Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.     

Regulation of peptide amidation in cultured pituitary cells

The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.     

Isolation of peptide containing essential tyrosine from lobster arginine kinase]

The essential tyrosine residue of Lobster muscle arginine kinase, which is part of an antigenic determinant, has been modified by tetranitromethane. Cleavage of the S-carboxymethylated nitrated enzyme with cyanogen bromide gives rise to eight peptides, one of which containing the labelled essential tyrosyl group. Ion exchange chromatography on sulfoethyl-Sephadex C-25 in urea medium has been used with success for isolation and purification of the nitrated peptide. From its amino acid composition and end groups structure this peptide is the N-terminal fragment of the protein.     

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.     

Drosophila signal peptide peptidase is an essential protease for larval development

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.     

Native chemical ligation derived method for recombinant peptide/protein C-terminal amidation

C-terminal amidation is often a requisite structural feature for peptide hormone bio-activity. We report a chemical amidation method that converts peptide/protein thioesters into their corresponding C-terminal amides. The peptide/protein thioester is treated with 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliary (1b) in a native chemical ligation (NCL) reaction to form an intermediate, which upon removal of the auxiliary with TFA, yields the peptide/protein amide. We have demonstrated the general utility of the approach by successfully converting several synthetic peptide thioesters to peptide amides with high conversion rates. Preliminary results of converting a recombinant peptide thioester to its amide form are also reported.     

Ability of cofactors to support peptide amidation is cell-type specific

The ability of various cofactors to substitute for ascorbate in the biosynthesis of alpha-aidated peptides from pro-ACTH/endorphin (PAE) was compared in corticotrope tumor cells (AtT-20) and in primary anterior and intermediate pituitary cultures. In all three systems, ascorbate was the most potent cofactor tested. In AtT-20 cells, dopamine, norepinephrine, epinephrine, dehydroascorbate and dihydro- and tetrahydrobiopterin supported significant alpha-amidation of joining peptide [PAE(77-94)NH2]. In contrast, amidation of joining peptide by primary corticotropes was stimulated only slightly by catecholamines and not by tetrahydrobiopterin. Neither catecholamines nor tetrahydrobiopterin stimulated peptide amidation by melanotropes. The ability of cofactors to support the synthesis of alpha-amidated peptides is cell-type specific.     

Synthetic peptide model of an essential region of an aminoacyl-tRNA synthetase

A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme. The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups. The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated. In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix. These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation. Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385. The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability.     

Peptide amidation: Production of peptide hormonesin vivo andin vitro

Over half of all biologically active peptides and peptide hormones are α-amidated at their C-terminus, which is essential for their full biological activities. Amidation is accomplished through the sequential reaction of the two enzymes encoded by the single bifunctional, peptidylglycine α-amidating monooxygenase (PAM or an α-amidating enzyme). PAM catalyzes the formation of a peptide amide from peptide precursors that include a C-terminal glycine, and requires copper, molecular oxygen, and ascorbate. PAM is the only enzyme that produces peptide amidesin vivo. However, various strategies utilizing PAM, carboxypeptidase-Y enzymes, and chemical synthesis have been developed for producing peptide amidesin vitro. The growing need and importance of peptide amide drugs has highlighted the necessity for an efficientin vitro amidating system for industrial application. In recent years, recombinant systems for enzymatic amidation have received growing attention for the production of peptide hormones, like calcitonin and oxytocin. This review presents the current situation regarding amidation, with a special emphasis on the industrial production of peptide hormones.     

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.     

Synthetic peptide substrates for a tyrosine protein kinase

Immunoprecipitates containing the transforming protein of the avian sarcoma virus, Y73, together with its associated tyrosine-specific protein kinase, have an activity which will phosphorylate the synthetic peptide Lys-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg at the tyrosine residue. This peptide corresponds to 10 out of 11 amino acids surrounding the phosphorylated tyrosine in both pp60src and P90, the transforming proteins of Rous sarcoma virus and Y73 virus, respectively. The apparent Km for phosphorylation of the peptide was about 5 mM. A second peptide with the sequence Lys-Leu-Ile-Asp-Asn-Glu-Tyr-Thr-ala-Arg differing from the first peptide only by the absence of the glutamic acid 4 residues from the tyrosine was also phosphorylated, but the apparent Km for the reaction was 40 mM. Several sites of tyrosine phosphorylation in viral transforming proteins have been found to have one or more glutamic acids close to the phosphorylated tyrosine on the NH2-terminal side. Taken together with our in vitro phosphorylation studies, this suggests that the primary sequence surrounding target tyrosines may play a role in recognition of substrates by tyrosine protein kinases, and in particular, that glutamic acid residues on the NH2-terminal side may be important.     

Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat

In the present studies we examined the distribution, release, and biological actions of peptide tyrosine tyrosine (PYY) in the rat. The concentration and distribution of PYY was highest in the ileum and colon as determined by both radioimmunoassay of rat tissue extracts and immunocytochemistry. An ultrastructural comparison of rat and dog colonic PYY cells revealed a bipolar distribution of peptide-containing secretory granules in both species. Serum PYY and pancreatic exocrine secretory responses were monitored after presentation of a meal to meal-trained rats (n = 12). A significant increase in PYY concentrations was not observed until 120 min after meal presentation, a delayed response similar to that previously observed in the dog. PYY responses were also observed in rats after perfusion of the intestine at the level of the duodenum and ileum with an 80 mOsm micellar solution of sodium oleate. Duodenal instillations of the fatty acids resulted in a maximum PYY response after 120 min, whereas rats subject to ileal perfusion of fat exhibited maximum PYY release within the first hour. In other experiments, infusion of exogenous PYY at 100 pmol.kg-1.h-1, which reproduced plasma PYY levels observed after a meal and perfusion of the gut with fat, significantly inhibited CCK-stimulated bile pancreatic volume (P less than 0.02), protein (P less than 0.01), and amylase (P less than 0.01) output. These studies demonstrate a bipolar distribution of PYY-containing secretory granules in cells of the jejunal, ileal, and colonic mucosa, and show that PYY is released in response to a meal in amounts sufficient to inhibit cholecystokinin-stimulated pancreatic secretion. Evidence is presented that PYY may mediate the delayed inhibition of pancreatic secretion that is observed in the rat after ingestion of a meal.     

Discoidin domain receptor 1 tyrosine kinase has an essential role in mammary gland development

Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor tyrosine kinases, DDR1 and DDR2. Here, we used a recombinant fusion protein between the extracellular domain of DDR1 and alkaline phosphatase to detect specific receptor binding sites during mouse development. Major sites of DDR1-binding activity, indicative of ligand expression, were found in skeletal bones, the skin, and the urogenital tract. Ligand expression in the uterus during implantation and in the mammary gland during pregnancy colocalized with the expression of the DDR1 receptor. The generation of DDR1-null mice by gene targeting yielded homozygous mutant animals that were viable but smaller in size than control littermates. The majority of mutant females were unable to bear offspring due to a lack of proper blastocyst implantation into the uterine wall. When implantation did occur, the mutant females were unable to lactate. Histological analysis showed that the alveolar epithelium failed to secrete milk proteins into the lumen of the mammary gland. The lactational defect appears to be caused by hyperproliferation and abnormal branching of mammary ducts. These results suggest that DDR1 is a key mediator of the stromal-epithelial interaction during ductal morphogenesis in the mammary gland.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.     

Synthesis and peptide incorporation of an unnatural amino acid containing activity-based probe for protein tyrosine phosphatases

An unnatural amino acid was synthesized to incorporate a quinone methide-generating activity-based probe for protein tyrosine phosphatases (PTPs) and then integrated into a PTP1B-specific substrate. The resulting probe led to preferential labeling of PTP1B in cell lysates in the presence of PTP4A3.     

PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP^C to scrapie isoform PrP^Sc). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126_CONH2 and PrP106-126_COOH) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126_COOH displayed a higher peptide-lipid binding affinity than PrP106-126_CONH2 (∼2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.