A hydroxyproline-containing glycopeptide released from the walls of sycamore tissue-culture cells by hydrazinolysis.
Ontology highlight
ABSTRACT:
SUBMITTER: Heath MF
PROVIDER: S-EPMC1165826 | biostudies-other | 1973 Oct
REPOSITORIES: biostudies-other
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Project description:1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.
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Project description:Incubation of a particulate preparation from potato tissue culture cells with UDP-beta-L-[1-3H] arabinose yielded a glycoprotein fraction containing labelled material with the characteristics of hydroxyproline arabinosides. The sugar-protein linkage was resistant to hot alkaline hydrolysis, and the hydrolytic products showed similar electrophoretic and chromatographic behavior to authentic hydroxyproline-arabinosides prepared from potato tissue culture cell walls. Incorporation of arabinose into glycoprotein was stimulated by the addition of de-arabinosylated potato lectin. The product of the incubation co-migrated with native potato lectin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The subcellular distribution of the arabinosyl-transferase was investigated by fractionating potato tissue culture membranes on a discontinuous sucrose gradient in the presence or absence of Mg2+. Under both fractionation conditions the highest specific activity of the enzyme was found in the Golgi-enriched fraction. The results are discussed in relation to the synthesis of the hydroxy-proline-rich glycoprotein component of plant cell walls.
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Project description:Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.
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Project description:Identification of hydroxyproline-containing proteins and hydroxylation of proline in rice
2020-06-09 | PXD017798 |
Project description:This manuscript describes the enrichment and mass spectrometric analysis of intact glycopeptides from mouse liver, which yielded site-specific N- and O-glycosylation data for ? 130 proteins. Incorporation of different sialic acid variants in both N- and O-linked glycans was observed, and the importance of using both collisional activation and electron transfer dissociation for glycopeptide analysis was illustrated. The N-glycan structures of predicted lysosomal, endoplasmic reticulum (ER), secreted and transmembrane proteins were compared. The data suggest that protein N-glycosylation differs depending on cellular location. The glycosylation patterns of several mouse liver and mouse brain glycopeptides were compared. Tissue-specific differences in glycosylation were observed between sites within the same protein: Some sites displayed a similar spectrum of glycan structures in both tissues, whereas for others no overlap was observed. We present comparative brain/liver glycosylation data on 50 N-glycosylation sites from 34 proteins and 13 O-glycosylation sites from seven proteins.
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Project description:We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a 'mitocytoplast' cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.
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Project description:The hydroxyproline-containing proteins (HCPs) among secretory and vacuolar proteins play important roles in growth and development of higher plants. Many hydroxyproline-rich glycoproteins (HRGPs), including Arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs), are identified as HCPs by bioinformatics approaches. The experimental evidence for validation of novel proline hydroxylation sites is vital for understanding their functional roles. In this study, the 62 HCPs containing 114 hydroxyproline (O, Hyp) residues were identified, and it was found that hydroxylation of proline residues in the HCPs could either constitute attachment sites for glycans or have other biological function in rice. The glycomodules of AO, OA, OG, VO, LO, and OE were abundant in the 62 HCPs. Further analysis showed that the 22 of 62 HCPs contained both signal peptides and transmembrane domains, and the 19 HCPs only contained transmembrane domains, while 21 HCPs contained neither. This study indicated the feasibility of mass spectrometry-based proteomics combined with bioinformatics approaches for the large-scale characterization of Hyp sites from complex protein digest mixtures. Furthermore, the expression of AGPs in rice was detected by using ?-GlcY reagent and JIM13 antibody. The results displayed that the AGPs were widely distributed in different tissues and organs of rice, especially expressed highly in lateral root, pollen and embryo. In conclusion, our study revealed that the HCPs and Hyp residues in rice were ubiquitous and that these Hyps could be candidates for linking to glycans, which laid the foundation for further studying the functions of HCPs and hydroxylation of proline residues in rice.
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Project description:Adipose-derived stem cells (ASCs) can differentiate into smooth muscle cells and have been engineered into elastic small diameter blood vessel walls in vitro. However, the mechanisms involved in the development of three-dimensional (3D) vascular tissue remain poorly understood. The present study analyzed protein expression profiles of engineered blood vessel walls constructed by human ASCs using methods of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). These results were compared to normal arterial walls. A total of 1701±15 and 1265±26 protein spots from normal and engineered blood vessel wall extractions were detected by 2DE, respectively. A total of 20 spots with at least 2.0-fold changes in expression were identified, and 38 differently expressed proteins were identified by 2D electrophoresis and ion trap MS. These proteins were classified into seven functional categories: cellular organization, energy, signaling pathway, enzyme, anchored protein, cell apoptosis/defense, and others. These results demonstrated that 2DE, followed by ion trap MS, could be successfully utilized to characterize the proteome of vascular tissue, including tissue-engineered vessels. The method could also be employed to achieve a better understanding of differentiated smooth muscle protein expression in vitro. These results provide a basis for comparative studies of protein expression in vascular smooth muscles of different origin and could provide a better understanding of the mechanisms of action needed for constructing blood vessels that exhibit properties consistent with normal blood vessels.
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