The HNK-1 carbohydrate antigen, which recognized by a monoclonal antibody HNK-1, was originally noticed as a maker of human natural killer (HNK) cells. Subsequent studies revealed that HNK-1 carbohydrate was highly expressed in the nervous system and was commonly expressed on cell adhesion molecules belonging to the immunoglobulin superfamily. Therefore, it was suggested the involvement of HNK-1 carbohydrate in migration of neural crest cells, adhesion of neurons to glial cells, and axon outgrowth. I was interested in this epitope and investigated the biosynthetic enzymes for this epitope. I fortunately received the JSCR Young Investigator Award 1998. In this review, I illustrated a series of our studies including recent findings about the HNK-1 carbohydrate.

In 1999, I received the Young Scientist Award from the Japanese Society of Carbohydrate Research for my research leading to the identification of the tumor suppressor EXT-like gene family member EXTL2. Specifically, I identified an N-acetylhexosaminyltransferase that transfers not only N-acetylgalactosamine but also N-acetylglucosamine to the glycosaminoglycan-protein linkage region. Since that award, my laboratory has shown that EXTL2 functions as a suppressor of glycosaminoglycan biosynthesis and that this suppression is enhanced by the xylose kinase FAM20B. Furthermore, we have found that an EXTL2-dependent mechanism that regulates glycosaminoglycan biosynthesis is important for maintaining tissue homeostasis under pathological conditions, and that lack of EXTL2 causes glycosaminoglycan overproduction and structural changes in glycosaminoglycans associated with pathological processes.

Sialic acids modify the outermost termini of glycan chains and show an extremely large structural diversity. While ubiquitous in vertebrates, they show a sparse distribution in other organisms. It is thus difficult to define the origin of sialic acids during evolution. We have carried out identification and comparative biochemistry of the metabolic enzymes of sialic acids to understand their structural diversity in evolution. In the end, we have faced an era of chaotic situations of knowledge, and at the same time might be close to the better understanding of the survival strategies of organisms through the sialic acid research.

My research was initiated when studying oligosaccharide synthesis. Besides my poor attitude toward this area of study, too many synthetic steps are required for the functional group protection and key coupling reactions in oligosaccharide synthesis. When starting an actual experiment, another difficulty that arises is the complex techniques used in the synthesis of oligosaccharides, which is experienced by most students. Despite these difficulties, oligosaccharide synthesis has already become a widely studied research topic; yet, its successful automation is still to be accomplished. An ideal oligosaccharide synthesis utilizes a simple yet flexible synthetic strategy, which is urgently required. Thus, to overcome the limitations associated with its synthesis and to satisfy the need of an ideal process, I came up with an idea of orthogonal coupling strategy for the synthesis of oligosaccharides.

Lipid rafts, glycosphingolipid microdomains, are compositionally and functionally heterogenous in the cell membrane. Recent reports have demonstrated that lipid rafts are spatially heterogeneous in the single cell membrane. In central nervous system, ganglioside GD3 rafts are involved in migration of granule cells during the early stage of development. In blood coagulation system, clot retraction is mediated by fibrin- integrin αIIbβ3-myosin axis in platelet sphingomyelin rafts.

During my tenure as a graduate student at the Faculty of Science, University of Tokyo, the experimental system for my research comprised eggs from trout fish or sea urchin! My current experimental material comprises plasma, serum, or cerebrospinal fluid from human patients and mice. Notwithstanding differences in the research materials, my research strategy regarding biochemical analysis remains unchanged.

Hyaluronan acts as a major extracellular matrix component in regulation of tissue organization and cellular functions, and its production is markedly increased with cancer progression. Since the discovery of hyaluronan synthase (HAS) genes, manipulation of HAS gene expression has led to better understanding of the altered hyaluronan biosynthesis in cancer. We have demonstrated that hyaluronan overproduction promotes cancer progression by modulating tumor microenvironment. Furthermore, we recently discovered that hyaluronan production regulates cancer stem cell (CSC) properties via the metabolic reprogramming of glycolysis and hexosamine biosynthetic pathway. These findings strongly suggest that hyaluronan plays a regulatory role in cellular metabolism coupled with its biosynthesis, in addition to its function as a conventional extracellular signal. In this review, I discuss multifaceted roles of hyaluronan in CSC regulation.

Sialic acid (Sia) is known to present as a monosialyl residue at a non-reducing terminal end of glycan in nature. In some cases, Sias occur as polymerized sialyl structures, di/oligo/polysialic acids (diSia/oligoSia/polySias). Here, we focus on the structure, function and biological significance of diSia/oligoSia/polySias. Following the definition of di/oligo/polySia structures and establishment of chemical and immunochemical detection methods, we analyzed these structures and their function based on a set of working hypotheses. So far, the biological meanings of degree of polymerization (DP) were shown and they lead to the change the concept of polySia. In addition, this research is applicable in the study of diseases such as mental disorder and cancer. However, research on the biological significance of the diversity in di/oligo/polySia structures is still ongoing.

Microheterogeneity has long been regarded a distinctive feature of glycans, but the details of its biological function remain unclear. I wrote this essay in hopes that it will be an opportunity to think about microheterogeneity again by describing how I was involved with it before and after receiving the Encouragement Award of the Japanese Society of Carbohydrate Research.

N-Glycosylation of secretory and membrane-bound proteins is an essential and highly conserved protein modification of eukaryotes. In the endoplasmic reticulum (ER), a combination of various enzymes, chaperones, lectins and cargo receptors constitutes the “glycoprotein quality control” (GQC) system, which elaborately regulates folding, transport and degradation of glycoproteins. Thus, function of asparagine linked sugar chains in the GQC process has been attracting attention. Understanding of these phenomena has progressed as a result of interaction analyses and substrate specificity studies of glycan related enzymes using synthetic sugar chains. In this review, our approach to the systematic synthesis of the ER type of asparagine linked sugar chains (N-glycans) and recent progress of this field is described.

Characterization of mucins are a difficult task because of their large molecular sizes, highly heterogeneity of glycosylation, and resistance to proteases. For the discovery of mucin biomarkers, supported molecular matrix electrophoresis (SMME), a relatively easy technique for mucin separation, has been developed. Using SMME, mucins and their O-linked glycans from pancreatic juice, cultured cells, and salivary glands were characterized. Furthermore, chemical inertness of the SMME membranes was effectively utilized to develop a novel staining method and glycan stripping method for mucins located on the membrane.

We studied enzymatic activities and biochemical properties of sulfo/glycotransferases responsible for the biosynthesis of sulfated glycans in normal colonic mucosa and colonic adenocarcinomas. We found the presence of a GlcNAc6ST which can catalyze 6-O-sulfation on GlcNAc residues of core 3 structure, in colonic mucinous adenocarcinomas. The presence of β1,4-galactosyltransferase (β4GalT) and β1,3-N-acetylglucosaminyltransferase (β3GnT) specific for GlcNAc 6-O-sulfate and Galβ1-4GlcNAc(6S), respectively, was also shown. These results will serve as studying physiological functions of sulfated glycans.

The identification of distinct saccharide sequences that have specific functions, such as a sulfated pentasaccharide sequence in heparin for antithrombin III-binding, is rare. Based on structural studies of sulfated glycosaminoglycans, a set of oligosaccharide sequences with different sulfation patterns, or “wobble motifs,” recognized by a single protein were found. However, there are still questions about the structure–function relationships of sulfated glycosaminoglycans. The glucosamine 3-O-sulfate structure, which is a minor component (less than 10%) in heparan sulfate/heparin, is especially of interest. Although glucosamine 3-O-sulfate is a rare structure, heparan sulfate 3-O-sulfotransferase is the most varied among heparan sulfate sulfotransferases. Therefore, other saccharide sequences including a 3-O-sulfated glucosamine residue may be needed for binding to proteins similar to the sequence for antithrombin-III-binding. Oligosaccharides isolated from naturally occurring polysaccharides after depolymerization have been used for the analysis of their biological activities to elucidate structure–function relationships. However, only the major sequences in the polysaccharide were tested under this oligosaccharide preparation method. Relatively minor and unique structures may influence the selectivity and interaction of glycosaminoglycans with functional proteins. Chemoenzymatic methods have used as an effective approach for the synthesis of variously sulfated oligosaccharides of glycosaminoglycans. These synthetic oligosaccharide libraries will become a major resource for the functional analysis of sulfated glycosaminoglycans.

Sialic acids are acidic 9 monosaccharides having a carboxylic acid and play a very important biological role. In addition, they are important and attractive targets in synthetic carbohydrate chemistry due to the difficulty of formation of α-sialyl linkage. We found that sialic acid units having a 5N,4O-carbonyl protecting group acted not only as an efficient glycosyl donor undergoing α-silylation without use of nitrile effects but also as glycosyl acceptor for sialylation of hydroxyl groups at C8 and 9 positions. By using it, we successfully prepared α(2,8)oligosialic acids and an epitope of of ganglioside (GP1c) containing five sialic acids. Furthermore, we successfully developed an efficient method for preparation of oligosialic acid based on a combination of one pot glycosylation and solid-phase assisted deprotection. We also found that a sialic acid sugar donor having free hydroxyl groups except for that at the 9 position underwent α-selective glycosidation in dichloromethane. These results indicated that the remote neighboring participation of the carbonyl group on protecting groups reduced the α-selectivity of sialylation.

The exceptional structural diversity of gangliosides has long piqued interest in chemistry and biology. This article describes the results of our continuing study of the chemical synthesis and functional elucidation of gangliosides over the last decade.

Eleven years ago, I moved from Nagoya City University to RIKEN soon after receiving a JSCR young scientist award. For me, the past 11 years is the activity at RIKEN itself. Looking back on the past years, I was thinking how I address the remaining issues in the glycoscience. Sometimes I felt a guilty for not contributing to the glycoscience field so much. Nonetheless, I can say with my confidence, that we tried to tackle with the unsolved issues in the glycoscience field. Here I would like to write down our study on lectin specificity, N-glycosylation/N-glycan maturation, and separation of isometric glycans as a proof of our activity.

Glycosylation is generally carried out repeatedly as a key reaction while chemically synthesizing oligosaccharides and polysaccharides. Thus, both efficiency and stereoselectivity of the key reaction is important for required quantities of the target product to be highly optimized. Although 1,2-cis glycoside is found in various bioactive glycan structures in both prokaryotes and eukaryotes, a few reports have shown a definite approach to achieving stereoselective formation of these linkages. Our previous developments were used as a key reaction for the synthesis and the further developing of bacterial glycans from Campylobacter jejuni and Mycobacterium tuberculosis, the fragments of the novel antifreeze, xylomannan, isolated from Upis ceramboides, and the post-translationally-modified plant glycoproteins.

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that acts on asparagine-linked (N-linked) glycans. I was involved in the discovery of this enzyme activity in 1993, as well as the identification of the gene encoding this enzyme in 2000. In 2007, I proposed the existence of a novel “non-lysosomal” glycan degradation pathway in which cytoplasmic PNGase plays pivotal roles. In 2008, I was fortunate to receive a Young Investigator Award from the Japanese Society of Carbohydrate Research. Since that time remarkable progress has been made regarding the functional analysis of the cytoplasmic PNGase (NGLY1 in mammals). After the discovery of a human genetic disorder, an NGLY1-deficiency, caused by the genetic mutations of NGLY1 gene, the functional importance of this enzyme has attracted widespread interest. In this article, I briefly summarize recent research progress on NGLY1.

My group has discovered extracellular O-GlcNAc on the epidermal growth factor repeats of Notch receptors. Unlike OGT-dependent O-GlcNAcylation, extracellular O-GlcNAc occurs in the endoplasmic reticulum by the action of EOGT. Genetic inactivation of eogt in Drosophila revealed that extracellular O-GlcNAc is essential for developmental processes and appears to regulate the function of apical extracellular molecules such as Dumpy. In contrast, extracellular O-GlcNAc is required for the precise control of Notch signaling in mammals. Eogt mutant mice show defects in retinal vascular integrity. Importantly, mutations in EOGT cause Adams-Oliver syndrome, a congenital disorder in humans. These findings indicate diverse but essential biological functions of extracellular O-GlcNAc in Drosophila and mammals. In this article, I will give an overview of the biochemical and biological studies of extracellular O-GlcNAc, summarizing previous findings and current developments.

Glycoprotein quality control is regulated by high-mannose glycans on the glycoprotein as signals. We found that the quality control mechanism is complementarily regulated by secondary factors other than glycan recognition through our glycoprobe-based analysis. We revealed that most related glycan-recognizing proteins discriminate the aglycon state difference of the substrate glycoprotein to regulate activity. We also found that the upstream of the glycan processing is accelerated and the downstream is decelerated under the molecular crowding conditions mimicking the inside of the cell. Furthermore, the effect of diseases such as obesity, type 2 diabetes, and osteoporosis on the operation status of glycoprotein quality control was clarified.

We achieved in functioning the “glycan pattern recognition” in vivo, by clusterizing various N-glycans through efficient synthetic transformation, i.e., RIKEN click reaction. Such “glycan pattern recognition” could control the interaction with the target cells, organs and cancers, as well as regulate the excretion pathways in mice. Furthermore, we performed for the first time the metal catalyzed reaction at the targeted organs in mice, by rapidly carrying the metal catalyst using the glycan-based drug delivery system. We thus developed the next-generation strategy from the glycochemical biology, termed as “Therapeutic In Vivo Synthetic Chemistry”, where the activities of drugs or other bioactive molecules could be elicited without off-targeting problems by directly synthesizing them at the targeted diseases.

We previously reported that a defect in O-mannosyl glycan is the primary cause of a group of congenital muscular dystrophies. Based on our pioneering findings, numerous studies have been performed and revealed various structures of O-mannosyl glycans. However, the glycan structure associated with muscular dystrophies remained unclear for a long time. Recently, we described the complete structure of an O-mannosyl glycan containing ribitol-phosphate (RboP), which had not previously been identified as a glycan component in mammals. In addition, its unique biosynthetic pathway was elucidated by identifying the functions of the gene products associated with muscular dystrophies. Here, we review recent findings regarding the mechanisms of O-mannosyl glycan biosynthesis in mammals.

Polylactosamine (pLN) is a basic structure of glycan in glycoproteins and glycolipids. The pLN structure itself is a functional carbohydrate antigen that acts as an endogenous lectin ligand. Various functional glycan epitopes are also known to be formed on pLN structures. Therefore, pLN glycan is believed to directly or indirectly regulate various molecular functions by interacting between glycan and lectin. To elucidate the biological function of pLN glycan, carrier glycoproteins of pLN glycan were identified using a newly developed glycoproteomics technique known as the Glyco-RIDGE (GR) method. As a result, many pLN carrier glycoproteins in HL-60 cells were identified. Enrichment analysis with gene ontology revealed that the pLN carrier glycoproteins contained many molecules related to signal transduction and other mechanisms. The biological functions of pLN glycan can be further elucidated by analyzing the carrier glycoproteins using the GR method.

Recent advances in synthesizing methodologies and purification techniques significantly increase the accessibility of the major glycan structures, which are heterogeneously present on the glycoproteins and glycolipids. In contrast, it is often difficult to obtain sufficient amounts of minor structures consisting of a number of sugar units with reasonable purity sufficient for biological studies. In terms of the accessibility of the desired glycan sequences, it is useful in certain instances to deal with not the full sequence but with the required oligosaccharides, called glycan epitopes. Glycan epitopes have been used for the structural and functional analysis of the proteins that recognize particular glycans. For instance, lectins usually recognize the glycan epitope of a few sugar units, not in a “key-and-keyhole” manner but in a “pattern recognition receptor” mode. Such oligosaccharide epitopes can be prepared through regular chemical synthesis. This report describes recent progress on the NMR analysis of glycan–glycan and protein–glycan interactions using chemically synthesized glycan epitopes.

N-glycan acts as quality control tag for determination of intracellular glycoprotein fates. I have demonstrated the structural basis for N-glycan-dependent determination of glycoprotein fates at atomic level by using several biophysical methods including X-ray crystallography. In 2012, I received a JSCR young science award entitled “Structural biology study of intracellular lectins involved in glycoprotein transport and degradation.” In recent years, I have focused on ER glycoprotein folding system, i.e., calnexin/calreticulin cycle, and successfully determined 3D structures of ER glucosidase II (GII) and UDP-glucose:glycoprotein glucosyltransferase (UGGT), which are respectively responsible for glucose trimming and attachment, providing structural insights into ER quality control mediated by glucose tagging.

A number of proteins on the plasma membrane are modified by the glycolipid, glycosylphosphatidylinositol (GPI). These proteins are called GPI-anchored proteins (GPI-APs) and approximately 150 proteins exist as GPI-anchored forms in mammals. Characteristics of GPI-APs include: 1) forming membrane domains with specific lipids such as sphingolipids and cholesterol on the cell membrane and 2) cleavage at the GPI moiety to release the protein from the cell surface. Since complete structures of GPI anchors were determined in 1988, genes involved in biosynthetic pathways and structural remodeling of GPI anchors have been identified. In recent years, structural remodeling of GPI has been shown to regulate quality control, transport and localization of GPI-APs. In addition, particular GPI-cleaving enzymes cause shedding of GPI-APs from the cell surface. Here, I introduce recent progress about the structural chemistry and cleaving mechanisms of GPI-anchors.

It was more than six years ago almost at the same time as I moved to Tottori University. I won the incentive award of Japanese Society of Carbohydrate Research by “The Control of Chemical Glycosylation Based on Organic Electrochemical Method” which was directed towards nowhere. After moving the current position we have achieved automated solution-phase synthesis of oligosaccharides based on the electrochemical method. My students and co-workers played crucial roles to develop the automated electrochemical synthesizer. We are not satisfied with yield and variety of oligosaccharides; however, we are updating the machine and providing thus-obtained oligosaccharide to our collaborators. This short review introduces our achievements during the course of the study.

Dystroglycanopathy (DGpathy) is a group of muscular dystrophies that is caused by abnormalities in the sugar chains on dystroglycan. We contributed to the understanding of structure, mechanism of modification, and function of sugar chains of dystroglycan. In particular, we found modification with ribitol phosphate, a new type of post-translational modification, and showed that FKTN, FKRP, ISPD, and TMEM5 are involved in ribitol phosphate modification. We also clarified the pathological mechanism using DGpathy model mice and proposed treatment strategies. Here, we discuss the recent advancements related to ribitol phosphate modification and the pathological mechanism and therapeutic strategies for DGpathies.

Glycosphingolipids are not uniformly present in cell membranes and form microdomains called glycolipid microdomains together with sphingomyelin and cholesterol. Many signaling molecules accumulate there, and glycolipid composition greatly influences signal transmission efficiency. Therefore, analyzing the dynamics and structure of glycolipid microdomains is very important in understanding life phenomena. In recent glycolipid microdomain studies, along with biochemical methods, analysis of membrane molecular dynamics by fluorescence microscopy, localization analysis using an electron microscope, and lipid structure analysis by mass spectrometry have been conducted. Here I outlined a super-resolution microscope that can be widely used for glycolipid microdomain analysis.

Influenza A virus (IAV)-binding receptors are classified mainly to two molecular species of sialic acids at the terminals of glycoconjugates, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). We investigated the function of Neu5Gc in IAV infection by using Neu5Gc binding-modified IAVs and Neu5Gc-expressed cells in an Neu5Gc biosynthesis-deficient human cell line. We compared substrate specificity of IAV enzyme sialidase for Neu5Ac and Neu5Gc among human IAVs and equine IAVs. In this review, we introduce our studies about the function of Neu5Gc in IAV infection and substrate specificity of IAV sialidase for Neu5Gc. In addition, we developed a fluorescence imaging technique for detecting IAV and the infected cells using a sialidase imaging probe. The combination use of the probe and anti-influenza drugs enabled selective detection and isolation of drug-resistant IAVs. In this review, we also introduce our recent studies about virus detection using the sialidase imaging probe and its applications.

We analyzed the glycan structure expressed in human pluripotent stem cells (hPSCs) using high density lectin microarray and mass spectrometry. α2-6Sia, α1-2Fuc, type1 lactosamine structure was found to be increased in hPSCs as compared with the differentiated somatic cells. Dramatic changes were found in sialic acid on N-glycans. While human induced pluripotent stem cells (hiPSCs) were all of α2-6 type, human fibroblasts were all of α2-3 type. Examination of the glycan structure of human mesenchymal stem cells (hMSCs) and cartilage tissue-derived chondrocytes revealed that the proportion of α2-6sialyl N-glycans is high in cells capable of differentiation. Then, H type 3 (Fucα1-2Galβ1-3GaINAc) was found as a new cell surface marker characterizing hPSCs. This carbohydrate epitope was expressed on type1 transmembrane protein, podocalyxin. In addition, we discovered a recombinant lectin named rBC2LCN as a probe specifically recognizing this structure. By utilizing rBC2LCN, we developed three key technologies to overcome the risk of tumorigenicity of hPSCs: live staining method, detection method using cell culture media, and elimination method. In this mini-review, we will overview our research findings from structural elucidation of glycans expressed on hPSCs to social implementation.

Formamidinium-type dehydrating reagents such as 2-chloro-1,3-dimthylimidazolinium chloride are useful as selective activators toward hemiacetal hydroxy groups at the sugar-reducing end in an aqueous media. This synthetic method enables us to obtain various sugar-containing compounds directly from the corresponding free saccharides using corresponding nucleophiles. Azide and aryl thiol act as suitable nucleophiles for this method and the resulting compounds are used as intermediates for glycoconjugates. This study aims to describe the progress of direct modification of the sugar-reducing end using a formamidinium-type electrophile.

Recent advances in total chemical synthesis of proteins enable us to prepare complex glycoproteins. Herein, the total chemical synthesis of antifreeze glycoprotein, which is a mucin type O-glycoprotein, is introduced as an example of the latest total chemical synthesis of glycoproteins. The functional analysis of the structurally-defined form of the synthetic O-glycoproteins revealed a unique function of O-GalNAcylation of a protein.

Glycans on glycoproteins are structurally diverse. The number of glycan branches and formation of glycan epitopes at non-reducing ends afford a wide variety of glycan structures. In particular, in the nervous system, many structurally unique glycans are expressed, which ensures that complex neural functions are maintained. We have focused on neural specific glycans such as HNK-1, branched O-mannose glycans, and bisecting GlcNAc, and revealed that expression of these neural glycans is tightly regulated. Moreover, studies by us and other groups have clarified that aberrant expression of these glycans is associated with various neural diseases, including dementia. In this review, the regulated expression and disease relevance of these glycans are discussed.

Neural stem cells (NSCs) possess high proliferative potential and capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. The glycoprotein glycans expressed on NSCs are associated with the maintenance of neural cell stemness and differentiation via several signaling pathways. Here we outline current knowledge of the possible functional mechanisms of glycoprotein glycans to determine cell fates, which are associated with their expression patterns and structural characteristic features.

1,2-cis-Glycosides are frequently found in various biologically active natural products. However, the stereoselective synthesis of these glycosides is still difficult due to the lack of neighboring group participation. Therefore, the development of efficient synthetic methods has been required. In this context, we have reported novel regio- and 1,2-cis-α-stereoselective glycosylations using 1,2-anhydroglycosyl donors and diol acceptor-derived boronic ester catalysts without additives under mild conditions. In addition, development of 1,2-cis-stereoselective glycosylations using mono-ol acceptor-derived borinic esters and their application to the total synthesis of natural glycolipids were also reported. In this mini-review, further extended studies on boronic-acid-catalyzed regio- and stereoselective glycosylations are introduced.

Asparagine (N)-linked glycosylation occurs on most secretory and membrane proteins synthesized in the endoplasmic reticulum (ER), which functions to modulate protein folding, degradation and intracellular trafficking. However, the regulatory mechanisms underlying N-linked glycosylation remain largely elusive, hindering our integrative understanding of the physiological and pathological functions of this posttranslational modification. In this article, I will introduce recent advances on our understanding of regulation of N-linked glycosylation and a newly discovered inhibitor of N-linked glycosylation reaction.

We have been working to develop unique and practical synthetic methods for biologically relevant carbohydrate molecules. In particular, we have addressed the synthesis of sialoglycans, including mammalian and echinodermatous gangliosides and O-glycans on glycoproteins. This review presents our recent achievements in sialoglycan syntheses.

Glycosaminoglycans (GAGs) are linear polysaccharide chains covalently attached to core proteins to form proteoglycans (PGs). GAG chain assembly begins with the synthesis of the common protein linkage region tetrasaccharide attached to the serine residues of specific core proteins. Previous structural analyses have shown that the GAG-protein linkage region tetrasaccharide of PGs is transiently modified by xylose 2-O-phosphorylation. We previously identified a Xyl kinase that phosphorylates C-2 of the xylose residue and phosphoxylose phosphatase. This review presents recent advances in the biological significance of phosphorylation and dephosphorylation of the Xyl residues in the linkage region tetrasaccharide of PGs.