The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase

The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.     

Purification of a peptidylglycine alpha-amidating enzyme from transplantable rat medullary thyroid carcinomas

A peptidyl glycine alpha-amidating activity has been isolated from total tissue extracts of rat medullary thyroid carcinoma (MTC). Purification of the activity by ammonium sulfate fractionation, Sephacryl S-300 chromatography, and strong anion-exchange chromatography at pH 6.0 has resolved at least four peaks of activity. The activity associated with peak III has been further purified to apparent homogeneity by strong anion-exchange chromatography at pH 8.0. The purified peak III enzyme has an apparent molecular mass of 75,000 Da as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the 75,000-Da band as the alpha-amidating enzyme has been confirmed by recovery of activity from a nondenaturing polyacrylamide gel. The enzyme is catalytically active as a monomer, exhibits a pH optimum between 5.0 and 5.5, and has a turnover number of 300 min-1 for N-dansyl-Tyr-Val-Gly amidation at pH 5.5. The larger size, more acidic pH optimum, and higher specific activity distinguish the purified peak III rat MTC enzyme from the enzymes isolated from bovine and porcine pituitary or from frog skin.     

Rat peptidylglycine alpha-amidating enzyme: the relation between activities at neutral and alkaline pH Values

A substantially high level of alpha-amidating activity at an alkaline pH (8-9.5), often seen as another pH optimum peak in addition to the neutral one, has been observed in various rat tissues. We have also found that crude enzymes from rat brain, pituitary, and small intestine showed a pH profile with two pH optima at neutral pH (6.5-7) and alkaline pH (8.5-9) when D-Tyr-Val-Gly was used as substrate. With a combination of ion-exchange and gel filtration chromatographies, we obtained two fractions, S-1 and S-2, from rat brain; S-1 contained an alpha-amidating enzyme of an apparent molecular weight of 36,000 (36K enzyme) exhibiting a single pH optimum at 8.5. On the other hand, S-2 apparently showed almost no or only marginal activity at either pH 7 or 8.5, but when S-2 was combined with S-1, a neutral pH optimum at 7 could be elicited. The factor in S-2 that was responsible for this combined action was a protein of an apparent molecular weight of 41,000 (41K protein). Both proteins were found to be colocalized in the same subcellular organelle, probably in the secretory granule. It seems likely, then, that the pH profiles characterized by two optimal peaks seen in crude rat enzymes can be attributed to the presence at an appropriate ratio of the 41K protein and 36K enzyme.     

18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase.

The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.     

Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine alpha-amidating enzyme.

Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine alpha-amidating enzyme (alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the alpha-AE-catalyzed conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-alpha-hydroxyglycine lyase active sites.     

Characterization of a bifunctional peptidylglycine alpha-amidating enzyme expressed in Chinese hamster ovary cells.

Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.     

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.     

Isolation and functional expression of pituitary peptidylglycine alpha-amidating enzyme mRNA. A variant lacking the transmembrane domain

We demonstrate that, in rat pituitary, peptidylglycine alpha-amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain-coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS-7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.     

Catalytically active peptidylglycine alpha-amidating monooxygenase in the media of androgen-independent prostate cancer cell lines

Peptidylglycine alpha-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated alpha-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAMactivated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures. Androgen-independent DU145 and PC-3 prostate cancer cell lines exhibited readily detectable levels of PAM activity in extracts and media, whereas the androgen-dependent LNCaP cell line showed little or no activity. Because of the much larger volume of media versus cell extracts, more than 90% of the total PAM activity was located in the media for both the PC-3 and DU145 cell lines, providing a readily accessible source of CaP PAM. A simple, scalable method to obtain PAM from the culture media of androgen-independent human prostate cancer cell lines is described in this article. This approach provides a much easier means of collecting CaP-derived PAM than previously described cell fractionation procedures and should facilitate the investigations of the role and targeting of PAM in hormone-independent CaP.     

Formation of a pyridinium derivative by reaction of 4-hydroxypentenal with glycine

4-Hydroxypentenal reacts with glycine in a slightly alkaline (pH 8.8) aqueous solution to 1-(1-carboxymethyl)-3-(2-hydroxypropyl)-pyridinium betaine. The yield of the pyridinium betaine isolated by preparative HPLC was 20 mol% and its structure was ascertained by 13C-NMR, IR, UV and mass spectroscopy. Several other products could be detected by HPLC in the 4-hydroxpentenal-glycine reaction mixture, their instability, however, impeded their preparative isolation. 4-Hydroxyalkenals are toxic products generated during lipid peroxidation and the results described explain in part the mechanism how these aldehydes react with nucleophilic amino groups in tissue.     

The membrane-bound bifunctional peptidylglycine alpha-amidating monooxygenase protein. Exploration of its domain structure through limited proteolysis

The biosynthesis of alpha-amidated peptides from their glycine-extended precursors is catalyzed by the sequential action of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The two enzymes are part of a bifunctional, integral membrane protein precursor, peptidylglycine alpha-amidating monooxygenase (PAM). The major forms of PAM mRNA in the adult rat atrium differ by the presence or absence of optional exon A, a 315-nucleotide segment separating the PHM and PAL domains. Using antipeptide antibodies specific to the PHM, exon A, PAL, and cytoplasmic domains of rat PAM, carbonate-washed atrial membranes were found to contain proteins corresponding to rPAM-1 and rPAM-2. Digestion of atrial membranes with a variety of endoproteinases released PHM and PAL catalytic activities. Dose-response curves indicated that both catalytic activities were extremely resistant to inactivation by trypsin. Endoproteolytic digestion of atrial membranes with trypsin, chymotrypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 35-kDa PHM fragment. Digestion with trypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 42-kDa PAL fragment. In contrast to the stability exhibited by the PHM and PAL domains, the cytoplasmic domain of PAM was destroyed by most of the enzymes; only digestion with endoproteinase Lys-C generated a stable fragment. Digestion with endoproteinase Arg-C removed the carboxyl-terminal tail from PAM but failed to release the PHM or PAL domains from the membranes. The PHM fragments generated by some of the endoproteinases showed a tendency to adhere to the membranes. Thus the bifunctional PAM protein consists of independent catalytic domains separated from each other and from the putative transmembrane domain by flexible regions accessible to attack by a wide variety of endoproteinases.     

Manipulation of neuropeptide biosynthesis through the expression of antisense RNA for peptidylglycine alpha-amidating monooxygenase.

Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.     

A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.     

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.     

Peptidylglycine alpha-amidating reaction: evidence for a two-step mechanism involving a stable intermediate at neutral pH

In our previous study of the rat brain alpha-amidating activity, we suggested that a protein of 41 kdal (41K protein) that shows no alpha-amidating activity is required for the reaction at neutral pH in addition to an alpha-amidating enzyme of 36 kdal (36K enzyme). Here we report on the purification of both proteins to near homogeneity and provide evidence that alpha-amidation proceeds via a two-step mechanism involving a stable intermediate at neutral pH, which is initially formed by the 36K enzyme and then readily converted into an amide product by the 41K protein.     

The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.     

Direct evidence that the carboxyl-terminal sequence of a bacterial chemoreceptor is an unstructured linker and enzyme tether

Sensory adaptation in bacterial chemotaxis involves reversible methylation of specific glutamyl residues on chemoreceptors. The reactions are catalyzed by a dedicated methyltransferase and dedicated methylesterase. In Escherichia coli and related organisms, control of these enzymes includes an evolutionarily recent addition of interaction with a pentapeptide activator located at the carboxyl terminus of the receptor polypeptide chain. Effective enzyme activation requires not only the pentapeptide but also a segment of the receptor polypeptide chain between that sequence and the coiled-coil body of the chemoreceptor. This segment has features consistent with a role as a flexible and presumably unstructured linker and enzyme tether, but there has been no direct information about its structure. We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to characterize structural features of the carboxyl-terminal 40 residues of E. coli chemoreceptor Tar. Beginning ～ 35 residues from the carboxyl terminus and continuing to the end of the protein, spectra of spin-labeled Tar embedded in native membranes or in reconstituted proteoliposomes, exhibited mobilities characteristic of unstructured, disordered segments. Binding of methyltransferase substantially reduced mobility for positions in or near the pentapeptide but mobility for the linker sequence remained high, being only modestly reduced in a gradient of decreasing effects for 10-15 residues, a pattern consistent with the linker providing a flexible arm that would allow enzyme diffusion within defined limits. Thus, our data identify that the carboxyl-terminal linker between the receptor body and the pentapeptide is an unstructured, disordered segment that can serve as a flexible arm and enzyme tether.