Heptasaccharide (X₃Glc₄ - borohydride reduced)

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different β-glucans we present a range of analytical tools crucial for validation of possible immune-stimulatory effects. Two yeast (MacroGard and Zymosan) and one cereal β-glucan (CBG40) increased IL-8 secretion by HT-29 cells considerably. Degradation of the β-glucan samples with β-glucan specific enzymes did hardly influence the effect of Zymosan and CBG40 but significantly decreased the effect of MacroGard. Stimulation of IL-8 secretion by CBG40 and Zymosan was hence not due to their β-glucan content. Instead, the effect of the CBG40 sample was due to low levels of LPS despite the inability of the known LPS inhibitor Polymyxin B to supress its stimulatory effect. We conclude that targeted enzymatic degradation of samples is a powerful validation tool to investigate carbohydrate specific immune-modulation.

α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-D-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.