Chemical synthesis of sugar chains to unravel the mysteries of their roles in biological phenomena
15 August 2008 (Volume 3 Issue 8)
In the field of life sciences, compounds known as glycan chains are attracting considerable attention. This is because sugar chains are known to be mediators of the complex biological phenomena of higher organisms, and they are associated with immune responses and various diseases. Yukishige Ito, Chief Scientist of the Synthetic Cellular Chemistry Laboratory, which is involved in the development of original techniques to synthesize various sugar chains, thereby seeking to accelerate the research on sugar chains, states, “compared with the research conducted on DNA and proteins, the results of the research conducted on sugar chains are quite often less clearcut because it is difficult to synthesize and understand the complex structures of sugar chains.”
Sugar chains that serve as the key to biological phenomena and the mechanism of diseases
Sugar chains consist of various components called monosaccharides. For example, starch is a chain of glucose units, which are constituents of the food we eat. In a more general sense, sugar chains are chains of various monosaccharides arranged in different orders. They are classified into various categories depending on the types and numbers of sugar units, the order of connection of the units, their three-dimensional structure, and the manner in which their branches are formed.
The majority of the proteins that constitute higher organisms such as human beings exist in the form of glycoproteins, which are proteins into which sugar chains are incorporated. Glycoproteins can interact with other proteins by virtue of the ability of sugar chains to act as markers, which are involved in biological phenomena. For example, sugar chains serve as labels when the activities of immune cells are controlled or when cells recognize each other (Fig. 1). Regarding the importance of sugar chains, Ito explains, “various functions of proteins based on a large variety of sugar chains support complex vital activities of higher organisms.”
Figure 1: Sugar chains on cell surfaces.
Cell surfaces are covered with sugar chains, which are also called ‘the face of cells.’ They act as markers when cells encounter each other.
Many viruses initially combine with sugar chains on the surface of a cell before breaking into it. For example, hemagglutinin (HA) proteins on the surface of the influenza virus bind with sugar chains that contain special sugars, called sialic acids, on the surface of a cell. Then the influenza virus breaks into the cell and multiplies inside it before bursting out to break into another cell. This infectious process is repeated continuously.
However, the influenza virus cannot burst out of the cell when the cell membrane contains sugar chains that contain sialic acids. The influenza virus therefore uses neuraminidase (NA) proteins on its surface to break off the sialic acids from the cell membrane so it can escape from the cell. Tamiflu, a therapeutic agent for influenza, can interfere with the this function of NA proteins and prevent the influenza virus from leaving the cell, thereby preventing the virus from infecting one cell after another. It is not well known that Tamiflu is a result of research into sugar chains (glycobiology).
The structures of sugar chains on the surface of a cancer cell are known to be significantly altered. The studies conducted in glycobiology are attracting more attention for cancer diagnosis and treatment-oriented research studies.
Importance of experts in chemical synthesis
In comparison with the research conducted into DNA and proteins, research on sugar chains has been less developed, although it is important for elucidating biological phenomena and in fighting diseases. In the research field of molecular biology, chemically synthesized DNA or parts of proteins (peptides) are used in many experiments. The development of synthetic techniques is facilitated by advances in organic chemistry, although biologists are not fully aware of this. The development of synthetic techniques has a very long history; this has enabled the automated synthesis of samples, which are being used in current experiments. Research in biology has witnessed rapid progress as a result of advances in organic chemistry.
In comparison with DNA and peptides, sugar chains cannot easily be synthesized because they have complex structures, with many branched chains and various stereoisomers (Fig. 2). In the present circumstances, not everybody can synthesize sugar chains, and therefore the expertise of professionals is required.
Figure 2: Example of sugar chain structure.
Sugar chains have various complex structures, including those with many branch chains that are combinations of various sugars in different orders and those with various stereoisomers that are slightly different from each other in their three-dimensional structures.
Quality control system of glycoproteins
The Synthetic Cellular Chemistry Laboratory at RIKEN has synthesized almost all the sugar chains in a cellular organelle, the endoplasmic reticulum, that are involved in protein folding and has conducted advanced research to elucidate their roles in various biological phenomena.
Proteins are chains of amino acids arranged as defined by genetic information. However, proteins cannot function normally unless they are folded into proper structures. It is coming to be understood that sugar chains have an important role in the process of protein folding.
In a cell, a high-mannose-type sugar chain, containing many mannose sugars, binds to a chain of amino acids in the endoplasmic reticulum, thereby forming a glycoprotein (see figures on page 6). Some glucose units, which are different from mannose, are attached to the end of the high-mannose-type sugar chain. The process is thought to continue with the removal of all glucose units except one from the high-mannose-type sugar chain; this is then combined with a protein (chaperone) that facilitates folding. Once folding is complete, the remaining single glucose unit is removed. The folding process does not always succeed. However, it is wasteful to discard misfolded glycoproteins, so a mechanism exists for recycling misfolded glycoproteins.
UGGT (a glucose transfer enzyme) verifies whether or not the folding has been performed properly, and it attaches a single glucose unit to the original sugar chain of the misfolded glycoprotein. A chaperone protein then binds with the glycoprotein, activating the folding process again. UGGT attempts to attach a single glucose unit exclusively to misfolded proteins or ‘loser’ proteins, and ignores successfully folded ‘winner’ proteins. Thus, UGGT serves as a guard to check whether folding has been properly performed.
The mechanism of the ‘quality control system of glycoproteins’ has not yet been studied in detail. This is because of the large variety of sugar chains, and it is almost impossible to select individual sugar chains with slightly different structures for detailed investigation from natural sources. Even if some sugar chains required for investigation are selected, their amounts will be too small to be used in experiments.
Ito says, “we have focused on high-mannose-type sugar chains, and we have extended our research to synthesize these sugar chains chemically.” Ito and his team have devised a method for dealing with different shapes of raw materials for the synthesis of sugar chains and for determining the order in which the chemical reactions must take place. They have successfully developed an efficient technique to synthesize only the sugar chains that have the required structures. In other words, they have succeeded in chemically synthesizing all the high-mannose-type sugar chains that are related to the quality control system of glycoproteins.
The number of chemical reactions available for the synthesis of sugar chains is limited. The most interesting part of this study is the development, by Ito and his team, of a new synthetic method based on their own unique ideas that can be applied under severe constraints.
How to distinguish between ‘winner’ and ‘loser’ proteins
One of the mysteries of the quality control system of glycoproteins is the manner in which UGGT, a guardian, can distinguish between the winner and loser proteins. Amino acids, which are components of proteins, are divided into two groups—hydrophilic amino acids, which are very soluble in water, and hydrophobic amino acids, which are insoluble in water, just as oil is. Since proteins are surrounded by water, they become stable when hydrophilic amino acids are exposed on the exterior and hydrophobic amino acids are hidden in the interior. However, in some proteins, hydrophobic amino acids may be exposed to the exterior. A hypothesis proposes that UGGT distinguishes between the winner and the loser proteins according to whether or not a cluster of hydrophobic amino acids is exposed on the outside of a glycoprotein. However, it has been difficult to prove the hypothesis experimentally.
Ito and his team have artificially combined chemically synthesized sugars with hydrophobic molecules, and they have investigated the reactivity of UGGT to this compound. The investigation has shown that UGGT causes a glucose sugar to be attached rapidly to the compound. The compound is considered to mimic misfolded proteins, or loser proteins. Thus, it is assumed that the experiment supports the hypothesis that UGGT distinguishes between the winner and the loser glycoproteins depending on whether or not hydrophobic amino acids are exposed. The investigation has also shown that the reactivity of UGGT depends significantly on a slight difference in the sugar chain structure (Fig. 3). A complex mechanism seems to be involved in the process of distinguishing between winner and loser proteins. In this manner, the mechanism of biological phenomena can be elucidated in detail by synthesizing artificial compounds.
Figure 3: Effects of the difference in sugar chain structure on the reactivity of UGGT.
Compounds composed of a hydrophobic molecule (MTX) and various types of high-mannose-type sugar chains are used to investigate the reactivity of UGGT. The investigation results show that its reactivity depends significantly on slight differences in sugar-chain structure.
Defective proteins are degraded
In fact, some loser proteins cannot have glucose units reattached after repeated attempts. UGGT ignores such defective proteins.
It remains unknown how UGGT distinguishes between loser and defective proteins. This is one of the very important points that remains to be investigated.
Defective proteins are attached to proteins called ubiquitins with the assistance of an enzyme, and they are finally decomposed inside an intracellular organelle called proteasome. A variety of enzymes cause ubiquitins to be attached to defective proteins. In 2004, a group at the Tokyo Metropolitan Institute of Medical Science discovered for the first time that some enzymes use sugar chains as a marker to cause ubiquitins to be attached to defective proteins. Ito’s team used various sugar chains and identified the types of chains that serve as markers in this process.
Sugar chains have been known to be involved in the decomposition of proteins in proteasomes. A proteasome is a type of narrow tunnel, and the protein molecules are disentangled and broken down when they enter it. However, proteins with sugar chains cannot enter the tunnel. Thus, the sugar chains are though to be chopped off before the protein enters the proteasome. In fact, experiments conducted in Ito’s laboratory confirmed that proteins with sugar chains cannot be decomposed in proteasomes. Tadashi Suzuki and the members of the Glycometabolome Team at the Advanced Science Institute are attempting to elucidate the decomposition processes related to such sugar chains. Ito continues his study in cooperation with the Glycometabolome Team. Suzuki and his team are using the sugar chains that Ito and his team synthesized in order to analyze the key mechanism of enzymes.
Most glycoproteins pass through the above-mentioned quality control system of glycoproteins. If the system is not working, the number of defective proteins increases. These proteins may remain in cells without being decomposed, thereby causing serious diseases. For example, Alzheimer’s disease and Parkinson’s disease are considered to be caused by the accumulation of defective proteins, although the involvement of sugar chains remains unclear. Disorders of glycosylation, which means that defective sugar chains are produced, is thought to cause various symptoms. Progress in the study of sugar chains may establish that disorders of glycosylation are at the root of various diseases. In the future, therapeutic agents aimed at correcting disorders in the biosynthesis of sugar chains will be developed.
Establishing efficient methods for synthesizing specified sugar chains
Many genes that produce enzymes for assembling sugar chains have been discovered by Japanese researchers. Japan is now the world leader in the study of sugar chains. However, the number of chemists engaged in the study of the chemical synthesis of sugar chains is small. Ito’s laboratory is one of the few laboratories in the world in which complex sugar chains can be synthesized.
Ito and his team are developing simpler synthetic techniques for sugar chains. Theoretically, any high-mannose-type sugar chains can be synthesized by chemically synthesizing long sugar chains in large quantities and using them as raw materials to obtain shorter lengths of sugar chains, either by chopping up the longer chains with the assistance of enzymes or by combining the chopped small pieces with each other. Such techniques can help biologists synthesize the required chains by themselves. In fact, cells use enzymes to create various sugar chains from long chains.
It is difficult to precisely recreate in a test-tube the phenomena that occur in cells. Furthermore, some enzymes are difficult to synthesize. Ito’s team has devised a method of using raw materials with different structures in developing special techniques for creating any high-mannose-type sugar chain by using a combination of enzymes that are relatively easy to use in synthesis. They have already succeeded in synthesizing about 70% of all high-mannose-type sugar chains.
In April 2008, the Chemical Biology Department, which is a new organization within the Advance Science Institute, was opened. In this newly formed organization, researchers in biology and chemistry work jointly to elucidate the mechanisms of biological phenomena and to facilitate drug discovery. The above-mentioned Glycometabolome Team belongs to the Systems Glycobiology Research Group in the Chemical Biology Department. “We intend to conduct further joint researches with the Chemical Biology Department,” says Ito.
Research at the Synthetic Cellular Chemistry Laboratory will bring about a significant breakthrough in the study of sugar chains.