Biochemistry 2005, 44, 4939-4948
4939
Neuropeptide Processing Profile in Mice Lacking Prohormone Convertase-1† Hui Pan,‡ Daniela Nanno,‡ Fa-Yun Che,§ Xiaorong Zhu,| Stephen R. Salton,⊥ Donald F. Steiner,| Lloyd D. Fricker,§ and Lakshmi A. Devi*,‡ Department of Pharmacology and Biological Chemistry and Department of Neuroscience, Mount Sinai School of Medicine, One GustaVe L. LeVy Place, New York 10029, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, and Howard Hughes Medical Institute, UniVersity of Chicago, Chicago, Illinois 60637 ReceiVed October 5, 2004; ReVised Manuscript ReceiVed December 23, 2004
ABSTRACT: Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of MetEnkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.
Neuroendocrine peptides are usually synthesized as large precursors and need to undergo limited proteolytic steps as well as other posttranslational modifications to gain biological activity. In most cases the cleavage sites within the peptide precursors contain multiple basic sequences such as Lys-Arg, Arg-Arg, or Arg-X-X-Arg (1-4). Two members of the subtilisin-like prohormone convertase (PC)1 family, PC1 (also known as PC3) and PC2, are broadly expressed in brain and neuroendocrine tissues and localized to secretory vesicles along with the neuropeptide precursors (5-7). It is believed that these two PCs play crucial roles in neuroendocrine peptide precursor processing at dibasic or multibasic sites (1, 2, 4). Following this endopeptidase cleavage, peptide intermediates with C-terminal basic residue extensions are processed by a carboxypeptidase (CP) such as CPE or CPD (8). The enzymatic activities of PC1 and PC2 as well as the roles they play in neuroendocrine peptide processing have † This work was supported by NIH grants NS 26880 and DA 00458 (to L.A.D.), NIH grants DA04494 (to L.D.F.) and DK57702 and the American Heart Association AHA0350085N (to S.R.S.), and NIH grants DK13914 and 20595 and the Howard Hughes Medical Institute (to D.F.). * To whom correspondence should be addressed. Phone: (212) 2418345. Fax: (212) 996-7214. E-mail:
[email protected]. ‡ Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine. § Albert Einstein College of Medicine. | University of Chicago. ⊥ Department of Neuroscience, Mount Sinai School of Medicine.
been extensively studied using in vitro techniques including purified recombinant enzymes, vaccinia virus overexpression systems, and neuroendocrine cell lines (9-17). The studies have suggested that these two enzymes play crucial roles in the processing of several neuropeptide precursors including proenkephalin and proopiomelanocortin (POMC) (10, 11, 14-19). Although both PC1 and PC2 cleave at sites containing multiple basic residues, they each have their own preferences. Moreover, because of the different expression patterns of these two enzymes in neuroendocrine tissue, they are believed to be involved in tissue-specific processing of the same precursor molecule. For example, in the anterior pituitary PC1 alone is expressed at a high level and generates ACTH from POMC. In the intermediate lobe both PC1 and PC2 are expressed at a high level and it appears that the presence of PC2 leads to the further processing of ACTH into R-MSH and CLIP (20). In the case of proenkephalin it has been shown that PC1 mainly processes the precursor into higher molecular weight intermediates, while PC2 is more efficient in performing the cleavages that produce the shorter, bioactive products (9, 14, 19). 1 Abbreviations: ACTH, adrenocorticotrophic hormone; R-MSH, alpha-melanocyte stimulating hormone; CLIP, corticotropin-like intermediate lobe peptide; CP, carboxypeptidase; ESI-TOF, electrospray ionization time-of-flight; GHRH, growth hormone releasing hormone; KO, knockout; LC-MS, liquid chromatography mass spectrometry; LPH, lipotropin; Met-Enk, methionine enkephalin; PC1, prohormone convertase 1; PC2, prohormone convertase 2; POMC, proopiomelanocortin.
10.1021/bi047852m CCC: $30.25 © 2005 American Chemical Society Published on Web 03/02/2005
4940 Biochemistry, Vol. 44, No. 12, 2005 Apart from the well-studied precursor molecules, several other less understood neuropeptide precursors have emerged as potential substrates for PC1 and PC2. One example is the peptide precursor VGF which was initially identified as an NGF-induced cDNA clone and is believed to be a neuropeptide precursor (21). VGF has a neuroendocrine tissue distribution and is essential for the regulation of energy balance since disruption of the mouse VGF gene produces a lean phenotype (21, 22). The primary sequence of this precursor reveals a number of dibasic or multibasic sites that are consistent with the consensus processing sites recognized by PC1 or PC2, and several of these sites have been shown to be cleaved in GH3 cells expressing ectopic PC1 or PC2 (23). Because VGF has a similar tissue and cellular distribution as PC1 and PC2, it is likely that VGF is a physiologically relevant substrate for these enzymes (21, 24). Another interesting precursor is proSAAS, a neuropeptide precursor that has a potent inhibitory effect on PC1 and a broad neuroendocrine distribution (25). The inhibitory region is located in a small region near the C-terminus of proSAAS (26-28). Cleavage at a Lys-Arg sequence within this region and subsequent removal of the basic residues by a CP eliminates the inhibition of PC1 (28). PC1 is able to cleave this site, although it is very slow. Because cleavage at this site is efficient in mouse brain and cell lines (25, 29), it was of interest to see whether PC1 itself is involved in this cleavage under physiological condition. Although the enzymatic activities of the two PCs have been extensively studied using in vitro biochemical and cell biological methods, it is important to evaluate the roles of the two enzymes in physiological conditions. Mice with a disruption of the PC2 gene (i.e. PC2 KO mice) have a number of neuropeptide processing defects including the processing of proinsulin, proglucagon, as well as prosomatostatin (30-35). Also, the processing of endogenous opioid peptide precursors, prodynorphin, and proenkephalin are affected (19, 36). The processing of POMC in pituitary is impaired in that the conversion of β-lipotropin into γ-lipotropin and β-endorphin is greatly diminished. The carboxy shortening of β-endorphin is completely abolished, and the processing of ACTH into R-MSH and CLIP is hampered (32, 33, 37). Therefore, the question to be asked is what role does PC1 play in the processing of these molecules in vivo. The PC1 KO mice have recently been generated, and they display impairments in the processing of a number of neuroendocrine peptide precursors including hypothalamic growth hormone releasing hormone (GHRH), POMC in the anterior pituitary lobe, pancreatic proinsulin and islet amyloid polypeptide precursor, intestinal proglucagon, hypothalamic CART, and cholecystokinin in several brain regions (3843). In the present study RIA was used to study changes in the processing of three peptide precursor molecules (proenkephalin, VGF, and proSAAS) in PC1 KO mice. In addition, a quantitative peptidomic method was used to analyze changes in the levels of a large number of pituitary peptides in the PC1 KO mice. MATERIALS AND METHODS: Western Blot Analysis of PC1 Protein. Brains from the PC1 KO mice or control littermates were frozen in isopentane
Pan et al. chilled with dry ice right after sacrificing. For extraction brains were put into liquid nitrogen and ground to a fine powder. The powder was homogenized in 50 mM Tris-Cl pH 7.4 with 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The homogenate was kept on ice for 30 min and then centrifuged at 12 000g for 30 min at 4 °C. The protein concentrations of the supernatants were determined using the Bradford method, and approximately 1 µg of solubilized protein was subjected to SDS-polyacrylamide gel electrophoresis. The proteins were then electrotransferred to a nitrocellulose membrane and probed with a 1:1000 dilution of a rabbit antiserum raised against the N-terminal region of PC1 or PC2 as described (44). Equal loading was confirmed by probing the blot with anti-tubulin antiserum (Sigma). The PC1 antiserum recognizes a ∼68 kDa protein representing the mature enzyme and a nonspecific band at 78 kDa. The PC2 antiserum recognizes ∼68-71 kDa proteins representing mature forms of PC2 (44). Brain Extraction, Gel-Filtration, and ReVerse-PhaseHPLC (RP-HPLC) Procedures. Brains from age- and sexmatched PC1 KO mice or control littermates were frozen in liquid nitrogen and ground into a fine powder. Ten volumes of 0.1 M boiling acetic acid were added. Samples were homogenized and incubated at 100 °C for 15 min. After cooling on ice, samples were centrifuged at 12 000g for 30 min at 4 °C. The supernatants were transferred to fresh tubes, dried in a vacuum centrifuge, and stored at -80 °C. For gel-filtration chromatography the samples were rehydrated in 100 mM sodium phosphate buffer, pH 7.4, containing 0.1% Triton X-100, adjusted to 30% acetonitrile and 0.1% trifluoroacetic acid, and then applied to a Superdex Peptide HR 10/30 gel-filtration column (Amersham Bioscience). The samples were fractionated in 30% acetonitrile and 0.1% trifluoroacetic acid with a flow rate of 0.5 mL/ min. One-minute fractions were collected. For the reverse-phase (RP)-HPLC analysis the gel-filtration fractions containing the immunoreactive peptides (fractions 20-23) were pooled, and the volume of samples was reduced in a vacuum centrifuge. A portion of the material was then subjected to RP-HPLC using a C18 column (length ) 250 mm; inner diameter ) 4.6 mm, VyDac). The samples were fractionated with a linear gradient from 5% acetonitrile/0.1% TFA to 75% acetonitrile/0.1% TFA over 70 min with a flow rate of 0.5 mL/min. One-minute fractions were collected. Radioimmunoassay (RIA) and Trypsin/Carboxypeptidase B (CPB) Treatment. Gel-filtration fractions were analyzed by RIAs to determine the levels of Met-Enk, the VGF C-terminal peptide AQEE, and the proSAAS-derived peptide PEN. For Met-Enk RIA gel-filtration fractions were either tested directly using antiserum against Met-Enk or subjected to digestion with trypsin/CPB. For this an aliquot (60 µL) of each fraction was treated with 5 µg/mL tosylphenylalanylchloromethyl ketone-treated trypsin (Sigma) for 16 h followed by treatment with 5 ng/mL carboxypeptidase B (CPB, Sigma) for 120 min. The reaction was terminated by boiling for 20 min. Another aliquot of 60 µL (“untreated”) was subjected to the same incubation and boiling conditions as above except that enzymes were omitted. This trypsin/ CPB treatment releases the Met-Enk moiety from the higher molecular weight precursors without affecting the levels of mature Met-Enk. The Met-Enk antiserum (Bachem Inc. San
Peptide Processing in PC1 K/O Mice Carlos, CA) is specific for the Met-Enk pentapeptide; it exhibits
