Development and exploitation of non-vertebrate glycan microarrays

A basic premise in glycobiology is that glycoconjugates must be recognised by carbohydrate-binding proteins so that the encoded information has a biological meaning. Therefore, adequate systems are required to test the interactions between glycans and their receptors, whether these be lectins, antibodies or enzymes. In recent years, a number of different glycan array formats have been developed, but these tend only to reflect the range of glycan structures present in mammals. However, with many non-mammalian eukaryotic species being either important genetic model organisms, such as Caenorhabditis, Dictyostelium or Drosophila, or being agriculturally- or medically-relevant protozoal or helminth parasites, there is a requirement that glycan arrays should reflect the glycomic potential of these species. To this end, it is proposed to develop glycan arrays based on the natural glycomes of these organisms - using glycans released from natural sources as well as forms remodelled using appropriate glycosidases and recombinant glycosyltransferases. The procedure for conjugating the glycan to the array should be compatible with prior HPLC purification - therefore, a novel linker incorporating a fluorophore for detection, an oxyamine for reaction with unprotected glycans and an amine or azide moiety for functionalisation of array surfaces is proposed. The linker will be tested using small model saccharides and purified N-glycans prior to use in protocols for modifying and fractionating non-mammalian glycomes. Backed up with the approach of enzymatic remodelling glycoproteins to mimic invertebrate-type glycans, the new non-mammalian glycan array will be employed to examine the binding of a range of lectins and anti-carbohydrate antibodies with a focus on carbohydrate-binding proteins from nematodes.

A basic premise in glycobiology is that glycoconjugates must be recognised by carbohydrate-binding proteins so that the encoded information has a biological meaning. Therefore, adequate systems are required to test the interactions between glycans and their receptors, whether these be lectins, antibodies or enzymes. In recent years, a number of different glycan array formats have been developed, but these tend only to reflect the range of glycan structures present in mammals. However, with many non-mammalian eukaryotic species being either important genetic model organisms or being agriculturally- or medically-relevant protozoal or helminth parasites, there is a requirement that glycan arrays should reflect the glycomic potential of these species. To this end, it was proposed to develop glycan arrays based on the natural glycomes of these organisms - using glycans released from natural sources as well as forms remodelled using appropriate glycosidases and recombinant glycosyltransferases. A new procedure for conjugating glycans was developed using a linker incorporating a fluorophore for detection, an oxyamine for reaction with unprotected glycans and an amine or azide moiety for functionalisation of array surfaces. The linker was tested using various small model saccharides and was found to be compatible with enzymatic modifications, HPLC purification, MALDI-TOF mass spectrometry and immobilisation on modified glass surfaces prior to probing with various lectins. The linker was also compatible with conjugation to natural N-glycans and thereby has promise to deliver interesting results about protein-carbohydrate interactions in the future.