Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F (PNGase F) is an amidase/amidohydrolase that hydrolyzes the β-aspartylglycosylamine bond of asparagine-linked glycopeptides and glycoproteins. The enzyme recognizes mainly the polypeptide region around the glycosylation site and the inner core of the carbohydrate chain. All classes of oligosaccharides are released by PNGase F, making it the enzyme of choice for deglycosylation of most asparagine-linked glycans. Used in conjunction with current biotechnology, PNGase F is an important tool for structure/function analysis of glycoproteins, structural analysis of oligosaccharides, and biosynthetic studies of asparagine-linked oligosaccharides. PNGase F is available from several commercial sources, but is relatively expensive. Alternatively, the enzyme can be purified in large amounts by most laboratories with minimal work using the one-step protocol described herein.
In order to further understand the biological significances and regulatory mechanisms for expression of gangliosides, it is essential to isolate glycosyltransferase genes responsible for the synthesis of their carbohydrate structures. Expression cloning and analysis of the expression of β1, 4 N-acetylgalactosaminyltransferase gene were reported. The cDNAs cloned by the eukaryotic cell expression system using CDM8 vector predicted the structure of type II transmembrane protein which is common among the glycosyltransferase genes reported so far. When appropriate cells were transfected with these cDNAs, they newly expressed GM2, or GM2 and GD2 suggesting that the cDNAs actually derived from GM2/GD2 synthase gene. The characteristic profile of the structure and function of this transferase gene was summarized, and the importance of isolation of another glycosyltransferase genes was emphasized.
The analysis of oligosaccharides and polysaccharides takes on increased importance with the discovery of each new biological activity attributable to this important class of biopolymers. Our understanding of other biopolymers, proteins and nucleic acids, has relied heavily on electrophoresis. This minireview describes the application of capillary electrophoresis and gradient polyacrylamide gel electrophoresis to saccharide compositional analysis, oligosaccharide mapping and oligosaccharide and polysaccharide sequencing. These approaches are applicable to both acidic polysaccharides (i.e., glycosaminoglycans) and neutral, glycoprotein-derived oligosaccharides. Separation by electrophoresis can be driven by the inherent charge of a carbohydrate, through the complexation of a carbohydrate with a charged molecule, or through derivatization reactions to prepare charged carbohydrate conjugates. New approaches including fluorescence labeling provide the high detection sensitivity required for microanalysis. The high resolution necessary for oligosaccharide analysis can be achieved using capillary electrophoresis or two-dimensional gel electrophoresis. Preparative gradient polyacrylamide gel electrophoresis can also be used to obtain pure oligosaccharides for structural studies. Future prospects for the complete sequence analysis of oligosaccharides and polysaccharides by electrophoresis are discussed.
Aldolase which catalyzes aldol cleavage in the process of glycolysis also catalyzes reverse reaction, aldol condensation, to afford carbohydrates. From this point of view, aldolases are useful catalysts to synthesize novel carbohydrates. The substrate specificity for the enolate source of aldolase catalyzed reactions is very high, but, the enzymes accept many kinds of aldehyde as acceptor substrates of aldol condensation. FDP-aldolase whose enolate source is dihydroxyacetone phosphate and sialic acid aldolase whose enolate source is pyruvate are purchased cheaply and many papers on the syntheses of carbohydrates using these aldolases have been published during the last ten years. Especially, the method of synthesizing fluorosugars and azasugars from aldehydes including fluoro or azido group by using aldolases has been well established. It is needless to say that the aldol condensation catalyzed by aldolases is stereospecific and the optical purities of the condensed products are extremely high. Details of the application of the aldolase catalyzed reactions for carbohydrate synthesis will be shown below.
A number of compounds have been identified over the past 10 years that inhibit specific glycosidases involved in the processing of N-linked oligosaccharides. These compounds include specific α-glucosidase and α-mannosidase inhibitors that block removal of either glucose or mannose residues at specific steps in the processing pathway, and cause the formation of altered carbohydrate structures. Many of these inhibitors have been widely used in cell culture to assess the role of specific oligosaccharide chains in the function of a given glycoprotein. In addition, these inhibitors have been useful tools to distinguish various glycosidases, as well as for producing affinity ligands for the purification of specific glycosidases. In particular, there are now several potent and selective inhibitors that show such activities towards the various α-mannosidases. With these inhibitors, a clear distinction can be made between the processing α-1, 2 specific mannosidases (i.e., mannosidase I), mannosidase II that cleaves both α1, 3 and α1, 6 linked mannoses from the GlcNAcMan5 (GlcNAc)2 substrate, and the broad-specificity α-mannosidases that release all of the α1, 2-, α1, 3- and α1, 6-linked mannoses from an9-4(GlcNAc)2 structures. Thus, deoxymannojirimycin or kifunensine strongly inhibit the Golgi mannosidase I, but have no effect on the Golgi mannosidase II, aryl or lysosomal α-mannosidases, and broad-specificity mannosidases. On the other hand, swainsonine and mannostatin A strongly inhibit mannosidase II, but are inactive on mannosidase I or the ER α-mannosidase. In addition, a new inhibitor, D-mannonolactam amidrazone, is a general mannosidase inhibitor that has been synthesized chemically, and this compound should provide new insight into the design of other useful and more specific inhibitors for other α-mannosidases.