Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum Ryegrass Endosperm Cells. I. Hydroxylation of Peptidyl Proline

1981 ◽  
Vol 8 (2) ◽  
pp. 121
Author(s):  
PC Pollard ◽  
GB Fincher
Keyword(s):  
Lolium Multiflorum ◽  
Total Radioactivity ◽  
Ferrous Ion ◽  
Arabinogalactan Protein ◽  
Trichloracetic Acid ◽  
Radioactive Label ◽  
Insoluble Material ◽  
Imino Acid ◽  
Proline Hydroxylation ◽  
Protein Moiety

Suspension-cultured endosperm cells from L. multiflorum secrete into the medium an arabinogalactan-protein in which the protein moiety is rich in hydroxyproline. When cells are grown in the presence of proline labelled with 14C or 3H, the imino acid is rapidly removed from the medium and radio- activity can subsequently be detected in extracellular trichloracetic acid-soluble, ethanol-insoluble material. In this fraction, which contains the arabinogalactan-protein and other polysaccharides, radioactive label is distributed between proline and hydroxyproline. α,α'-Dipyridyl, a chelator of ferrous ion, has no effect on the total radioactivity secreted but markedly alters the distribution of radioactivity in favour of peptidyl proline. Although this inhibition of peptidyl proline hydroxylation can be reversed by ferrous or zinc ions, it is not possible to conclude that ferrous ion, which is required for hydroxylation in other systems, participates specifically in the reaction in ryegrass endosperm cells. Concomitant with the inhibition of proline hydroxylation, α,α'-dipyridyl suppresses the biosynthesis or secretion of extracellular arabinogalactan-protein and arabinoxylan.

scholarly journals Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells

1989 ◽  
Vol 264 (3) ◽  
pp. 857-862 ◽  
Author(s):  
P A Gleeson ◽  
M McNamara ◽  
R E H Wettenhall ◽  
B A Stone ◽  
G B Fincher
Keyword(s):  
Polyclonal Antibodies ◽  
Lolium Multiflorum ◽  
Italian Ryegrass ◽  
Arabinogalactan Protein ◽  
Myeloma Protein ◽  
Protein Core ◽  
Liquid Suspension ◽  
Peptide Component ◽  
Immunodominant Epitopes

An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.

Fine structure of the arabinogalactan-protein from Lolium multiflorum

1987 ◽  
Vol 162 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Antony Bacic ◽  
Shirley C. Churms ◽  
Alistair M. Stephen ◽  
Peter B. Cohen ◽  
Geoffrey B. Fincher
Keyword(s):  
Fine Structure ◽  
Lolium Multiflorum ◽  
Arabinogalactan Protein

scholarly journals Three Decades of Advances in Arabinogalactan-Protein Biosynthesis

2020 ◽  
Vol 11 ◽  
Author(s):  
Jessy Silva ◽  
Ricardo Ferraz ◽  
Paul Dupree ◽  
Allan M. Showalter ◽  
Sílvia Coimbra
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Protein Biosynthesis ◽  
Arabinogalactan Protein ◽  
The Past ◽  
Outer Leaflet ◽  
The Family ◽  
Development Processes ◽  
Proline Hydroxylation ◽  
Genetics And Genomics

Arabinogalactan-proteins (AGPs) are a large, complex, and highly diverse class of heavily glycosylated proteins that belong to the family of cell wall hydroxyproline-rich glycoproteins. Approximately 90% of the molecules consist of arabinogalactan polysaccharides, which are composed of arabinose and galactose as major sugars and minor sugars such as glucuronic acid, fucose, and rhamnose. About half of the AGP family members contain a glycosylphosphatidylinositol (GPI) lipid anchor, which allows for an association with the outer leaflet of the plasma membrane. The mysterious AGP family has captivated the attention of plant biologists for several decades. This diverse family of glycoproteins is widely distributed in the plant kingdom, including many algae, where they play fundamental roles in growth and development processes. The journey of AGP biosynthesis begins with the assembly of amino acids into peptide chains of proteins. An N-terminal signal peptide directs AGPs toward the endoplasmic reticulum, where proline hydroxylation occurs and a GPI anchor may be added. GPI-anchored AGPs, as well as unanchored AGPs, are then transferred to the Golgi apparatus, where extensive glycosylation occurs by the action of a variety glycosyltransferase enzymes. Following glycosylation, AGPs are transported by secretory vesicles to the cell wall or to the extracellular face of the plasma membrane (in the case of GPI-anchored AGPs). GPI-anchored proteins can be released from the plasma membrane into the cell wall by phospholipases. In this review, we present an overview of the accumulated knowledge on AGP biosynthesis over the past three decades. Particular emphasis is placed on the glycosylation of AGPs as the sugar moiety is essential to their function. Recent genetics and genomics approaches have significantly contributed to a broader knowledge of AGP biosynthesis. However, many questions remain to be elucidated in the decades ahead.

Uridine and guanosine incorporation by the mouse one-cell embryo

Development ◽  
1978 ◽  
Vol 44 (1) ◽  
pp. 133-148
Author(s):  
R. J. Young ◽  
K. Sweeney ◽  
J. M. Bedford
Keyword(s):  
Cell Stage ◽  
Trichloracetic Acid ◽  
Insoluble Material ◽  
Embryonic Genome ◽  
Internal Position ◽  
Cell Embryo ◽  
The One ◽  
Sugar Derivatives ◽  
Hydrolysis Of ◽  
Derivatives Of

The activity of the embryonic genome prior to the first cleavage has been assessed by studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold trichloracetic acid (TCA) insoluble material at a low level 1–9 h post fertilization. The incorporation of [3H]guanosine was also low but followed a biphasic curve which had a steeper slope at 1–3 h than during the period 4–9 h post fertilization. Unfertilized mouse ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of [3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no effect on the incorporation of [3H]uridine by them, or of [3H] uridine and [3H]guanosine by unfertilized ova. The uptake and incorporation of [3H] guanosine by one-cell embryos were enhanced by DSMO, but only during the period 1–3 h post fertilization. Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been identified in the soluble fraction obtained from mouse one-cell embryos incubated with [3H] uridine 1–3 h post fertilization. Very little of the [3H] uridine taken up by the embryos is present as [3H] UTP, or [3H] CTP; most is found as [3H] UMP or [3H] UDP or as the sugar derivatives. Alkaline or ribonuclease (A, T1 and T2) hydrolysis of the 3H-labeled ethanol insoluble material precipitated from the lysate of one-cell embryos incubated with [3H] uridine 1–3 h post fertilization liberated radioactive cytidine and uridine-3'-phosphates. This demonstrates that [3H] uridine is incorporated into an internal position in RNA and suggests that RNA synthesis does occur in the one-cell embryo 1–3 h post fertilization. Since pronuclei of one-cell embryos incubated with [3H] uridine were not labeled it appears, however, that the RNA synthesized at the one-cell stage is not a product of the embryonic genome.

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells. II. In vitro Incorporation of Galactosyl Residues From UDPgalactose Into Polymeric Products

1982 ◽  
Vol 9 (1) ◽  
pp. 31 ◽  
Author(s):  
T Mascara ◽  
GB Fincher
Keyword(s):  
Cell Walls ◽  
Membrane Fraction ◽  
Lolium Multiflorum ◽  
Apparent Molecular Weight ◽  
Arabinogalactan Protein ◽  
Methylation Data ◽  
Myeloma Protein ◽  
Mouse Myeloma ◽  
Galactosyl Residues

When mixed-membrane fractions from suspension-cultured Lolium multiflorum endosperm cells are incubated in vitro with UDP-[14C]galactose, 66% ethanol-insoluble products of apparent molecular weight greater than 60 000 are labelled in both galactosyl and glucosyl residues, suggesting that an active UDPgalactose 4-epimerase is present on the membrane fraction. The epimerase can be inhibited with ADPribose, to produce polymeric material in which [14C]galactosyl residues pre-dominate. While some of these residues appear to be associated with glycoproteins, affinity chromatography of the products on mouse myeloma protein J539-Sepharose provides evidence that β-galactans containing 1,6-linkages are amongst the products. Monosaccharide analyses and methylation data indicate that the mixed-membrane preparations contain associated polysaccharide of structure analogous to the 1,3;1,4-β-glucans, arabinoxylans and arabino-3,6-galactans normally found in cell walls or secreted into the medium.

CHARACTERIZATION OF LIGNIN-CARBOHYDRATE COMPLEXES OF ITALIAN RYEGRASS AND ALFALFA

1990 ◽  
Vol 70 (1) ◽  
pp. 193-201 ◽  
Author(s):  
TSUNEO KONDO ◽  
KAZUHIKO MIZUNO ◽  
TADASHI KATO ◽  
TADAKAZU HIROI
Keyword(s):  
Enzymatic Hydrolysis ◽  
Ferulic Acid ◽  
Enzymatic Degradation ◽  
Produced Water ◽  
Lolium Multiflorum ◽  
Complex Structure ◽  
Italian Ryegrass ◽  
Syringyl Lignin ◽  
Insoluble Material ◽  
Ferulic Acids

Lignin-carbohydrate complexes (LCC) were isolated from Italian ryegrass (Lolium multiflorum Lam.) and alfalfa (Medicago sativa L.) stems and their chemical and physical properties, and enzymatic degradation examined. The LCCs, soluble in water, were composed of guaiacyl-syringyl lignin and hemicellulosic carbohydrate mostly consisting of xylose, glucose and arabinose. Enzymatic hydrolysis of the LCCs produced water-insoluble materials which were markedly rich in lignin. In both plant species, the carbohydrate of the insoluble material had a high proportion of arabinose. The insoluble material of Italian ryegrass contained two to three times more xylose and arabinose than that of alfalfa. The Italian ryegrass LCC released significant amounts of p-coumaric and ferulic acids by alkaline and acid hydrolysis, but the alfalfa LCC did not, indicating a more complex structure of the cell wall matrix of Italian ryegrass. The ferulic acid in ryegrass lignin may be responsible for restricting enzymatic degradation of hemicellulose in ryegrass more than occurs in legumes.Key words: Lignin-carbohydrate complex, enzymatic hydrolysis, hemicellulose, ferulic acid, Italian ryegrass, alfalfa

Uptake and Metabolism of Hydroxyproline in Endosperm Cells of Lolium multiflorum (Ryegrass)

1981 ◽  
Vol 8 (6) ◽  
pp. 535 ◽  
Author(s):  
PC Pollard ◽  
PW Way ◽  
GB Fincher
Keyword(s):  
Lolium Multiflorum ◽  
Metabolic Energy ◽  
Arabinogalactan Protein ◽  
Intracellular Protein ◽  
Pyrrolidine Ring ◽  
Peptide Linkage ◽  
Imino Acids ◽  
Hydroxyproline Metabolism ◽  
Uptake And Metabolism ◽  
Extracellular Fraction

Hydroxyproline is rapidly removed from the medium by suspension-cultured endosperm cells of ryegrass (Lolium multiflorum) in a process which exhibits Michaelis-Menten kinetics and is dependent on temperature and metabolic energy. Following uptake by the cells, hydroxyproline is rapidly converted to proline by a route which appears to be more direct than the pathways of hydroxyproline metabolism in mammalian and bacterial systems and which results in substantial conservation of the pyrrolidine ring. Four hours after providing the cells with 5.5 nM [3H]hydroxyproline, approximately 50% of the radioactivity is recovered as intracellular imino acids, intracellular protein and extracellular peptide material. The radioactivity is associated predominantly with proline and hydroxyproline residues. In common with other plant and animal systems, hydroxyproline is not incorporated directly into protein. However, radioactivity originating in extracellular hydroxyproline is detected eventually in peptide linkage, where it appears first as peptidyl proline. In the extracellular fraction which contains arabinogalactan-protein, peptidyl proline is hydroxylated to form peptidyl hydroxyproline and, after 4 h, approximately 18% of the original 3H label is found in this fraction.

scholarly journals Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells

1983 ◽  
Vol 72 (3) ◽  
pp. 754-758 ◽  
Author(s):  
Peter B. Cohen ◽  
Angelo Schibeci ◽  
Geoffrey B. Fincher
Keyword(s):  
Lolium Multiflorum ◽  
Arabinogalactan Protein
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