Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2.
Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.
Han
Mei
M
Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, Missouri, USA.
Park
Dongkook
D
Vanderzalm
Pamela J
PJ
Mains
Richard E
RE
Eipper
Betty A
BA
Taghert
Paul H
PH
D000577:Amides / Q000378:metabolism*; D019760:Amidine-Lyases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation / Q000502:physiology; D002460:Cell Line; D002490:Central Nervous System / Q000166:cytology / Q000201:enzymology; D004330:Drosophila / Q000201:enzymology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007814:Larva / Q000201:enzymology; D009474:Neurons / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D051381:Rats; D014162:Transfection
D000577:Amides; D029721:Drosophila Proteins; D009479:Neuropeptides; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase