This review presents mass spectrometric methods for glycoprotein identification, determination of glycosylation sites, structural elucidation of carbohydrates, and their applications to glycomics and proteomics.
Free N-glycans are present at micromolar concentrations in plant cells during their differentiation, growth and maturation stages, and might play a role in processes such as seed germination and fruit ripening. The structure of free N-glycans, which are found in hypocotyls and developing seeds and fruit, can be classified into two types: a high-mannose type (HMT) and a plant complex type (CT); the former, in most cases, has only one GlcNAc residue, while the latter has a chitobiose unit. It is thought that the enzyme endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) is involved in the production of HMT sugar chains, whereas the enzyme peptide: N-glycanase (PNGase) is involved in the production of plant CT sugar chains. However, the mechanism and significance of free N-glycan production in plant cells remain obscure. To characterize N-glycan metabolism and the physiological function of free sugar chains, we have investigated the substrate specificities, intracellular distributions, and gene structures of endo-β-GlcNAc-ase, PNGase, and α-mannosidase in various plants. Here, we report our discovery that endo-β-GlcNAcase activity begins to increase at a specific stage of tomato ripening, and that the amount of free N-glycans dramatically increases in conjunction with this event. In addition, the structural properties of free N-glycans also change notably as the fruit ripens. This review describes N-glycan metabolism in plant cells, and proposes a role for free sugar chains in the differentiation and growth of plants. The recent finding that plant CT sugar chains are immunoactive is also discussed.
Analysis of the substrate specificity of some sugar chain-related enzymes using homogeneous sugar chains (mainly fluorescently labeled sugar chains) can reveal the in vivo substrate of the enzyme. Such analyses have shown that these enzymes have the ability to recognize not only the sites of hydrolysis or glycosyl transfer but also other regions far apart from them. They can recognize a region consisting of pentasaccharides, hexasaccharides and, in some cases, decasaccharides or larger saccharides. The enzymes recognize part of the substrate strictly, and another part of it tolerantly. These analyses have also identified pairs of enzymes with substrate specificities that are complementary to each other. Here we describe some sugar chain-related enzymes with these properties. The biological significance of the recognition of a large substrate area and its strictness and tolerance will be discussed.
N-Linked glycans are common in eukaryotes, as they are attached to many different proteins. They are classified into three categories termed high mannose, paucimannose, and complex (include hybrid) types. Each phylogenetic group of living organisms has characteristic glycans. In most cases their full significance is unknown, although several functions may be known. For example high mannose-type glycans take part in quality control during the synthesis of proteins. In this review, N-linked glycans are looked at from the viewpoint of their phylogeny and ontogeny.