Structural characterization of endo-glycanase-generated oligoglycosyl side chains of rhamnogalacturonan I.
Rhamnogalacturonan I (RG-I) has been isolated from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus), and additional structural features of the polysaccharide were elucidated. Treatment of RG-I with a purified endo-(1-->5)-alpha-L-arabinanase released a series of arabinose-containing oligosaccharides with degrees of polymerization (dp's) between 2 and 20. These oligosaccharides were shown, by glycosyl-linkage composition analysis, to contain terminal, 5-, and (3-->5)-linked Araf residues. These results provide evidence that a branched arabinan is attached to the backbone of RG-I. RG-I was freed of 95% of its arabinosyl residues by treating the polysaccharide with a combination of endo-(1-->5)-alpha-L-arabinanase and alpha-L-arabinosidase. No galacturonic acid was released by these enzymes, which is evidence that the arabinosyl-containing portions of the side chains do not contain galactosyluronic acid residues. The galactose-containing portions of the side chains of RG-I were not fragmented by an endo-(1-->4)-beta-D-galactanase. However, approximately 85% of the galactose and small amounts of galacturonic acid were released by digestion of arabinose-depleted RG-I with a combination of endo- and exo-beta-D-galactanases. The galacturonic acid may have been released by small amounts of an exo-alpha-galactosyluronidase contaminating the galactanases. Treatment of RG-I with this mixture of endo- and exo-glycanases resulted in a relatively size-homogeneous, almost side chain-free backbone composed of the O-acetylated diglycosyl repeating unit -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap. A combination of 1H NMR spectroscopy and periodate oxidation established that the backbone repeating unit contained a single O-acetyl substituent on C-2 or C-3 of each galactosyluronic acid residue.
Lerouge
P
P
Complex Carbohydrate Research Center, University of Georgia, Athens 30602-4712.
O'Neill
M A
MA
Darvill
A G
AG
Albersheim
P
P
D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000737:chemistry; D002478:Cells, Cultured; D002850:Chromatography, Gel; D008401:Gas Chromatography-Mass Spectrometry; D006026:Glycoside Hydrolases; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010368:Pectins / Q000737:chemistry* / Q000302:isolation & purification; D014197:Trees / Q000737:chemistry*; D005644:alpha-L-Fucosidase
D009844:Oligosaccharides; C042491:rhamnogalacturonan I; D010368:Pectins; D006026:Glycoside Hydrolases; D005644:alpha-L-Fucosidase; C082526:arabinan endo-1,5-alpha-L-arabinosidase