The plasma membrane establishes the interface for the communication of cells with the environment. Thus, surface determinants govern the reactivity and capacity of cells to respond to external signals. Changes in their profile, for example in malignant transformation, and manifestation of cell-type-specific features apparently hold inspiring lessons in store for us on how they are translated into cellular responses. But before turning to the signaling routes the biochemical modes for coding signals warrant a comment, as proteins are often unduly portrayed as the decisive hardware. In contrast, and actually prominent among the biochemical systems to store information, carbohydrate epitopes of cellular glycoconjugates favorably combine high-density coding with strategic positioning, rendering them readily accessible for interactions with adaptor molecules. The interaction with lectins is the ignition key to start glycoconjugate-mediated biosignaling. Several plant lectins, especially due to their mitogenicity, have become a popular type of laboratory tool to elicit cell responses and to analyze biochemical pathways leading from initial binding to measured activity such as enhanced proliferation. With emerging insights into the roles of mammalian (endogenous) lectins and the promising perspective for medical applications, emphasis in this area is shifting from model studies with plant proteins toward work with the physiological effectors. By targeting branchend epitopes of glycan chains two classes of endogenous lectins, i.e. galectins and selectins, are remarkably well suited to establish initial contacts with the cell surface. Indeed, these lectins-in their interplay with certain cognate binding partners-are being defined as potent signal inducers. Consequently, we can take aspects of their activity profiles as incentive to dissect underlying routes of signal transmission with an eye more on principles than on intricate case-specific details. Hence, regulation of cell growth by cascades of mitogen-activated protein kinases (MAPKs), cyclins/cyclindependent kinases and inhibitors thereof, of cell survival by the phosphatidylinositol 3'-OH kinase (PI3K)/Akt pathway, the remodeling of the cytoskeleton by integrin-mediated cell adhesion, the implication of p53 in regulating cell fate and details of programmed cell death by the intrinsic and extrinsic routes for induction of apoptosis will be discussed. Moreover, we will look at selectin-induced signaling during leukocyte homing. Explicitly, it is the aim of our review to familiarize glycoscientists, whose main interest is to scrutinize the structural aspects or to develop applications, with basic concepts of cellular signaling triggered by these interactions.
Endo-β-mannosidase (EC 126.96.36.199), which hydrolyzes Manβ1-4GlcNAc linkages in the core structure of N-glycans, has been found in higher plants. Recently, purification of this enzyme and molecular cloning of its gene have been accomplished. Orthologues of endo-β-mannosidase gene have been found only in plant species, suggesting that this enzyme has plant-specific functions. This endoglycosidase has unique substrate specificity, hydrolyzing the Manβ1-4GlcNAc linkage in (Man)nManα1-6Manβ1-4GlcNAcβ1-4GlcNAc (n=0-2). These substrates are generated from high-mannose type N-glycans by the action of a jack bean α-mannosidase-like enzyme, which prefers to hydrolyze Manα1-3Manβ linkages. It is therefore likely that endo-β-mannosidase and jack bean α-mannosidase-like enzyme cooperatively hydrolyze highmannose type N-glycans to N, N'-diacetylchitobiose in plant cells. In addition, endo-β-mannosidase has transglycosylation activity that can form a β-mannoside linkage, for which anomeric regulation during chemical synthesis reaction is difficult.
Many glycosidases exhibit transglycosylation activities that involve the transfer of a carbohydrate moiety to the hydroxyl groups of various compounds, in addition to hydrolytic activities. We established the chemo-enzymatic synthesis of a glycopeptide using the transglycosylation activity of endo-β-N-acetylglucosaminidase of Mucor hiemalis (Endo-M). This method consists of the chemical synthesis of N-acetylglucosaminyl peptide and the transglycosylation of N-linked sugar chain from oligosaccharide donor to an N-acetylglucosaminyl peptide by Endo-M. We could add the sialo-complex-type oligosaccharide to bioactive peptides such as Peptide T and calcitonin by this method. We were also able to add the oligosaccharide to the glutamine residue of the Substance P neuropeptide and yeast α-mating factor. These glycosylated bioactive peptides showed a higher degree of resistance to protease digestion than original peptides. By means of transglycosylation of Endo-M, we prepared glycopolymer containing multivalent oligosaccharides which can inhibit infection of influenza viruses to host cells. We also could exchange the high-mannose type of oligosaccharides in glycoproteins/glycopeptides to the complex types by the transglycosylation of Endo-M, and could synthesize novel glycolipids having a sugar chain of glycoprotein using the transglycosylation activity of Endo-M followed by preparation of a monoclonal antibody against the oligosaccharide of glycoprotein using the obtained glycolipid as immunogen. Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum exhibits transglycosylation activity. We succeeded in adding Galβ1, 3GalNAc from Galβ1, 3GalNAcα1pNP to 1-alchanol, monosaccharide and bioactive peptides containing a serine/threonine residue using this enzyme.
We investigated the transglycosylation activity of the recombinant endo-β-N-acetylglucosaminidase (Endo-M) from Mucor hiemalis expressed in Candida boidinii in media containing organic solvents, using a disialo biantennary complex-type oligosaccharide from the hen egg yolk glycopeptide as the glycosyl donor and p-nitrophenyl N-acetyl-β-D-glucosaminide as the glycosyl acceptor. The recombinant Endo-M had sufficient transglycosylation activity to transfer the oligosaccharide to pNP-GlcNAc in up to 30-40% organic solvents (v/v) such as acetone, dimethyl sulfoxide and methanol. The transglycosylation activity in each organic solvent differed from each other. The investigation of the donor concentrations in the organic solvents revealed that the increase in the concentrations significantly increased the transglycosylation yields. Using the Endo-M transglycosylation system in organic solvents, we successfully demonstrated the transglycosylation reaction to the glycosyl acceptors which were sparingly soluble in water such as artificial N-acylated D-glucosamines and a 2-O-glycosylated disaccharide.
The enzyme which catalyzed deglycosylation of human salivary α-amylase family A(HSA-A) to form salivary α-amylase family B(HSA-B) in saliva was revealed to be an endo-β-N-acetylglucosaminidase. It was named endo-β-N-acetylglucosaminidase HS(Endo HS). Endo HS is specific for complex type asparagine-linked oligosaccharides and can release bi, tri and tetrantennary complex type oligosaccharides from native glycoproteins, glycopeptides and asparaginyl oligosaccharides regardless of the existence of fucosyl residue attached to the proximal GlcNAc residue of oligosaccharides. Endo HS can also transfer these oligosaccharides to various monosaccharides. Furthermore, HSA having oligosaccharides at the two potential glycosylation sites was also found, purified, and named human salivary α-amylase family C(HSA-C). It showed the similar antigenicity for HAS-A and HAS-B. HSA-C has two biantennary complex type oligosaccharides. HSA-C was converted to HSA-B via HSA-A by the action of Endo HS. The multiple form pattern of HSA-C was also converted to the same pattern as that of HSA-B. The existence of HSA-C and Endo HS, and the conversion of HSA-C to HSA-B via HSA-A by the action of Endo HS in human saliva suggest that HSA is glycosylated at the two potential glycosylation sites, secreted into saliva, as HSA-C and then deglycosylated by Endo HS to be the oligosaccharide-deficient glycoisoforms such as HAS-A and HAS-B present in human saliva.