Mode of Action of β-Glucuronidase fromAspergillus nigeron the Sugar Chains of Arabinogalactan-Protein

Abstract

A beta-glucuronidase purified from a commercial pectolytic enzyme preparation of Aspergillus niger hydrolyzed about half of the 4-O-methyl-glucuronic acid (4-Me-GlcA) residues located at the nonreducing terminals of (1-->6)-linked beta-galactosyl side chains of the carbohydrate portion of a radish arabinogalactan-protein (AGP) modified by treatment with fungal alpha-L-arabinosidase. Digestion of the alpha-L-arabinosidase-treated AGP with exo-beta-(1-->3)-galactanase released, by exo-fission of beta-(1-->3)-galactosidic bonds in the backbone chains of the AGP, neutral beta-(1-->6)-galactooligosaccharides with various chain lengths and their acidic derivatives substituted at their nonreducing terminals with 4-Me-beta-GlcA groups. In contrast, successive digestion of the alpha-L-arabinosidase-treated AGP with beta-glucuronidase followed by exo-beta-(1-->3)-galactanase liberated much higher amounts of beta-(1-->6)-galactooligomers together with a small portion of short acidic oligomers, mainly 4-Me-beta-GlcA-(1-->6)-Gal and 4-Me-beta-GlcA-(1-->6)-beta-Gal-(1-->6)-Gal. These results indicate that beta-glucuronidase acts upon 4-Me-beta-GlcA residues in long (1-->6)-linked beta-galactosyl side chains of the AGP, whereas short acidic side chains survive the attack of the enzyme.

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

“…7). The model is similar to that proposed for radish root (Haque et al, 2005) and wheat flour AGPs but very different from the modular structure proposed for AG on heterologously expressed proteins in tobacco and Arabidopsis (Tan et al, 2004(Tan et al, , 2010. The AG structures identified here and shown in our model come from many different AGPs in the leaf extracts, and it will be interesting to determine if some of these structures are found specifically on particular AGP protein backbones.…”

“…Most characterized b-(1→6)-galactan side chains in AGs are reported to be short, of one or two residues (Neukom and Markwalder, 1975;Gane et al, 1995;Gaspar et al, 2001). On the contrary, there are reports of long b-(1→6)-galactan side chains in radish root AGPs (Haque et al, 2005). Similarly, we recently found evidence that wheat (Triticum aestivum) flour endosperm AGP extracts contained long b-(1→6)-galactan side chains heavily substituted with L-Araf at C-3 .…”

“…The series of 1,5 X and Y ions (Domon and Costello, 1988) show the distribution of Gal residues, while the nonreducing end 0,4 A 3 and 0,4 A 2 ions confirm the sequence and also indicate the presence of (1→6)-glycosidic linkages not only between the two Gal residues but also between the GlcA and Gal residues. The methyl group on GlcA is likely to be attached to C-4, as 4-O-Me-GlcA has been found in radish AGP (Haque et al, 2005). This is supported by the presence of the H 3 "elimination" ion (Maslen et al, 2007).…”

“…1A), indicating that many oligosaccharides are substituted with terminal b-GlcA residues. Since both GlcA and 4-O-Me-GlcA (Haque et al, 2005) have been reported in AGPs, we performed per-deuteromethylation to distinguish by MS between the methylated and nonmethylated forms ( were present in the spectra. The relative amounts of the oligosaccharides carrying the 4-O-Me-GlcA modifications were much higher than their nonmethylated counterparts (Fig.…”

Characterization of an Exo-β-1,3-D-galactanase fromStreptomyces avermitilisNBRC14893 Acting on Arabinogalactan-Proteins

“…This is the unique property of exo--1,3-galactanases. [6][7][8][9]20) The catalytic efficiency of Sa1,3Gal43A against -1,3-linked galactooligosaccharides with different degrees of polymerization (dp) was determined ( Table 3). The catalytic efficiency increased from galactobiose (dp2) to galactotetraose (dp4), similarly to that of Ct1,3Gal43A.…”