Department of Chemistry, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
PLoS One. 2011;6(12):e28679. doi: 10.1371/journal.pone.0028679. Epub 2011 Dec 16.
Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.
酰胺化神经肽在神经系统和内分泌系统中发挥着重要作用。缺乏肽基甘氨酸 α-酰胺化单加氧酶（PAM）的小鼠（唯一能够产生酰胺化肽的酶）不能存活。在酰胺化反应中，反应物（甘氨酸延伸肽）被 PAM 的铜依赖性肽基甘氨酸-α-羟化单加氧酶（PHM）结构域转化为反应中间体（羟甘氨酸延伸肽）。然后，羟甘氨酸延伸肽被 PAM 的肽基-α-羟甘氨酸酰胺化裂解酶（PAL）结构域转化为酰胺化产物。PHM 和 PAL 在脊椎动物中缝合在一起，但在某些无脊椎动物（如果蝇和水螅）中分开。除了其腔内腔催化结构域外，PAM 还包括一个胞质结构域，通过 γ-分泌酶从膜释放后可以进入细胞核。在这项工作中，通过基于液相色谱-质谱的方法，从野生型小鼠和携带 Pam 基因（PAM(+/-)）单个拷贝的小鼠的垂体中定性和定量评估了几种甘氨酸和羟甘氨酸延伸肽以及酰胺化肽。我们首次提供了体内存在肽基-α-羟甘氨酸的证据，表明反应中间体成为游离体，在脊椎动物中不会直接从 PHM 传递到 PAL。喂食铜缺乏饮食的野生型小鼠和 PAM(+/-)小鼠表现出相似的行为缺陷。虽然 PAM(+/-)小鼠中积累了甘氨酸延伸反应中间体，并反映了饮食铜的可用性，但在检查的条件下，酰胺化产物更为普遍，这表明观察到的行为缺陷并非仅仅反映缺乏酰胺化肽。
