An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles.
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ABSTRACT:
SUBMITTER: Madsen TD
PROVIDER: S-EPMC7441158 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we executed a focused discovery strategy to provide an extensive map of O-glycans on peptides hormones. We found that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites were conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the NPY and Glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for this important class of molecules with potential opportunities for new drug designs.
2020-04-15 | MSV000085289 | MassIVE
Project description:Matrix metalloproteinase-9 (MMP-9) is a secreted endoproteinase with a critical role in the regulation of the extracellular matrix and proteolytic activation of signaling molecules. Human (h)MMP-9 has two well-defined N-glycosylation sites at residues N38 and N120; however, their role has remained mostly unexplored partly because expression of the N-glycosylation-deficient N38S has been difficult due to a recently discovered single nucleotide polymorphism-dependent miRNA-mediated inhibitory mechanism. hMMP-9 cDNA encoding amino acid substitutions at residues 38 (modified-S38, mS38) or 120 (N120S) were created in the background of a miRNA-binding site disrupted template and expressed by transient transfection. hMMP-9 harboring a single mS38 replacement secreted well, whereas N120S, or a double mS38/N120S hMMP-9 demonstrated much reduced secretion. Imaging indicated endoplasmic reticulum (ER) retention of the non-secreted variants and co-immunoprecipitation confirmed an enhanced strong interaction between the non-secreted hMMP-9 and the ER-resident protein calreticulin (CALR). Removal of N-glycosylation at residue 38 revealed an amino acid-dependent strong interaction with CALR likely preventing unloading of the misfolded protein from the ER chaperone down the normal secretory pathway. As with other glycoproteins, N-glycosylation strongly regulates hMMP-9 secretion. This is mediated, however, through a novel mechanism of cloaking an N-glycosylation-independent strong interaction with the ER-resident CALR.
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Project description:Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A(12)) in muscle, while complexes with PRiMA represent tetrameric globular forms (G(4)) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.
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Project description:Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.
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Project description:Here we report a facile and efficient method for site-directed glycosylation of peptide/protein. The method contains two sequential steps: generation of a GlcNAc-O-peptide/protein, and subsequent ligation of a eukaryotic N-glycan to the GlcNAc moiety. A pharmaceutical peptide, glucagon-like peptide-1 (GLP-1), and a model protein, bovine ?-Crystallin, were successfully glycosylated using such an approach. It was shown that the GLP-1 with O-linked N-glycan maintained an unchanged secondary structure after glycosylation, suggesting the potential application of this approach for peptide/protein drug production. In summary, the coupled approach provides a general strategy to produce homogeneous glycopeptide/glycoprotein bearing eukaryotic N-glycans.
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Project description:Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyse the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer -than -expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesised to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.
2017-10-31 | PXD007614 | Pride
Project description:Eukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5' caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export, and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined during 3' end formation. Most lncRNAs are targeted for nuclear RNA surveillance, but a subset with 3' cleavage and polyadenylation features resembling the mRNA consensus can be exported to the cytoplasm. The Hrp1 and Nab2 proteins act at this decision point, with dual roles in mRNA cleavage/polyadenylation and lncRNA surveillance. Our data also reveal the dynamic and heterogeneous nature of mRNA maturation, and highlight a subset of "lncRNA-like" mRNAs regulated by the nuclear surveillance machinery.
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Project description:Proteins targeted to the plasma membrane (PM) of cells are degraded at different rates. Sorting motifs contained within the cytoplasmic domains of transmembrane proteins, post-translational modifications (e.g. ubiquitination), and assembly into multiprotein or protein-lipid complexes all may affect the efficiency of endocytosis and recycling and influence the delivery to degradative compartments. Using the SNAP-tag labeling system, we examined the turnover of a model PM protein, the ? chain of the interleukin-2 receptor (Tac). The surface lifetimes of SNAP-Tac fusions were influenced by their mode of entry into cells (clathrin-dependent versus clathrin-independent), their orientation in the PM (transmembrane versus glycosylphosphatidylinositol-anchored), and ubiquitination in their cytosolic domains. In addition, shedding of SNAP-Tac into the medium was greatly influenced by its O-linked glycosylation status. For a number of PM proteins, delivery to lysosomes and ectodomain shedding represent distinct parallel mechanisms to determine protein half-life.
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Project description:Sulfated peptides are plant hormones that are active at nanomolar concentrations. The sulfation at one or more tyrosine residues is catalysed by tyrosylprotein sulfotransferase (TPST), which is encoded by a single-copy gene. The sulfate group is provided by the co-substrate 3´-phosphoadenosine 5´-phosphosulfate (PAPS), which links synthesis of sulfated signaling peptides to sulfur metabolism. The precursor proteins share a conserved DY-motif that is implicated in specifying tyrosine sulfation. Several sulfated peptides undergo additional modification such as hydroxylation of proline and glycosylation of hydroxyproline. The modifications render the secreted signaling molecules active and stable. Several sulfated signaling peptides have been shown to be perceived by leucine-rich repeat receptor-like kinases (LRR-RLKs) but have signaling pathways that, for the most part, are yet to be elucidated. Sulfated peptide hormones regulate growth and a wide variety of developmental processes, and intricately modulate immunity to pathogens. While basic research on sulfated peptides has made steady progress, their potential in agricultural and pharmaceutical applications has yet to be explored.
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