Plant glycosidases acting on protein-linked oligosaccharides

Glycosidases have been used as invaluable tools in glycobiology research for decades, and their role in glycoprotein maturation has been amply studied. The molecular biological coverage of this large group of enzymes has only recently reached an appreciable level. In this review, we present an overview of plant glycosidases, whose DNA/protein sequence has been identified and for which recombinant enzymes have been characterized. The physiological role in the maturation of glycoproteins is discussed as well as the biotechnological prospects arising from knowing the enzymes responsible for the removal of terminal N-acetylglucosamine residues. The current knowledge on plant fucosidases and of the first bits of information on glycosidases acting on arabinogalactan proteins is presented.     

Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells

Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions. The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration. Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E. freundii), as judged from the change in the gel filtration pattern. These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure.     

A Novel alpha1,2-L-fucosidase acting on xyloglucan oligosaccharides is associated with endo-beta-mannosidase

Endo-beta-mannosidase, which hydrolyses the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, was discovered in plants. During the course of the purification of the enzyme from lily flowers, we found a higher molecular mass form of the enzyme (designated as EBM II). EBM II was purified by column chromatography to homogeneity and its molecular composition revealed EBM II to be comprised of endo-beta-mannosidase and an associated protein. The cDNA of this associated protein encodes a protein with slight homology to the fucosidase domain of bifidus AfcA. EBM II has alpha1,2-L-fucosidase activity and acts on a fucosylated xyloglucan nonasaccharide. The amino acid sequence of this associated protein has no similarity to known plant alpha-L-fucosidases. These results show that EBM II is a novel alpha1,2-L-fucosidase and a protein complex containing endo-beta-mannosidase.     

Typing of core and backbone domains of mucin-type oligosaccharides from human ovarian-cyst glycoproteins by 500-MHz 1H-NMR spectroscopy

Human blood-group A active glycoproteins from ovarian-cyst fluid were subjected to Smith degradation and subsequent beta-elimination. The resulting oligosaccharide-alditols represent the core and backbone domains of the O-linked carbohydrate chains. Nine of these, ranging in size from disaccharides to hexasaccharides, were investigated by 1H-NMR spectroscopy. Their primary structures could be adequately characterized. In particular, the core types, i.e. the substitution patterns of N-acetylgalactosaminitol (GalNAc-ol) as well as the types of backbone, i.e. the linkage types of alternating Gal-GlcNAc sequences, were unambiguously identified. The core type GlcNAc beta(1-3)GalNAc-ol is described for the first time as occurring in ovarian-cyst glycoprotein.     

O-glycosylation pathway for mucin-type glycoproteins

O-glycosylation is the post-translational process whereby carbohydrate is added to hydroxylated amino acids of proteins. The major O-glycosylation pathway in animal cells is involved in the synthesis of oligosaccharides linked by N-acetylgalactosamine to serine or threonine residues in 'mucin-type' proteins or their analogs. In this review, we discuss the evidence for the cellular localization of the biosynthetic steps in this pathway and propose a simplified, consensus version. We also propose variations of the simple pathway to account for its heterogeneity and variability in different cell types and differentiation states.     

Engineering mammalian mucin-type O-glycosylation in plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.     

Roles of mucin-type O-glycans in cell adhesion

Mucin-type O-glycans containing Core2 branches have distinctly different functions from those O-glycans that contain Core1 structures. Core2 branched O-glycans can have terminal structures that function as ligands for carbohydrate binding proteins. However, sialylated Core2 branched O-glycans without additional modifications exhibit anti-adhesive properties. These results demonstrate that certain mucin-type O-glycans can either facilitate or attenuate cell adhesion depending on the core structures and the structures of the non-reducing termini.     

Isolation and structural characterization of the smaller-size oligosaccharides from desialylated human kappa-casein. Establishment of a novel type of core for a mucin-type carbohydrate chain

Alkaline borohydride reductive cleavage (beta-elimination) of desialylated human kappa-caseinoglycopeptide resulted in the release of a series of oligosaccharides. The smaller-size compounds among them were purified to virtual homogeneity by gel filtration followed by high-performance liquid chromatography. The structures of 9 oligosaccharides were determined by 1H-NMR spectroscopy in conjunction with sugar analysis. The tetrasaccharide Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol and various partial structures thereof were characterized. Notably, the disaccharide GlcNAc beta(1----6)GalNAc-ol and the trisaccharide Gal beta(1----4)GlcNAc beta(1----6)GalNAc-ol were identified; they represent a novel type of core structure for mucin-type carbohydrate chains, namely a peptide-linked GalNAc that is mono-substituted at C-6. In addition, some oligosaccharides ending in GlcNAc-ol could be characterized. Their possible origin is discussed.     

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.     

Cell surface mucin-type glycoproteins and mucin-like domains.

Cell surface mucins and mucin-like domains comprise a diverse and heterogeneous group of cell surface glycoproteins. The heterogeneity results from both genetic variations in the polypeptides and carbohydrate differences. Mucins form extended rods from the cell surface. The mucin domains apparently serve a protective function, protecting the glycoproteins from cell surface proteolysis and protecting the cells from attack by other cells. Biosynthesis of mucin oligosaccharides is initiated near the transit of the proteins from the endoplasmic reticulum to the Golgi and proceeds rapidly during passage to the cell surface. In some carcinomas a second O-glycosylation pathway adds new oligosaccharides to the glycoproteins after they have reached the cell surface, presumably during recycling.     

Robust in-gel fluorescence detection of mucin-type O-linked glycosylation

Mucin-type O-linked glycosylation is a common post translational modification of cell-surface and secretory pathway proteins and is implicated in vital biological processes as well as human disease. We report here the use of the metabolic chemical reporter GalNAz along with Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition conditions for the robust, in-gel fluorescent visualization of mucin-type O-linked glycoproteins.     

The chemistry and biology of mucin-type O-linked glycosylation

Mucin-type O-linked glycosylation is a fundamental post-translational modification that is involved in a variety of important biological processes. However, the lack of chemical tools to study mucin-type O-linked glycosylation has hindered our molecular understanding of O-linked glycans in many biological contexts. The review discusses the significance of mucin-type O-linked glycosylation initiated by the polypeptide N-acetylgalactosaminyltransferases in biology and development of chemical tools to study these enzymes and their substrates.     

The role of mucin-type O-glycans in eukaryotic development

Newly emerging genetic studies have revealed that a subset of the family of glycosyltransferases responsible for the formation of mucin-type O glycans is essential for normal development. As additional genetic, biochemical and physical tools are developed to interrogate the complex structure and surface location of this under-studied class of carbohydrate, no doubt additional roles will be elucidated.