CM-Linear 1,5-α-L-Arabinan (Sugar Beet)

Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of β-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.

Comparison of Salvia miltiorrhiza polysaccharides (SMPs) from different geographical origins in China (Henan, Hebei, Shandong, Sichuan, Shaanxi) was performed using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID), saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and combined with principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Based on the results of HPSEC-MALLS/RI, the relative content of SMPs showed a significant difference between different geographical origins, however, the molecular weight of SMPs showed almost no significance. SMPs can be discriminated as five regions after PACE coupled with OPLS-DA models analysis of endo-1,5-α-arabinanase hydrolysates. Moreover, all the PACE fingerprint indicated that 1,4-β-D-Galp, 1,5-α-Araf, 1,4-α-D-GalAp and 1,4-β-D-Glcp linkages existed in SMPs.

A novel endo-α-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45-50°C with stability between pH 5.0-8.0 and at temperatures up to 40°C. The recombinant ARN2 catalyzed internal cleavage of α-1,5 glycosidic bonds of CM-arabinan, debranched arabinan, linear arabinan, and sugar beet (native) arabinan at rates of decreasing order, and was inactive on wheat arabinoxylan and p-nitrophenyl-α-L-arabinofuranoside. Kinetic analysis showed that branching in the arabinan did not significantly affect the apparent K_m values, and the difference in the reaction rates was likely due to the chemical step after substrate binding. The enzyme hydrolyzed arabino-oligosaccharides of DP 6 to smaller oligomers and mostly arabinotriose. Natural and modified arabinans were cleaved to oligomers of various chain lengths, which were progressively hydrolyzed to yield arabinotriose. The pattern of degradation revealed an endo-acting mechanism with arabinotriose as the end product.