Barbara S. Valent

A general and sensitive chemical method for sequencing the glycosyl residues of complex carbohydrates

This paper describes a new glycosyl-sequencing method. This method was made possible by the ability to fractionate complex mixtures of peralkylated oligosaccharides by reversed-phase, high-pressure liquid chromatography. The fractionation ability of the reversed-phase system allows the isolation and subsequent unambiguous identification by g.l.c.-m.s. of disaccharides, almost all trisaccharides, and, in some cases, tetrasaccharides generated by successive partial acid hydrolysis, reduction, and ethylation of a permethylated, complex carbohydrate. As these small oligosaccharides overlap within the unhydrolyzed, complex carbohydrate, the oligosaccharide sequences may be pieced together, and, with the glycosyl-linkage composition of the intact complex carbohydrate, can be used to determine the glycosyl sequence of the complex carbohydrate. The details of the sequencing method are illustrated by the elucidation of the glycosyl sequences of three complex carbohydrates. These examples demonstrate the wide variety of complex carbohydrates whose structures can be ascertained by the new sequencing technique. Two of the examples are the commercially available polysaccharides, lichenan and xanthan, whose structures have already been reported. The other example is a nonasaccharide derived from xyloglucan, a structural polymer of plant cell-walls. The glycosyl residues of the complex carbohydrates studied include hexosyl, deoxyhexosyl, pentosyl, glycosyluronic, and pyruvic acetal-substituted hexosyl residues. It will be demonstrated that the new glycosyl-sequencing technique is not compromised by the presence, in the carbohydrate to be analyzed, of glycosyl linkages possessing very different acid labilities. Two major advantages of this sequencing technique are that it is relatively rapid and that it requires only milligram quantities of carbohydrate.

Oligosaccharins: Naturally Occurring Carbohydrates with Biological Regulatory Functions

Complex carbohydrates have many functions but, until recently, their functions were thought to be limited to serving as structural polymers and energy reserves. It is now well established that complex carbohydrates play an important role in biological recognition. In their role as recognition agents, complex carbohydrates are: receptors for phage and bacteriocins; specific surface antigens that can determine the pathogenicity of microbes, and the mating type, the blood group type, and tissue type of eukaryotic cells; highly specific receptors in eukaryotes for viruses, bacteria, hormones, and toxins; and determinants of where glycoproteins go within cells, when they are secreted, and when they are taken up. We have now come to recognize that certain complex carbohydrates are chemical messengers, that they are, in fact, biological regulatory molecules. Results of research in our laboratory have led us to believe that these chemical messengers are especially important in regulating growth, development, reproduction, and disease resistance in plants.

Structure and function of complex carbohydrates active in regulating plant-microbe interactions

A key regulatory role of complex carbohydrates in the interactions between plants and microbes has been estab— lished. The complex carbohydrates act as regulatory mole— cules or hormones in that the carbohydrates induce de novo protein synthesis in receptive cells. [11 The first complex carbohydrate recognized to possess such regulatory proper— ties is a polysaccharide (PS) present in the walls of fungi (2). Hormonal concentrations of this PS elicit plant cells to accumulate phytoalexins (antibiotics). [2] More recently we have recognized that a PS in the walls of growing plant cells also elicits phytoalexin accumulation; microbes and viruses may cause the release of active fragments of this endogenous elicitor. [3] Another PS in plant cell walls is the Proteinase Inhibitor Inducing Factor (PIIF) (53). This hormone appears to protect plants by inducing synthesis in plants of proteins which specifically inhibit digestive enzymes of insects and bacteria. [4] Glycoproteins secreted by incompatible races (races that do not infect the plant) of a fungal pathogen of soybeans protect seedlings from attack by compatible races. Glycoproteins from compatible races do not protect the seedlings (61). [5] The acidic PS secreted by the nitrogen—fixing rhizobia appear to function in the infection of legumes by the rhizobia. W.D. Bauer and his co—workers have evidence that these PS are required for the development of root hairs capable of being infected by symbiont rhizobia. Current knowledge of the structures of these biologically active complex carbohydrates will be presented.

Structure and Function of Complex Carbohydrates Active in Regulating the Interactions of Plants and Their Pests

Our laboratory has recently come to realize that complex carbohydrates of higher plants, fungi, and bacteria can act as regulatory molecules, that is, as molecules which in minute quantities alter the metabolism of receptive cells by causing the synthesis of specific proteins. It is not surprising that these structurally complex and exquisitely specific molecules can possess regulatory properties, as many diverse classes of molecules including glycoproteins, proteins, peptides, steroids and a variety of smaller molecules such as epinephrine, indoleacetic acid, gibberellic acid, cytokinins, and even ethylene, are known to possess regulatory properties.