Immunological and biochemical characterization of extracellular polysaccharides of mucoralean moulds

Abstract

In this thesis the characterization is described of the antigenic determinants (epitopes) of the extracellular polysaccharides (EPSs) from moulds belonging to the order of Mucorales. Detailed knowledge of the structure of these epitopes allows for further development of a new generation of methods for reliable detection of moulds in food. These immunoassays, such as the ELISA (Enzyme-linked Immunosorbent Assay), the latex agglutination assay and the dot- blot assay, are based on the specific recognition of antigenic EPSs by IgG antibodies raised against these polysaccharides.As described in Chapter 2, the water-soluble extracellular polysaccharides which are excreted under various conditions of growth by the mucoralean moulds tested, including the genera Mucor, Rhizopus, Rhizomucor, Absidia, Syncephalastrum and Thamnidium, consist mainly of carbohydrate residues and some protein. The polyclonal IgG antibodies raised in rabbits against EPS of Mucor racemosus were very specific for species of Mucorales; as no cross-reactivity with other moulds was observed. In Chapter 10, the production and characterization of mouse-monoclonal IgG antibodies against the same EPS is described. These antibodies are also very specific for mucoralean moulds but based on different epitopes. However, as shown in Chapter 10, monoclonal antibodies are not necessarily more specific and may be less sensitive than polyclonal antibodies. The immunogenic specificity provides a taxonomic value to EPS as was shown by analysis of EPS preparations of species belonging to the Mortierella isabellina group, which pointed to a classification of this group into the Mucoraceae and not to the Mortierellaceae (Chapter 9).A new method was developed (Chapter 4) for accurate sugar analysis of complex carbohydrate structures with acid sugar residues. This method includes the successive use of methanolysis and TFA hydrolysis followed by high-performance anion-exchange chromatography (HPAEC) analysis of the monosaccharides. This method provided a rapid, accurate and sensitive assay to determine the exact carbohydrate composition of the uronic-acid containing mucoralean EPSs on the microgram scale without any derivatisation. With this method the presence of fucose, mannose, glucose, galactose, and glucuronic acid residues in the mucoralean EPSs was established as well as their relative amounts.A rapid method was developed in which high-performance size-exclusion chromatography (HPSEC) was combined with ELISA detection (Chapter 3). This method allowed the rapid screening and determination of fractions for the presence of antigenic polysaccharides, and enabled an optimal choice for column material to be used for isolation of the antigenic fractions. With this method a common rabbit antigenic fraction, characteristic for moulds of Mucorales, was found. It had an apparent molecular mass of approx. 30 kDa. This fraction accounted for only a minor part of the total EPSs and was mainly composed of mannose residues. The major part, a fraction containing approx.50% glucuronic acid which is known in the literature as mucoran, was found to be antigenic in mice (monoclonal IgG) but not antigenic in rabbits (polyclonal IgG). A β(1-4)-linked D- glucuronan polymer isolated from the mucoralean EPS preparations did not react with the antibodies raised in mice and rabbits (Chapter 6). The antigenic polysaccharides could also be separated from the non-antigenic polysaccharides with immobilized antibodies. As demonstrated for polyclonal rabbit IgG tile antigenic polysaccharides could be separated in one step with an immunoaffinity column with covalently linked IgG antibodies (Chapter 5).A valuable approach to reveal the structure of fungal carbohydrate epitopes, is the use of purified enzymes which are able to degrade the epitopes, which can then be screened with ELISA. In Chapter 7, such an enzyme was purified from a commercial enzyme preparation from Trichoderma harzianum. It appeared to be an exo-α-D-mannanase. EPSs were treated with this enzyme and the products were analysed by highperformance anion-exchange chromatography and by gas-liquid chromatography/mass spectrometry after derivatisation to alditol acetates. The exo-α-D-mannanase removed the ELISA activity of polysaccharides from Mucorales by hydrolysing the terminal α-D-mannose residues from α(1-2)-linked chains of D-mannose and terminal 2- O -methylmannose residues. This 2- O -methyl-mannose residue was identified in all mucoralean EPS preparations. It represents less than 0.5% (w/w) in all samples tested, and was initially overlooked with all other methods. In particular, determination of glycosidic linkages of carbohydrate residues is often performed by methylation analysis, a method which leaves 2- O -methyl-mannose residues undetected. To our knowledge, this compound has never been reported to occur in fungi. It was proved that the epitopes reactive with rabbit-IgG of Mucorales carry this 2- O -methyl-mannose residue at the non- reducing terminal.As shown in Chapter 8, the antigenic activity of the mucoralean moulds with polyclonal IgG antibodies is mainly based on the 2- O -methyl-mannose residues. Therefore, it can be assumed that the results of the methylation analyses on antigenic mucoralean oligosaccharides performed by Miyazaki and coworkers were erroneously interpreted as proof of α(1-6)-linked mannose residues. It is most likely, that the oligomers they isolated indeed carried 2- O -methyl- mannose residues at the non-reducing end which were not recognised. Finally, the structure of the epitopes reactive with rabbit-IgG raised against mucoralean EPSs was consolidated by hapten-inhibition experiments with synthetic oligosaccharides, which unequivocally proved that 2- O -methyl-mannose residues play a vital role in this immunochemical reaction (Chapter 8).This thesis is a contribution to the basic knowledge of the structure of extracellular polysaccharides from moulds. This knowledge can be used in the development of immunochemical methods for moulds, particularly in food products. A protocol was developed to elucidate the antigenic determinant of fungal polysaccharides. This approach appeared fruitful in discovering that the immunochemical reactivity of EPSs from Mucorales moulds reside mainly in non-reducing terminal 2- O -methyl-mannose residues.Furthermore, this thesis can be considered as a contribution to the development of the knowledge of immunochemistry of sugars in general. This approach may be used to investigate structural features of any polysaccharide or glycoconjugate which is or can be made immunologically active. The methodology is particularly suitable in detecting minor sugars with unusual structure which occur at non-reducing terminals of biopolymers and may have specific biological functions. Specific enzymatic removal of such sugars may uncover structures of which the biological function or specificity (e.g. immunochemical reactivity) may be studied subsequently