ANNIVERSARY
COMM
25thP

anel Discussion

Trends of Glycolipid Researchthat Emerged fromAnalysis of Grant Applications forMizutani Foundation forGlycoscience

M akoto Ito

Kyushu University, Japan

Makoto Ito received his Ph.D. in 1981 from Kyushu University, Fukuoka, Japan, under
the supervision of Prof. Manabu Kitamikado. He studied an endo-β-galactosidase
capable of cleaving the internal β-galactosidic linkage in keratan sulfate as wellas lacto/neolacto-series glycosphingolipids in his thesis study. Between 1982 and1993, he worked at the laboratory of Glycoconjugate Research (Dr. Tatsuya Yamagata) at the Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan, first as a
postdoctoral fellow, then as a senior researcher. Dr. Ito discovered the endoglycoceramidase that cleaves the β-glycosidic linkage between oligosaccharide and ceramide of various glycosphingolipids at this institute. He received the Young Scientist
Award for Biochemists from the Japanese Biochemical Society in 1991. He thenreturned to Kyushu University in 1993 as an associate professor. He cloned andcharacterized many novel enzymes involved in metabolism of glycolipids and sphingolipids, including sphingolipid ceramide N-deacylase (SCDase), neutral ceramidase(from bacteria to humans), human cytosolic glucocerebraosidase (Klotho-relatedprotein, GBA3), endogalactosylceramidase (EGALC), fungus glucocerebrosidase
and sterylglucosidase (EGCrP1 and EGCrP2, respectively), glycolipid-specific β-Nacetylgalactosaminidase (NgaP), and zebrafish GM4 synthase. He was appointed afull professor in 2002 and is currently a professor of Molecular Bioscience, Department of Bioscience and Biotechnology, Faculty of Agriculture, and a professor ofBio-Architecture Center (BAC), Kyushu University. He served as a president of theJapanese Society of Carbohydrate Research from 2015 to 2017, and as a directorof the Japanese Biochemical Society from 2017 to 2019. He is interested in the metabolism and functions of glycolipids from bacteria to humans.

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the 25th Anniversary of Mizutani Foundation for Glycoscience

MEMORATIVE SYMPOSIUM
Glycolipids are complex carbohydrates in which monosaccharides
or oligosaccharides are linked to lipids. Depending on the type oflipid, they are mainly classified as sphingoid-based glycosphingolipids, glycerol-based glycoglycerolipids, and sterol-based sterylglycosides. In addition to these glycolipids, GPI-anchored proteins
are sometimes regarded as glycolipids. Glycosphingolipids are
ubiquitous components of plasma membranes of vertebrates,
invertebrates, plants, yeasts, and some bacteria while glycoglycerolipids are mainly present in plants and bacteria especiallyabundant in Archaea. The latter are rare in mammals but a sulfatedglycoglycerolipid named seminolipid is the most abundant glycolipid in the testis and sperm. Sterylglycosides are major glycolipids
in plants and fungi; however, they are also present in mammals.
Every year there are over 150 applications to the Mizutani Foundation from all over the world. In this symposium, the author willreport and discuss the trends of glycolipid research that have been
analyzed from Grant applications over the past three years. Applications targeting glycolipids are approximately 15 to 20% of all
applications. This trend has not changed significantly in the past
three years. The most common theme is immunity/infectious diseases, followed by themes such as metabolism, neuron/brain functions, lysosomal storage diseases, and chemical synthesis (Figure 1).
Given the classification of glycolipids, the most common theme isrelated to glycosphingolipids, but those targeting glycoglycerolipids and sterylglycosides are also found.
Until recently, almost all enzymes involved in the synthesis anddegradation of glycosphingolipids have been identified in mammals, including humans. Knockout mice of individual genes havebeen generated and analyzed. Deficiency of synthases that form
the core structure of glycolipids, such as glucosylceramide andlactosylceramide, has decisive effects on early mouse development and leads to death in the fetal stage. However, the molecularmechanism of why they died because of the lack of these synthases is not clear. Zebrafish and medaka can be used to visualize theearly development process, and may be useful in clarifying how
these glycolipids play a role in early developments. Analyses of theGM3 synthase knockout mice revealed that this simple gangliosideis important for maintaining the normal function of the lipid microdomain and various membrane receptor functions. Failure ofGM3-mediated cellular functions may exhibit severe diseases suchas diabetes and hearing loss 1). On the other hand, GM3 only mice
have been found to cause neurodegeneration due to complement
activation and inflammation, suggesting that the normal ganglioside composition is critical for neural tissue integrity and repair 2).
With respect to immunity/infectious diseases, research on how theglycolipids of pathogens activate the host’s innate immune system
is an important and interesting topic. It is reported that Mincle(macrophage-inducible C-type lectin) uses glycolipids as ligands,
and activates the host innate immune system, e.g., Mincle recog-nizes a glycolipid called trehalose dimycolate of Mycobacterium
tuberculosis, which is the causative bacterium of tuberculosis. Veryrecently, glucosyl diacylglycerol of Streptococcus pneumoniae has
also been shown to be a potent ligand of Mincle 3).
In recent years, lipid rafts consisting of sphingolipid and cholesterol has been actively studied. Lactosylceramide is enriched in
the specific microdomain of mouse neutrophils and mediates thecell migration and phagocytosis 4). It was shown that there is a new
type of microdomain that constitutes phosphatidyl glucoside inbrain cells. Since this unique glycolipid is composed only of saturated fatty acids, it phase-separates from the phospholipid bilayer
consisting of unsaturated fatty acids and forms a specific lipid microdomain. Lyso-form of phosphatidyl glucoside, a metabolite ofphosphatidyl glucoside, regulates modality-specific sensory axonal induction of the spinal cord 5). With regard to chemical synthesis, progress has been made in the synthesis of chemical probesthat enable visualization of glycolipid-rich microdomains.
The lysosomal degradation of glycosphingolipids is performed bya combination of lysosomal glycosidases and noncatalytic activator proteins and, in some cases, specific anion phospholipids suchas bis(monoacylglycero)phosphate. Lysosomal storage diseases
are caused by a deficiency of one of the glycosidases or activator
proteins. GBA1 is an acid β-glucocerebrosidase in lysosomes. The
deficiency of GBA1, a causative gene of Gautier’s disease, resultsin the accumulation of SNCN/α-synuclein as well as GlcCer, and increases the risk of Parkinson’s disease 6). Inhibition of the autophagy pathway by inactivation of protein phosphatase 2A may be one
important scenario; however, its molecular mechanism is not wellunderstood. Interestingly, GBA1 and GBA2 synthesize a cholesteryl
glucoside using glucosylceramide and cholesterol as donor andacceptor substrates, respectively 7). However, the functional relationship between cholesteryl glucoside and these diseases is notclear at present.
Lyso-forms of glycosphingolipids are detected in normal tissues
and cells at very low levels; however, they accumulate in tissues ofindividuals with inherited sphigolipid storage diseases and may
contribute to the dysfunction of various cellular events including
cytokinesis 8). The enzyme (SCDase) that produces lyso-foms of glycosphingolipids has been found in bacteria but not in mammals.
Very recently, acid ceramidase was found to be involved in the formation of lyso-forms of glycosphinoglipids in lysosomes 9).
Regarding the structural analysis of glycolipids, the recent progress of mass spectrometry is remarkable. Although more than 200
glycosphingolipids with structurally distinct glycans are known,
ceramide moieties are also quite heterogeneous in structures. The
comprehensive profiling glycosphingolipid glycans using mass
spectrometry after cleaving ceramide moiety by endoglycoceramidase is shown to be useful for quantitative cellular glycomics 10).

Commemorative Symposium

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ANNIVERSARY
COMM
25thFigure 1

Classification of subjects of glycolipid research in grant applications for Mizutani Foundation for glycoscience from 2015 to 2017.

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2)	Furukawa K. (2011) Regulatory mechanisms of nervous systems with glycosphingolipids. Neurochem Res. 36(9) 1578-1586. Doi:10.1007/s11064-011-0494-2.
3)	Behlere-Janbeck F et al. (2016) C-type lectin Mincle recognizes glucosyl-diacylglycerol of Streptococcus pneumoniae and plays a protective role in Pneumococcal
pneumonia. PLoS Pathog. 12(12) e1006038. Doi: 10.1371/journal.ppat. 1006038.
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5)	Guy AT et al. (2015) Glycerophospholipid regulation of modality-specific sensory axon guidance in the spinal cord. Science 349, 974-977. Doi:10.1126/science.aab3516.
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7)	Akiyama H and Hirabayashi Y. (2017) A novel function for glucocerebrosidase as a regulator of sterylglucoside metabolism. Biochim Biophys Acta, in press8)	Watanabe H et al. (2016) Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow. Mol Biol Cell. 27, 2037–2050. Doi:10.1091/mbc.E15-08-0555.
9)	Ferraz MJ et al. (2016) Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases. FEBS Lett.
590, 716-725. Doi:10.1002/1873-3468.12104.
10)	Fujitani N et al. (2011) Qualitative and quantitative cellular glycomics of glycosphingolipids based on rhodococcal endoglycosylceramidase-assisted glycan cleavage, glycoblotting-assisted sample preparation, and matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry analysis. J Biol Chem 286,41669–41679. Doi:10.1074/jbc.M111.301796.

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the 25th Anniversary of Mizutani Foundation for Glycoscience