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The Production of Biologically Active Peptides in Brain Tissues
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
It had been considered that amidation might be achievable through the action of an oxidizing (hydroxylating) enzyme followed by chemical cleavage of the resultant hydroxylated peptide intermediate. Such an intermediate has been identified.59,60 It has also been shown that dismutation of the hydroxyglycine intermediate to peptide amide and glyoxylate can occur non-enzymatically, but that at physiological pH this reaction is assisted by a second enzyme (Figure 2).59–63 Thus, peptide amidation in vivo is a two-step process catalyzed by: (1) peptidylglycine monooxygenase (PGM), i.e., peptidyl α-amidating enzyme; peptidylglycine 2-hydroxylase (EC 1.14.17.3) and (2) peptidylamidoglycolate lyase(PAL); α-hydroxyglycine amidating dealkylase (EC 4.3.2.5).61 It has been demonstrated that PGM and PAL have matching stereoselectivities.64,65
Ascorbate as an Enzyme Cofactor
Published in Qi Chen, Margreet C.M. Vissers, Vitamin C, 2020
Margreet C.M. Vissers, Andrew B. Das
Many neuropeptide hormones and neurotransmitters contain a C-terminal amide that is necessary for biological activity [241,255]. The final step in the amidation of glycine-extended precursors of these neuropeptides is catalyzed by peptidylglycine α-amidating monooxygenase (PAM), in a reaction that requires copper, ascorbate, and molecular oxygen (Figure 5.5 and Table 5.1) [241,248]. PAM is a multifunctional enzyme with two distinct catalytic domains that generate the amidated products in two sequential steps (Figure 5.5). In the first step, a hydroxylated intermediate is generated by peptidylglycine α-hydroxylating monooxygenase (PHM). This reaction is Cu-dependent, consumes ascorbate and O2 (Figure 5.5), and is followed by conversion of the intermediate to the α-amidated peptide product and glyoxylate by peptidyl-α-hydroxyglycine α-amidating lyase (PAL) (Figure 5.5) [241,248,256].
Regulation of Gastrointestinal Neuropeptide Gene Expression and Processing
Published in Edwin E. Daniel, Neuropeptide Function in the Gastrointestinal Tract, 2019
Amidation is a posttranslational modification process which is essential for bioactivity of several peptides occurring in the mature secretory granule. It occurs after the proteolytic processing at double basic residues and involves an enzyme that converts a C-terminal glycine (Gly) residue and the peptide bond between the ultimate residue preceding Gly to-CO-NH2; the enzyme is dependent upon copper and ascorbate and has recently been cloned.15,73,83–86
Recent advances in proteolytic stability for peptide, protein, and antibody drug discovery
Published in Expert Opinion on Drug Discovery, 2021
Xianyin Lai, Jason Tang, Mohamed E.H. ElSayed
About 50% of the bioactive peptides found in the nervous and endocrine systems possess a C-terminal α-amide group (-X-NH2). For most of these peptides, the presence of the α-amide moiety is important to their bioactivities. Peptide tyrosine tyrosine (PYY), amylin, calcitonin, oxytocin, vasopressin, and many others naturally have α-amidation at the C-terminal [114,115]. Besides the bioactivity benefits from the C-amidation, the modification prevents degradation by proteases. In a study to examine the effect of C-amidation against a periplasmic endoprotease Tsp, two pairs of peptides were used. Peptides BAS7 and BAS9 differed only in whether the C-terminus was a carboxyl or a carboxamide group. The only difference between Peptides BAS8 and BAS10 was that BAS8 was C-amidated. After the peptides were incubated with Tsp for 5 h, BAS7, and BAS8 had less than 5% cleavage, while BAS9 and BAS10 showed 50% and 45% cleavage, respectively [116].
Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry
Published in mAbs, 2019
Yutong Jin, Ziqing Lin, Qingge Xu, Cexiong Fu, Zhaorui Zhang, Qunying Zhang, Wayne A. Pritts, Ying Ge
Previous middle-down MS analysis of mAb glycosylation focused on highly abundant Fc/2 glycoforms, such as G0F and G1F.26,33 In this study, we not only analyzed the high-abundance glycoforms, but also characterized the micro glycoforms with confident bond cleavages. We obtained 58% bond cleavage of Fc/2-G0 and 44% bond cleavage of Fc/2-G2F in offline MS/MS analysis with the localization of glycosylation site at Asn61. Moreover, we characterized a micro-variant with C-terminal glycine clipping and proline amidation. Previously, Johnson et al.39 used weak cation exchange-high performance LC separation and bottom-up MS to detect the C-terminal amidation in an IgG1 heavy chain. Here we demonstrated the characterization of C-terminal amidation by middle-down MS, which avoids the lengthy sample preparation and improves the confidence of identification by analyzing large polypeptide fragment of ~ 25 kDa. It was reported that C-terminal amidation is a general PTM of therapeutic mAbs and it could be catalyzed by peptidylglycine alpha-amidating monooxygenase.40–42 However, no effect of C-terminal amidation on activity of therapeutic mAbs was observed so far.43
Developing mass spectrometry for the quantitative analysis of neuropeptides
Published in Expert Review of Proteomics, 2021
Christopher S. Sauer, Ashley Phetsanthad, Olga L. Riusech, Lingjun Li
Neuropeptides and other bioactive peptides are formed after enzymatic cleavage of larger precursors by peptidases. Additional enzymes can alter these peptides with PTMs, altering their structure, function, and stability, among other effects, contributing to the vast diversity of neuropeptides. Neuropeptide modifications can include amidation, phosphorylation, acetylation, glycosylation, and sulfation, to name a few [87]. Identifying and differentiating between these forms is crucial to understand molecular mechanisms in neurobiology, and thus these modified neuropeptides are investigated using MS by a variety of labs [10,88–90], thoroughly summarized in a few recent reviews [91]. The quantification of post-translationally modified neuropeptides faces additional challenges. Labeling approaches often target specific residues and moieties, so post-translational modification of these residues often inhibits quantitation via labeling. For example, many tags target primary amines and are therefore ineffective or less effective for peptides with acetylated N-termini. Further challenging analysis of modified peptides is the decreased ion signal intensity due to poor ionization efficiency of the modified peptides or from distribution of already low abundance neuropeptides across the differentially modified forms. This leads to the need for targeted analyses and enrichment strategies to detect and quantify peptides with PTMs [92–94], especially for those modified by highly dynamic glycosylation [94–97]. As MS considerations are more prominent for peptides modified by glycosylation [93,98,99], we will focus on the discussion of glycosylated peptides.