Peptidylglycine-alpha-hydroxylating monooxygenase
Staining of larval CNS with the affinity-purified antisera indicates widespread expression of Phm-like protein throughout all levels of the CNS, as well as in other tissues, including endocrine glands and gut. Antibody specificity was deduced by comparison with tissues that were stained with preimmune serum. This expression is limited to a small number of CNS neurons (approximately a few hundred) that displayed very high levels of Phm-like immunoreactivity. Among stained neurons, immunoreactivity is seen both in cell bodies and within neuropil regions. The latter represents stained neuronal processes and also may include glial staining. Phm antibody staining is also prevalent in secretory cells of the Ring Gland, salivary gland, and in diverse cells at all levels of the midgut . In the CNS, several strongly stained cells were identifiable as neuroendocrine neurons because they project immunoreactive axons to defined neurohemal organs like the Ring Gland or the dorsal neurohemal organs of the ventral ganglion. Many Phm-positive neurons have positions similar to those of peptidergic cells. Such identity has been verified in the case of several FMRFamide-expressing neurons using a Drosophila stock containing a FMRFamide-beta-galactosidase construct called pWF3. The lacZ-positive SE2 interneurons of the subesophageal neuromeres and the neuroendocrine Tv and Tva neurons of thoracic neuromeres are among the strong Phm-staining neurons. Thus, in the case of certain identified neurons, high levels of Phm expression correlates with a peptidergic neuronal phenotype (Kolhekar, 1997a).
The P[07623] insertion lies within the coding region of the Phm gene and the lethality associated with the stock is reverted when the element undergoes precise excision (Kolhekar, 1997a). A screen was performed for imprecise excisions of the P[07623] element to generate a small deletion of the Phm locus. Revertants of the white+ phenotype were selected, and these stocks were analyzed by PCR and by Southern blotting, and one deletion was selected called Df(2R)PhmP(29) [the P(29) allele]. PhmP(29) animals retain homozygous lethality and fail to complement large deficiencies of the region. From blot analysis, it was inferred that the deletion lost part of Exon I, and all of Exons II, III, and IV, and that it ended within intron D. Sequence analysis confirms that the deletion extends over 1309 bp from the P[07623] insertion to 561 bp 59 of Exon V. There are 301 bp of the P[07623] element remaining within the P(29) allele. The initial segment of Exon I also remains, lying to the retained side of the P element. The portions of the Phm coding region lost in the P(29) deletion include motifs that are essential for activity of the enzyme (e.g., one of the two dihistidine motifs known to bind copper; Eipper, 1995). The sequence analysis indicates that at least one other predicted gene (CG17263) is mutated in the P(29) chromosome (Jiang, 2000).
The two mutant alleles of Phm -- the P[07623] insertion and the P(28) deletion -- both display complete lethality, as indicated by the absence of homozygous and hemizygous adults. Occasional putative adult escapers (~0.1% at 25oC) were identified as balanced heterozygotes by multiplex, single-fly PCR. Marked mutant stocks were studied following various genetic crosses to determine when Phm mutant animals died. The hatching rate of mutant animals was first determined to evaluate their ability to complete embryogenesis. Compared to Mendelian predictions, ~58% of PhmP[07623] homozygotes and ~76% of balanced PhmP[07623] heterozygotes hatched from the egg. With very few exceptions, PhmP(28) homozygotes failed to hatch; those that did were nearly immobile and died within a few hours. PhmP[07623]/PhmP(28) trans-heterozygotes hatched at a rate comparable to that of PhmP[07623] homozygotes (55% of the predicted value). When analyzed in trans to a large deficiency of the region [Df(2R)or-Br11], PhmP[07623] appeared less severe than did PhmP(28). These results indicate that the PhmP(28) allele produces nearly complete embryonic lethality (failure to hatch) and acts as a null in this assay. PhmP[07623] acts like a strong hypomorph, with approximately 50% of animals failing to hatch (Jiang, 2000).
While the PhmP(28) mutant animals fail to hatch, most complete the large part of embryogenesis prior to their death. Homozygotes and hemizygotes were scored by cuticular markers and/or by single embryo PCR: More than 70% of PhmP(28) mutant embryos complete stage 16 and more than 40% reached stage 17. These embryos display little evidence of tissue disorganization or morphological abnormality. For example, BP102 staining of PhmP(28) homozygotes reveals proper axonal organization within the CNS by stage 15. A minority of embryos (15/79) die at early stages without evidence of cellularization. Single-embryo PCR does not yield signals with these embryos, so they remain unidentified as to genotype. If these represent Phm mutant animals, the embryonic phenotype would include an early lethal phase. Those Phm mutant embryos that hatch as larvae display normal morphology when they first emerge, as indicated by anti-Synaptotagmin staining of neuropil tissues in PhmP[07623] homozygotes (Jiang, 2000).
The ability of hs-Phm induction to rescue the lethality associated with PhmP(28) homozygotes was tested. The effect of hs-Phm-A5 induction on Phm levels in WT animals was measured. Eggs were collected from balanced P[hs-Phm-A5];PhmP(28)/SM6 adults and then given daily 30-min inductions through the embryonic and postembryonic stages, until the day the first adults emerged. Thirty-five percent (89 of 253) of adult flies emerging from cultures that had received this induction schedule were homozygous PhmP(28) mutant animals. This indicates that the absence of Phm activity is responsible for the lethality associated with homozygous PhmP(28) mutant animals (Jiang, 2000).
Phm immunoreactivity is present heterogeneously in most larval tissues (Kolhekar, 1997a). In heterozygous and wild-type animals, Phm-like immunostaining in the CNS consists of strong staining in about 200 scattered neuronal cell bodies and in widespread neuropil regions. Other cell bodies and neuropil regions display lower levels of staining. In both homozygous and hemizygous PhmP[07623] and PhmP(28) mutants, CNS staining is severely reduced although not absent. Neuropil staining is weak and diffuse. Most cell body staining is eliminated, with the exception of a few lightly stained cells: a single protocerebral brain cell and ~5 cells in lateral positions of anterior abdominal segments. The weak neuropil staining may represent unrelated, cross-reacting species similar to non-Phm proteins that are recognized by anti-Phm in tissue immunoblots. Moderate signals in specific abdominal neuronal cell bodies may represent maternally derived Phm that could be enriched and retained in peptide-containing secretory granules of certain peptidergic neurons in mutant animals. In the gut, wild-type and heterozygous Phm mutant animals display strong staining of scattered, putative endocrine cells in the anterior and middle regions of the midgut. In homozygous and hemizygous mutant animals, all gut staining was eliminated. Likewise, the normally strong Phm immunostaining of the salivary gland was eliminated in all Phm homozygous and hemizygous mutants (Jiang, 2000).
The survival of some PhmP[07623] homozygotes into early larval stages afforded the opportunity to evaluate levels of Phm enzyme and protein in these mutants. Homozygous first-instar larvae contain an amount of Phm activity that is at least 10 times less than heterozygotes and that is equal to or just above control values. By Western blot analyses, anti-Phm recognizes a doublet of immunoreactive proteins of ~40 kDa. PhmP[07623] homozygous animals contain no detectable ~40-kDa immunoreactive species compared to a sizable amount present in heterozygous animals. By both measures therefore, the PhmP[07623] allele contains very little or no Phm protein in young larvae. In parallel experiments, Phm levels were measured in adult heads of wild-type animals that are transgenic for a Phm cDNA under the control of an hsp70 heat-inducible promoter. A 30-min 37°C induction in the P[hs-Phm-A5] line produces an ~10-fold induction of Phm enzyme levels within 4 h; 24 h after induction, induced levels remain about 5-fold higher than baseline. By Western blot analysis, the same induction paradigm also produced a sizable increase in staining of an ~40-kDa protein doublet (Jiang, 2000).
The behavior of the heterozygous PhmP[07623] mutant larvae is normal with respect to their general coordination, their locomotory steps and feeding movements, and their response to tactile stimulation and molting behavior. By casual inspection, homozygous and hemizygous PhmP[07623] larvae resemble their heterozygous siblings immediately following their emergence. However, over the course of the next 24-48 h, homozygous and hemizygous mutant animals become lethargic and typically die at or near the end of the first larval instar. When reared at room temperature, the longest-lived mutants reach 7 days of age and have just molted to the third larval instar. 780 PhmP[07623] mutant larvae were segregated within 12 h of hatching and their lethal phase was examined by scoring the structures of mouthparts and of the anterior spiracles displayed by corpses. Among corpses recovered, more than 75% display a double-mouthparts phenotype, with tooth structures characteristic of animals molting to the second larval instar. Nearly 20% of the corpses were animals that had died as nonmolting first-instar larvae. A small number of animals were recovered that had successfully completed the first and/or second larval molts and that died as second-instar animals, as third-instar animals, or while attempting to molt to the third instar. These data indicate that more than half of the PhmP[07623] mutants that survive embryogenesis die while attempting to molt to the second larval instar (Jiang, 2000).
Abbreviated schedules of hs-Phm transgene induction were tested to begin defining developmental stages when Phm expression was required to reverse lethality in PhmP(28) homozygotes. Limiting inductions (one per day) to just the first 4 days after egg-laying is sufficient to rescue most or all mutant animals. Conversely, delaying inductions for as few as the first 2 days after egg-laying severely lowers the number of rescued PhmP(28) homozygotes, and no rescue is observed when inductions are delayed until 5 days after egg-laying. Together these results suggest that a minimal Phm induction schedule limited to early developmental stages is both necessary and sufficient to rescue adult lethality (Jiang, 2000).
The minimal Phm induction schedule produced PhmP(28) homozygous adults (here called G1 mosaic animals) that were normal in many respects, but that also showed stereotyped behavioral abnormalities. Rescued G1 adults were both fecund and fertile. Their G2 progeny could live at restrictive temperatures and a small percentage of these reached the adult stage. Thus a minimal rescue schedule provided the opportunity to study animals homozygous for the PhmP(28) mutation past their normal lethal phase. Immunostaining with anti-FMRFamide revealed a largely normal pattern in the CNS of G2 larvae. G2 animals were produced and reared at 25°C and these G2 PhmP(28) mutants remained homozygous for the inducible hs-Phm-A5 transgene. Approximately 76% of G2 PhmP(28) homozygous animals completed embryogenesis and hatched, and approximately 61% of G2 PhmP(28) first-instar larval homozygotes became pupae. Only 22% of G2 PhmP(28) homozygous prepupae completed metamorphosis. Puparium formation was often aberrant: puparia typically displayed a slight curvature, a failure to shorten normally, and a failure to evert anterior spiracles properly. The gas bubble failed to move anteriorly, leaving pupae in anterior positions with abnormal posterior gas spaces. A subset of the noneclosing puparia were dissected to evaluate their lethal phases. The majority of these (28/57) died after completing pupation and proper eversion of the head, wings, and legs, but they revealed no further adult differentiation (e.g., died between stages P4ii and P7). Almost as many animals (25/57) reached this same stage, but displayed aberrant disc eversion, retention of larval mouthparts, and imprecise elimination of apolysed tracheal cuticle. In the entire group of PhmP(28) homozygotes examined, only 2 of the 255 prepupae that failed to eclose differentiated past stage P7. Of these two, one had normal adult features, but was cryptocephalic. The other displayed heterochronic differentiation: scattered bristles were tanned prior to other developmental events that normally occur earlier (Jiang, 2000).
Daily 37°C inductions of PhmP(28) G2 animals did not increase the percentage of mutant animals that completed larval development: the observed larval mortality appears due to the experimental manipulation of the young larvae. Inductions did rescue nearly all mutant prepupae through their transitions to adult eclosion. In addition, the puparial shapes of G2 animals receiving hs-Phm inductions were largely normal. This indicates that the defects of metamorphosis observed in G2 PhmP(28) homozygotes reflect the absence of normal Phm functions (Jiang, 2000).
P[hs-Phm-A5]; PhmP(28) homozygous G1 mutant adults, produced by the minimal abbreviated schedules of hs-Phm induction, contain lowered levels of Phm enzymatic activity. When animals experienced daily inductions through the day of adult eclosion, heads from P[hs-Phm-A5]; PhmP(28)/SM6 animals (Phm heterozygotes) and P[hs-Phm-A5]; PhmP(28)/PhmP(28) (Phm homozygotes) both contained large amounts of enzyme activity on adult day 1. Levels subsequently declined in each genotype over the next 20 days, but homozygotes exhibited ~50% less enzyme than heterozygotes. A value of ~2.5 pmol/mg/h is found in PhmP[07623] heterozygotes; the approximately twofold higher values seen in these PhmP(28) heterozygotes animals presumably reflect the contributions of basal and induced (perduring) levels of P[hs-Phm-A5] transgene expression. Similar though more dramatic results were observed in animals that experienced the minimal rescue schedule (4 early inductions). Throughout all adult stages assayed, P[hs-Phm-A5];PhmP(28)/SM6 animals that had received the minimal induction schedule displayed levels comparable to older P[hs-Phm-A5];PhmP(07623)/SM6 adults that had received the long induction schedule (i.e., ~5 nmol/mg/h). In contrast, sibling P[hs-Phm-A5];PhmP(28)/PhmP(28) animals had reduced levels throughout all adult days tested (Jiang, 2000).
The expression of amidated neuropeptides was examined in G1 P[hs-Phm-A5];PhmP(28) heterozygous and homozygous adults that had experienced the minimal induction schedule. The homozygotes are referred to as 'rescued adults.' In particular, the spatial profile of FMRFamide-like immunoreactivity, which in the wild-type adult brain has been categorized within 13 principal cell types, was studied. In adult brains (adult days 1 through 10), 6 of 13 FMRFamide-immunoreactive cell types were absent or in-frequently stained in rescued G1 PhmP(28) homozygotes. Most of the 'missing cells' correspond to neurons that are 'adult-specific' for the FMRFamide neuropeptide phenotype, e.g., the MP2 and OL2 neurons that appear only during or after metamorphosis. To ask whether this mutant phenotype represents incomplete cellular differentiation or a defect in neuropeptide biosynthesis, comparable animals were examined 24 h after they received a single additional hs-Phm induction as adults. It was reasoned that a neuropeptide biosynthesis defect should be reversible within a short period following such a late transgene induction. In separate experiments, single inductions were given on adult days 4 and 10. After 24 h, tissues were analyzed by comparison to age-matched P[hs-Phm-A5]; PhmP(28)/SM6 given the same induction schedule and to rescued P[hs-Phm-A5];PhmP(28)/PhmP(28) not given an additional induction. A reversal of the phenotype for OL2, MP2, and CC neuronal cell types was observed. For example, OL2 neurons were detected in 20 of 20 hemispheres of P[hs-Phm-A5]; PhmP(28)/PhmP(28) animals that received the additional induction. In 12 hemispheres of the age- and genotype-matched controls (day 5 rescued adults that did not experience additional induction), no OL2 neurons were seen (Jiang, 2000).
Following induction, OL2 immunolabeling included both cell bodies and axonal projections, indicating that previously 'missing' neurons displayed considerable morphological differentiation in rescued Phm mutant adults. A comparison to staining in age-matched P[hs-Phm-A5]; PhmP(28)/SM6 animals suggests the phenotype reversal is not complete: OL2 neurons in Phm heterozygotes are more intensely stained in 18 of 18 hemispheres (Jiang, 2000).
In Drosophila, the amidated neuropeptide Pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. It was asked whether other neuropeptides contribute to such auxiliary mechanisms. The gal4/UAS system was used to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restrict PHM expression to different numbers of peptidergic neurons. These mosaics display aberrant locomotor rhythms to degrees that parallel the apparent complexity of the spatial patterns. Certain PHM mosaics are less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms (Taghert, 2001).
These results support the hypothesis that daily locomotor rhythms in flies require signaling by neuropeptides that are C-terminally amidated. The gal4/UAS-PHM system predictably controls PHM spatial expression. It was found that certain PHM mosaic flies (e.g., 36Y-gal4- and c929-gal4-rescued) are largely arrhythmic under conditions of constant darkness. The working hypothesis is that such a behavioral disruption is attributable to changes in the normal patterns of peptide amidation. The last point has not been demonstrated directly. It is a premise based on the previous demonstration that manipulation of PHM produces large-scale changes in peptide amidation in both larval and adult stages (Taghert, 2001).
A second result supports the conclusion that amidated peptides contribute to daily locomotor rhythms; increasing PHM expression by combining pdf(N)-gal4 with c929-gal4 improves rhythmic behavior over that displayed by c929-gal4 alone. Although the combination does not completely restore wild-type behavior, the improvement indicates that PHM activity in PDF neurons does contribute to display of rhythmic daily locomotion. It is presumed that this indicates a requirement for amidation of PDF because the pigment-dispersing activity of PDH on crustacean melanophores is highly dependent (~300-fold) on the C-terminal amide. Whether PDF must be modified as such to display circadian signaling activity is unknown (Taghert, 2001).
The strongest evidence for this conclusion comes from the performance of 36Y-gal4- and c929-gal4-rescued flies under constant conditions. Unlike pdf01 flies, these populations displayed little rhythmicity behavior during the first cycle of constant conditions. Also, the average signal-to-noise ratio of 36Y-gal4-rescued flies was much less than that of pdf01 and very comparable with that of per01. Are such deficits attributable to a loss of PHM function or a gain of deleterious function? The pdf neuropeptide gene can produce a gain-of-function phenotype: When pdf was misexpressed by certain gal4 drivers in WT flies, rhythmic locomotor behavior was degraded. In the case of PHM, however, two results suggest it is the absence of the enzyme that causes arrhythmicity: (1) increasing PHM expression (by adding tim-gal4 to 36Y-gal4 or c929-gal4) restored near-normal rhythmicity to both arrhythmic lines; (2) misexpression of PHM by driving it with tim-gal4 or with c929-gal4 in a WT did not degrade behavioral rhythmicity. In general, the greater the extent of PHM gene expression in a PHM mutant background, the more predictable is the degree of behavioral improvement. Interestingly, some combinations restored considerable PHM expression (e.g., Appl-gal4) but improved behavioral performance only moderately. Together, those experiments suggest that normal PHM expression is required in specific neurons and/or secretory cells for the behavior examined. The tim-gal4 line produced a broad expression pattern of great complexity. That amount of expression precludes clear definition of places or times by which 'additional' PHM restores the functions of circadian regulatory circuits (Taghert, 2001).
The behavior of stocks which each contained multiple transposons was compared. To improve the scope of the study, five control genotypes were tested that combined subsets of the multiple transposons used in the experimental genotypes. In general, these controls displayed a level of rhythmicity lower than that of WT but greater than those of PHM mosaics. gal4 lines were used primarily to create spatial differences in gene expression. Three gal4 lines for this study (36Y, c929, and 386Y) were used because their expression patterns prominently feature peptidergic neurons of the CNS and secretory cells of peripheral tissues. The three patterns are very similar: the fact that all successfully reverted PHM lethality is probably a reflection of such anatomical similarities. The gal4 patterns also included clear differences in cell number (386Y > c929 > 36Y) (Taghert, 2001).
These pattern differences are of interest, because they may reveal specific neurons (or non-neuronal cells) that produce secretory peptides required for circadian behaviors. However, it is concluded that the interpretation of where 'critical PHM expression' occurs in these experiments is problematic, because there are several ways by which such patterns may defy simple interpretation. For example, two gal4 patterns could appear similar and stable in the adult stage, yet be different because of a transient event during development. In fact, the c929-gal4 pattern is relatively stable in the adult stage but transiently includes the VA neuroendocrine neurons for only a brief period during adult development. In such a case, behavioral rescue may reflect temporal, not spatial, differences in gal4-dependent gene expression. A separate problem in the interpretation could arise when two patterns are spatially similar but differ in levels of expression by specific neurons. In that case, the extent of behavioral rescue may reflect quantitative, not spatial, differences in gal4-dependent gene expression (Taghert, 2001).
Given these complexities, it is currently not possible to specify in which neurons, beyond the LN-V, PHM activity is required for daily locomotor rhythms. Instead, considering candidate amidated neuropeptides directly is favored for subsequent analysis. From scans of the Drosophila genome, there are at least 23 neuropeptide-encoding genes. This is likely an underestimate because of the difficulty in predicting neuropeptide precursor sequences with accuracy. Of the identified genes, ~20 encode peptides are known or are predicted to display C-terminal amidation. Thus, it may be reasonable to systematically address the roles of each of the ~20 precursors using Drosophila genetics (Taghert, 2001).
Genetic studies of circadian behaviors traditionally strive to establish that a mutant phenotype does not simply degrade the ability to produce movement. It this study, the behavior of animals with large-scale alterations in transmitter profiles throughout the entire nervous system has been analyzed. In one genotype (36Y-gal4/UAS-PHM), rhythmicity under constant conditions was extremely poor (as low as that of per01 animals); activity levels were also lower than in other genotypes tested. Nevertheless, when the locomotor rhythm of 36Y-gal4-rescued animals was restored by addition of tim-gal4, activity levels were not also increased. It is proposed that the 36Y-gal4 transmitter mosaic contains disruptions of distinct neural centers, ones that control the general level of activity and ones that organize rest-activity cycles. A similar point is made considering the results seen with tim-gal4. Addition of tim-gal4 to 36Y-gal4-rescued flies produced the greatest restoration of rhythmicity. However, tim-gal4 was by itself unable to revert the lethality of PHM mutants. Therefore, places and times of PHM expression that promote normal vitality do not necessarily equal those promoting circadian behavioral rhythmicity (Taghert, 2001).
Flies lacking clock gene function (e.g., per0) display light-driven behavior under cycling conditions, then become arrhythmic during the first cycle of constant conditions. Arrhythmic PHM mosaics are different. For example, 36Y-gal4-rescued flies, whose rhythmicity under constant conditions is quantitatively as weak as that of per01 animals, entrained well during LD. Therefore, it is concluded that even the most severely arrhythmic PHM mosaic animals studied have levels of circadian clock function and output greater than that present in authentic clock mutants (Taghert, 2001).
The average activity histograms for behavior under constant conditions indicates that different gal4 drivers produce graded levels in circadian locomotor performance. Evidence has been found for at least three levels. The lowest level is represented by single gal4 flies (e.g., 36Y); they have the weakest measures of rhythmicity (by periodogram or MESA) and display little reproducible variation in the average activity histogram. An intermediate level is seen in certain gal4-combination flies (e.g., c929/D42); these display moderate rhythmicity and an average activity peak during early subjective day. The strongest level is seen in other gal4-combination flies (e.g., c929/tim); these are strongly rhythmic, and they display a large average activity peak during late subjective day and a rapid decrease in activity during early subjective night. Presumably, these graded levels of performance reflect incremental contributions by different amidated peptides to one or more circuit components. Relating specific peptide systems to separate levels of behavioral performance represents a challenge for future studies (Taghert, 2001).
Ciccotosto, G. D., Hand, T. A., Mains, R. E. and Eipper, B. A. (2000). Breeding stock-specific variation in peptidylglycine {alpha}-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary. Endocrinology 141: 476-486. 10650926
Eipper, B. A., et al. (1992). Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM). J. Biol. Chem. 267(6): 4008-15. 1740449
Eipper, B. A., Quon, A. S., Mains, R. E., Boswell, J. S., and Blackburn, N. J. (1995). The catalytic core of peptidylglycine alpha-hydroxylating monooxygenase: Investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance. Biochemistry 34: 2857-2865. 7893699
Henrich, V. C., Rybczynski, R. and Gilbert, L. I. (1999). Peptide hormones, steroid hormones, and puffs: Mechanisms and models in insect development. Vitam. Horm. 55: 73-125. 9949680
Jiang, N., et al. (2000). Phm is required for normal developmental transitions and for biosynthesis of secretory peptides in Drosophila. Dev. Biol. 226: 118-136. 10993678
Kolhekar, A. S., Roberts, M. S., Jiang, N., Johnson, R. C., Mains, R. E., Eipper, B. A., and Taghert, P. H. (1997a). Neuropeptide amidation in Drosophila: Separate genes encode the two enzymes catalyzing amidation. J. Neurosci. 17: 1363-1376. 9006979
Kolhekar, A. S., et al. (1997b). Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core. Biochemistry 36(36): 10901-9. 9283080
Kolhekar, A. S., Quon, A. S. W., Berard, C. A., Mains, R. E. and Eipper, B. A. (1998). Post-translational N-glycosylation of a truncated form of a peptide processing enzyme. J. Biol. Chem. 273: 23012-23018. 9722525
Prigge, S. T., et al. (1997). Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase. Science 278(5341): 1300-5. 9360928
Prigge, S. T., et al. (1999). Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase. Nat. Struct. Biol. 6(10): 976-83. 10504734
Siekhaus, D. E. and Fuller, R. S. (1999). A role for amontillado, the Drosophila homolog of the neuropeptide precursor processing protease PC2, in triggering hatching behavior. J Neurosci. 19(16): 6942-54. 10436051
Spijker, S., et al. (1999). A molluscan peptide {alpha}-amidating enzyme precursor that generates five distinct enzymes. FASEB J. 13: 735-748. 10094934
Taghert, P. H., et al. (2001). Multiple amidated neuropeptides are required for normal circadian locomotor rhythms in Drosophila. J. Neurosci. 21(17): 6673-6686. 11517257
date revised: 5 December 2001
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.