown in the diagram included as supplemental data. The top panels represent the proenkephalin-derived “octapeptide” with a monoisotopic mass (after subtraction of the TMAB tag and one proton) of 929.45 Da, as determined from MS/MS sequence analysis (data in supplement). This peptide incorporated 1 TMAB group and is doubly charged, with the resulting m/z values of approximately 529.19 and 533.72 for the light and heavy isotopic forms, respectively. Note that levels of this peptide are much lower in the Cpefat/fat mice, as compared to the WT mice. The bottom panels represent the peptide-processing intermediate of the octapeptide (YGGFMRSLKR), as determined by MS/MS analysis, which incorporated 2 TMAB tags and 2 protons to produce m/z ions of 367.91 and 372.43 for the light and heavy isotopic forms, respectively. Note that this peptide cannot be detected in the WT samples.

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