Sugar Composition Analysis of a ConA-binding Glycoprotein Isolated from the Novikoff Hepatoma Cell Surface

This study reports on the ultrastructural location and biophysical properties of cell-associated glycosaminoglycans of AH-130 cells, an azo dye-induced ascites hepatoma. Earlier studies have shown that a low-sulfated heparan sulfate, which comprises 93% of their total glycosaminoglycan (GAG) content, is associated with these cells. High-iron diamine, an ultrastructural stain for sulfated glycoconjugates, stained the hepatoma cell surfaces heavily. With the exception of occasional light staining in a few cytoplasmic granules, intracellular organelles did not stain with this method. The lack of an extensive pool of intracellular GAG was confirmed by quantitative fluorescence microscopy of cells vitally stained with acridine orange. The nature of the binding of the cell-surface heparan sulfate was explored by competitive binding studies with exogenous heparin. When cells were incubated with exogenous heparin, release of heparan sulfate into the medium was not detected, although heparin was bound. We conclude that low-sulfated heparan sulfate is an integral component of the AH-130 hepatoma cell surface and is bound at a site different than heparin.

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Intracellular interactions of transferrin and its receptor during biosynthesis.

The Journal of Cell Biology ◽

10.1083/jcb.111.4.1383 ◽

1990 ◽

Vol 111 (4) ◽

pp. 1383-1392 ◽

Cited By ~ 9

Author(s):

J J Neefjes ◽

T Hengeveld ◽

O Tol ◽

H L Ploegh

Keyword(s):

Cell Surface ◽

Cell Line ◽

Transferrin Receptor ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Human Hepatoma ◽

Human Hepatoma Cell ◽

Human Hepatoma Cell Line ◽

Hepatoma Cell Line Hepg2 ◽

Acidic Compartment

The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfide-linked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and is ultimately delivered to an acidic compartment, where Tf releases its Fe3+. We did not observe conversion of Tf to apoTf in the TGR, showing that the part of the TGR passed by secreted Tf has a pH higher than 5.5. We conclude that when a ligand-receptor combination is synthesized by one and the same cell, ligand and receptor can interact during biosynthesis and be transported to the cell surface.

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Flagellin Glycans from Two Pathovars of Pseudomonas syringae Contain Rhamnose in d and l Configurations in Different Ratios and Modified 4-Amino-4,6-Dideoxyglucose

Journal of Bacteriology ◽

10.1128/jb.00500-07 ◽

2007 ◽

Vol 189 (19) ◽

pp. 6945-6956 ◽

Cited By ~ 34

Author(s):

Kasumi Takeuchi ◽

Hiroshi Ono ◽

Mitsuru Yoshida ◽

Tadashi Ishii ◽

Etsuko Katoh ◽

...

Keyword(s):

Host Specificity ◽

Pseudomonas Syringae ◽

Molar Ratio ◽

Composition Analysis ◽

Resonance Spectroscopy ◽

Mass Analysis ◽

Sugar Composition ◽

Enantiomeric Ratio ◽

Race 4

ABSTRACT Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, β-d-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely of l-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both l-Rha and d-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae.

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