The structure of the extracellular polysaccharide from Rhizobium meliloti, a microsymbiont in the nitrogen fixing symbiosis, has been investigated. The polysaccharide contains a terminal @-D-glucopyranosyl group with pyruvic acid ketalically linked to its 4 and 6 positions. After removal of this substituent from the methylated polysaccharide the four sugar residues in the side chain were removed sequentially by specific degradations; each of these steps involved oxidation, p-elimination by treatment with base, and, when necessary, acid hydrolysis under mild conditions. The result of each degradation was followed by trideuteriomethylation, hydrolysis, and analysis of the product by GLC/MS. The sequence of the sugar residues in the main chain was determined using a modified Smith degradation in which the polyalcohol, obtained after periodate oxidation-borohydride reduction, was methylated before the mild acid hydrolysis. As a result of these studies, it is concluded that the polysaccharide is composed of octasaccharide repeating units with the structure 25.Biosynthetic and structural studies have revealed that many bacterial polysaccharides are composed of oligosaccharide repeating units* and the largest of these which have been conclusively demonstrated are hexasaccharide repeating units (cf. ref 2-4). There is strong justification for this occurrence of small repeating units since larger ones would require more complex systems of enzymes for their biosynthesis. Despite this, however, preliminary studies on the extracellular polysaccharide from Rhizobium meliloti have indicated that it should either be composed of octasaccharide repeating units or have a less regular s t r~c t u r e .~,~ We now report the structural elucidation of this polysaccharide, using specific degradations.
The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (Mr = 86,000) possesses two carbohydrate-binding sites for N,N'N",N"'- tetraacetylchitotetritol /mol protein, with an apparent Ka = 8.7 X 10(3) M-1 at 4 degrees C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N'- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N',N"'- triacetylchitotriose was over 6-fold more potent than the disaccharide (N,N'- diacetylchitobiose ) which, in turn, was 90 times more reactive than N-acetyl-D-glucosamine. N-Acetyllactosamine [beta-D-Gal-(1----4)-D-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N'- diacetylchitobiose . The requirement for an N-acetyl-D-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.
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