Various biological functions of carbohydrate chains learned from glycosyltransferase-deficient mice (2024)

Abstract

Carbohydrate chains are attached to various proteins and lipids and modify theirfunctions. The complex structures of carbohydrate chains, which have various biologicalfunctions, are involved not only in regulating protein conformation, transport, andstability but also in cell–cell and cell–matrix interactions. These functionalcarbohydrate structures are designated as “glyco-codes.” Carbohydrate chains areconstructed through complex reactions of glycosyltransferases, glycosidases, nucleotidesugars, and protein and lipid substrates in a cell. To elucidate the functions ofcarbohydrate chains, I and my colleagues generated and characterized knockout (KO) mice ofgalactosyltransferase family genes. In this review, I introduce our studies aboutgalactosyltransferase family genes together with related studies performed by otherresearchers, which I presented in my award lecture for the Ando-Tajima Prize of theJapanese Association for Laboratory Animal Science (JALAS) in 2019.

Introduction of Myself and My Research

In 1982, when I was a fourth-year student in the Faculty of Science, Kyoto University, Iwas very impressed with the giant mouse reported in the top pages of newspapers. Mice thatwere twice their regular size were generated by microinjecting the gene for rat growthhormone into fertilized mouse eggs. These findings were reported by Drs. Palmitter andBrinster in Nature [28]. At thatmoment I ambiguously thought that I would like to undertake that kind of research infuture.

I went on to the Graduate School of Science, Kyoto University, and fortunately had a chanceto learn about transgenic technology under the supervision of Dr. Iwakura and Prof. Kawadeat the Virus Research institute Kyoto University. We started to develop transgenictechnology through trial and error and could finally generate transgenic (Tg) mice carryingthe interferon-β (IFN-β) gene under the control of the metallothionein promoter andenhancer. The male IFN-β Tg mice were sterile, without germ cells after the pachytene stage,probably because the metallothionein promoter and enhancer is constitutively active in thetestis and IFN-β has a harmful effect on cell growth, especially on germ cells [17]. Furthermore, I unexpectedly found that IFN-α as wellas IFN-β were expressed in the IFN-β Tg mice. Several experiments revealed that IFN-α wasinduced by IFN-β in the mouse body [5]. I obtained myPh.D. from Kyoto University in 1989.

After obtaining my Ph.D., I moved to Osaka Bioscience Institute and joined Dr. Nagata’slaboratory as a postdoctoral fellow. Here, I was engaged in working on G-CSF projects [6, 7] and learned alot about molecular biology. Then, I relocated abroad to Prof. Gruss’s laboratory at the MaxPlanck Institute for Biophysical Chemistry in Goettingen, Germany. Here, I studiedPax genes during mouse development [4] and tried to make gene knockout (KO) mice using ES cells. In 1993, I returnedto Japan and began work in Prof. Iwakura’s laboratory at the Institute of Medical Science,the University of Tokyo, as a research associate. Here, I studied interleukin (IL)-1function by generating IL-1α/β double KO mice [13]and IL-1 receptor antagonist KO mice [14]. So far, myresearch remained focused on studying the biology of cytokines such as IFN, G-CSF, and IL-1.Before I turned 40, I decided to change my research interests and started novel research inthe field of glycobiology.

Glycobiology Research by Generating KO Mice of Various Glycosyltransferases

Carbohydrate chains are attached to various proteins and lipids and modify their functions.Although amino acid sequences of proteins are strictly encoded by DNA sequences,carbohydrate chain sequences are not encoded by the genetic code. The only attachment ruleis that Asn-linked N-glycans and Ser/Thr-linked O-glycansare attached to Asn in the Asn-X-Ser/Thr motif and Ser/Thr without any motif, respectively.Three dimensional complex structures of carbohydrate chains are constructed by complexreactions of glycosyltransferases, glycosidases, nucleotide sugars, and protein and lipidsubstrates in a cell. Complex structures of carbohydrate chains have various biologicalfunctions, not only in regulating protein conformation, transport, and stability but alsofor cell–cell and cell–matrix interactions. The functions of many proteins are modified bythese glycosylation patterns. Therefore, functional carbohydrate structures are designatedas glyco-codes. My ultimate goal is to elucidate the glyco-code. The importance ofcarbohydrate chains in the body has been revealed by generating KO mice ofglycosyltransferases. Notably, KO mice of GlcNAcT-I, which transfersN-acetylglucosamine (GlcNAc) to high mannose-typeN-glycans to synthesize complex- and hybrid-typeN-glycans, showed embryonic lethality [15, 23]. This indicated that complex- andhybrid-type N-glycans are essential for mouse development.

My colleagues and I are interested in galactose residues that are suggested to have variousbiological functions. There are four kinds of galactosyltransferase families: α-1,3, α-1,4,β-1,3, and β-1,4-galactosyltransferases, containing 3, 1, 7, and 7 genes, respectively,according to the linkage of sugar chains [12]. Amongthem we focused on β-1,4-galactosyltransferases (β4GalTs; gene name,B4galts) containing B4galt-1 toB4galt-7 genes (Fig. 1). Using gene targeting methods in ES cells, we first generated KO mice of β4GalT-1,which transfers galactose to the terminal GlcNAc of complex-type N-glycansin the Golgi apparatus. While B4galt-1 KO mice were fertile and bornnormally, they showed growth retardation after birth and had short life spans. Epithelialcell proliferation in the skin and small intestine was enhanced, and cell differentiation inintestinal villi was abnormal. These observations suggest that β4GalT-1 plays critical rolesin the regulation of proliferation and differentiation of epithelial cells [3]. At around the same time, Dr. Shur reportedB4galt-1 KO mice which showed similar growth retardation with hypoplasiaof the posterior pituitary [21].

Carbohydrate Chains in Inflammation and Hematopoietic Stem Cells

One of the well-known functions of carbohydrate chains is the interaction of selectins andtheir carbohydrate ligands, which are involved in adhesion of leukocytes to endothelialcells during inflammation and lymphocyte homing to peripheral lymph nodes (PLNs) underphysiological conditions (Fig. 2) [18, 20]. One of these selectin ligands is sialyl Lewis x (sLe^x), which ismainly presented at the terminus of N-acetyl lactosamine repeats on core 2O-glycans (Fig. 3). Another selectin ligand is sialyl 6-sulfo Lewis x (Le^x), which is mainlypresented at the terminus on core 1 O-glycans (Fig. 3). The former is involved in leukocyte adhesion to endothelialcells during inflammation, while the latter is involved in lymphocyte homing to PLNs. Mostof the core 2 O-glycans on the leukocyte membrane glycoproteins ofB4galt-1 KO mice lacked galactose residues, and soluble P-selectinbinding to their neutrophils and monocytes was significantly reduced, indicating animpairment of selectin-ligand biosynthesis. B4galt-1 KO mice exhibitedblood leukocytosis but normal lymphocyte homing to PLNs because core 1O-glycans were normally expressed. Acute and chronic inflammatoryresponses, including the contact hypersensitivity (CHS) and delayed-type hypersensitivity(DTH) responses, were suppressed in these mice. Our results demonstrate that β4GalT-1 is amajor galactosyltransferase responsible for the selectin-ligand biosynthesis and thatinflammatory responses of B4galt-1 KO mice are impaired because of defectsin selectin-ligand biosynthesis [3]. We also examinedthe effect of β4GalT-1-deficiency in skin wound healing. B4galt-1 KO miceshowed significantly delayed wound healing with reduced re-epithelialization, collagensynthesis, and angiogenesis compared with control mice. Neutrophil and macrophagerecruitment at wound sites was also impaired because of selectin-ligand deficiency inB4galt-1 KO mice [24].

Very recently, we reported about the role of carbohydrate chains in the homing andengraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). We foundthat transplanted BM cells deficient in β4GalT-1 could not support survival in mice exposedto a lethal dose of irradiation. BM cells obtained from B4galt-1 KO miceshowed normal colony-forming activity and hematopoietic stem cell (HSC) numbers. However,colony-forming cells were markedly reduced in the BM of recipient mice 24 h aftertransplantation of β4GalT-1-deficient BM cells, suggesting that β4GalT-1-deficiency severelyimpairs HSPC homing [29]. Although E/P-selectindouble KO mice also show partially impaired HSPC homing as recipient mice [10], the phenotype of B4galt-1 KO miceis much more severe than that of E/P-selectin double KO mice, suggesting that carbohydratechains other than sLe^x play an important role in HSPC homing.

In addition, HSC differentiation is also disturbed in B4galt-1 KO mice(Fig. 4). Although the HSC number was normal, the number of multipotent progenitors (MPPs)increased 3-fold, while the number of common myeloid progenitors (MEPs) and common lymphoidprogenitors (CLPs) decreased 0.5-fold compared with control in the BM [29]. In particular, erythrocytes and platelets were markedly reduced inthe peripheral blood (unpublished data). Further analysis of HSC differentiation is inprogress.

Carbohydrate Chains in Human Diseases

Recent studies indicate that aberrant glycosylation causes various human diseases, such asmetastasis of tumor cells [11], muscular dystrophy[32], and dyserythropoietic anemia [8]. The congenital disorders of glycosylation (CDG) arealso known to be inherited in multisystemic disorders characterized by the defectiveglycosylation of glycoproteins [22].B4galt-1 KO mice spontaneously developed human immunoglobulin Anephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangialmatrix. The mice also showed high serum IgA levels with increased polymeric forms as inhuman IgAN [26]. IgAN is the most common form ofglomerulonephritis, and a significant proportion of patients progress to renal failure.However, pathological molecular mechanisms of IgAN are poorly understood. In humans, serumIgA1 showed aberrant galactosylation and sialylation of O-glycans in itshinge region that is thought to contribute to the pathogenesis of IgAN (Fig. 5) [1]. Mouse IgA has N-glycansbut not O-glycans, and β4-galactosylation and sialylation of theN-glycans on the serum IgA from B4galt-1 KO mice wascompletely absent [26]. We propose that carbohydratechains of serum IgA are involved in the development of IgAN, regardless of whether thecarbohydrates are O-glycans or N-glycans.

Mutations in the key enzyme of sialic acid biosynthesis,UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosaminekinase (GNE), result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusionbody myopathy (HIBM) in humans [9]. Among the variousGNE mutations, one GNE founder mutation (V572L) has been reported in Japanese familiesaffected by DMRV [2]. We generated mice with a V572Lpoint mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparentmyopathies or motor dysfunctions. However, they had a short life span and exhibited renalimpairment with massive albuminuria. Histological analysis showed enlarged glomeruli withmesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte footprocess morphologies in the kidneys. Glycan analysis using several lectins revealedglomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin,which is an important molecule for the glomerular filtration barrier. Furthermore,administering sialic acid, Neu5Ac, to the mutant mice from embryonic stages significantlysuppressed the albuminuria and renal pathology and partially restored the glomerularglycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observedin these mutant mice resulted from impaired glomerular filtration due to the hyposialylationof podocyte glycoproteins, including podocalyxin [16].

Carbohydrate Chains in Development

Next, we generated B4galt-5 KO mice. Although most widely and stronglyexpressed β4GalT-1 is dispensable for mouse embryogenesis, B4galt-5 KO miceunexpectedly showed embryonic lethality. While β4GalT-1 is responsible forN- and O-galactosylation of glycoproteins, we found thatβ4GalT-5 is responsible for glycosphingolipids (GSLs) synthesis. β4GalT-5 islactosylceramide (LacCer) synthetase, which transfers galactose to glucosylceramide (GlcCer)to synthesize LacCer, a core structure of GSLs, including gangliosides [25] (Fig. 6). LacCer synthetase activity and the amounts of LacCer and GM3 ganglioside inB4galt-5 KO embryos were markedly reduced. B4galt-5 KOembryos showed developmental retardation from E7.5 and died by E10.5 [25], as reported by Furukawa’s group [19]. Hematoma, hemorrhage, and abnormal localization of trophoblast giant cellswere observed in extraembryonic tissues, in contrast to normal formation of three embryoniclayers. B4galt-5 KO embryos developed until E12.5 as chimeras withwild-type tetraploid cells, which could form the extraembryonic membranes (Fig. 7), indicating that extraembryonic defects caused the early embryonic lethality [25]. Our results suggest that β4GalT-5 is essential forextraembryonic development during early mouse embryogenesis.

Carbohydrate Chains in the Nervous System

B4galt-5 is strongly expressed in the central nervous system (CNS), andvarious gangliosides are abundantly accumulated in the brain. We generatedB4galt-5 conditional KO (cKO) mice using Nestin-Cre mice. Unexpectedly,B4galt-5 cKO mice developed normally and exhibited normal behavior,although B4galt-5 expression was markedly reduced in the brain.B4galt-6, most homologous to B4galt-5 (Fig. 1), was reported to encode LacCer synthetase inthe rat brain [27]. However,B4galt-6 KO mice were reported to appear normal [30]. To elucidate whether β4GalT-5 and/or β4GalT-6 are responsible forLacCer synthetase in the brain, we generated double KO (DKO) mice by crossingB4galt-5 cKO mice and B4galt-6 KO mice. LacCer synthaseactivity and major brain gangliosides were completely absent in brain homogenates from theDKO mice, although LacCer synthase activity was about half its normal level inB4galt-5 cKO mice and B4galt-6 KO mice. The DKO micewere born normally but they showed growth retardation and motor deficits at 2 weeks and diedby 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarelyfound localized in axons in the cerebral cortex, and axonal and myelin formation wereremarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated fromneurospheres (neural stem cells) that were prepared from the DKO embryos, showed impairmentsin neurite outgrowth and branch formation, which can be explained by the fact thatneurospheres from DKO mice could not strongly interact with laminin due to a lack ofgangliosides, such as GM1a. Furthermore, the neurons were immature, and perineuronal nets(PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCersynthase is encoded by B4galt-5 and -6 genes in the mouseCNS (Fig. 6) and that gangliosides areindispensable for neuronal maturation, PNN formation, and axonal and myelin formation [33]. Since β4GalT-6 was reported to be responsible forLacCer synthetase in rat brains [27], we arecurrently examining rat LacCer synthetases to reveal the difference between rats andmice.

While B4galt-1 is widely and strongly expressed in various tissues exceptthe CNS, B4galt-2, most homologous to B4galt-1 (Fig. 1), is strongly expressed in the CNS. Toelucidate the role of carbohydrate chains on proteins in the CNS, we generatedB4galt-2 KO mice. B4galt-2 KO mice were born and grownnormally and were fertile. In a behavioral test battery, the B4galt-2 KOmice showed normal spontaneous activity in a novel environment but impaired spatiallearning/memory and motor coordination/learning. Immunohistochemistry showed that the amountof HNK-1 carbohydrate was markedly decreased in the brain of B4galt-2 KOmice, whereas the expression of polysialic acid was not affected [34]. Glucuronyltransferase (GlcAT-P) KO mice lacking the HNK-1carbohydrate also showed impaired spatial learning/memory, although their motorcoordination/learning was normal [31]. Histologicalexamination showed an abnormal alignment and reduced number of Purkinje cells in thecerebellum of B4galt-2 KO mice. These results suggest that theGalβ1–4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by β4GalT-2 and thatthe glycans synthesized by β4GalT-2 have essential roles in higher brain functions,including some that are HNK-1 dependent and some that are not [34].

Conclusions

For the last twenty years, our group has studied carbohydrate chain functions in an animalbody by generating and examining KO mice of various galactosyltransferases. To our surprise,each galactosyltransferase has an individual function, and carbohydrate chains synthesizedby it have essential biological functions in inflammation, hematopoiesis, development, andthe nervous system. In some cases, aberrant glycosylation causes human diseases. In thepresent situation, although we cannot draw a whole picture of the glyco-code, I believeaccumulation of these kinds of studies by many researchers will elucidate the glyco-code inthe near future.

Acknowledgments

It is my great honor to be awarded the Ando-Tajima Prize of the JALAS. I would like tothank all the members of the JALAS. I am very grateful to my former supervisors, Profs.Yoshimi Kawade, Shigekazu Nagata, Peter Gruss, and Yoichiro Iwakura, for their continuoussupport and encouragement. My glycobiology studies started in Prof. Iwakura’s laboratory atthe Institute of Medical Science, the University of Tokyo. After I became a principalinvestigator, my studies continued at the Kanazawa University Graduate School of MedicalScience and Advanced Science Research Center, and at present, they continue at the KyotoUniversity Graduate School of Medicine. I appreciate all my colleagues, students, andcollaborators. In particular, I am thankful to my laboratory staff, Drs. Chie Naruse, ToruYoshihara, Kazushi Sugihara, Noriyoshi Hashimoto, and Eikichi Kamimura. This study wassupported in part by Grants-in-Aid for Scientific Research from the Ministry of Education,Culture, Sports, Science and Technology of Japan and a research grant from MizutaniFoundation for Glycoscience. I would like to thank Editage for English language editing.

References