Menkes Protein Contributes to the Function of Peptidylglycine -Amidating Monooxygenase
Abstract., http://www.100md.com
Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine {alpha} -amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.
Introductiondp44/y5, 百拇医药
COPPER IS REQUIRED for the survival of all aerobic organisms and is essential for the function of several enzymes (1, 2, 3). Copper is not stored in a significant amount in the body and must be obtained from dietary sources before delivery to the appropriate tissues (4, 5). Although many proteins involved in fundamental biological functions rely on copper ions to serve as catalytic cofactors for redox reactions, the conversion of Cu(II) to Cu(I) generates reactive oxygen species that can damage susceptible cellular components (4, 6). Therefore, organisms have evolved a complex system of metal ion transporters and chaperones to regulate copper homeostasis and ensure that copper is provided to essential proteins without causing cellular damage (for review, see Refs. 7 and 8).dp44/y5, 百拇医药
Copper transporters are highly conserved from yeast to humans, and several of the key players have been cloned (9, 10, 11, 12, 13). In most cells, copper transporter 1 (Ctr1) (1) has been shown to be essential for the uptake of copper across the plasma membrane (Fig. 1) (4, 13, 14). Mice lacking Ctr1 are smaller than wild-type (WT) mice by embryonic d 7.5 and do not develop past embryonic d 12.5 (15). Specific cytosolic chaperones deliver copper to cytosolic copper-requiring enzymes such as the Cu/Zn superoxide dismutase (SOD) and to organelles, including mitochondria, for incorporation into enzymes such as cytochrome c oxidase (Fig. 1) (16, 17).
fig.ommitteed^, http://www.100md.com
Figure 1. The key players in copper metabolism. Key players in the uptake and distribution of copper in mammalian cells are indicated. CCS1, Copper chaperone for SOD; COX17, cytochrome c oxidase chaperone; Cyt Ox, cytochrome c oxidase; ER, endoplasmic reticulum.^, http://www.100md.com
A subset of the copper-dependent enzymes, including peptidylglycine {alpha} -amidating monooxygenase (PAM) and dopamine ß-monooxygenase, are localized to the lumen of the secretory pathway (18, 19, 20, 21). Both enzymes generate signaling molecules that are stored in large dense core vesicles before release in response to appropriate stimuli. It is not clear, however, how copper is delivered to these regulated secretory pathway copper-requiring enzymes. Two cation-translocating P-type ATPases, ATP7A (Menkes protein) and ATP7B (Wilson’s disease protein), transport cytosolic copper into the secretory pathway (22). Though ATP7B is most prevalent in the liver, ATP7A is more widely expressed (4, 23, 24).
Over 50% of all neuropeptides are {alpha} -amidated by PAM, and the glycine (Gly)-extended precursors from which {alpha} -amidated peptides are produced have vastly reduced biological activity (1). By limiting the availability of copper in vitro or in vivo, the production of {alpha} -amidated peptides such as cholecystokinin (CCK) (25), {alpha} -MSH (25, 26), LHRH, and substance P (27) is decreased. PAM catalyzes the conversion of peptidylglycine precursors into {alpha} -amidated products in a two-step process. First, peptidylglycine {alpha} -hydroxylating monooxygenase (PHM) catalyzes hydroxylation of the {alpha} -carbon of the carboxylterminal Gly in a reaction requiring copper, ascorbate, and molecular oxygen. Second, peptidyl {alpha} -hydroxyglycine {alpha} -amidating lyase (PAL), a zinc-dependent enzyme, converts the peptidyl {alpha} -hydroxyglycine intermediate into the {alpha} -amidated peptide with release of glyoxylate (28). The P-element-mediated deletion of PHM results in larval lethality in Drosophila and blocks the production of amidated peptides (29, 30).
The highest levels of PAM are found in the pituitary, adrenal medulla, atrium of the heart, and central nervous system (31, 32). Generation of peptidylglycine substrates for PHM requires prohormone cleavage by the appropriate prohormone convertases and carboxypeptidase E (33). Immunoelectron microscopy indicates that {alpha} -amidated peptides are first detected in the trans-most cisternae of the Golgi (34), with most {alpha} -amidated peptides localized to secretory granules (35). Although crystallographic studies have clearly demonstrated that two moles of copper are bound per mole of PHM (36), it remains unknown how or where in the cell PHM acquires the copper it needs to perform its catalytic role. Whether ATP7A, with its ability to pump copper into the lumen of the secretory pathway, is essential and a candidate to provide copper to PHM remains largely unexplored. Recently, it was shown that the expression of ATP7A is essential for the activity of tyrosinase, a copper-dependent secretory pathway enzyme that functions in melanosomes (37), thus providing evidence that ATP7A may be essential for other copper-dependent enzymes, including PHM. However, the expression of ATP7A has not been examined in tissues producing large amounts of {alpha} -amidated peptides.
Menkes disease is an X-linked recessive disorder of copper metabolism caused by mutations in the ATP7A gene (38). Patients with Menkes disease have abnormally low levels of copper in most of their organs except for the kidney and small intestine. In the most severe form of this disorder, affected males die in early childhood (for review, see Ref. 5). The hemizygous mottled-brindled male mouse (referred to here as the Atp7a mouse) has a six-nucleotide deletion in Atp7a, and serves as a model for human Menkes disease (39, 40). In this study, we used tissue from WT and Atp7a mice to determine whether ATP7A plays a role in the delivery of copper to PHM. First, we determined whether ATP7A is expressed at high levels in tissues expressing high levels of PAM. We then used cultured primary pituitary cells and frozen pituitary tissue sections to evaluate the localization of ATP7A. Atp7a mice were used to establish whether the function of PAM is compromised by the inactivation of ATP7A. Finally, PAM activity was evaluated by measuring levels of {alpha} -amidated peptides, using in vitro assays to assess the enzymatic activity of PHM.
Materials and Methods)x8kki, 百拇医药
Animals)x8kki, 百拇医药
A colony of mottled-brindled (Atp7a) mice was established by mating heterozygous mottled-brindled females (C57BL/6J Atp7aMo-br/+) with WT C57BL/6J males purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were bred and maintained, in the animal care facility at the University of Connecticut Health Center, on a 12-h light, 12-h dark cycle according to the guidelines set forth by the University of Connecticut Health Center Animal Care and Use Committee. In this study, only male hemizygous mice carrying the defective gene (Atp7a) and control littermates (male and female) were used. Male hemizygous Atp7a mice were easily identified because of the lack of pigmentation in their coats. Both WT and hemizygous Atp7a mice were killed, between postnatal d 10 and 12, by decapitation. The mean weight of the control animals was 5.4 ± 0.2 g (n = 22), compared with 3.8 ± 0.1 g (n = 26) for the male Atp7a hemizygous mice. To prevent tissue contamination, a dissecting microscope was used to excise the cortex, hypothalamus, atria, adrenals, and intact pituitary from each animal. Adult Sprague Dawley rats were purchased from Charles River Laboratories Inc. (Wilmington, MA) maintained as described for the mice, and killed by decapitation before excision of the pituitary. All animal experimentation described was conducted in accordance with the accepted standards of humane animal care as deemed appropriate by the Animal Care and Use Committee at the University of Connecticut Health Center.
Tissue extraction6&}{fjm, http://www.100md.com
Extracts of each tissue were prepared by homogenization in ice-cold 20-mM Na N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid [10 mM mannitol and 1% Triton X-100 (pH 7.4)] containing protease inhibitors (41). After three cycles of freezing and thawing, the samples were centrifuged at 13,000 rpm, and the supernatant was separated from the pellet and retained. The protein concentration for each sample was determined using the bicinchoninic acid protein reagent kit according to manufacturer’s instructions (Pierce Chemical Co., Rockford, IL).6&}{fjm, http://www.100md.com
Primary pituitary cell cultures and immunofluorescence microscopy6&}{fjm, http://www.100md.com
Primary pituitary cultures were prepared as previously described, with a few modifications (42, 43, 44). Intact pituitaries, dissected from WT male C57BL/6J or Atp7a male hemizygous mice, were dissociated (37 C) for 30 min in Liebovitz L-15 medium (Mediatech, Inc., Herndon, VA) containing 4 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, NJ), 1 mg/ml hyaluronidase (Sigma, St. Louis, MO), 0.1 U/ml benzonase (EM Industries, Darmstadt, Germany), and 10 mg/ml fatty acid-free BSA. Further dissociation was carried out in medium containing freshly dissolved 3 mg/ml trypsin (TO646; Sigma). The dissociated cells were plated on protamine and Nu-serum-coated, four-well glass slides and grown in DMEM/Ham’s F-12 supplemented with 10% fetal clone III (HyClone Laboratories, Inc., Logan, UT) and 10% Nu-serum IV (Collaborative Research, Bedford, MA); 10 µM cytosine arabinoside (Sigma) was added after 24 h. After 4–5 d, the pituitary cells were fixed either with 100% ice-cold methanol or 4% formaldehyde (37 C) in isotonic PBS. To assess the localization of various organelles, ATP7A, and PAM function, cells were incubated with antibodies to the following antigens: ATP7A(1475–1492) (CT77; 1:1,000; see below), ACTH (Kathy, 1:32,000; or monoclonal, 1:500; Novocastra Laboratories, Newcastle, UK) (44), joining peptide-NH2 (JP-NH2; Jamie, 1:16,000) (45), trans Golgi network (TGN) (38) (1:1,000) (20), or synaptotagmin (1:200; BD Biosciences Transduction Laboratories, Lexington, KY). The cells were then incubated with fluorescein isothiocyanate-conjugated goat antibody to rabbit IgG or Cy3-conjugated donkey antibody to mouse IgG (46). Cells were observed under epifluorescence optics on an Axioskop microscope (Carl Zeiss, Thornwood, NY) and photographed with a Spot RT camera (Diagnostic Instruments, Sterling Heights, MI) (21).
Generation of the COOH-terminal-specific ATP7A antibody6|, 百拇医药
A peptide, Asp1475-Lys-His-Ser-Leu-Leu-Val-Gly-Asp-Phe-Arg-Glu-Asp-Asp-Asp-Thr-Thr-Leu1492 [mouse (NM 009726) and rat (NM 052803) ATP7A are identical], from the COOH-terminal of ATP7A was synthesized on the Accurate Surgical \|[amp ]\| Scientific Instruments Corp. (Westbury, NY) Model 396 multiple peptide synthesizer using Fmoc chemistry. Peptide (4 mg in 4 ml 100-mM Na phosphate, pH 6.8) was mixed with keyhole limpet hemocyanin (8 mg in 1.0 ml 1-mM NaOH); half of the sample was linked using glutaraldehyde (10 µl of a 25% stock), and half was linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (2 ml of 4 mg/ml in 100-mM Na phosphate, pH 6.8; Bio-Rad Laboratories, Inc., Hercules, CA). After 3 h at room temperature, the two conjugates were mixed and dialyzed against 100 mM Na phosphate, pH 6.8. Two rabbits were each immunized with 120 µg peptide, followed by booster injections of 60 µg peptide (Covance Laboratories, Inc., Denver, PA). The serum from rabbit CT77 was used for the work presented in this manuscript. Specificity was verified by preincubating a 1:100 dilution of the antiserum in Tween 20 and Tris-buffered saline (or phosphate buffer for immunostaining) with 0.1 mg/ml peptide for 30 min at room temperature before diluting the mixture 10-fold and applying it to the polyvinylidene difluoride membrane or using it for immunostaining.
PHM enzyme assaysqw+]o$p, http://www.100md.com
To measure PHM activity, aliquots of tissue extracts or serum were assayed using 1.0 µM CuSO4 (5.0 µM for cortex and hypothalamus tissue extracts), 0.5 mM ascorbate, 0.5 µM Ac-Tyr-Val-Gly, trace amounts of [125I]–labeled Ac-Tyr-Val-Gly, 0.1 mg/ml catalase, and 150 mM Na MES (pH 5.5) (47, 48).qw+]o$p, http://www.100md.com
Western blot analysisqw+]o$p, http://www.100md.com
Aliquots of tissue extracts were fractionated on 4–15% polyacrylamide, 0.25% N,N'-methylene-bis-acrylamide/sodium dodecyl sulfate gels (49). Proteins transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) were visualized with one of the following primary antisera and the Amersham Enhanced Chemiluminescence Kit (Amersham Biosciences, Piscataway, NJ): Exon A [JH629; rPAM-1 (409–497);1:1000], PAL [JH471; rPAM-1 (463–864); 1:1000], PHM [JH1764; rPAM-1 (37–382);1:1000], or ATP7A (CT77; 1:1000) (28, 41).qw+]o$p, http://www.100md.com
Immunohistochemistry of pituitary tissue
Rats were anesthetized with ketamine (10 mg/100 g body weight, Sigma) and perfused transcardially with saline, followed by 4% paraformaldehyde in PBS (pH 7.4). The pituitary was removed and post-fixed overnight in 4% paraformaldehyde (4 C), followed by incubation in 25% sucrose in PBS containing 0.02% NaN3 (4 C). Sections (12 µm) were cut on a cryostat and collected on gelatin-coated slides.4, 百拇医药
Whole pituitaries from postnatal d 10–12 WT or Atp7a mice were fixed in Bouin’s solution for 20 h (4 C) and paraffin embedded. Sections of 6 µm were mounted on precoated microscope slides, dried at 50 C (30 min), dewaxed, and rehydrated. Endogenous peroxidase activity was blocked with 0.1% H2O2 in methanol (15 min). Pituitary sections from control and mutant animals were washed; blocked in phosphate buffer containing 1% BSA, 2% normal goat serum, and 0.2% Triton X-100 (1 h); and incubated overnight (4 C) with primary antibody (ACTH, 1:2000; JP-NH2 1:500; {alpha} -MSH, 1:8000; CT77, 1:500). Sections then were washed, and incubated (1 h) in biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA). After an additional wash step, sections were incubated in ABC reagent (Vector Laboratories, Inc.) before visualization with diaminobenzidine. Finally, sections were dehydrated with ethanol and cleared with xylene, and slides were coverslipped in Permount. Substitution of each primary antibody with phosphate buffer eliminated staining.
RIAs of proopiomelanocortin (POMC)-related peptidesko.p@, 百拇医药
Pituitaries from WT and Atp7a mice were extracted in 5 N acetic acid/2 mg/ml BSA with protease inhibitors, lyophilized, and dissolved in 50 mM Na phosphate buffer (pH 7.6) with 0.1% Triton X-100 added to measure immunoreactive ACTH and {alpha} -amidated joining peptide (JP-NH2). A COOH-terminal ACTH antibody (Kathy; 1:15,000) that detects ACTH biosynthetic intermediate, ACTH(1–39), and corticotropin-like intermediate lobe peptide [ACTH(1–39)] but not intact POMC, was used in the ACTH RIA (34, 50, 51). Synthetic human ACTH(1–39) (Ciba Pharmaceutical Co., Summit, NJ) was used to generate a standard curve with [125I]-ACTH(1–39) (NEN Life Science Products, Boston, MA). Antibody Jamie (1:1000), which detects {alpha} -amidated joining peptide but not Gly-extended joining peptide, was used in the JP-NH2 RIA (45). Synthetic D-Tyr-Joining-Peptide-(12–18)-NH2 (Vega Biotechnologies, Tucson, AZ) was used to generate a standard curve with [125I]-D-Tyr-Joining-Peptide-(12–18)-NH2 (45).
RIAs of CCK-related peptidesf$7[.7, 百拇医药
WT and Atp7a cortex and duodenum were extracted in 90% methanol and assayed for immunoreactive {alpha} -amidated CCK-8 (CCK-8-NH2) and Gly-extended CCK (CCK-Gly)-related peptides as previously described (52). Briefly, CCK-8 (CCK 26–33-NH2) was analyzed with rabbit polyclonal antiserum P-45, which has an absolute preference for C-terminal {alpha} -amidation and recognizes the N-terminal of CCK without preference to sulfation state. However, N-terminal shortening of CCK-8 by one or two amino acids reduces cross-reactivity to less than 8% and 1%, respectively (25). CCK-Gly was measured using antiserum no. 22 raised against CCK-8-Gly and exhibits less than 2% cross-reactivity with CCK-8-NH2, CCK-8-Gly-Arg, or CCK-8-Gly-Arg-Arg (52).f$7[.7, 百拇医药
mRNA analysisf$7[.7, 百拇医药
Total RNA was isolated using RNA Stat-60 reagent (Tel-Test B) (53). RT-PCR was used to determine relative levels of ATP7A mRNA in different tissues, using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as a loading control (53). Primers for ATP7A were based on the murine sequence in GenBank U71091: exon 11 sense (2443–2467); exon 11 antisense (2549–2525); downstream-sense (1950–1974); upstream-antisense (3043–3018). PCR conditions for ATP7A used an annealing step at 54 C (1 min; 35 cycles). G3PDH was annealed at 60 C (21 cycles); the same normalization was found using actin instead of G3PDH (53).
Results'5, 百拇医药
ATP7A is expressed highly in the pituitary and adrenal'5, 百拇医药
Although the tissue expression of ATP7A is known to be more widespread than that of ATP7B (4, 23, 24), data on the expression of ATP7A in endocrine tissues are not available. To address this issue, a rabbit polyclonal antibody was raised against the 18-amino-acid peptide at the COOH terminus of ATP7A [ATP7A(1475–1492)]. Notably, the sequence homology in this region of ATP7A is identical in rat and mouse, with a single nonconservative change in human. The specificity of this ATP7A antibody was tested first on crude soluble (S) and particulate (P) fractions prepared from rat liver and anterior pituitary (Fig. 2A). A single major band of 180 ± 5 kDa was detected in the particulate fraction of the rat anterior pituitary. As expected, for an integral membrane protein, no cross-reactive material was detected in the soluble fraction. Furthermore, no cross-reactive material was detected in a particulate fraction prepared from the rat liver, a tissue in which ATP7A has limited expression, if any (9, 10, 12). Preincubation of the ATP7A antiserum with the peptide antigen completely eliminated the signal from the rat anterior pituitary (Fig. 2B), demonstrating its specificity for ATP7A.
fig.ommitteed3, 百拇医药
Figure 2. The ATP7A protein is expressed highly in pituitary. A, Adult rat liver and anterior pituitary were separated into crude soluble (S) and particulate (P) fractions before fractionation of 30 µg protein, by SDS-PAGE. ATP7A was visualized using antiserum CT77 (diluted 1:1000). B, The ATP7A antiserum was preincubated with 0.1 mg/ml antigen in phosphate buffer before application ("Blocked"). C, Detergent extracts (20 µg protein) were prepared from the indicated WT, postnatal d 10–12 mouse tissues (Pit., whole pituitary; Hypo., hypothalamus) and fractionated by SDS-PAGE, and ATP7A was visualized using antiserum CT77. The mobilities of molecular weight markers analyzed simultaneously are indicated.3, 百拇医药
Next, we examined the protein expression of ATP7A in WT postnatal d 10–12 mice by preparing detergent extracts of tissues known to express high levels of PAM or dopamine ß-monooxygenase; pituitary, atrium, and adrenal, as well as cerebral cortex and hypothalamus, were examined (Fig. 2C). A single major cross-reactive protein of 180 ± 5 kDa was detected in each tissue examined. Interestingly, the levels of ATP7A protein were highest in pituitary and adrenal tissues rich in large dense core vesicles containing amidated peptide products and/or catecholamines, respectively. The presence of high levels of ATP7A in the pituitary, compared with the other tissues examined, suggests that ATP7A plays a role in delivering copper to PAM.
RT-PCR was used to compare the levels of ATP7A mRNA in a variety of mouse tissues (Fig. 3A). The selected ATP7A primers amplified a single product in all tissues examined. Levels of G3PDH were evaluated to enable normalization of the data across the tissues (Fig. 3B). When normalized to the levels of G3PDH product, the levels of ATP7A were 2- to 3-fold higher in the pituitary than in any other tissue examined (Fig. 3C). Although the levels of ATP7A mRNA were higher in pituitary than in adrenal, the levels of ATP7A protein were comparable in these tissues (Figs. 2C and 3C). This observation may reflect the extremely long lifetime of adrenal secretory granules (54, 55).k7, 百拇医药
fig.ommitteedk7, 百拇医药
Figure 3. ATP7A mRNA is expressed highly in pituitary. Tissues dissected from adult male mice (n = 2; experiments repeated six times) were used to prepare RNA: pit, whole pituitary; hyp, hypothalamus; adr, adrenal; hrt, heart; cor, cortex; cbl, cerebellum; o.b., olfactory bulb; spl, spleen; liv, liver. After RT, ATP7A transcripts (A) and G3PDH transcripts (B) were amplified, as described in Materials and Methods, using the exon 11 sense/upstream antisense primer pair for ATP7A. C, Expression of ATP7A was normalized to levels of G3PDH using scion image analysis software to quantify the scans for two analyses. The downstream sense/exon 11 antisense primer pair for ATP7A gave a similar distribution, and primers entirely within exon 11 (the exon with the 6-nucleotide deletion in the MoBr mice) gave the same tissue pattern in two experiments. There were no differences in ATP7A tissue distribution for WT and Atp7a mice. Results are expressed as the mean ± range.
ATP7A is localized to the TGN region in pituitary endocrine cells?[qy&hw, 百拇医药
To begin to determine the subcellular localization of ATP7A in pituitary, adult rats were perfused, and the pituitary was removed, fixed, and sectioned on a cryostat. As shown in Fig. 4A, all the cells in both the anterior and intermediate lobes of the pituitary were visualized with COOH-terminal-specific antibody for ATP7A, with no apparent differences in staining intensity. At higher magnification (Figs. 4, B and C), staining is seen localized to a distinct, compact perinuclear region in most cells in both the anterior and intermediate lobes. This pattern of staining resembles that of the TGN. A higher intensity of staining was observed in cells of the anterior pituitary.?[qy&hw, 百拇医药
fig.ommitteed?[qy&hw, 百拇医药
Figure 4. ATP7A is concentrated in the TGN area of pituitary endocrine cells. A–C, Sections from pituitaries of perfused rats were stained with antiserum specific for the COOH-terminus of ATP7A. B and C are high-power magnifications of A. Scale bar for A, 180 µm; scale bar for B and C, 20 µm. Ant, Anterior lobe; Int, intermediate lobe; Neural, neural lobe. D–I, Dissociated cells from whole pituitaries from postnatal d 12 C57BL/6J mice were fixed after 3–5 d in culture and immunostained for ATP7A (D and H) using the ATP7A antibody, for vesicles using a synaptotagmin monoclonal antibody (E), or for TGN using a TGN38 antibody (I). F shows the phase contrast micrograph of a pituitary cell incubated with the blocked ATP7A antibody (G). The scale bar for micrographs D–I is shown in I. N, Nucleus; arrows, staining in TGN region; arrowheads, staining in cell body.
In fibroblasts, ATP7A is localized to the TGN (56, 57, 58, 59, 60) and cycles to the plasma membrane in response to high levels of copper (61, 62, 63). To further explore the subcellular localization of ATP7A, primary cultures were prepared from whole pituitaries of WT postnatal d 10–12 C57BL/6J mice. After 3–5 d in culture, cells were fixed and stained simultaneously with the ATP7A and synaptotagmin antibodies (Fig. 4, D and E). The synaptotagmin antibody, a vesicle protein marker, was used to identify endocrine cells, and punctate staining was distributed throughout the cytosol of the endocrine cells (Fig. 4E). Intense staining in the perinuclear region of all the pituitary endocrine cells was observed using the ATP7A antibody (Fig. 4, D and H). To further confirm the specificity of the ATP7A antibody, pituitary cultures were immunostained with the ATP7A antibody preincubated with the ATP7A(1475–1492) peptide; staining of the pituitary cells did not occur (Fig. 4G; phase contrast of this cell is shown in Fig. 4F). Finally, pituitary cultures were immunostained singly with the TGN38 antibody to determine whether ATP7A is localized to the TGN (Fig. 4I). Based on the similar immunostaining patterns of the TGN38 and ATP7A antibodies (Fig. 4, H and I), we conclude that ATP7A is localized to the TGN in pituitary cells.
ATP7A and PAM expression and processing are unaltered in Atp7a mice\62*[s, 百拇医药
Having established that ATP7A can be detected in various endocrine tissues from adult and early postnatal mice, we compared the expression of ATP7A and PAM in WT and Atp7a mice with nonfunctional Atp7a. Because the hemizygous male mice bearing the mottled-brindled mutation (Atp7a) survive to about postnatal d 15 (64, 65, 66), WT and Atp7a mice were killed between postnatal d 10 and 12. Using Western blot analysis, it was established that ATP7A protein levels in pituitary, cortex, hypothalamus, atrium, and adrenal were similar in WT and Atp7a mice and that the mutant protein was not degraded (Fig. 5A).\62*[s, 百拇医药
fig.ommitteed\62*[s, 百拇医药
Figure 5. Levels of ATP7A and PAM protein are unaltered in Atp7a mice. Control, WT mice, and Atp7a mice were killed at postnatal d 10–12, and the tissues indicated (Hypoth., hypothalamus) were extracted in 20 mM Na N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 10 mM mannitol, 1% Triton X-100, pH 7.4. Aliquots (20 µg protein) were fractionated by SDS-PAGE. A, For the analysis of ATP7A, samples were fractionated on 4–15% gradient gels, transferred for 3 h, and visualized using the COOH-terminal ATP7A antibody (Ab) at 1:1000. B, For the analysis of PAM, samples were fractionated on 4–15% gradient gels, and products derived from PAM-1 were identified using an Ab to the Exon A region that separates PHM and PAL. The major products derived from PAM-1 are shown diagrammatically on the right, and the specificity of the Exon A Ab is depicted. The apparent molecular masses of the PAM products are indicated to the left of the blot.
Because the ATP7A protein in the Atp7a mice is nonfunctional, we wondered whether PAM synthesized in the presence of decreased copper supplies might be differentially processed or perhaps unstable. Levels of PAM protein were unaltered in Atp7a mutant mice (Fig. 5B). PAM is known to undergo tissue-specific endoproteolytic cleavages to generate smaller, soluble forms of PHM and membrane or soluble forms of PAL (67). Using Western blot analysis with a PAM antibody specific to the linker region between PHM and PAL (Exon A), we determined that the levels and types of PAM cleavage products were indistinguishable in each of the tissues extracted from the WT and Atp7a mice (Fig. 5B).o{, http://www.100md.com
Although these results clearly indicate that the PAM protein is expressed and processed correctly in the Atp7a mouse pituitary, these data do not indicate whether the PAM protein is catalytically competent. To address this issue, tissue extracts were prepared from WT and Atp7a mice (Fig. 6), protein concentrations were measured, and in vitro PHM assays were performed after the addition of optimal amounts of copper. Overall, the weak binding of copper to PHM means that the apoenzyme is recovered from tissue extracts (68, 69). Thus, for maximal levels of PHM activity to be detected (70), exogenous copper must be added to the tissue extracts during the assay reaction. Using these established assay conditions, no differences were observed in levels of PHM activity between the WT and Atp7a mice. Despite a deficit in functional ATP7A, no change occurred in the level of PAM protein expression or in its ability to catalyze the first step in the {alpha} -amidation reaction when assayed in the presence of exogenous copper.
fig.ommitteed*d*, 百拇医药
Figure 6. PHM Activity in vitro is unaltered in Atp7a mice pituitary. Detergent extracts of WT and Atp7a pituitary, cortex, hypothalamus, atrium, and adrenal were prepared as described in Fig. 5 (n = 3–21, depending on tissue). Assays for PHM activity were done either in the presence of 1 µM CuSO4 (pituitary, atrium, adrenal) or 5 µM CuSO4 (cortex, hypothalamus). Samples were assayed in duplicate at several different dilutions, and the data shown are the mean ± SEM. None of the pairs of samples are significantly different (P > 0.30).*d*, 百拇医药
The levels of immunoreactive -amidated joining peptide are reduced in the pituitary of Atp7a mice*d*, 百拇医药
Because the levels of -amidated peptides processed and stored in the pituitary should be a reliable indicator of how efficiently PAM functions in the pituitary, we assessed the levels of -amidated POMC end-products by RIA. POMC is the precursor of ACTH, which is not {alpha} -amidated, as well as of two -amidated products, JP-NH2 and ACTH (1–13)-NH2 or -MSH (Fig. 7A). As a control, ACTH levels were first evaluated using a COOH-terminal ACTH-specific antibody (C-ACTH) in whole pituitaries dissected from postnatal d 10–12 WT and Atp7a mice (Fig. 7B. The anterior and intermediate pituitary lobes were not separated for analysis, because the pituitaries in these young animals are too small and fragile for reliable dissection. As shown in Fig. 7B, pituitary extracts from WT and Atp7a mice contained similar levels of C-ACTH, whereas JP-NH2 levels were reduced in the Atp7a mice. To control for sample-to-sample variability, levels of JP-NH2 were normalized to levels of C-ACTH. Based on this ratio, -amidation of JP was reduced to half of control levels in the pituitaries of the Atp7a mice.
fig.ommitteed:i, 百拇医药
Figure 7. The ability of the Atp7a mice to produce -amidated peptides is compromised. A, Proopiomelanocortin is converted into a variety of smaller end-products. The specificities of the antisera used, in this study, for these products are indicated (JP-NH2; ACTH(1–13)NH2 or -MSH; C-terminal ACTH, C-ACTH). B, Pituitaries from individual mice or pools of two to three mice were extracted for analysis of peptide content by RIA. Levels of C-ACTH and JP-NH2 are plotted per pituitary (pmol/pituitary) on the left side of the graph, and the ratios of JP-NH2 to ACTH in individual pituitaries are shown on the right side of the graph. The C-ACTH values are not different (P > 0.22); the JP-NH2 values are quite different (P < 0.0016), as are the JP-NH2/C-ACTH ratios (P < 0.00004). C, Pituitaries dissected from WT and Atp7a mice were dissociated and plated onto glass slides. The primary cultures were fixed and stained with antisera to ACTH or JP-NH2. Micrographs of WT and Atp7a cultures were photographed using the same parameters. The scale bar for all micrographs is shown in the last panel. LPH, -Lipotropin; ß-End, ß-endorphin.
The decreased JP-NH2 content of the Atp7a mouse pituitary could be attributable to low circulating levels of copper resulting from its poor uptake of copper in the small intestine and poor suckling. Therefore, we examined dissociated cells in culture prepared from whole pituitaries of WT and Atp7a mice so that the effects of circulating copper could be eliminated. The DMEM/F-12 medium minus serum in which the cultures were grown was measured to contain 0.1 µM copper. After incubation for 3–5 d, the primary pituitary cultures were fixed and immunostained with antiserum to ACTH or JP-NH2 (Fig. 7C). The ACTH-producing endocrine cells exhibited similar staining intensity for ACTH in both the WT and Atp7a mice. In contrast, POMC cells from WT mice stained intensely with the JP-NH2 antibody, whereas POMC cells from Atp7a mice did not. The ability of PAM to produce -amidated POMC products was compromised in dissociated pituitary cells from mice expressing a nonfunctional ATP7A protein.
Peptide -amidation is compromised in POMC cells of Atp7a micep;, 百拇医药
To examine POMC processing in anterior pituitary corticotropes and intermediate pituitary melanotropes, whole pituitaries (0.3–0.5 mg) from WT and Atp7a mice were fixed and sectioned. Antiserum specific for the COOH-terminus of ACTH visualized a subset of the cells in the anterior pituitary and all of the cells in the intermediate lobe (Fig. 8, A and D). No differences in staining intensity were apparent, in a comparison of WT with Atp7a animals, for either the intermediate (Fig. 8, B and E) or anterior lobe (Fig. 8, C and F) of the pituitary.p;, 百拇医药
fig.ommitteedp;, 百拇医药
Figure 8. The levels of immunoreactive -MSH and JP-NH2, but not ACTH, are reduced in the pituitaries of Atp7a mice. Pituitaries from WT and Atp7a mice were fixed in Bouin’s solution and embedded in paraffin. Sections were stained either with antisera specific for the COOH-terminus of ACTH or -amidated MSH (-MSH) or -amidated joining peptide (JP-NH2). B and C are high-power magnifications of A. E and F are high-power magnifications of D. H and J are high-power magnifications of panels G and I, respectively. Scale bar for A, D, G, and I, 120 µm. Scale bar for B, C, E, F, H, J, K, and L, 50 µm. The intermediate lobes in D and I (Atp7a mice) appear larger than that in A and G (WT mice), but this reflects the plane of sectioning and is not a consistent finding. N, Neural lobe; I, intermediate lobe; Ant Pit, anterior lobe.
The presence of -MSH was evaluated using an antiserum specific for the {alpha} -amidated COOH-terminus of this peptide (Fig. 7A). The -MSH antiserum does not cross-react with ACTH or the Gly-extended form of the peptide, -MSH-Gly. Intermediate pituitary melanotropes make {alpha} -MSH and corticotropin-like intermediate lobe peptide. In young mice, as in young rats, ACTH is cleaved into an MSH-sized peptide in anterior pituitary corticotropes; in adult animals, little of this cleavage occurs in corticotropes (70, 71). Intense staining for -MSH was apparent in the entire intermediate lobe (Fig. 8G) and in many of the anterior pituitary corticotropes of the WT animals (Fig. 8, G and H). In contrast, substantially less staining was observed in the anterior pituitaries of the tp7a mice (Fig. 8, I and J), and staining in the melanotropes was also less intense (Fig. 8I). Finally, the presence of JP-NH2 was evaluated using an antiserum specific for its {alpha} -amidated COOH-terminus. JP-NH2 is produced in both corticotropes and melanotropes. Consistent with the immunoassay data for whole pituitaries (Fig. 7B), staining for JP-NH2 in the corticotropes of Atp7a mice (Fig. 8J) was substantially lower in intensity than in the corticotropes of the WT mice (Fig. 8K). Although PAM protein is present at normal levels in the pituitary of the Atp7a mouse and is fully functional when copper is added in test tube assays, PAM does not function properly in the secretory pathway of endocrine cells in the pituitaries of these mice.
The levels of immunoreactive {alpha} -amidated CCK are reduced in the cerebral cortex of Atp7a mice}, 百拇医药
CCK is derived from preproCCK, and the major form of immunoreactive CCK in the nervous system is CCK4 (72). Extracts of cerebral cortex from WT and Atp7a mice were assayed for {alpha} -amidated CCK (CCK-NH2) and for the CCK-Gly (Fig. 9A). In the WT mice, most of the immunoreactive CCK present in cerebral cortex extracts was in the -amidated form. In contrast, in the Atp7a animals, levels of immunoreactive CCK-Gly were higher than levels of CCK-NH2. The fraction of {alpha} -amidated CCK was 4-fold lower in the cerebral cortex of Atp7a mice than in control mice. Levels of CCK mRNA were similar in WT and Atp7a mice (data not shown). In contrast to the CCK processing observed in the cortex, in the duodenum of both WT and Atp7a mice, very little of the CCK stored in the tissue was -amidated, whereas the levels of CCK-Gly greatly exceeded the levels of CCK-NH2 (Fig. 9B). Although the PAM present in Atp7a animals is capable of functioning when provided with exogenous copper, its function is impaired in the tissues of the Atp7a mice.
fig.ommitteed(, 百拇医药
Figure 9. The levels of immunoreactive CCK-NH2 are reduced in the cerebral cortex, but not in the duodenum, of the Atp7a mice. WT and Atp7a mice were killed at postnatal d 10–12, and cortex and duodenum were dissected and frozen before extraction in 90% methanol. Extracts were analyzed for CCK content by RIA. Levels of immunoreactive {alpha} -amidated CCK (CCK-NH2) and CCK-Gly are plotted per milligram of tissue (pmol/mg) or as the fraction amidated for (A) cerebral cortex and (B) duodenum, mean ± SE. All the differences in the cortex samples are statistically significant (CCK-NH2, P < 0.003; CCK-Gly, P < 0.03; fraction, P < 0.000001), whereas none of the differences in the duodenum rise significantly (CCK-NH2, P > 0.78; CCK-Gly, P > 0.33; fraction, P > 0.11).(, 百拇医药
Discussion(, 百拇医药
Copper is required for the survival of organisms from yeast to humans (2, 3). Presumably, because of the unique redox properties of copper, copper transporter proteins have evolved to maintain cellular copper homeostasis (4, 6). A proposed model for mammalian copper uptake and distribution at the cellular level is shown in Fig. 1 (4, 73). After copper is reduced from Cu(II) to Cu(I) by a membrane reductase (Fig. 1, step 1), Ctr1 mediates the passive uptake of Cu(I) across the plasma membrane (Fig. 1; step 2) (15, 74). Intracellular free copper concentrations are maintained at extremely low levels by small cytosolic chaperones that bind and distribute Cu(I) to specific subcellular compartments. Copper is delivered to cytochrome c oxidase in the mitochondria by cytochrome c oxidase chaperone 17 (Fig. 1, step 3), to cytoplasmic Cu/Zn SOD by CCS1 (copper chaperone for SOD) (Fig. 1, step 4), and to the TGN/secretory compartment by ATX1 homolog HAH1 (Fig. 1, step 5) (17, 75, 76, 77, 78).
In this work, we explored the role of ATP7A in delivering copper to PAM, a secretory granule enzyme (Fig. 1, step 6). Recently, ATP7A was shown to be required for the activity of tyrosinase, a secreted copper-dependent enzyme, which is synthesized in the secretory pathway and involved in melanogenesis (37). The results of our study provide evidence that ATP7A is involved in the delivery of copper to the membrane-bound copper-dependent enzyme, PAM, in the mammalian pituitary and brain. Furthermore, our data suggest that many of the developmental failures observed in patients with Menkes disease may reflect the lack of amidated peptides at specific target sites.:\8, 百拇医药
Using RT-PCR, we established that ATP7A mRNA is present in both endocrine and brain tissues that have high PAM expression (Fig. 3) (31, 32). Overall, ATP7A mRNA levels are highest in the pituitary. Western blot analyses confirmed that the ATP7A protein is highly expressed in both the pituitary and adrenal and is present in the cerebral cortex, hypothalamus, and atrium of the heart (Fig. 2). Both the mRNA and protein levels for ATP7A are very high in pituitary, where PAM is crucial to the production of several amidated peptides, including {alpha} -MSH, galanin, and pituitary adenylate cyclase-activating polypeptide (79, 80, 81). Interestingly, though ATP7A mRNA is detected in the rat liver, the ATP7A protein is not well expressed in this tissue, where ATP7B acts as the predominant copper-transporting ATPase (82). Discrepancies between mRNA and protein levels have been observed for other proteins early in development (83, 84).
PAM is active in the TGN and immature secretory granules and is localized predominantly to these organelles at steady-state in mammalian cells (20, 34, 35, 45). However, it is not known when newly synthesized PAM acquires the copper it needs to become functional. In cultured fibroblasts, both ATP7A and PAM are localized to the TGN (56, 57, 58, 59, 60) and cycle from the TGN to the plasma membrane and back (61, 62, 63, 85). Immunohistochemistry of whole pituitary, using the ATP7A antibody and double immunostaining of cultured pituitary cells from postnatal d 10–12 WT mice with ATP7A and TGN38 antibodies, demonstrates that the ATP7A is localized to the TGN region of endocrine cells (Fig. 4, A and B). Colocalization of PAM and ATP7A to the TGN suggests that copper could become available to PAM in this compartment.j;sq4:, http://www.100md.com
To explore a role for ATP7A in the delivery of copper to PAM, we used the mottled-brindled mutant mouse (Atp7a). This mouse serves as a good model for human Menkes disease, with mutations in the murine homolog of the Menkes gene (40, 86). The Atp7a protein is nonfunctional because of deletion of Ala799-Leu800 (39, 86). The X-linked mutant male mice have severe neurological problems, hypopigmentation, curly whiskers, ataxia, and tremors and are emaciated and do not survive past about postnatal d 15 (3, 65, 66). Although the causes of neurodegeneration, a hallmark of Menkes disease, are not fully understood, it seems that the reduced activities of several copper-dependent enzymes within the secretory pathway of neuroendocrine cells, such as PAM, could contribute.
Using the ATP7A antibody and Western blot analysis (Fig. 5), we detected mutant Atp7a in several endocrine tissues of the Atp7a mouse. The mutant Atp7a protein is the correct size and is not degraded, although malfolded proteins are often rapidly degraded (for review, see Ref. 87). Alternatively, the presence of a negative feedback loop monitoring copper availability could result in an elevation in levels of ATP7A, as seen with POMC expression after adrenal failure (88). We then examined PAM, which requires copper to function properly, in extracts of Atp7a tissue, finding that the levels and processing of PAM were normal. We also determined, using an in vitro assay in which exogenous copper was supplied, that the PAM protein is fully active in endocrine and brain tissues from Atp7a mice (Fig. 6). Thus, PAM in the Atp7a mice is synthesized correctly and has the ability to amidate peptides in vitro as long as it receives copper.9m11--i, 百拇医药
The activity of PAM in the secretory granules of the Atp7a mice was assessed by monitoring levels of {alpha} -MSH and JP-NH2, two amidated peptides derived from POMC (33). It has been suggested that -MSH plays a role in development, because it is produced as a major end-product in the anterior pituitary lobe of the postnatal rat, instead of intact ACTH as in the adult anterior pituitary (89, 90). RIAs, comparing levels of immunoreactive amidated JP-NH2 to levels of ACTH, established that amidated JP-NH2 is reduced 2-fold in the Atp7a mice pituitary (Fig. 7). Additionally, immunohistochemistry showed that -MSH and JP-NH2 are reduced in the pituitary of Atp7a mice when compared with ACTH (Fig. 8). Overall, these data demonstrate that ATP7A plays an important role in the delivery of copper to PAM sequestered in the secretory pathway, lending support to the idea that a reduction of amidated peptides could contribute to the developmental problems associated with Menkes disease.
Levels of CCK-NH2 were used to assess how well PAM can amidate peptides in the cerebral cortex of mice with nonfunctional Atp7a. We assayed levels of CCK-NH2 and compared them with levels of the CCK-Gly in the mouse cerebral cortex, where low-molecular-weight forms of amidated CCK are produced (72, 91, 92). We also compared levels of immunoreactive CCK-NH2 with CCK-Gly in the duodenum, where mostly high molecular weight forms of CCK are found (92). As expected from previous studies (93), a large fraction of the total CCK in the duodenum in both the WT and Atp7a mouse remains in the Gly-extended form. However, in the cerebral cortex, the usually high levels of amidated CCK-NH2 are dramatically reduced in the Atp7a mice. Because CCK-NH2 has an important role as a neurotransmitter or neuromodulator in the brain (72), the underproduction of amidated CCK could contribute to some of the debilitating effects observed in patients with Menkes disease.
Based on these results, we conclude that the ability of PAM to produce -amidated peptides is greatly compromised in Atp7a mice, evidenced by the major loss of several of the amidated neuropeptides involved in neuronal growth and development (79, 80, 81, 94, 95, 96, 97, 98, 99, 100). If the ability of PAM to amidate these important peptides is affected by reduced levels of copper, as occurs in the pituitary and cortex of the Atp7a mouse, then the targets of these amidated peptides could also be greatly affected. Thus, the lack of amidated peptides affecting many target tissues at crucial periods in development could contribute to the Menkes disease phenotype.2], 百拇医药
Acknowledgments2], 百拇医药
We wish to thank Dr. Henry Keutmann (Endocrine Unit, Massachusetts General Hospital, Boston, MA) for synthesizing the ATP7A peptide, Dr. Carolyn Worby (Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI) for providing rat CCK cDNA, and Drs. Jonathan Gitlin and Iqbal Hamza (Department of Pediatrics, Washington University School of Medicine, St. Louis, MO) for providing antisera to ATP7A that were used to initiate this work. We also wish to thank Dr. Michael Petris (University of Missouri-Columbia, Columbia, MO) for Chinese hamster ovary cells expressing myc-ATP7A and Dr. Margery C. Beinfeld (Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA) for providing the no. 22 anti-CCK-Gly antiserum. We also express our thanks to Shana Berger for assistance with the mRNA analysis. Finally, we wish to thank Marie Bell and Lixian Jin for laboratory assistance in Baltimore, MD, and Darlene D’Amato for maintaining the mottled-brindled mouse colony and for general laboratory assistance in Farmington, CT.
Received July 15, 2002.!i, 百拇医药
Accepted for publication September 9, 2002.!i, 百拇医药
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Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine {alpha} -amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.
Introductiondp44/y5, 百拇医药
COPPER IS REQUIRED for the survival of all aerobic organisms and is essential for the function of several enzymes (1, 2, 3). Copper is not stored in a significant amount in the body and must be obtained from dietary sources before delivery to the appropriate tissues (4, 5). Although many proteins involved in fundamental biological functions rely on copper ions to serve as catalytic cofactors for redox reactions, the conversion of Cu(II) to Cu(I) generates reactive oxygen species that can damage susceptible cellular components (4, 6). Therefore, organisms have evolved a complex system of metal ion transporters and chaperones to regulate copper homeostasis and ensure that copper is provided to essential proteins without causing cellular damage (for review, see Refs. 7 and 8).dp44/y5, 百拇医药
Copper transporters are highly conserved from yeast to humans, and several of the key players have been cloned (9, 10, 11, 12, 13). In most cells, copper transporter 1 (Ctr1) (1) has been shown to be essential for the uptake of copper across the plasma membrane (Fig. 1) (4, 13, 14). Mice lacking Ctr1 are smaller than wild-type (WT) mice by embryonic d 7.5 and do not develop past embryonic d 12.5 (15). Specific cytosolic chaperones deliver copper to cytosolic copper-requiring enzymes such as the Cu/Zn superoxide dismutase (SOD) and to organelles, including mitochondria, for incorporation into enzymes such as cytochrome c oxidase (Fig. 1) (16, 17).
fig.ommitteed^, http://www.100md.com
Figure 1. The key players in copper metabolism. Key players in the uptake and distribution of copper in mammalian cells are indicated. CCS1, Copper chaperone for SOD; COX17, cytochrome c oxidase chaperone; Cyt Ox, cytochrome c oxidase; ER, endoplasmic reticulum.^, http://www.100md.com
A subset of the copper-dependent enzymes, including peptidylglycine {alpha} -amidating monooxygenase (PAM) and dopamine ß-monooxygenase, are localized to the lumen of the secretory pathway (18, 19, 20, 21). Both enzymes generate signaling molecules that are stored in large dense core vesicles before release in response to appropriate stimuli. It is not clear, however, how copper is delivered to these regulated secretory pathway copper-requiring enzymes. Two cation-translocating P-type ATPases, ATP7A (Menkes protein) and ATP7B (Wilson’s disease protein), transport cytosolic copper into the secretory pathway (22). Though ATP7B is most prevalent in the liver, ATP7A is more widely expressed (4, 23, 24).
Over 50% of all neuropeptides are {alpha} -amidated by PAM, and the glycine (Gly)-extended precursors from which {alpha} -amidated peptides are produced have vastly reduced biological activity (1). By limiting the availability of copper in vitro or in vivo, the production of {alpha} -amidated peptides such as cholecystokinin (CCK) (25), {alpha} -MSH (25, 26), LHRH, and substance P (27) is decreased. PAM catalyzes the conversion of peptidylglycine precursors into {alpha} -amidated products in a two-step process. First, peptidylglycine {alpha} -hydroxylating monooxygenase (PHM) catalyzes hydroxylation of the {alpha} -carbon of the carboxylterminal Gly in a reaction requiring copper, ascorbate, and molecular oxygen. Second, peptidyl {alpha} -hydroxyglycine {alpha} -amidating lyase (PAL), a zinc-dependent enzyme, converts the peptidyl {alpha} -hydroxyglycine intermediate into the {alpha} -amidated peptide with release of glyoxylate (28). The P-element-mediated deletion of PHM results in larval lethality in Drosophila and blocks the production of amidated peptides (29, 30).
The highest levels of PAM are found in the pituitary, adrenal medulla, atrium of the heart, and central nervous system (31, 32). Generation of peptidylglycine substrates for PHM requires prohormone cleavage by the appropriate prohormone convertases and carboxypeptidase E (33). Immunoelectron microscopy indicates that {alpha} -amidated peptides are first detected in the trans-most cisternae of the Golgi (34), with most {alpha} -amidated peptides localized to secretory granules (35). Although crystallographic studies have clearly demonstrated that two moles of copper are bound per mole of PHM (36), it remains unknown how or where in the cell PHM acquires the copper it needs to perform its catalytic role. Whether ATP7A, with its ability to pump copper into the lumen of the secretory pathway, is essential and a candidate to provide copper to PHM remains largely unexplored. Recently, it was shown that the expression of ATP7A is essential for the activity of tyrosinase, a copper-dependent secretory pathway enzyme that functions in melanosomes (37), thus providing evidence that ATP7A may be essential for other copper-dependent enzymes, including PHM. However, the expression of ATP7A has not been examined in tissues producing large amounts of {alpha} -amidated peptides.
Menkes disease is an X-linked recessive disorder of copper metabolism caused by mutations in the ATP7A gene (38). Patients with Menkes disease have abnormally low levels of copper in most of their organs except for the kidney and small intestine. In the most severe form of this disorder, affected males die in early childhood (for review, see Ref. 5). The hemizygous mottled-brindled male mouse (referred to here as the Atp7a mouse) has a six-nucleotide deletion in Atp7a, and serves as a model for human Menkes disease (39, 40). In this study, we used tissue from WT and Atp7a mice to determine whether ATP7A plays a role in the delivery of copper to PHM. First, we determined whether ATP7A is expressed at high levels in tissues expressing high levels of PAM. We then used cultured primary pituitary cells and frozen pituitary tissue sections to evaluate the localization of ATP7A. Atp7a mice were used to establish whether the function of PAM is compromised by the inactivation of ATP7A. Finally, PAM activity was evaluated by measuring levels of {alpha} -amidated peptides, using in vitro assays to assess the enzymatic activity of PHM.
Materials and Methods)x8kki, 百拇医药
Animals)x8kki, 百拇医药
A colony of mottled-brindled (Atp7a) mice was established by mating heterozygous mottled-brindled females (C57BL/6J Atp7aMo-br/+) with WT C57BL/6J males purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were bred and maintained, in the animal care facility at the University of Connecticut Health Center, on a 12-h light, 12-h dark cycle according to the guidelines set forth by the University of Connecticut Health Center Animal Care and Use Committee. In this study, only male hemizygous mice carrying the defective gene (Atp7a) and control littermates (male and female) were used. Male hemizygous Atp7a mice were easily identified because of the lack of pigmentation in their coats. Both WT and hemizygous Atp7a mice were killed, between postnatal d 10 and 12, by decapitation. The mean weight of the control animals was 5.4 ± 0.2 g (n = 22), compared with 3.8 ± 0.1 g (n = 26) for the male Atp7a hemizygous mice. To prevent tissue contamination, a dissecting microscope was used to excise the cortex, hypothalamus, atria, adrenals, and intact pituitary from each animal. Adult Sprague Dawley rats were purchased from Charles River Laboratories Inc. (Wilmington, MA) maintained as described for the mice, and killed by decapitation before excision of the pituitary. All animal experimentation described was conducted in accordance with the accepted standards of humane animal care as deemed appropriate by the Animal Care and Use Committee at the University of Connecticut Health Center.
Tissue extraction6&}{fjm, http://www.100md.com
Extracts of each tissue were prepared by homogenization in ice-cold 20-mM Na N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid [10 mM mannitol and 1% Triton X-100 (pH 7.4)] containing protease inhibitors (41). After three cycles of freezing and thawing, the samples were centrifuged at 13,000 rpm, and the supernatant was separated from the pellet and retained. The protein concentration for each sample was determined using the bicinchoninic acid protein reagent kit according to manufacturer’s instructions (Pierce Chemical Co., Rockford, IL).6&}{fjm, http://www.100md.com
Primary pituitary cell cultures and immunofluorescence microscopy6&}{fjm, http://www.100md.com
Primary pituitary cultures were prepared as previously described, with a few modifications (42, 43, 44). Intact pituitaries, dissected from WT male C57BL/6J or Atp7a male hemizygous mice, were dissociated (37 C) for 30 min in Liebovitz L-15 medium (Mediatech, Inc., Herndon, VA) containing 4 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, NJ), 1 mg/ml hyaluronidase (Sigma, St. Louis, MO), 0.1 U/ml benzonase (EM Industries, Darmstadt, Germany), and 10 mg/ml fatty acid-free BSA. Further dissociation was carried out in medium containing freshly dissolved 3 mg/ml trypsin (TO646; Sigma). The dissociated cells were plated on protamine and Nu-serum-coated, four-well glass slides and grown in DMEM/Ham’s F-12 supplemented with 10% fetal clone III (HyClone Laboratories, Inc., Logan, UT) and 10% Nu-serum IV (Collaborative Research, Bedford, MA); 10 µM cytosine arabinoside (Sigma) was added after 24 h. After 4–5 d, the pituitary cells were fixed either with 100% ice-cold methanol or 4% formaldehyde (37 C) in isotonic PBS. To assess the localization of various organelles, ATP7A, and PAM function, cells were incubated with antibodies to the following antigens: ATP7A(1475–1492) (CT77; 1:1,000; see below), ACTH (Kathy, 1:32,000; or monoclonal, 1:500; Novocastra Laboratories, Newcastle, UK) (44), joining peptide-NH2 (JP-NH2; Jamie, 1:16,000) (45), trans Golgi network (TGN) (38) (1:1,000) (20), or synaptotagmin (1:200; BD Biosciences Transduction Laboratories, Lexington, KY). The cells were then incubated with fluorescein isothiocyanate-conjugated goat antibody to rabbit IgG or Cy3-conjugated donkey antibody to mouse IgG (46). Cells were observed under epifluorescence optics on an Axioskop microscope (Carl Zeiss, Thornwood, NY) and photographed with a Spot RT camera (Diagnostic Instruments, Sterling Heights, MI) (21).
Generation of the COOH-terminal-specific ATP7A antibody6|, 百拇医药
A peptide, Asp1475-Lys-His-Ser-Leu-Leu-Val-Gly-Asp-Phe-Arg-Glu-Asp-Asp-Asp-Thr-Thr-Leu1492 [mouse (NM 009726) and rat (NM 052803) ATP7A are identical], from the COOH-terminal of ATP7A was synthesized on the Accurate Surgical \|[amp ]\| Scientific Instruments Corp. (Westbury, NY) Model 396 multiple peptide synthesizer using Fmoc chemistry. Peptide (4 mg in 4 ml 100-mM Na phosphate, pH 6.8) was mixed with keyhole limpet hemocyanin (8 mg in 1.0 ml 1-mM NaOH); half of the sample was linked using glutaraldehyde (10 µl of a 25% stock), and half was linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (2 ml of 4 mg/ml in 100-mM Na phosphate, pH 6.8; Bio-Rad Laboratories, Inc., Hercules, CA). After 3 h at room temperature, the two conjugates were mixed and dialyzed against 100 mM Na phosphate, pH 6.8. Two rabbits were each immunized with 120 µg peptide, followed by booster injections of 60 µg peptide (Covance Laboratories, Inc., Denver, PA). The serum from rabbit CT77 was used for the work presented in this manuscript. Specificity was verified by preincubating a 1:100 dilution of the antiserum in Tween 20 and Tris-buffered saline (or phosphate buffer for immunostaining) with 0.1 mg/ml peptide for 30 min at room temperature before diluting the mixture 10-fold and applying it to the polyvinylidene difluoride membrane or using it for immunostaining.
PHM enzyme assaysqw+]o$p, http://www.100md.com
To measure PHM activity, aliquots of tissue extracts or serum were assayed using 1.0 µM CuSO4 (5.0 µM for cortex and hypothalamus tissue extracts), 0.5 mM ascorbate, 0.5 µM Ac-Tyr-Val-Gly, trace amounts of [125I]–labeled Ac-Tyr-Val-Gly, 0.1 mg/ml catalase, and 150 mM Na MES (pH 5.5) (47, 48).qw+]o$p, http://www.100md.com
Western blot analysisqw+]o$p, http://www.100md.com
Aliquots of tissue extracts were fractionated on 4–15% polyacrylamide, 0.25% N,N'-methylene-bis-acrylamide/sodium dodecyl sulfate gels (49). Proteins transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) were visualized with one of the following primary antisera and the Amersham Enhanced Chemiluminescence Kit (Amersham Biosciences, Piscataway, NJ): Exon A [JH629; rPAM-1 (409–497);1:1000], PAL [JH471; rPAM-1 (463–864); 1:1000], PHM [JH1764; rPAM-1 (37–382);1:1000], or ATP7A (CT77; 1:1000) (28, 41).qw+]o$p, http://www.100md.com
Immunohistochemistry of pituitary tissue
Rats were anesthetized with ketamine (10 mg/100 g body weight, Sigma) and perfused transcardially with saline, followed by 4% paraformaldehyde in PBS (pH 7.4). The pituitary was removed and post-fixed overnight in 4% paraformaldehyde (4 C), followed by incubation in 25% sucrose in PBS containing 0.02% NaN3 (4 C). Sections (12 µm) were cut on a cryostat and collected on gelatin-coated slides.4, 百拇医药
Whole pituitaries from postnatal d 10–12 WT or Atp7a mice were fixed in Bouin’s solution for 20 h (4 C) and paraffin embedded. Sections of 6 µm were mounted on precoated microscope slides, dried at 50 C (30 min), dewaxed, and rehydrated. Endogenous peroxidase activity was blocked with 0.1% H2O2 in methanol (15 min). Pituitary sections from control and mutant animals were washed; blocked in phosphate buffer containing 1% BSA, 2% normal goat serum, and 0.2% Triton X-100 (1 h); and incubated overnight (4 C) with primary antibody (ACTH, 1:2000; JP-NH2 1:500; {alpha} -MSH, 1:8000; CT77, 1:500). Sections then were washed, and incubated (1 h) in biotinylated antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA). After an additional wash step, sections were incubated in ABC reagent (Vector Laboratories, Inc.) before visualization with diaminobenzidine. Finally, sections were dehydrated with ethanol and cleared with xylene, and slides were coverslipped in Permount. Substitution of each primary antibody with phosphate buffer eliminated staining.
RIAs of proopiomelanocortin (POMC)-related peptidesko.p@, 百拇医药
Pituitaries from WT and Atp7a mice were extracted in 5 N acetic acid/2 mg/ml BSA with protease inhibitors, lyophilized, and dissolved in 50 mM Na phosphate buffer (pH 7.6) with 0.1% Triton X-100 added to measure immunoreactive ACTH and {alpha} -amidated joining peptide (JP-NH2). A COOH-terminal ACTH antibody (Kathy; 1:15,000) that detects ACTH biosynthetic intermediate, ACTH(1–39), and corticotropin-like intermediate lobe peptide [ACTH(1–39)] but not intact POMC, was used in the ACTH RIA (34, 50, 51). Synthetic human ACTH(1–39) (Ciba Pharmaceutical Co., Summit, NJ) was used to generate a standard curve with [125I]-ACTH(1–39) (NEN Life Science Products, Boston, MA). Antibody Jamie (1:1000), which detects {alpha} -amidated joining peptide but not Gly-extended joining peptide, was used in the JP-NH2 RIA (45). Synthetic D-Tyr-Joining-Peptide-(12–18)-NH2 (Vega Biotechnologies, Tucson, AZ) was used to generate a standard curve with [125I]-D-Tyr-Joining-Peptide-(12–18)-NH2 (45).
RIAs of CCK-related peptidesf$7[.7, 百拇医药
WT and Atp7a cortex and duodenum were extracted in 90% methanol and assayed for immunoreactive {alpha} -amidated CCK-8 (CCK-8-NH2) and Gly-extended CCK (CCK-Gly)-related peptides as previously described (52). Briefly, CCK-8 (CCK 26–33-NH2) was analyzed with rabbit polyclonal antiserum P-45, which has an absolute preference for C-terminal {alpha} -amidation and recognizes the N-terminal of CCK without preference to sulfation state. However, N-terminal shortening of CCK-8 by one or two amino acids reduces cross-reactivity to less than 8% and 1%, respectively (25). CCK-Gly was measured using antiserum no. 22 raised against CCK-8-Gly and exhibits less than 2% cross-reactivity with CCK-8-NH2, CCK-8-Gly-Arg, or CCK-8-Gly-Arg-Arg (52).f$7[.7, 百拇医药
mRNA analysisf$7[.7, 百拇医药
Total RNA was isolated using RNA Stat-60 reagent (Tel-Test B) (53). RT-PCR was used to determine relative levels of ATP7A mRNA in different tissues, using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as a loading control (53). Primers for ATP7A were based on the murine sequence in GenBank U71091: exon 11 sense (2443–2467); exon 11 antisense (2549–2525); downstream-sense (1950–1974); upstream-antisense (3043–3018). PCR conditions for ATP7A used an annealing step at 54 C (1 min; 35 cycles). G3PDH was annealed at 60 C (21 cycles); the same normalization was found using actin instead of G3PDH (53).
Results'5, 百拇医药
ATP7A is expressed highly in the pituitary and adrenal'5, 百拇医药
Although the tissue expression of ATP7A is known to be more widespread than that of ATP7B (4, 23, 24), data on the expression of ATP7A in endocrine tissues are not available. To address this issue, a rabbit polyclonal antibody was raised against the 18-amino-acid peptide at the COOH terminus of ATP7A [ATP7A(1475–1492)]. Notably, the sequence homology in this region of ATP7A is identical in rat and mouse, with a single nonconservative change in human. The specificity of this ATP7A antibody was tested first on crude soluble (S) and particulate (P) fractions prepared from rat liver and anterior pituitary (Fig. 2A). A single major band of 180 ± 5 kDa was detected in the particulate fraction of the rat anterior pituitary. As expected, for an integral membrane protein, no cross-reactive material was detected in the soluble fraction. Furthermore, no cross-reactive material was detected in a particulate fraction prepared from the rat liver, a tissue in which ATP7A has limited expression, if any (9, 10, 12). Preincubation of the ATP7A antiserum with the peptide antigen completely eliminated the signal from the rat anterior pituitary (Fig. 2B), demonstrating its specificity for ATP7A.
fig.ommitteed3, 百拇医药
Figure 2. The ATP7A protein is expressed highly in pituitary. A, Adult rat liver and anterior pituitary were separated into crude soluble (S) and particulate (P) fractions before fractionation of 30 µg protein, by SDS-PAGE. ATP7A was visualized using antiserum CT77 (diluted 1:1000). B, The ATP7A antiserum was preincubated with 0.1 mg/ml antigen in phosphate buffer before application ("Blocked"). C, Detergent extracts (20 µg protein) were prepared from the indicated WT, postnatal d 10–12 mouse tissues (Pit., whole pituitary; Hypo., hypothalamus) and fractionated by SDS-PAGE, and ATP7A was visualized using antiserum CT77. The mobilities of molecular weight markers analyzed simultaneously are indicated.3, 百拇医药
Next, we examined the protein expression of ATP7A in WT postnatal d 10–12 mice by preparing detergent extracts of tissues known to express high levels of PAM or dopamine ß-monooxygenase; pituitary, atrium, and adrenal, as well as cerebral cortex and hypothalamus, were examined (Fig. 2C). A single major cross-reactive protein of 180 ± 5 kDa was detected in each tissue examined. Interestingly, the levels of ATP7A protein were highest in pituitary and adrenal tissues rich in large dense core vesicles containing amidated peptide products and/or catecholamines, respectively. The presence of high levels of ATP7A in the pituitary, compared with the other tissues examined, suggests that ATP7A plays a role in delivering copper to PAM.
RT-PCR was used to compare the levels of ATP7A mRNA in a variety of mouse tissues (Fig. 3A). The selected ATP7A primers amplified a single product in all tissues examined. Levels of G3PDH were evaluated to enable normalization of the data across the tissues (Fig. 3B). When normalized to the levels of G3PDH product, the levels of ATP7A were 2- to 3-fold higher in the pituitary than in any other tissue examined (Fig. 3C). Although the levels of ATP7A mRNA were higher in pituitary than in adrenal, the levels of ATP7A protein were comparable in these tissues (Figs. 2C and 3C). This observation may reflect the extremely long lifetime of adrenal secretory granules (54, 55).k7, 百拇医药
fig.ommitteedk7, 百拇医药
Figure 3. ATP7A mRNA is expressed highly in pituitary. Tissues dissected from adult male mice (n = 2; experiments repeated six times) were used to prepare RNA: pit, whole pituitary; hyp, hypothalamus; adr, adrenal; hrt, heart; cor, cortex; cbl, cerebellum; o.b., olfactory bulb; spl, spleen; liv, liver. After RT, ATP7A transcripts (A) and G3PDH transcripts (B) were amplified, as described in Materials and Methods, using the exon 11 sense/upstream antisense primer pair for ATP7A. C, Expression of ATP7A was normalized to levels of G3PDH using scion image analysis software to quantify the scans for two analyses. The downstream sense/exon 11 antisense primer pair for ATP7A gave a similar distribution, and primers entirely within exon 11 (the exon with the 6-nucleotide deletion in the MoBr mice) gave the same tissue pattern in two experiments. There were no differences in ATP7A tissue distribution for WT and Atp7a mice. Results are expressed as the mean ± range.
ATP7A is localized to the TGN region in pituitary endocrine cells?[qy&hw, 百拇医药
To begin to determine the subcellular localization of ATP7A in pituitary, adult rats were perfused, and the pituitary was removed, fixed, and sectioned on a cryostat. As shown in Fig. 4A, all the cells in both the anterior and intermediate lobes of the pituitary were visualized with COOH-terminal-specific antibody for ATP7A, with no apparent differences in staining intensity. At higher magnification (Figs. 4, B and C), staining is seen localized to a distinct, compact perinuclear region in most cells in both the anterior and intermediate lobes. This pattern of staining resembles that of the TGN. A higher intensity of staining was observed in cells of the anterior pituitary.?[qy&hw, 百拇医药
fig.ommitteed?[qy&hw, 百拇医药
Figure 4. ATP7A is concentrated in the TGN area of pituitary endocrine cells. A–C, Sections from pituitaries of perfused rats were stained with antiserum specific for the COOH-terminus of ATP7A. B and C are high-power magnifications of A. Scale bar for A, 180 µm; scale bar for B and C, 20 µm. Ant, Anterior lobe; Int, intermediate lobe; Neural, neural lobe. D–I, Dissociated cells from whole pituitaries from postnatal d 12 C57BL/6J mice were fixed after 3–5 d in culture and immunostained for ATP7A (D and H) using the ATP7A antibody, for vesicles using a synaptotagmin monoclonal antibody (E), or for TGN using a TGN38 antibody (I). F shows the phase contrast micrograph of a pituitary cell incubated with the blocked ATP7A antibody (G). The scale bar for micrographs D–I is shown in I. N, Nucleus; arrows, staining in TGN region; arrowheads, staining in cell body.
In fibroblasts, ATP7A is localized to the TGN (56, 57, 58, 59, 60) and cycles to the plasma membrane in response to high levels of copper (61, 62, 63). To further explore the subcellular localization of ATP7A, primary cultures were prepared from whole pituitaries of WT postnatal d 10–12 C57BL/6J mice. After 3–5 d in culture, cells were fixed and stained simultaneously with the ATP7A and synaptotagmin antibodies (Fig. 4, D and E). The synaptotagmin antibody, a vesicle protein marker, was used to identify endocrine cells, and punctate staining was distributed throughout the cytosol of the endocrine cells (Fig. 4E). Intense staining in the perinuclear region of all the pituitary endocrine cells was observed using the ATP7A antibody (Fig. 4, D and H). To further confirm the specificity of the ATP7A antibody, pituitary cultures were immunostained with the ATP7A antibody preincubated with the ATP7A(1475–1492) peptide; staining of the pituitary cells did not occur (Fig. 4G; phase contrast of this cell is shown in Fig. 4F). Finally, pituitary cultures were immunostained singly with the TGN38 antibody to determine whether ATP7A is localized to the TGN (Fig. 4I). Based on the similar immunostaining patterns of the TGN38 and ATP7A antibodies (Fig. 4, H and I), we conclude that ATP7A is localized to the TGN in pituitary cells.
ATP7A and PAM expression and processing are unaltered in Atp7a mice\62*[s, 百拇医药
Having established that ATP7A can be detected in various endocrine tissues from adult and early postnatal mice, we compared the expression of ATP7A and PAM in WT and Atp7a mice with nonfunctional Atp7a. Because the hemizygous male mice bearing the mottled-brindled mutation (Atp7a) survive to about postnatal d 15 (64, 65, 66), WT and Atp7a mice were killed between postnatal d 10 and 12. Using Western blot analysis, it was established that ATP7A protein levels in pituitary, cortex, hypothalamus, atrium, and adrenal were similar in WT and Atp7a mice and that the mutant protein was not degraded (Fig. 5A).\62*[s, 百拇医药
fig.ommitteed\62*[s, 百拇医药
Figure 5. Levels of ATP7A and PAM protein are unaltered in Atp7a mice. Control, WT mice, and Atp7a mice were killed at postnatal d 10–12, and the tissues indicated (Hypoth., hypothalamus) were extracted in 20 mM Na N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 10 mM mannitol, 1% Triton X-100, pH 7.4. Aliquots (20 µg protein) were fractionated by SDS-PAGE. A, For the analysis of ATP7A, samples were fractionated on 4–15% gradient gels, transferred for 3 h, and visualized using the COOH-terminal ATP7A antibody (Ab) at 1:1000. B, For the analysis of PAM, samples were fractionated on 4–15% gradient gels, and products derived from PAM-1 were identified using an Ab to the Exon A region that separates PHM and PAL. The major products derived from PAM-1 are shown diagrammatically on the right, and the specificity of the Exon A Ab is depicted. The apparent molecular masses of the PAM products are indicated to the left of the blot.
Because the ATP7A protein in the Atp7a mice is nonfunctional, we wondered whether PAM synthesized in the presence of decreased copper supplies might be differentially processed or perhaps unstable. Levels of PAM protein were unaltered in Atp7a mutant mice (Fig. 5B). PAM is known to undergo tissue-specific endoproteolytic cleavages to generate smaller, soluble forms of PHM and membrane or soluble forms of PAL (67). Using Western blot analysis with a PAM antibody specific to the linker region between PHM and PAL (Exon A), we determined that the levels and types of PAM cleavage products were indistinguishable in each of the tissues extracted from the WT and Atp7a mice (Fig. 5B).o{, http://www.100md.com
Although these results clearly indicate that the PAM protein is expressed and processed correctly in the Atp7a mouse pituitary, these data do not indicate whether the PAM protein is catalytically competent. To address this issue, tissue extracts were prepared from WT and Atp7a mice (Fig. 6), protein concentrations were measured, and in vitro PHM assays were performed after the addition of optimal amounts of copper. Overall, the weak binding of copper to PHM means that the apoenzyme is recovered from tissue extracts (68, 69). Thus, for maximal levels of PHM activity to be detected (70), exogenous copper must be added to the tissue extracts during the assay reaction. Using these established assay conditions, no differences were observed in levels of PHM activity between the WT and Atp7a mice. Despite a deficit in functional ATP7A, no change occurred in the level of PAM protein expression or in its ability to catalyze the first step in the {alpha} -amidation reaction when assayed in the presence of exogenous copper.
fig.ommitteed*d*, 百拇医药
Figure 6. PHM Activity in vitro is unaltered in Atp7a mice pituitary. Detergent extracts of WT and Atp7a pituitary, cortex, hypothalamus, atrium, and adrenal were prepared as described in Fig. 5 (n = 3–21, depending on tissue). Assays for PHM activity were done either in the presence of 1 µM CuSO4 (pituitary, atrium, adrenal) or 5 µM CuSO4 (cortex, hypothalamus). Samples were assayed in duplicate at several different dilutions, and the data shown are the mean ± SEM. None of the pairs of samples are significantly different (P > 0.30).*d*, 百拇医药
The levels of immunoreactive -amidated joining peptide are reduced in the pituitary of Atp7a mice*d*, 百拇医药
Because the levels of -amidated peptides processed and stored in the pituitary should be a reliable indicator of how efficiently PAM functions in the pituitary, we assessed the levels of -amidated POMC end-products by RIA. POMC is the precursor of ACTH, which is not {alpha} -amidated, as well as of two -amidated products, JP-NH2 and ACTH (1–13)-NH2 or -MSH (Fig. 7A). As a control, ACTH levels were first evaluated using a COOH-terminal ACTH-specific antibody (C-ACTH) in whole pituitaries dissected from postnatal d 10–12 WT and Atp7a mice (Fig. 7B. The anterior and intermediate pituitary lobes were not separated for analysis, because the pituitaries in these young animals are too small and fragile for reliable dissection. As shown in Fig. 7B, pituitary extracts from WT and Atp7a mice contained similar levels of C-ACTH, whereas JP-NH2 levels were reduced in the Atp7a mice. To control for sample-to-sample variability, levels of JP-NH2 were normalized to levels of C-ACTH. Based on this ratio, -amidation of JP was reduced to half of control levels in the pituitaries of the Atp7a mice.
fig.ommitteed:i, 百拇医药
Figure 7. The ability of the Atp7a mice to produce -amidated peptides is compromised. A, Proopiomelanocortin is converted into a variety of smaller end-products. The specificities of the antisera used, in this study, for these products are indicated (JP-NH2; ACTH(1–13)NH2 or -MSH; C-terminal ACTH, C-ACTH). B, Pituitaries from individual mice or pools of two to three mice were extracted for analysis of peptide content by RIA. Levels of C-ACTH and JP-NH2 are plotted per pituitary (pmol/pituitary) on the left side of the graph, and the ratios of JP-NH2 to ACTH in individual pituitaries are shown on the right side of the graph. The C-ACTH values are not different (P > 0.22); the JP-NH2 values are quite different (P < 0.0016), as are the JP-NH2/C-ACTH ratios (P < 0.00004). C, Pituitaries dissected from WT and Atp7a mice were dissociated and plated onto glass slides. The primary cultures were fixed and stained with antisera to ACTH or JP-NH2. Micrographs of WT and Atp7a cultures were photographed using the same parameters. The scale bar for all micrographs is shown in the last panel. LPH, -Lipotropin; ß-End, ß-endorphin.
The decreased JP-NH2 content of the Atp7a mouse pituitary could be attributable to low circulating levels of copper resulting from its poor uptake of copper in the small intestine and poor suckling. Therefore, we examined dissociated cells in culture prepared from whole pituitaries of WT and Atp7a mice so that the effects of circulating copper could be eliminated. The DMEM/F-12 medium minus serum in which the cultures were grown was measured to contain 0.1 µM copper. After incubation for 3–5 d, the primary pituitary cultures were fixed and immunostained with antiserum to ACTH or JP-NH2 (Fig. 7C). The ACTH-producing endocrine cells exhibited similar staining intensity for ACTH in both the WT and Atp7a mice. In contrast, POMC cells from WT mice stained intensely with the JP-NH2 antibody, whereas POMC cells from Atp7a mice did not. The ability of PAM to produce -amidated POMC products was compromised in dissociated pituitary cells from mice expressing a nonfunctional ATP7A protein.
Peptide -amidation is compromised in POMC cells of Atp7a micep;, 百拇医药
To examine POMC processing in anterior pituitary corticotropes and intermediate pituitary melanotropes, whole pituitaries (0.3–0.5 mg) from WT and Atp7a mice were fixed and sectioned. Antiserum specific for the COOH-terminus of ACTH visualized a subset of the cells in the anterior pituitary and all of the cells in the intermediate lobe (Fig. 8, A and D). No differences in staining intensity were apparent, in a comparison of WT with Atp7a animals, for either the intermediate (Fig. 8, B and E) or anterior lobe (Fig. 8, C and F) of the pituitary.p;, 百拇医药
fig.ommitteedp;, 百拇医药
Figure 8. The levels of immunoreactive -MSH and JP-NH2, but not ACTH, are reduced in the pituitaries of Atp7a mice. Pituitaries from WT and Atp7a mice were fixed in Bouin’s solution and embedded in paraffin. Sections were stained either with antisera specific for the COOH-terminus of ACTH or -amidated MSH (-MSH) or -amidated joining peptide (JP-NH2). B and C are high-power magnifications of A. E and F are high-power magnifications of D. H and J are high-power magnifications of panels G and I, respectively. Scale bar for A, D, G, and I, 120 µm. Scale bar for B, C, E, F, H, J, K, and L, 50 µm. The intermediate lobes in D and I (Atp7a mice) appear larger than that in A and G (WT mice), but this reflects the plane of sectioning and is not a consistent finding. N, Neural lobe; I, intermediate lobe; Ant Pit, anterior lobe.
The presence of -MSH was evaluated using an antiserum specific for the {alpha} -amidated COOH-terminus of this peptide (Fig. 7A). The -MSH antiserum does not cross-react with ACTH or the Gly-extended form of the peptide, -MSH-Gly. Intermediate pituitary melanotropes make {alpha} -MSH and corticotropin-like intermediate lobe peptide. In young mice, as in young rats, ACTH is cleaved into an MSH-sized peptide in anterior pituitary corticotropes; in adult animals, little of this cleavage occurs in corticotropes (70, 71). Intense staining for -MSH was apparent in the entire intermediate lobe (Fig. 8G) and in many of the anterior pituitary corticotropes of the WT animals (Fig. 8, G and H). In contrast, substantially less staining was observed in the anterior pituitaries of the tp7a mice (Fig. 8, I and J), and staining in the melanotropes was also less intense (Fig. 8I). Finally, the presence of JP-NH2 was evaluated using an antiserum specific for its {alpha} -amidated COOH-terminus. JP-NH2 is produced in both corticotropes and melanotropes. Consistent with the immunoassay data for whole pituitaries (Fig. 7B), staining for JP-NH2 in the corticotropes of Atp7a mice (Fig. 8J) was substantially lower in intensity than in the corticotropes of the WT mice (Fig. 8K). Although PAM protein is present at normal levels in the pituitary of the Atp7a mouse and is fully functional when copper is added in test tube assays, PAM does not function properly in the secretory pathway of endocrine cells in the pituitaries of these mice.
The levels of immunoreactive {alpha} -amidated CCK are reduced in the cerebral cortex of Atp7a mice}, 百拇医药
CCK is derived from preproCCK, and the major form of immunoreactive CCK in the nervous system is CCK4 (72). Extracts of cerebral cortex from WT and Atp7a mice were assayed for {alpha} -amidated CCK (CCK-NH2) and for the CCK-Gly (Fig. 9A). In the WT mice, most of the immunoreactive CCK present in cerebral cortex extracts was in the -amidated form. In contrast, in the Atp7a animals, levels of immunoreactive CCK-Gly were higher than levels of CCK-NH2. The fraction of {alpha} -amidated CCK was 4-fold lower in the cerebral cortex of Atp7a mice than in control mice. Levels of CCK mRNA were similar in WT and Atp7a mice (data not shown). In contrast to the CCK processing observed in the cortex, in the duodenum of both WT and Atp7a mice, very little of the CCK stored in the tissue was -amidated, whereas the levels of CCK-Gly greatly exceeded the levels of CCK-NH2 (Fig. 9B). Although the PAM present in Atp7a animals is capable of functioning when provided with exogenous copper, its function is impaired in the tissues of the Atp7a mice.
fig.ommitteed(, 百拇医药
Figure 9. The levels of immunoreactive CCK-NH2 are reduced in the cerebral cortex, but not in the duodenum, of the Atp7a mice. WT and Atp7a mice were killed at postnatal d 10–12, and cortex and duodenum were dissected and frozen before extraction in 90% methanol. Extracts were analyzed for CCK content by RIA. Levels of immunoreactive {alpha} -amidated CCK (CCK-NH2) and CCK-Gly are plotted per milligram of tissue (pmol/mg) or as the fraction amidated for (A) cerebral cortex and (B) duodenum, mean ± SE. All the differences in the cortex samples are statistically significant (CCK-NH2, P < 0.003; CCK-Gly, P < 0.03; fraction, P < 0.000001), whereas none of the differences in the duodenum rise significantly (CCK-NH2, P > 0.78; CCK-Gly, P > 0.33; fraction, P > 0.11).(, 百拇医药
Discussion(, 百拇医药
Copper is required for the survival of organisms from yeast to humans (2, 3). Presumably, because of the unique redox properties of copper, copper transporter proteins have evolved to maintain cellular copper homeostasis (4, 6). A proposed model for mammalian copper uptake and distribution at the cellular level is shown in Fig. 1 (4, 73). After copper is reduced from Cu(II) to Cu(I) by a membrane reductase (Fig. 1, step 1), Ctr1 mediates the passive uptake of Cu(I) across the plasma membrane (Fig. 1; step 2) (15, 74). Intracellular free copper concentrations are maintained at extremely low levels by small cytosolic chaperones that bind and distribute Cu(I) to specific subcellular compartments. Copper is delivered to cytochrome c oxidase in the mitochondria by cytochrome c oxidase chaperone 17 (Fig. 1, step 3), to cytoplasmic Cu/Zn SOD by CCS1 (copper chaperone for SOD) (Fig. 1, step 4), and to the TGN/secretory compartment by ATX1 homolog HAH1 (Fig. 1, step 5) (17, 75, 76, 77, 78).
In this work, we explored the role of ATP7A in delivering copper to PAM, a secretory granule enzyme (Fig. 1, step 6). Recently, ATP7A was shown to be required for the activity of tyrosinase, a secreted copper-dependent enzyme, which is synthesized in the secretory pathway and involved in melanogenesis (37). The results of our study provide evidence that ATP7A is involved in the delivery of copper to the membrane-bound copper-dependent enzyme, PAM, in the mammalian pituitary and brain. Furthermore, our data suggest that many of the developmental failures observed in patients with Menkes disease may reflect the lack of amidated peptides at specific target sites.:\8, 百拇医药
Using RT-PCR, we established that ATP7A mRNA is present in both endocrine and brain tissues that have high PAM expression (Fig. 3) (31, 32). Overall, ATP7A mRNA levels are highest in the pituitary. Western blot analyses confirmed that the ATP7A protein is highly expressed in both the pituitary and adrenal and is present in the cerebral cortex, hypothalamus, and atrium of the heart (Fig. 2). Both the mRNA and protein levels for ATP7A are very high in pituitary, where PAM is crucial to the production of several amidated peptides, including {alpha} -MSH, galanin, and pituitary adenylate cyclase-activating polypeptide (79, 80, 81). Interestingly, though ATP7A mRNA is detected in the rat liver, the ATP7A protein is not well expressed in this tissue, where ATP7B acts as the predominant copper-transporting ATPase (82). Discrepancies between mRNA and protein levels have been observed for other proteins early in development (83, 84).
PAM is active in the TGN and immature secretory granules and is localized predominantly to these organelles at steady-state in mammalian cells (20, 34, 35, 45). However, it is not known when newly synthesized PAM acquires the copper it needs to become functional. In cultured fibroblasts, both ATP7A and PAM are localized to the TGN (56, 57, 58, 59, 60) and cycle from the TGN to the plasma membrane and back (61, 62, 63, 85). Immunohistochemistry of whole pituitary, using the ATP7A antibody and double immunostaining of cultured pituitary cells from postnatal d 10–12 WT mice with ATP7A and TGN38 antibodies, demonstrates that the ATP7A is localized to the TGN region of endocrine cells (Fig. 4, A and B). Colocalization of PAM and ATP7A to the TGN suggests that copper could become available to PAM in this compartment.j;sq4:, http://www.100md.com
To explore a role for ATP7A in the delivery of copper to PAM, we used the mottled-brindled mutant mouse (Atp7a). This mouse serves as a good model for human Menkes disease, with mutations in the murine homolog of the Menkes gene (40, 86). The Atp7a protein is nonfunctional because of deletion of Ala799-Leu800 (39, 86). The X-linked mutant male mice have severe neurological problems, hypopigmentation, curly whiskers, ataxia, and tremors and are emaciated and do not survive past about postnatal d 15 (3, 65, 66). Although the causes of neurodegeneration, a hallmark of Menkes disease, are not fully understood, it seems that the reduced activities of several copper-dependent enzymes within the secretory pathway of neuroendocrine cells, such as PAM, could contribute.
Using the ATP7A antibody and Western blot analysis (Fig. 5), we detected mutant Atp7a in several endocrine tissues of the Atp7a mouse. The mutant Atp7a protein is the correct size and is not degraded, although malfolded proteins are often rapidly degraded (for review, see Ref. 87). Alternatively, the presence of a negative feedback loop monitoring copper availability could result in an elevation in levels of ATP7A, as seen with POMC expression after adrenal failure (88). We then examined PAM, which requires copper to function properly, in extracts of Atp7a tissue, finding that the levels and processing of PAM were normal. We also determined, using an in vitro assay in which exogenous copper was supplied, that the PAM protein is fully active in endocrine and brain tissues from Atp7a mice (Fig. 6). Thus, PAM in the Atp7a mice is synthesized correctly and has the ability to amidate peptides in vitro as long as it receives copper.9m11--i, 百拇医药
The activity of PAM in the secretory granules of the Atp7a mice was assessed by monitoring levels of {alpha} -MSH and JP-NH2, two amidated peptides derived from POMC (33). It has been suggested that -MSH plays a role in development, because it is produced as a major end-product in the anterior pituitary lobe of the postnatal rat, instead of intact ACTH as in the adult anterior pituitary (89, 90). RIAs, comparing levels of immunoreactive amidated JP-NH2 to levels of ACTH, established that amidated JP-NH2 is reduced 2-fold in the Atp7a mice pituitary (Fig. 7). Additionally, immunohistochemistry showed that -MSH and JP-NH2 are reduced in the pituitary of Atp7a mice when compared with ACTH (Fig. 8). Overall, these data demonstrate that ATP7A plays an important role in the delivery of copper to PAM sequestered in the secretory pathway, lending support to the idea that a reduction of amidated peptides could contribute to the developmental problems associated with Menkes disease.
Levels of CCK-NH2 were used to assess how well PAM can amidate peptides in the cerebral cortex of mice with nonfunctional Atp7a. We assayed levels of CCK-NH2 and compared them with levels of the CCK-Gly in the mouse cerebral cortex, where low-molecular-weight forms of amidated CCK are produced (72, 91, 92). We also compared levels of immunoreactive CCK-NH2 with CCK-Gly in the duodenum, where mostly high molecular weight forms of CCK are found (92). As expected from previous studies (93), a large fraction of the total CCK in the duodenum in both the WT and Atp7a mouse remains in the Gly-extended form. However, in the cerebral cortex, the usually high levels of amidated CCK-NH2 are dramatically reduced in the Atp7a mice. Because CCK-NH2 has an important role as a neurotransmitter or neuromodulator in the brain (72), the underproduction of amidated CCK could contribute to some of the debilitating effects observed in patients with Menkes disease.
Based on these results, we conclude that the ability of PAM to produce -amidated peptides is greatly compromised in Atp7a mice, evidenced by the major loss of several of the amidated neuropeptides involved in neuronal growth and development (79, 80, 81, 94, 95, 96, 97, 98, 99, 100). If the ability of PAM to amidate these important peptides is affected by reduced levels of copper, as occurs in the pituitary and cortex of the Atp7a mouse, then the targets of these amidated peptides could also be greatly affected. Thus, the lack of amidated peptides affecting many target tissues at crucial periods in development could contribute to the Menkes disease phenotype.2], 百拇医药
Acknowledgments2], 百拇医药
We wish to thank Dr. Henry Keutmann (Endocrine Unit, Massachusetts General Hospital, Boston, MA) for synthesizing the ATP7A peptide, Dr. Carolyn Worby (Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI) for providing rat CCK cDNA, and Drs. Jonathan Gitlin and Iqbal Hamza (Department of Pediatrics, Washington University School of Medicine, St. Louis, MO) for providing antisera to ATP7A that were used to initiate this work. We also wish to thank Dr. Michael Petris (University of Missouri-Columbia, Columbia, MO) for Chinese hamster ovary cells expressing myc-ATP7A and Dr. Margery C. Beinfeld (Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA) for providing the no. 22 anti-CCK-Gly antiserum. We also express our thanks to Shana Berger for assistance with the mRNA analysis. Finally, we wish to thank Marie Bell and Lixian Jin for laboratory assistance in Baltimore, MD, and Darlene D’Amato for maintaining the mottled-brindled mouse colony and for general laboratory assistance in Farmington, CT.
Received July 15, 2002.!i, 百拇医药
Accepted for publication September 9, 2002.!i, 百拇医药
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