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Mammalian O-mannosyl glycans: Biochemistry and glycopathology

Mammalian O-mannosyl glycans: Biochemistry and glycopathology No. 1] Proc. Jpn. Acad., Ser. B 95 (2019) 39 Review 1,† By Tamao ENDO (Communicated by Kunihiko SUZUKI, M.J.A.) Abstract: Glycosylation is an important posttranslational modification in mammals. The glycans of glycoproteins are classified into two groups, namely, N-glycans and O-glycans, according to their glycan-peptide linkage regions. Recently, O-mannosyl glycan, an O-glycan, has been shown to be important in muscle and brain development. A clear relationship between O-mannosyl glycans and the pathomechanisms of some congenital muscular dystrophies has been established in humans. Ribitol-5-phosphate is a newly identified glycan component in mammals, and its biosynthetic pathway has been elucidated. The discovery of new glycan structures and the identification of highly regulated mechanisms of glycan processing will help researchers to understand glycan functions and develop therapeutic strategies. Keywords: O-mannosylation, congenital muscular dystrophy, dystroglycan, ribitol-5- phosphate 3) altered glycosylation was published in this journal, Introduction including our pioneering findings of muscular dys- The major glycans of glycoproteins are classified trophy and glycosylation. Since then, many bio- into two groups according to their glycan-peptide chemists, molecular biologists, pediatricians, neurol- linkages. Glycans linked to asparagine (Asn) residues ogists, and geneticists have entered this new research of proteins are termed N-glycans, whereas glycans field. This review will describe recent progress in linked to serine (Ser) or threonine (Thr) residues are establishing the biochemistry and glycopathology of called O-glycans. In N-glycans, the reducing terminal O-Man glycans in mammals. N-acetylglucosamine (GlcNAc) is linked to the amide Structure group of Asn via an aspartylglycosylamine linkage. In O-glycans, the reducing terminal N-acetylgalactos- O-Mannosylation is known as a yeast-type amine (GalNAc) is attached to the hydroxyl group modification, and all O-Man glycan structures that of Ser and Thr residues. In addition to the abundant have been elucidated in yeast are neutral linear O-GalNAc forms, several unique types of protein O- structures consisting of only Man residues. O- glycosylation have been identified, such as O-linked Mannosylation of proteins is essential for viability fucose (Fuc), glucose (Glc), GlcNAc, and mannose in yeast, and its absence is thought to affect the cell (Man), which have been shown to mediate diverse wall structure and rigidity. On the other hand, physiological functions. We and other researchers mammalian O-Man glycan is a unique type of protein have shown that O-Man glycan is important in modification that is present in a limited number of muscle and brain development, and its deficiency glycoproteins in the brain, nerves, and skeletal 4) leads to a group of congenital muscular dystrophies muscle. One of the best known O-Man-modified 1),2) known as ,-dystroglycanopathies. In 2004, a glycoproteins is ,-dystroglycan (,-DG), which is a review of human genetic diseases characterized by central component of the dystrophin-glycoprotein complex (DGC) isolated from skeletal muscle mem- Tokyo Metropolitan Institute of Gerontology, Tokyo, branes. ,-DG is heavily glycosylated, and its glycans Japan. have an important role in binding to proteins such Correspondence should be addressed: T. Endo, Tokyo as laminin, neurexin, perlecan, pikachurin, and agrin, Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan (e-mail: endo@tmig.or.jp). which contain laminin G (LG) domains. Recently, doi: 10.2183/pjab.95.004 ©2019 The Japan Academy 40 T. ENDO [Vol. 95, the binding mode of the LG4 and LG5 domains of tion of core M3, and its defect causes ,-dystrogly- laminin-,2 with the GlcAO1-3Xyl disaccharide re- canopathy. 5) peat was resolved using X-ray crystallography. Ribitol (Rbo) is a sugar alcohol, and the usage (1) Core M1 and core M2. We first iden- of Rbo or Rbo5P as a glycan component has not tified a sialylated O-Man glycan, Sia,2-3GalO1- been reported in mammals. However, Rbo5P is used 4GlcNAcO1-2Man, in ,-DG present in bovine pe- as a component of the teichoic acids present in the 6) 7) 14) ripheral nerves and then in rabbit skeletal muscle. cell walls of most gram-positive bacteria. Rbo5P Subsequently, many studies of the O-Man glycan was first detected as a glycan component in the 15) structure have been performed and various O-Man extended structure of core M3 by our group. glycan structures have been elucidated. Currently, Shortly thereafter, several groups independently these glycans are classified into three core O-Man reported that Rbo5P is a component of mammalian 16)–18) structures based on the linkage of GlcNAc to the glycans. Man residue: core M1 (GlcNAcO1-2Man), core Before a detailed glycan structure was deter- M2 [GlcNAcO1-6(GlcNAcO1-2)Man], and core M3 mined, importance of the glycan moiety of ,-DG was 2) (GalNAcO1-3GlcNAcO1-4Man). Furthermore, in well recognized by an antibody, IIH6, because the addition to Sia,2-3GalO1-4GlcNAc (sialyl glycan), IIH6 antibody recognizes glycosylated ,-DG and GalO1-4(Fuc,1-3)GlcNAc (Lewis X glycan) and functionally competes with DG-laminin binding. HSO -3GlcAO1-3GalO1-4GlcNAc (HNK-1 epitope Thus, IIH6 was considered to recognize laminin- glycan) are exclusively attached to core M1 and binding epitopes on sugar chains. The IIH6 epitope core M2. Notably, the core M2 structure is present in has been proposed to attach to core M3 on ,-DG via the brain. HNK-1 and Lewis X glycans on core M1 the phosphodiester linkage because hydrogen fluoride and core M2 are thought to play important roles in (HF) treatment, which cleaves the phosphodiester 8),9) 19) brain development. bond, ablates the laminin-binding activity. Origi- (2) Core M3. The extended complete core M3 nally the IIH6 epitope glycan was thought to be structure is novel and has recently been revealed linked to the 6-position of Man in core M3 but the 10) (bottom structure in Fig. 1). Characteristic fea- correct binding to the 3-position of GalNAc in 15) tures include 1) the phosphorylation of the 6-position core M3 was subsequently reported. of Man; 2) a tandem ribitol-5-phosphate (Rbo5P) The glycosaminoglycan-like (-3GlcAO1-3Xyl,1-) structure; 3) a (-3GlcAO1-3Xyl,1-) repeat; and 4) a (GlcA-Xyl) repeat is unique. The GlcA-Xyl repeat single GlcAO1-4XylO1-4 unit. was identified to be assembled as a result of the The addition of a phosphate to the monosac- enzymatic activity of LARGE (like-acetylglucosami- 20) charide of glycans is a glycan modification whose nyltransferase). LARGE overexpression drastically significance is partially understood. For example, enhances IIH6 reactivity and the laminin-binding Man 6-phosphate acts as a recognition marker of activity of ,-DG, whereas HF treatment induces the 11) lysosomal enzymes. In mammalian cells, newly loss of IIH6 reactivity and laminin-binding activity of synthesized lysosomal enzymes are modified with a ,-DG, suggesting that the IIH6 epitope is probably phosphate and acquire the Man 6-phosphate marker. the same as the GlcA-Xyl repeat structure. However, These enzymes bind to the lumenal domains of extensive data suggested that the GlcA-Xyl repeat sorting receptors (Man 6-phosphate receptors) is not directly linked to the 6-position of Man in through their Man 6-phosphate recognition markers core M3. For example, mutations in FKTN (fukutin) in the trans-Golgi network and are targeted to and FKRP (fukutin-related protein) are responsible acidified endosomes and lysosomes. Another case is for ,-dystroglycanopathy, and these patients show a xylose (Xyl) 2-phosphate of glycosaminoglycan as dramatic reduction in the reactivity of IIH6, suggest- a common linkage tetrasaccharide. Phosphorylation ing the presence of an unknown “scaffold moiety” and de-phosphorylation of the 2-position of the Xyl between the phosphate and GlcA-Xyl repeat. We residue are an important modification that regulates used small recombinant ,-DG containing the first 20 the formation of the linkage region and glycosami- amino acids of the mucin-like domain to determine 12) noglycan biosynthesis. This transient phosphoryl- the scaffold structure up to the GlcA-Xyl repeat. 12) ation is performed by FAM20B, and its deletion in Finally, we proposed that the scaffold glycan was 13) 15) mice results in embryonic lethality. As will be “GlcA-Xyl-Rbo5P-Rbo5P”. Rbo is a pentose alco- described later, the 6-phosphate of Man attached hol (pentitol) and has never been reported in to core M3 is required for the glycan chain elonga- mammalian glycans. No. 1] Mammalian O-mannosyl glycans 41 Ser/Thr Dol-P-Man POMT1/POMT2 core M3 Manα1 Ser/Thr GlcNAcβ1 4Manα1 Ser/Thr ER POMGNT2 Golgi B3GALNT2 core M1 POMGNT1 GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr GlcNAcβ1 2Manα1 Ser/Thr POMK core M2 GNT-IX (VB) PO GlcNAcβ1 Manα1 Ser/Thr GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr GlcNAcβ1 ER Golgi FKTN PO Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr FKRP PO Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr TMEM5 (RXYLT1) PO Xylβ1 4Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr B4GAT1 PO GlcAβ1 4Xylβ1 4Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr LARGE 3GlcAβ1 3Xylα1 n PO 3GlcAβ1 4Xylβ1 4Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr Fig. 1. Biosynthetic pathway of core M1, core M2, and core M3 O-Man glycans in the ER and Golgi. GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Man, mannose; GlcA, glucuronic acid; Rbo5P, ribitol-5-phosphate; Xyl, xylose; PO , phosphate; POMT1, protein O-mannosyltransferase 1; POMT2, protein O-mannosyltransferase 2; B3GALNT2, O-1,3-N-acetylgalactosaminyl- transferase 2; B4GAT1, O-1,4-glucuronosyltransferase 1; Dol-P-Man, dolichol-phosphate-mannose; FKTN, fukutin; FKRP, fukutin- related protein; GNT-IX(VB), O-1,6-N-acetylglucosaminyltransferase IX(VB); LARGE, acetylglucosaminyltransferase-like; POMGNT1, protein O-linked mannose O-1,2-N-acetylglucosaminyltransferase 1; POMGNT2, protein O-linked mannose O-1,4-N- acetylglucosaminyltransferase 2; POMK, protein O-mannose kinase; TMEM5 (RXYLT1), transmembrane protein 5 (Rbo5PO1,4- xylosyltransferase). In addition to ,-DG, several proteins have been Man-core and peripheral structures is present in shown to carry the core M1 and/or core M2 struc- mammals. The identification and characterization of tures, such as IgG2, phosphacan, CD24, neurofascin, enzymes involved in the biosynthesis of mammalian and lecticans. On the other hand, ,-DG is currently O-Man glycans are necessary to elucidate the the only known core M3-modified protein. function and regulation of the glycans. The biosynthesis of O-Man glycans begins with Biosynthesis the transfer of a Man residue from dolichol-phos- A series of O-Man glycans with heterogeneous phate-mannose (Dol-P-Man) to Ser/Thr residues of 42 T. ENDO [Vol. 95, certain proteins in the endoplasmic reticulum (ER) O-linked mannose O-1,4-N-acetylglucosaminyltrans- (Fig. 1). O-Mannosylation is essential for normal ferase 2), which transfers GlcNAc from a UDP- 21) development in Drosophila melanogaster, zebra- GlcNAc to the O-Man residue of ,-DG. B3GALNT2 22) 23) fish, and mice. Of note, both components, (O-1,3-N-acetylgalactosaminyltransferase 2) forms POMT1 (protein O-mannosyltransferase 1) and GalNAcO1-3GlcNAc by transferring GalNAc from POMT2 (protein O-mannosyltransferase 2), are UDP-GalNAc to the GlcNAc residue of the 24) necessary for O-mannosyltransferase activity. Re- POMGNT2 product. Then, the core M3 structure is cently, the presence of another protein in the O- phosphorylated at the 6-position of Man by POMK mannosylation machinery was suggested in addition (protein O-mannose kinase) in an ATP-dependent to the POMT1/POMT2 system. An O-mannosyla- manner and forms the phospho-core M3 structure tion pathway that selectively modifies cadherins GalNAcO1-3GlcNAcO1-4(phospho-6)Man in the ER. and protocadherins has been reported. According to POMK is regarded as a pseudokinase because it lacks 25) proteomics data, the initiation of the O-Man the functional motifs present in typical kinases, but glycosylation of cadherins and protocadherins does recent crystal structures have revealed the detailed not depend on the evolutionarily conserved POMT1/ mechanisms underlying POMK catalysis and sub- 5),33) POMT2 enzymes that initiate O-Man glycosylation strate recognition. Notably, POMGNT2 func- on ,-DG. Four TMTC (transmembrane and tetra- tions in the ER before glycosylation by POMGNT1 tricopeptide repeat containing) genes are predicted in the Golgi, and thus the site of ,-DG occupied by to encode distinct O-mannosyltransferases that core M3 should be determined by the substrate cooperatively mannosylate the common extracellular specificity of POMGNT2. A better understanding cadherin domains of cadherins and protocadherins, of the determinants of the site requires information suggesting the existence of another as yet undiscov- about the mechanism by which POMGNT2 recog- 26) ered O-Man glycosylation pathway. It is important nizes the peptide sequence and/or conformation to determine whether or not the TMTC products near the target O-Man. However, the details cur- actually exhibit enzymatic activity towards the rently remain unclear and information about the cadherin family. POMGNT2 structure will improve our understand- (1) Core M1 and core M2. After O-manno- ing of the site-recognition mechanism. sylation by POMT1/POMT2, POMGNT1 (protein Each glycosyltransferase responsible for synthe- O-linked mannose O-1,2-N-acetylglucosaminyltrans- sizing the extended complete core M3 structure has ferase 1) forms the GlcNAcO1-2Man (core M1) using been identified (Fig. 1). The first enzymes to be UDP-GlcNAc as a donor substrate in the Golgi identified that form the extended structure were the 27) (Fig. 1). The core M2 structure [GlcNAc O1- enzymes that synthesize the novel glycosaminogly- 6(GlcNAc O1-2)Man] is formed sequentially through can-like GlcA-Xyl repeat, which is essential for the the actions of POMGNT1 and GNT-IX(GNT-VB) binding of ,-DG to laminin. In 2012, Inamori et al. [O-1,6-N-acetylglucosaminyltransferase IX(VB)], an found that LARGE exhibits both xylosyltransferase enzyme that catalyzes the formation of the and glucuronyltransferase activities and produces 28) GlcNAcO1-6Man linkage. Because GNT-IX(GNT- repeating units of GlcA-Xyl using UDP-GlcA and 29),30) 20) VB) is specifically expressed in the brain, O- UDP-Xyl as donor substrates. LARGE is an ,- Man glycans with the core M2 structure are exclu- dystroglycanopathy [congenital muscular dystrophy sively detected in the brain. Notably, the presence of 1D (MDC1D)] gene product. The molecular mechan- the HNK-1 glycan on the core M2 of phosphacan/ ism by which LARGE determines the length of the RPTPO is an important regulator of re-myelination GlcA-Xyl repeat synthesized remains unclear. 31) in the brain. Peripheral structures on core M1 and LARGE2, a LARGE paralog, also exhibits both core M2 are synthesized by a series of glycosyltrans- enzyme activities and synthesizes GlcA-Xyl repeats 34),35) ferases, such as galactosyltransferase, sialyltransfer- on ,-DG. Although LARGE is likely specific for ase, glucuronyltransferase, sulfotransferase, and ,-DG, LARGE2-dependent glycosylation also elon- ,1,3-fucosyltransferase in the Golgi. gates a glycosaminoglycan chain on proteoglycans 36) (2) Core M3. On the other hand, the core such as glypican-4. 32) M3 structure is synthesized in the ER (Fig. 1). We assumed that FKTN and FKRP might be After the initial O-Man transfer is catalyzed by candidate enzymes that synthesize the tandem the POMT1/POMT2 complex, the GlcNAcO1- Rbo5P structure because: 1) FKTN and FKRP are 4Man linkage is catalyzed by POMGNT2 (protein responsible for ,-dystroglycanopathy; 2) both pro- No. 1] Mammalian O-mannosyl glycans 43 Table 1. Summary of genes responsible for ,-dystroglycanopa- teins exhibit similarities to glycosyltransferases con- thies and their functions taining the DXD motif, which is conserved in many glycosyltransferases; 3) both belong to the nucleoti- Gene name Function dyltransferase fold protein superfamily, and 4) both Primary have similarities to enzymes involved in phosphor- DAG1 Dystroglycan ylcholine modification or the mannosyl-phosphoryla- Secondary tion of glycans in bacteria and yeast. Actually, as POMT1 Protein O-mannosyltransferase 15) shown in our previous study, FKTN transfers the POMT2 Protein O-mannosyltransferase first Rbo5P to the 3-position of GalNAc from CDP- Protein O-mannose O-1, 2-N-acetylglucosami- POMGNT1 Rbo and FKRP transfers the second Rbo5P to the nyltransferase 1-position of the first Rbo5P (Fig. 1). Previously, the FKTN Ribitol-5-phosphate transferase glycan was assumed to extend from the 6-position of FKRP Ribitol-5-phosphate transferase the core Man, but it actually extends from the 3-posi- O-1, 3-glucuronyltransferase and ,-1, 3-xylo- 15) LARGE tion of GalNAc, as confirmed by an NMR study. syltransferase The sequential actions of FKTN and FKRP pro- Protein O-mannose O-1, 4-N-acetylglucosami- POMGNT2 duce the “Rbo5P-1Rbo5P-3GalNAcO1-3GlcNAcO1- nyltransferase 4(phosphate-6)Man” structure. Notably, the 6-phos- B3GALNT2 O-1, 3-N-acetylgalactosaminyltransferase phate of Man in the phospho-core M3 peptide is B4GAT1 O-1, 4-glucuronyltransferase required for FKTN activity because the non-phos- POMK Protein O-mannose kinase phorylated core M3 peptide does not serve as an TMEM5 15) Ribitol-5-phosphate O-1, 4-xylosyltransferase acceptor. Notably, FKTN does not transfer the (RXYLT1) second Rbo5P and does not form the tandem Rbo5P Tertiary structure by itself. On the other hand, FKRP does GMPPB GDP-mannose pyrophosphorylase not transfer the first Rbo5P and does not form a DPM1 Dolichol-phosphate-mannose synthase Rbo5P trimer or more. Based on the data, the DPM2 Dolichol-phosphate-mannose synthase synthesis of the tandem Rbo5P unit is highly DPM3 Dolichol-phosphate-mannose synthase regulated by the strict substrate specificities of DOLK Dolichol kinase FKTN and FKRP, although both enzymes share ISPD CDP-ribitol synthetase homology. Information about the FKTN and FKRP structures will facilitate the elucidation of the substrate recognition mechanism. We and other groups have observed an addi- produced by TMEM5(RXYLT1) and LARGE may tional GlcAO1-4XylO1-4 unit linked to the tandem be important for the formation of the functional O- Rbo5P structure. As described above, this GlcAO1- Man glycan on ,-DG. 4XylO1-4 unit was not formed by LARGE. Two ,-Dystroglycanopathy groups independently reported that the GlcAO1-4 unit was formed by the actions of B4GAT1 (O-1,4- Muscular dystrophies are genetic diseases that glucuronosyltransferase 1), which shares homol- cause progressive muscle weakness and wasting. ogy with the glucuronyltransferase domain of According to recent data, aberrant O-mannosylation 37),38) 10) LARGE. As shown in our previous study, of ,-DG is the primary cause of some forms of TMEM5 acts as a ribitol O1,4-xylosyltransferase to congenital muscular dystrophy known as ,-dystro- generate a XylO1-4Rbo5P linkage, in which the glycanopathy. To date, eighteen genes have been second Rbo5P in the tandem structure serves as identified as causative factors in these diseases the acceptor for Xyl. TMEM5 has been proposed to (Table 1). ,-Dystroglycanopathies are classified as be renamed RXYLT1 (Rbo5PO-1,4-Xyl transferase). primary (caused by mutations in the gene encoding Furthermore, the TMEM5 product serves as an DG, DAG1), secondary (caused by mutations in the acceptor substrate for B4GAT1, but not for genes that directly modify the O-Man glycan of ,- 10) LARGE. Because the glucuronosyltransferase ac- DG), or tertiary (caused by the genes that indirectly tivity of LARGE is specific for ,-linked Xyl and not modify the O-Man glycan of ,-DG). O-linked Xyl, the formation of GlcAO1-4Xyl by This section will briefly describe a history of the B4GAT1 is required for the LARGE reaction to identification of the traits of ,-dystroglycanopathy. form GlcA-Xyl repeats. The different Xyl linkages The best known and most common type of muscular 44 T. ENDO [Vol. 95, dystrophy is Duchenne-type muscular dystrophy, the predominant form of ,-dystroglycanopathy in and its causative gene, dystrophin, was identified in Japan, and FKRP-deficient MDC1C and LGMD2I 1987. Dystrophin encodes an actin-binding cytoske- are the most frequent forms of ,-dystroglycanopathy letal protein that is present inside the muscle in the USA and Europe. We revealed that the first membrane and is similar to spectrin. Later, many three gene products were glycosyltransferases them- proteins, including glycoproteins, form a large com- selves, but the functions of the remaining three plex called DGC. The function of DGC is thought to gene products were unclear until recently. FKTN 15) connect extracellular matrix components and intra- and FKRP are Rbo5P transferases and LARGE 35) cellular components with the actin cytoskeleton. is a xylosyltransferase and glucuronyltransferase, Notably, several components of the DGC have been whose mutations were identified to cause ,-dystro- identified as causative agents for different type glycanopathy. The mutations of all genes led to muscular dystrophies. DG is a component of the defects in O-Man glycan formation. Notably, not all DGC that is encoded by a single gene and is cleaved patients with WWS carry mutations in POMT1 or into two proteins, ,-DG and O-DG, by posttransla- POMT2. Furthermore, cohort studies of patients tional processing. with ,-dystroglycanopathy reported that two-thirds 44) (1) Primary ,-dystroglycanopathy. In a case of patients had no mutations in these six genes. study of primary ,-dystroglycanopathy, a mutation Clearly, other unidentified causative genes are (Thr192Met) in the N-terminal domain of ,-DG was present in these patients, and a molecular diagnosis identified in a patient with limb-girdle muscular of each patient is necessary to improve our under- 39) dystrophy and cognitive impairment. The muta- standing of ,-dystroglycanopathy because patients tion reduces the number of GlcA-Xyl repeats because with this syndrome exhibit an extremely broad this amino acid is required for the recognition by clinical spectrum of symptoms. The most severe form LARGE. This study reported the first case in which a is characterized by muscular dystrophy with struc- mutation in the DAG1 gene itself was shown to cause tural abnormalities in the brain and eye. The mildest muscular dystrophy via a defect in O-Man glycan form is limb-girdle muscular dystrophy without brain formation. Another patient with different mutations or eye involvement. Later, many previously uniden- 40) showed similar hypoglycosylation of ,-DG. tified ,-dystroglycanopathy causative genes were 45) (2) Secondary ,-dystroglycanopathy. We revealed through genetic analyses. As described identified and characterized the glycosyltransferases in the previous sections, recent studies have finally POMGNT1 and POMT1/POMT2. Mutations in revealed and characterized these causative gene POMGNT1 are responsible for muscle-eye-brain products involved in processing the entire core M3 disease (MEB), and mutations in POMT1/2 are glycan. Among them, the pathogenic role of the 6- responsible for Walker–Warburg syndrome (WWS). position of Man should be noted. It is phosphorylated 32) MEB and WWS are congenital muscular dystrophies by POMK, and POMK mutations cause ,- 45) characterized by brain malformations and structural dystroglycanopathy because the 6-phosphate of 15) abnormalities in the eye. At approximately the same Man is required for the FKTN activity. time, other groups reported abnormal glycosylation The entire structure of the core M3 glycan of ,-DG with substantial reductions in laminin- and its biosynthetic pathway have been identified. binding activity and IIH6 antibody reactivity in Mutations in enzymes involved in each glycosylation patients with MEB and WWS. Because the common step cause ,-dystroglycanopathy. However, a re- biochemical feature in MEB and WWS is abnormal maining question is that the GlcNAcO1-2 linkage is glycosylation of ,-DG, we proposed that MEB and lost in the proposed structure (bottom structure in WWS are glycan-deficient diseases. By the early Fig. 1). Approximately two decades ago, we re- 2000s, causative mutations in six genes (POMT1, ported that the POMGNT1 gene is responsible for 27) POMT2, POMGNT1, FKTN, FKRP, and LARGE) MEB. A selective deficiency in glycosylated ,-DG have been identified in patients with ,-dystroglycan- was observed in patients with MEB. MEB is opathy. Fukuyama-type congenital muscular dys- inherited as a loss-of-function of the POMGNT1 41) trophy (FCMD) results from mutations in FKTN. gene, and was one of the findings prompting the Congenital muscular dystrophy type 1C (MDC1C) development of this new research field, glycosylation and limb-girdle muscular dystrophy type 2I and muscular dystrophy. The lack of the core M1 (LGMD2I) are caused by a defect in fukutin-related structure (GlcNAcO1-2 elongation) is hypothesized 42),43) protein (FKRP), a homolog of FKTN. FCMD is to disturb the processing of the core M3 glycan in No. 1] Mammalian O-mannosyl glycans 45 (A) CDP-Rbo P 3 β3 P Man Man FKTN GalNAc GlcNAc RboP GalNAc GlcNAc β4 Catalytic Stem CBD DG DG POMGNT1 (B) CDP-Rbo P P P Man Man Man GalNAc GlcNAc GalNAc GlcNAc RboP GalNAc GlcNAc FKTN FKTN DG DG DG Catalytic Catalytic Stem CBD Stem CBD POMGNT1 POMGNT1 Man GlcNAc Man GlcNAc Man β2 UDP-GlcNAc Fig. 2. Proposed mechanisms underlying the efficient recruitment of FKTN to core M3 by the carbohydrate-binding domain (CBD) of POMGNT1. (A) Core M3 (GalNAcO1-3GlcNAcO1-4Man) binds to the CBD of POMGNT1. Because the POMGNT1 stem domain binds to core M3 via the CBD, FKTN is simultaneously recruited to the reaction site. (B) Core M1 (GlcNAcO1-2Man) binding to the CBD of POMGNT1. Glycosylated sites modified with core M3 are suggested to be located in close proximity to core M1. First, the FKTN-POMGNT1 complex forms core M1 (GlcNAcO1-2Man) on an O-linked Man near core M3 and then the complex binds this newly formed core M1 structure. In this situation, FKTN easily transfers Rbo5P to neighboring core M3 structures. GalNAc, N- acetylgalactosamine; GlcNAc, N-acetylglucosamine; Man, mannose; Rbo, ribitol; P, phosphate: DG, dystroglycan. the Golgi. We performed an X-ray crystallographic ensure the efficient synthesis of rare glycans in study of human POMGNT1 to answer this ques- mammals. In the case when the FKTN-POMGNT1 46) tion. complex binds core M3 (Fig. 2A), FKTN is simulta- 27),47) As shown in our previous study, neously recruited to reaction site. On the other hand, POMGNT1 is composed of a catalytic domain and in the case when the complex binds core M1, FKTN a stem domain, and the stem domain is unusually is recruited to the reaction site through a different longer than the same domain in other glycosyltrans- mechanism. Glycosylated sites modified with cor- ferases, but its function was unclear. As a result of eM3 in ,-DG include Thr317, Thr319, and Thr379, 46) the X-ray crystallographic study, we unexpectedly although other precise sites remain unclear. Accord- identified the presence of a carbohydrate-binding ing to the results from mass spectrometry-based domain (CBD) in the stem domain. After an in-depth analyses, the core M1 structure is located near the 15),18),19) investigation of the glycan binding specificity of this core M3 site. As shown in Fig. 2B, the domain, we found that it specifically recognizes the FKTN-POMGNT1 complex initially synthesizes GlcNAcO1-2Man and GalNAcO1-3GlcNAc linkages, GlcNAcO1-2Man on O-Man near core M3, and then corresponding to the core M1 and core M3 struc- the complex binds this formed core M1. Under this tures, respectively. Of note, the CBD did not bind to circumstance, FKTN easily transfers Rbo5P to any other types of glycans. In addition, we reported neighboring core M3 structures. Actually, we con- 10 years ago that FKTN and POMGNT1 formed a firmed that the sugar-binding activity of the CBD of complex in the Golgi, but its biological meaning POMGNT1 is necessary for the maturation of glycan 48) 46) was not determined at the time. Because the on the core M3 structure. Furthermore, the for- POMGNT1 stem domain binds to core M3 and mation of an FKTN, FKRP, and TMEM5 complex 18),49) core M1 via the CBD, FKTN may be recruited to was proposed. The formation of this multi- the reaction site to form a FKTN-POMGNT1 component complex may ensure the synthesis of complex, which leads to efficient Rbo5P transfer to tandem Rbo5P repeats and the subsequent efficient core M3 (Fig. 2). The formation of this complex may elongation of the core M3 glycan. 46 T. ENDO [Vol. 95, (3) Tertiary ,-dystroglycanopathy. Glyco- Rbo5P by the enzyme teichoic acid ribitol I (TarI). sylation is determined by the actions of many Notably, bacterial TarI shares homology with human glycosyltransferases that catalyze the transfer of a ISPD (isoprenoid synthase domain-containing), monosaccharide moiety from a nucleotide sugar which is known to be responsible for ,-dystroglycan- donor substrate to the acceptor substrate. In some opathy. According to data from our previous 15) cases, glycosyltransferases use a dolichol-pyrophos- study, human ISPD is a CDP-Rbo synthetase phate sugar instead of a nucleotide sugar. This (CDP-Rbo pyrophosphorylase) that synthesizes mechanism is used for the O-mannosylation of the CDP-Rbo from CTP and Rbo5P, and CDP-Rbo is ,-DG protein. POMT1/POMT2 catalyze the trans- required for the biosynthesis of functional O-Man fer of a Man residue from Dol-P-Man to Ser/Thr glycans. Shortly thereafter, two other groups inde- residues on certain proteins. Therefore, some defects pendently confirmed that human ISPD catalyzes 16),17) in glycosylation are not only mediated by glycosyl- CDP-Rbo synthesis. Thus, the amount of transferases but also by the presentation of nucleo- available CDP-Rbo affects the O-mannosylation of tide sugar (or dolichol-sugar). Thus, diseases caused ,-DG. Providing further support for this hypothesis, by mutations in these genes are categorized as the addition of CDP-Rbo restores the IIH6 epitope 15) tertiary ,-dystroglycanopathies. of ,-DG in ISPD knockout cells, suggesting that DPM1, DPM2, DPM3, DOLK, and GMPPB CDP-Rbo supplementation is a possible therapeutic encode proteins involved in the synthesis of Dol-P- strategy for ISPD-deficient patients. Furthermore, Man. Dol-P-Man is an essential Man donor that is dietary supplementation with Rbo is suggested to be required for mannosylation, including O-mannosyla- beneficial in mice with not only ISPD mutations but 55) tion, N-glycosylation, and C-mannosylation, as well also a FKRP mutation. 56) as the formation of glycosyl-phosphatidylinositol As shown in our recent study, CDP-glycerol anchors. DOLK (dolichol kinase) encodes the DOLK inhibits the RboP transfer activities of both FKTN activity responsible for the formation of Dol-P. A and FKRP, suggesting that CDP-glycerol inhibits combined N-glycosylation and O-mannosylation de- the synthesis of the functional O-Man glycan of ficiency has been observed in patients with a ,-DG by preventing the further elongation of the predominant presentation of dilated cardiomyopathy glycan chain. A remaining question is whether the 50) due to DOLK mutations has been observed. amount of CDP-glycerol is causative factor for GMPPB (GDP-mannose pyrophosphorylase B) cat- uncharacterized ,-dystroglycanopathy. alyzes the synthesis of GDP-Man from GTP and Glycosylation of Notch receptors regulates Man-1-P. GDP-Man is required for the synthesis ligand-induced Notch signaling in the cell-to-cell of Dol-P-Man. An O-mannosylation deficiency was communication system that is required for cell-fate 57) observed, but no evidence of abnormal N-glycosyla- decisions during development. Aberrant activation tion has been found in patients carrying mutant or inactivation of the Notch signaling pathway leads 51) GMPPB. Dol-P-Man is synthesized from Dol-P to human diseases, including many different cancers. and GDP-Man by the DPM (dolichol-phosphate- Notch receptors are known to be modulated by mannose) synthase complex, which comprises three unusual O-glycans: O-Fuc, O-Glc, and O-GlcNAc. subunits: DPM1, DPM2, and DPM3. Mutations in A notable glycan is O-Glc, the synthesis of which each component result in dystroglycanopathy-type is catalyzed by protein O-glucosyltransferase1 muscular dystrophy, but defects in N-glycosylation (POGLUT1). POGLUT1 is an enzyme that trans- 52)–54) levels differed. Because the DPM1, DPM2, fers Glc from UDP-Glc to a distinct Ser residue in DPM3, DOLK, and GMPPB proteins are all the epidermal growth factor (EGF)-like repeat. involved in the synthesis of Dol-P-Man, the amount Importantly, POGLUT1 is essential for Notch of available Dol-P-Man affects the O-mannosylation receptor function. Recently, a missense mutation of ,-DG. (Asp233Glu) in POGLUT1 was identified in a Rbo5P in a mammalian glycan component is patient showing an impairment in muscle develop- very unique and is incorporated by the sequential ment and hypoglycosylation of muscle ,-DG, but not actions by FKTN and FKRP using CDP-Rbo, as in fibroblasts. A detailed analysis revealed a clearly described above. However, the CDP-Rbo biosyn- different disorder from secondary ,-dystroglycanop- thetic pathway was unknown in mammals. In athy, suggesting that a pathomechanism for this form bacteria, this synthetic pathway has already been of muscular dystrophy is Notch signaling-dependent 58) elucidated. CDP-Rbo is synthesized from CTP and loss of satellite cells. Further studies are necessary No. 1] Mammalian O-mannosyl glycans 47 (α-Dystroglycanopathy) (α-Dystroglycanopathy + Sugars) (Normal) Sugars Laminin α-DG Basal lamina Membrane β β-DG Dystrophin Cytoplasm F-actin Muscle instability, destruction and necrosis Prevention of muscle instability Fig. 3. ,-Dystroglycanopathies caused by a defect in the glycosylation of ,-DG and possible new glycotherapeutic strategies for these diseases. ,-DG is an extracellular peripheral membrane glycoprotein that is anchored to the cell membrane by binding to a transmembrane glycoprotein, O-DG. ,-DG-O-DG is thought to stabilize the plasma membrane by acting as an axis through which the extracellular matrix is tightly linked to the cytoskeleton. In addition, the cytoplasmic domain of O-DG interacts with dystrophin, which subsequently binds to the F-actin cytoskeleton. ,-DG is heavily glycosylated, and its sugars play a role in binding to laminin. Abnormal glycosylation of ,-DG causes ,-dystroglycanopathies (see Table 1). New glycotherapeutic strategies for ,- dystroglycanopathies that induce the interaction laminin and ,-DG include glycan supplementation. ,-DG, ,-dystroglycan; O- DG, O-dystroglycan. to determine the detailed mechanism using data novel structure in mammals. However, the findings from other patients. Similar to O-Glc, O-Fuc and O- produce additional questions. For example, are any GlcNAc modifications also occur at specific positions other protein(s) modified with the core M3 glycan? within an EGF repeat if the appropriate O-glyco- Do proteins other than ,-DG contain the Rbo5P 59) sylation consensus sequence is present, suggesting modification? What is the biological meaning of a mechanism for fine-tuning the Notch signaling Rbo5P in mammalian glycosylation? How is Rbo5P pathway by O-glycan that is probably related to synthesized and degraded in mammals? Additionally, muscle formation. further studies are needed to clarify the distribution The transporters of nucleotide-sugars into the of O-Man glycans in various tissues and to examine Golgi are critical for the glycosylation of glycoconju- their changes during development and in response to gates, and mutations in the transporters may cause a pathological conditions. These questions should be group of genetic disorders named congenital disorders addressed in the future. A major challenge will be to of glycosylation. A missense mutation (Gln101His) in integrate the forthcoming structural, cell biological, one of these transporters, SLC35A1, was identified and genetic information to understand how ,-DG in a patient with intellectual disability, seizures, O-mannosylation contributes to muscular dystrophy ataxia, macrothrombocytopenia, and bleeding dia- and brain development. thesis, and the mutation displayed a reduction in The hypoglycosylation of ,-DG in muscle 60) the sialylation of N- and O-glycosylated glycans. substantially reduces its affinity for a number of Of note, SLC35A1-deficient cells showed a lack of extracellular ligands. Based on this finding, the ,-DG O-mannosylation with a concomitant reduc- defective glycosylation of ,-DG is reasonably ex- 61) tion in sialylation. The results indicate a role for plained to cause muscle cell degeneration and SLC35A1 in ,-DG O-mannosylation that is distinct abnormal brain structures in patients with ,- from sialic acid metabolism. In addition, SLC35A1- dystroglycanopathies. In other words, interference deficient patients present a combined disorder of with the glycosylation of ,-DG may lead to a ,-DG O-mannosylation and sialylation, which is a combination of muscle and brain phenotypes in novel variant of the tertiary ,-dystroglycanopathies. patients with these diseases. Because ,-DG hypo- glycosylation is a common feature of ,-dystroglyca- Future directions nopathies, ,-DG may be a potential target of new The O-Man glycan moiety including Rbo5P is a glycotherapeutic strategies for these diseases (Fig. 3). 48 T. ENDO [Vol. 95, ology 24, 314–324. The formation of the interaction between laminin 9) Yaji, S., Manya, H., Nakagawa, N., Takematsu, H., and ,-DG induced by glycan supplementation may Endo, T., Kannagi, R. et al. (2015) Major glycan improve the symptoms of ,-dystroglycanopathy in structure underlying expression of the Lewis X patients who carry mutations in any enzymes of the epitope in the developing brain is O-mannose- glycan processing pathway. linked glycans on phosphacan/RPTPO. Glycobiol- ogy 25, 376–385. Muscular dystrophy is a group of genetic 10) Manya, H., Yamaguchi, Y., Kanagawa, M., disorders characterized by progressive muscle weak- Kobayashi, K., Tajiri, M., Akasaka-Manya, K. ness and degeneration. Unfortunately, an effective et al. (2016) The muscular dystrophy gene treatment for the disease is not available. I hope our TMEM5 encodes a ribitol O1,4-xylosyltransferase findings will provide new opportunities for the required for the functional glycosylation of dystro- glycan. J. Biol. Chem. 291, 24618–24627. development of treatments for these diseases in the 11) Braulke, T. and Bonifacino, J.S. (2009) Sorting of future. lysosomal proteins. Biochim. Biophys. Acta 1793, 605–614. Acknowledgements 12) Izumikawa, T., Sato, B., Mikami, T., Tamura, J., I thank Dr. Tatsushi Toda and other colleagues Igarashi, M. and Kitagawa, H. (2015) GlcUAO1- 3GalO1-3GalO1-4Xyl(2-O-phosphate) is the pre- for fruitful and long-term collaborations and valuable ferred substrate for chondroitin N-acetylgalactosa- discussions. This work was supported by a grant from minyltransferase-1. J. Biol. Chem. 290, 5438– the National Center of Neurology and Psychiatry (NCNP; Intramural Research Grant 29-4) and a 13) Wen, J., Xiao, J., Rahdar, M., Choudhury, B.P., Japan Society for the Promotion of Science Grant Cui, J., Taylor, G.S. et al. (2014) Xylose phospho- rylation functions as a molecular switch to regulate (JP16K08262). I have no conflicts of interest with the proteoglycan biosynthesis. Proc. Natl. Acad. Sci. contents of this article. U.S.A. 111, 15723–15728. 14) Brown, S., Santa Maria, J.P. and Walker, S. (2013) References Wall teichoic acids of gram-positive bacteria. 1) Endo, T. 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(2013) results from abnormal dystroglycan O-mannosyla- Intellectual disability and bleeding diathesis due tion. PLoS Genet. 7, e1002427. to deficient CMP-sialic acid transport. Neurology 51) Carss, K.J., Stevens, E., Foley A.R., Cirak, S., 81, 681–687. Riemersma, M., Torelli, S. et al. (2013) Mutations 61) Riemersma, M., Sandrock, J., Boltje, T.J., Bull, in GDP-mannose pyrophosphorylase B cause con- C., Heise, T., Ashikov, A. et al. (2015) Disease genital and limb-girdle muscular dystrophies asso- mutations in CMP-sialic acid transporter ciated with hypoglycosylation of ,-dystroglycan. SLC35A1 result in abnormal ,-dystroglycan O- Am. J. Hum. Genet. 93,29–41. mannosylation, independent from sialic acid. Hum. 52) Lefeber, D.J., Schonberger, J., Morava, E., Guillard, Mol. Genet. 24, 2241–2246. M., Huyben, K.M., Verrijp, K. et al. (2009) Deficiency of Dol-P-Man synthase subunit DPM3 bridges the congenital disorders of glycosylation (Received Oct. 5, 2018; accepted Nov. 5, 2018) with the dystroglycanopathies. Am. J. Hum. No. 1] Mammalian O-mannosyl glycans 51 Profile Tamao Endo was born in Asahi-city, Chiba in 1954, and graduated from the Faculty of Pharmaceutical Sciences, The University of Tokyo in 1977 and obtained his Ph.D. at the same institution in 1982. He was a postdoctoral fellow at the Baylor College of Medicine, and a research associate in the Institute of Medical Science, The University of Tokyo. Since 1994, he has been the head of Department of Molecular Glycobiology, Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology (TMIG). He has served as the vice-director of TMIG since 2012. He served as the president of The Japanese Society of Carbohydrate Research (JSCR) and does so currently for the Japan Consortium for Glycobiology and Glycotechnology (JCGG). He is a member of the Science Council of Japan. He is now focusing on glycobiology in aging and diseases. He was honored to receive the Tokyo Metropolitan Governor’s Award (2002), The Pharmaceutical Society of Japan Award for Divisional Scientific Promotion (2003), The Asahi Award (2007), and The Japan Academy Prize (2017). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Proceedings of the Japan Academy. Series B, Physical and Biological Sciences Pubmed Central

Mammalian O-mannosyl glycans: Biochemistry and glycopathology

Proceedings of the Japan Academy. Series B, Physical and Biological Sciences , Volume 95 (1) – Jan 11, 2019

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DOI
10.2183/pjab.95.004
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Abstract

No. 1] Proc. Jpn. Acad., Ser. B 95 (2019) 39 Review 1,† By Tamao ENDO (Communicated by Kunihiko SUZUKI, M.J.A.) Abstract: Glycosylation is an important posttranslational modification in mammals. The glycans of glycoproteins are classified into two groups, namely, N-glycans and O-glycans, according to their glycan-peptide linkage regions. Recently, O-mannosyl glycan, an O-glycan, has been shown to be important in muscle and brain development. A clear relationship between O-mannosyl glycans and the pathomechanisms of some congenital muscular dystrophies has been established in humans. Ribitol-5-phosphate is a newly identified glycan component in mammals, and its biosynthetic pathway has been elucidated. The discovery of new glycan structures and the identification of highly regulated mechanisms of glycan processing will help researchers to understand glycan functions and develop therapeutic strategies. Keywords: O-mannosylation, congenital muscular dystrophy, dystroglycan, ribitol-5- phosphate 3) altered glycosylation was published in this journal, Introduction including our pioneering findings of muscular dys- The major glycans of glycoproteins are classified trophy and glycosylation. Since then, many bio- into two groups according to their glycan-peptide chemists, molecular biologists, pediatricians, neurol- linkages. Glycans linked to asparagine (Asn) residues ogists, and geneticists have entered this new research of proteins are termed N-glycans, whereas glycans field. This review will describe recent progress in linked to serine (Ser) or threonine (Thr) residues are establishing the biochemistry and glycopathology of called O-glycans. In N-glycans, the reducing terminal O-Man glycans in mammals. N-acetylglucosamine (GlcNAc) is linked to the amide Structure group of Asn via an aspartylglycosylamine linkage. In O-glycans, the reducing terminal N-acetylgalactos- O-Mannosylation is known as a yeast-type amine (GalNAc) is attached to the hydroxyl group modification, and all O-Man glycan structures that of Ser and Thr residues. In addition to the abundant have been elucidated in yeast are neutral linear O-GalNAc forms, several unique types of protein O- structures consisting of only Man residues. O- glycosylation have been identified, such as O-linked Mannosylation of proteins is essential for viability fucose (Fuc), glucose (Glc), GlcNAc, and mannose in yeast, and its absence is thought to affect the cell (Man), which have been shown to mediate diverse wall structure and rigidity. On the other hand, physiological functions. We and other researchers mammalian O-Man glycan is a unique type of protein have shown that O-Man glycan is important in modification that is present in a limited number of muscle and brain development, and its deficiency glycoproteins in the brain, nerves, and skeletal 4) leads to a group of congenital muscular dystrophies muscle. One of the best known O-Man-modified 1),2) known as ,-dystroglycanopathies. In 2004, a glycoproteins is ,-dystroglycan (,-DG), which is a review of human genetic diseases characterized by central component of the dystrophin-glycoprotein complex (DGC) isolated from skeletal muscle mem- Tokyo Metropolitan Institute of Gerontology, Tokyo, branes. ,-DG is heavily glycosylated, and its glycans Japan. have an important role in binding to proteins such Correspondence should be addressed: T. Endo, Tokyo as laminin, neurexin, perlecan, pikachurin, and agrin, Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan (e-mail: endo@tmig.or.jp). which contain laminin G (LG) domains. Recently, doi: 10.2183/pjab.95.004 ©2019 The Japan Academy 40 T. ENDO [Vol. 95, the binding mode of the LG4 and LG5 domains of tion of core M3, and its defect causes ,-dystrogly- laminin-,2 with the GlcAO1-3Xyl disaccharide re- canopathy. 5) peat was resolved using X-ray crystallography. Ribitol (Rbo) is a sugar alcohol, and the usage (1) Core M1 and core M2. We first iden- of Rbo or Rbo5P as a glycan component has not tified a sialylated O-Man glycan, Sia,2-3GalO1- been reported in mammals. However, Rbo5P is used 4GlcNAcO1-2Man, in ,-DG present in bovine pe- as a component of the teichoic acids present in the 6) 7) 14) ripheral nerves and then in rabbit skeletal muscle. cell walls of most gram-positive bacteria. Rbo5P Subsequently, many studies of the O-Man glycan was first detected as a glycan component in the 15) structure have been performed and various O-Man extended structure of core M3 by our group. glycan structures have been elucidated. Currently, Shortly thereafter, several groups independently these glycans are classified into three core O-Man reported that Rbo5P is a component of mammalian 16)–18) structures based on the linkage of GlcNAc to the glycans. Man residue: core M1 (GlcNAcO1-2Man), core Before a detailed glycan structure was deter- M2 [GlcNAcO1-6(GlcNAcO1-2)Man], and core M3 mined, importance of the glycan moiety of ,-DG was 2) (GalNAcO1-3GlcNAcO1-4Man). Furthermore, in well recognized by an antibody, IIH6, because the addition to Sia,2-3GalO1-4GlcNAc (sialyl glycan), IIH6 antibody recognizes glycosylated ,-DG and GalO1-4(Fuc,1-3)GlcNAc (Lewis X glycan) and functionally competes with DG-laminin binding. HSO -3GlcAO1-3GalO1-4GlcNAc (HNK-1 epitope Thus, IIH6 was considered to recognize laminin- glycan) are exclusively attached to core M1 and binding epitopes on sugar chains. The IIH6 epitope core M2. Notably, the core M2 structure is present in has been proposed to attach to core M3 on ,-DG via the brain. HNK-1 and Lewis X glycans on core M1 the phosphodiester linkage because hydrogen fluoride and core M2 are thought to play important roles in (HF) treatment, which cleaves the phosphodiester 8),9) 19) brain development. bond, ablates the laminin-binding activity. Origi- (2) Core M3. The extended complete core M3 nally the IIH6 epitope glycan was thought to be structure is novel and has recently been revealed linked to the 6-position of Man in core M3 but the 10) (bottom structure in Fig. 1). Characteristic fea- correct binding to the 3-position of GalNAc in 15) tures include 1) the phosphorylation of the 6-position core M3 was subsequently reported. of Man; 2) a tandem ribitol-5-phosphate (Rbo5P) The glycosaminoglycan-like (-3GlcAO1-3Xyl,1-) structure; 3) a (-3GlcAO1-3Xyl,1-) repeat; and 4) a (GlcA-Xyl) repeat is unique. The GlcA-Xyl repeat single GlcAO1-4XylO1-4 unit. was identified to be assembled as a result of the The addition of a phosphate to the monosac- enzymatic activity of LARGE (like-acetylglucosami- 20) charide of glycans is a glycan modification whose nyltransferase). LARGE overexpression drastically significance is partially understood. For example, enhances IIH6 reactivity and the laminin-binding Man 6-phosphate acts as a recognition marker of activity of ,-DG, whereas HF treatment induces the 11) lysosomal enzymes. In mammalian cells, newly loss of IIH6 reactivity and laminin-binding activity of synthesized lysosomal enzymes are modified with a ,-DG, suggesting that the IIH6 epitope is probably phosphate and acquire the Man 6-phosphate marker. the same as the GlcA-Xyl repeat structure. However, These enzymes bind to the lumenal domains of extensive data suggested that the GlcA-Xyl repeat sorting receptors (Man 6-phosphate receptors) is not directly linked to the 6-position of Man in through their Man 6-phosphate recognition markers core M3. For example, mutations in FKTN (fukutin) in the trans-Golgi network and are targeted to and FKRP (fukutin-related protein) are responsible acidified endosomes and lysosomes. Another case is for ,-dystroglycanopathy, and these patients show a xylose (Xyl) 2-phosphate of glycosaminoglycan as dramatic reduction in the reactivity of IIH6, suggest- a common linkage tetrasaccharide. Phosphorylation ing the presence of an unknown “scaffold moiety” and de-phosphorylation of the 2-position of the Xyl between the phosphate and GlcA-Xyl repeat. We residue are an important modification that regulates used small recombinant ,-DG containing the first 20 the formation of the linkage region and glycosami- amino acids of the mucin-like domain to determine 12) noglycan biosynthesis. This transient phosphoryl- the scaffold structure up to the GlcA-Xyl repeat. 12) ation is performed by FAM20B, and its deletion in Finally, we proposed that the scaffold glycan was 13) 15) mice results in embryonic lethality. As will be “GlcA-Xyl-Rbo5P-Rbo5P”. Rbo is a pentose alco- described later, the 6-phosphate of Man attached hol (pentitol) and has never been reported in to core M3 is required for the glycan chain elonga- mammalian glycans. No. 1] Mammalian O-mannosyl glycans 41 Ser/Thr Dol-P-Man POMT1/POMT2 core M3 Manα1 Ser/Thr GlcNAcβ1 4Manα1 Ser/Thr ER POMGNT2 Golgi B3GALNT2 core M1 POMGNT1 GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr GlcNAcβ1 2Manα1 Ser/Thr POMK core M2 GNT-IX (VB) PO GlcNAcβ1 Manα1 Ser/Thr GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr GlcNAcβ1 ER Golgi FKTN PO Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr FKRP PO Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr TMEM5 (RXYLT1) PO Xylβ1 4Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr B4GAT1 PO GlcAβ1 4Xylβ1 4Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr LARGE 3GlcAβ1 3Xylα1 n PO 3GlcAβ1 4Xylβ1 4Rbo5P 1Rbo5P 3GalNAcβ1 3GlcNAcβ1 4Manα1 Ser/Thr Fig. 1. Biosynthetic pathway of core M1, core M2, and core M3 O-Man glycans in the ER and Golgi. GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Man, mannose; GlcA, glucuronic acid; Rbo5P, ribitol-5-phosphate; Xyl, xylose; PO , phosphate; POMT1, protein O-mannosyltransferase 1; POMT2, protein O-mannosyltransferase 2; B3GALNT2, O-1,3-N-acetylgalactosaminyl- transferase 2; B4GAT1, O-1,4-glucuronosyltransferase 1; Dol-P-Man, dolichol-phosphate-mannose; FKTN, fukutin; FKRP, fukutin- related protein; GNT-IX(VB), O-1,6-N-acetylglucosaminyltransferase IX(VB); LARGE, acetylglucosaminyltransferase-like; POMGNT1, protein O-linked mannose O-1,2-N-acetylglucosaminyltransferase 1; POMGNT2, protein O-linked mannose O-1,4-N- acetylglucosaminyltransferase 2; POMK, protein O-mannose kinase; TMEM5 (RXYLT1), transmembrane protein 5 (Rbo5PO1,4- xylosyltransferase). In addition to ,-DG, several proteins have been Man-core and peripheral structures is present in shown to carry the core M1 and/or core M2 struc- mammals. The identification and characterization of tures, such as IgG2, phosphacan, CD24, neurofascin, enzymes involved in the biosynthesis of mammalian and lecticans. On the other hand, ,-DG is currently O-Man glycans are necessary to elucidate the the only known core M3-modified protein. function and regulation of the glycans. The biosynthesis of O-Man glycans begins with Biosynthesis the transfer of a Man residue from dolichol-phos- A series of O-Man glycans with heterogeneous phate-mannose (Dol-P-Man) to Ser/Thr residues of 42 T. ENDO [Vol. 95, certain proteins in the endoplasmic reticulum (ER) O-linked mannose O-1,4-N-acetylglucosaminyltrans- (Fig. 1). O-Mannosylation is essential for normal ferase 2), which transfers GlcNAc from a UDP- 21) development in Drosophila melanogaster, zebra- GlcNAc to the O-Man residue of ,-DG. B3GALNT2 22) 23) fish, and mice. Of note, both components, (O-1,3-N-acetylgalactosaminyltransferase 2) forms POMT1 (protein O-mannosyltransferase 1) and GalNAcO1-3GlcNAc by transferring GalNAc from POMT2 (protein O-mannosyltransferase 2), are UDP-GalNAc to the GlcNAc residue of the 24) necessary for O-mannosyltransferase activity. Re- POMGNT2 product. Then, the core M3 structure is cently, the presence of another protein in the O- phosphorylated at the 6-position of Man by POMK mannosylation machinery was suggested in addition (protein O-mannose kinase) in an ATP-dependent to the POMT1/POMT2 system. An O-mannosyla- manner and forms the phospho-core M3 structure tion pathway that selectively modifies cadherins GalNAcO1-3GlcNAcO1-4(phospho-6)Man in the ER. and protocadherins has been reported. According to POMK is regarded as a pseudokinase because it lacks 25) proteomics data, the initiation of the O-Man the functional motifs present in typical kinases, but glycosylation of cadherins and protocadherins does recent crystal structures have revealed the detailed not depend on the evolutionarily conserved POMT1/ mechanisms underlying POMK catalysis and sub- 5),33) POMT2 enzymes that initiate O-Man glycosylation strate recognition. Notably, POMGNT2 func- on ,-DG. Four TMTC (transmembrane and tetra- tions in the ER before glycosylation by POMGNT1 tricopeptide repeat containing) genes are predicted in the Golgi, and thus the site of ,-DG occupied by to encode distinct O-mannosyltransferases that core M3 should be determined by the substrate cooperatively mannosylate the common extracellular specificity of POMGNT2. A better understanding cadherin domains of cadherins and protocadherins, of the determinants of the site requires information suggesting the existence of another as yet undiscov- about the mechanism by which POMGNT2 recog- 26) ered O-Man glycosylation pathway. It is important nizes the peptide sequence and/or conformation to determine whether or not the TMTC products near the target O-Man. However, the details cur- actually exhibit enzymatic activity towards the rently remain unclear and information about the cadherin family. POMGNT2 structure will improve our understand- (1) Core M1 and core M2. After O-manno- ing of the site-recognition mechanism. sylation by POMT1/POMT2, POMGNT1 (protein Each glycosyltransferase responsible for synthe- O-linked mannose O-1,2-N-acetylglucosaminyltrans- sizing the extended complete core M3 structure has ferase 1) forms the GlcNAcO1-2Man (core M1) using been identified (Fig. 1). The first enzymes to be UDP-GlcNAc as a donor substrate in the Golgi identified that form the extended structure were the 27) (Fig. 1). The core M2 structure [GlcNAc O1- enzymes that synthesize the novel glycosaminogly- 6(GlcNAc O1-2)Man] is formed sequentially through can-like GlcA-Xyl repeat, which is essential for the the actions of POMGNT1 and GNT-IX(GNT-VB) binding of ,-DG to laminin. In 2012, Inamori et al. [O-1,6-N-acetylglucosaminyltransferase IX(VB)], an found that LARGE exhibits both xylosyltransferase enzyme that catalyzes the formation of the and glucuronyltransferase activities and produces 28) GlcNAcO1-6Man linkage. Because GNT-IX(GNT- repeating units of GlcA-Xyl using UDP-GlcA and 29),30) 20) VB) is specifically expressed in the brain, O- UDP-Xyl as donor substrates. LARGE is an ,- Man glycans with the core M2 structure are exclu- dystroglycanopathy [congenital muscular dystrophy sively detected in the brain. Notably, the presence of 1D (MDC1D)] gene product. The molecular mechan- the HNK-1 glycan on the core M2 of phosphacan/ ism by which LARGE determines the length of the RPTPO is an important regulator of re-myelination GlcA-Xyl repeat synthesized remains unclear. 31) in the brain. Peripheral structures on core M1 and LARGE2, a LARGE paralog, also exhibits both core M2 are synthesized by a series of glycosyltrans- enzyme activities and synthesizes GlcA-Xyl repeats 34),35) ferases, such as galactosyltransferase, sialyltransfer- on ,-DG. Although LARGE is likely specific for ase, glucuronyltransferase, sulfotransferase, and ,-DG, LARGE2-dependent glycosylation also elon- ,1,3-fucosyltransferase in the Golgi. gates a glycosaminoglycan chain on proteoglycans 36) (2) Core M3. On the other hand, the core such as glypican-4. 32) M3 structure is synthesized in the ER (Fig. 1). We assumed that FKTN and FKRP might be After the initial O-Man transfer is catalyzed by candidate enzymes that synthesize the tandem the POMT1/POMT2 complex, the GlcNAcO1- Rbo5P structure because: 1) FKTN and FKRP are 4Man linkage is catalyzed by POMGNT2 (protein responsible for ,-dystroglycanopathy; 2) both pro- No. 1] Mammalian O-mannosyl glycans 43 Table 1. Summary of genes responsible for ,-dystroglycanopa- teins exhibit similarities to glycosyltransferases con- thies and their functions taining the DXD motif, which is conserved in many glycosyltransferases; 3) both belong to the nucleoti- Gene name Function dyltransferase fold protein superfamily, and 4) both Primary have similarities to enzymes involved in phosphor- DAG1 Dystroglycan ylcholine modification or the mannosyl-phosphoryla- Secondary tion of glycans in bacteria and yeast. Actually, as POMT1 Protein O-mannosyltransferase 15) shown in our previous study, FKTN transfers the POMT2 Protein O-mannosyltransferase first Rbo5P to the 3-position of GalNAc from CDP- Protein O-mannose O-1, 2-N-acetylglucosami- POMGNT1 Rbo and FKRP transfers the second Rbo5P to the nyltransferase 1-position of the first Rbo5P (Fig. 1). Previously, the FKTN Ribitol-5-phosphate transferase glycan was assumed to extend from the 6-position of FKRP Ribitol-5-phosphate transferase the core Man, but it actually extends from the 3-posi- O-1, 3-glucuronyltransferase and ,-1, 3-xylo- 15) LARGE tion of GalNAc, as confirmed by an NMR study. syltransferase The sequential actions of FKTN and FKRP pro- Protein O-mannose O-1, 4-N-acetylglucosami- POMGNT2 duce the “Rbo5P-1Rbo5P-3GalNAcO1-3GlcNAcO1- nyltransferase 4(phosphate-6)Man” structure. Notably, the 6-phos- B3GALNT2 O-1, 3-N-acetylgalactosaminyltransferase phate of Man in the phospho-core M3 peptide is B4GAT1 O-1, 4-glucuronyltransferase required for FKTN activity because the non-phos- POMK Protein O-mannose kinase phorylated core M3 peptide does not serve as an TMEM5 15) Ribitol-5-phosphate O-1, 4-xylosyltransferase acceptor. Notably, FKTN does not transfer the (RXYLT1) second Rbo5P and does not form the tandem Rbo5P Tertiary structure by itself. On the other hand, FKRP does GMPPB GDP-mannose pyrophosphorylase not transfer the first Rbo5P and does not form a DPM1 Dolichol-phosphate-mannose synthase Rbo5P trimer or more. Based on the data, the DPM2 Dolichol-phosphate-mannose synthase synthesis of the tandem Rbo5P unit is highly DPM3 Dolichol-phosphate-mannose synthase regulated by the strict substrate specificities of DOLK Dolichol kinase FKTN and FKRP, although both enzymes share ISPD CDP-ribitol synthetase homology. Information about the FKTN and FKRP structures will facilitate the elucidation of the substrate recognition mechanism. We and other groups have observed an addi- produced by TMEM5(RXYLT1) and LARGE may tional GlcAO1-4XylO1-4 unit linked to the tandem be important for the formation of the functional O- Rbo5P structure. As described above, this GlcAO1- Man glycan on ,-DG. 4XylO1-4 unit was not formed by LARGE. Two ,-Dystroglycanopathy groups independently reported that the GlcAO1-4 unit was formed by the actions of B4GAT1 (O-1,4- Muscular dystrophies are genetic diseases that glucuronosyltransferase 1), which shares homol- cause progressive muscle weakness and wasting. ogy with the glucuronyltransferase domain of According to recent data, aberrant O-mannosylation 37),38) 10) LARGE. As shown in our previous study, of ,-DG is the primary cause of some forms of TMEM5 acts as a ribitol O1,4-xylosyltransferase to congenital muscular dystrophy known as ,-dystro- generate a XylO1-4Rbo5P linkage, in which the glycanopathy. To date, eighteen genes have been second Rbo5P in the tandem structure serves as identified as causative factors in these diseases the acceptor for Xyl. TMEM5 has been proposed to (Table 1). ,-Dystroglycanopathies are classified as be renamed RXYLT1 (Rbo5PO-1,4-Xyl transferase). primary (caused by mutations in the gene encoding Furthermore, the TMEM5 product serves as an DG, DAG1), secondary (caused by mutations in the acceptor substrate for B4GAT1, but not for genes that directly modify the O-Man glycan of ,- 10) LARGE. Because the glucuronosyltransferase ac- DG), or tertiary (caused by the genes that indirectly tivity of LARGE is specific for ,-linked Xyl and not modify the O-Man glycan of ,-DG). O-linked Xyl, the formation of GlcAO1-4Xyl by This section will briefly describe a history of the B4GAT1 is required for the LARGE reaction to identification of the traits of ,-dystroglycanopathy. form GlcA-Xyl repeats. The different Xyl linkages The best known and most common type of muscular 44 T. ENDO [Vol. 95, dystrophy is Duchenne-type muscular dystrophy, the predominant form of ,-dystroglycanopathy in and its causative gene, dystrophin, was identified in Japan, and FKRP-deficient MDC1C and LGMD2I 1987. Dystrophin encodes an actin-binding cytoske- are the most frequent forms of ,-dystroglycanopathy letal protein that is present inside the muscle in the USA and Europe. We revealed that the first membrane and is similar to spectrin. Later, many three gene products were glycosyltransferases them- proteins, including glycoproteins, form a large com- selves, but the functions of the remaining three plex called DGC. The function of DGC is thought to gene products were unclear until recently. FKTN 15) connect extracellular matrix components and intra- and FKRP are Rbo5P transferases and LARGE 35) cellular components with the actin cytoskeleton. is a xylosyltransferase and glucuronyltransferase, Notably, several components of the DGC have been whose mutations were identified to cause ,-dystro- identified as causative agents for different type glycanopathy. The mutations of all genes led to muscular dystrophies. DG is a component of the defects in O-Man glycan formation. Notably, not all DGC that is encoded by a single gene and is cleaved patients with WWS carry mutations in POMT1 or into two proteins, ,-DG and O-DG, by posttransla- POMT2. Furthermore, cohort studies of patients tional processing. with ,-dystroglycanopathy reported that two-thirds 44) (1) Primary ,-dystroglycanopathy. In a case of patients had no mutations in these six genes. study of primary ,-dystroglycanopathy, a mutation Clearly, other unidentified causative genes are (Thr192Met) in the N-terminal domain of ,-DG was present in these patients, and a molecular diagnosis identified in a patient with limb-girdle muscular of each patient is necessary to improve our under- 39) dystrophy and cognitive impairment. The muta- standing of ,-dystroglycanopathy because patients tion reduces the number of GlcA-Xyl repeats because with this syndrome exhibit an extremely broad this amino acid is required for the recognition by clinical spectrum of symptoms. The most severe form LARGE. This study reported the first case in which a is characterized by muscular dystrophy with struc- mutation in the DAG1 gene itself was shown to cause tural abnormalities in the brain and eye. The mildest muscular dystrophy via a defect in O-Man glycan form is limb-girdle muscular dystrophy without brain formation. Another patient with different mutations or eye involvement. Later, many previously uniden- 40) showed similar hypoglycosylation of ,-DG. tified ,-dystroglycanopathy causative genes were 45) (2) Secondary ,-dystroglycanopathy. We revealed through genetic analyses. As described identified and characterized the glycosyltransferases in the previous sections, recent studies have finally POMGNT1 and POMT1/POMT2. Mutations in revealed and characterized these causative gene POMGNT1 are responsible for muscle-eye-brain products involved in processing the entire core M3 disease (MEB), and mutations in POMT1/2 are glycan. Among them, the pathogenic role of the 6- responsible for Walker–Warburg syndrome (WWS). position of Man should be noted. It is phosphorylated 32) MEB and WWS are congenital muscular dystrophies by POMK, and POMK mutations cause ,- 45) characterized by brain malformations and structural dystroglycanopathy because the 6-phosphate of 15) abnormalities in the eye. At approximately the same Man is required for the FKTN activity. time, other groups reported abnormal glycosylation The entire structure of the core M3 glycan of ,-DG with substantial reductions in laminin- and its biosynthetic pathway have been identified. binding activity and IIH6 antibody reactivity in Mutations in enzymes involved in each glycosylation patients with MEB and WWS. Because the common step cause ,-dystroglycanopathy. However, a re- biochemical feature in MEB and WWS is abnormal maining question is that the GlcNAcO1-2 linkage is glycosylation of ,-DG, we proposed that MEB and lost in the proposed structure (bottom structure in WWS are glycan-deficient diseases. By the early Fig. 1). Approximately two decades ago, we re- 2000s, causative mutations in six genes (POMT1, ported that the POMGNT1 gene is responsible for 27) POMT2, POMGNT1, FKTN, FKRP, and LARGE) MEB. A selective deficiency in glycosylated ,-DG have been identified in patients with ,-dystroglycan- was observed in patients with MEB. MEB is opathy. Fukuyama-type congenital muscular dys- inherited as a loss-of-function of the POMGNT1 41) trophy (FCMD) results from mutations in FKTN. gene, and was one of the findings prompting the Congenital muscular dystrophy type 1C (MDC1C) development of this new research field, glycosylation and limb-girdle muscular dystrophy type 2I and muscular dystrophy. The lack of the core M1 (LGMD2I) are caused by a defect in fukutin-related structure (GlcNAcO1-2 elongation) is hypothesized 42),43) protein (FKRP), a homolog of FKTN. FCMD is to disturb the processing of the core M3 glycan in No. 1] Mammalian O-mannosyl glycans 45 (A) CDP-Rbo P 3 β3 P Man Man FKTN GalNAc GlcNAc RboP GalNAc GlcNAc β4 Catalytic Stem CBD DG DG POMGNT1 (B) CDP-Rbo P P P Man Man Man GalNAc GlcNAc GalNAc GlcNAc RboP GalNAc GlcNAc FKTN FKTN DG DG DG Catalytic Catalytic Stem CBD Stem CBD POMGNT1 POMGNT1 Man GlcNAc Man GlcNAc Man β2 UDP-GlcNAc Fig. 2. Proposed mechanisms underlying the efficient recruitment of FKTN to core M3 by the carbohydrate-binding domain (CBD) of POMGNT1. (A) Core M3 (GalNAcO1-3GlcNAcO1-4Man) binds to the CBD of POMGNT1. Because the POMGNT1 stem domain binds to core M3 via the CBD, FKTN is simultaneously recruited to the reaction site. (B) Core M1 (GlcNAcO1-2Man) binding to the CBD of POMGNT1. Glycosylated sites modified with core M3 are suggested to be located in close proximity to core M1. First, the FKTN-POMGNT1 complex forms core M1 (GlcNAcO1-2Man) on an O-linked Man near core M3 and then the complex binds this newly formed core M1 structure. In this situation, FKTN easily transfers Rbo5P to neighboring core M3 structures. GalNAc, N- acetylgalactosamine; GlcNAc, N-acetylglucosamine; Man, mannose; Rbo, ribitol; P, phosphate: DG, dystroglycan. the Golgi. We performed an X-ray crystallographic ensure the efficient synthesis of rare glycans in study of human POMGNT1 to answer this ques- mammals. In the case when the FKTN-POMGNT1 46) tion. complex binds core M3 (Fig. 2A), FKTN is simulta- 27),47) As shown in our previous study, neously recruited to reaction site. On the other hand, POMGNT1 is composed of a catalytic domain and in the case when the complex binds core M1, FKTN a stem domain, and the stem domain is unusually is recruited to the reaction site through a different longer than the same domain in other glycosyltrans- mechanism. Glycosylated sites modified with cor- ferases, but its function was unclear. As a result of eM3 in ,-DG include Thr317, Thr319, and Thr379, 46) the X-ray crystallographic study, we unexpectedly although other precise sites remain unclear. Accord- identified the presence of a carbohydrate-binding ing to the results from mass spectrometry-based domain (CBD) in the stem domain. After an in-depth analyses, the core M1 structure is located near the 15),18),19) investigation of the glycan binding specificity of this core M3 site. As shown in Fig. 2B, the domain, we found that it specifically recognizes the FKTN-POMGNT1 complex initially synthesizes GlcNAcO1-2Man and GalNAcO1-3GlcNAc linkages, GlcNAcO1-2Man on O-Man near core M3, and then corresponding to the core M1 and core M3 struc- the complex binds this formed core M1. Under this tures, respectively. Of note, the CBD did not bind to circumstance, FKTN easily transfers Rbo5P to any other types of glycans. In addition, we reported neighboring core M3 structures. Actually, we con- 10 years ago that FKTN and POMGNT1 formed a firmed that the sugar-binding activity of the CBD of complex in the Golgi, but its biological meaning POMGNT1 is necessary for the maturation of glycan 48) 46) was not determined at the time. Because the on the core M3 structure. Furthermore, the for- POMGNT1 stem domain binds to core M3 and mation of an FKTN, FKRP, and TMEM5 complex 18),49) core M1 via the CBD, FKTN may be recruited to was proposed. The formation of this multi- the reaction site to form a FKTN-POMGNT1 component complex may ensure the synthesis of complex, which leads to efficient Rbo5P transfer to tandem Rbo5P repeats and the subsequent efficient core M3 (Fig. 2). The formation of this complex may elongation of the core M3 glycan. 46 T. ENDO [Vol. 95, (3) Tertiary ,-dystroglycanopathy. Glyco- Rbo5P by the enzyme teichoic acid ribitol I (TarI). sylation is determined by the actions of many Notably, bacterial TarI shares homology with human glycosyltransferases that catalyze the transfer of a ISPD (isoprenoid synthase domain-containing), monosaccharide moiety from a nucleotide sugar which is known to be responsible for ,-dystroglycan- donor substrate to the acceptor substrate. In some opathy. According to data from our previous 15) cases, glycosyltransferases use a dolichol-pyrophos- study, human ISPD is a CDP-Rbo synthetase phate sugar instead of a nucleotide sugar. This (CDP-Rbo pyrophosphorylase) that synthesizes mechanism is used for the O-mannosylation of the CDP-Rbo from CTP and Rbo5P, and CDP-Rbo is ,-DG protein. POMT1/POMT2 catalyze the trans- required for the biosynthesis of functional O-Man fer of a Man residue from Dol-P-Man to Ser/Thr glycans. Shortly thereafter, two other groups inde- residues on certain proteins. Therefore, some defects pendently confirmed that human ISPD catalyzes 16),17) in glycosylation are not only mediated by glycosyl- CDP-Rbo synthesis. Thus, the amount of transferases but also by the presentation of nucleo- available CDP-Rbo affects the O-mannosylation of tide sugar (or dolichol-sugar). Thus, diseases caused ,-DG. Providing further support for this hypothesis, by mutations in these genes are categorized as the addition of CDP-Rbo restores the IIH6 epitope 15) tertiary ,-dystroglycanopathies. of ,-DG in ISPD knockout cells, suggesting that DPM1, DPM2, DPM3, DOLK, and GMPPB CDP-Rbo supplementation is a possible therapeutic encode proteins involved in the synthesis of Dol-P- strategy for ISPD-deficient patients. Furthermore, Man. Dol-P-Man is an essential Man donor that is dietary supplementation with Rbo is suggested to be required for mannosylation, including O-mannosyla- beneficial in mice with not only ISPD mutations but 55) tion, N-glycosylation, and C-mannosylation, as well also a FKRP mutation. 56) as the formation of glycosyl-phosphatidylinositol As shown in our recent study, CDP-glycerol anchors. DOLK (dolichol kinase) encodes the DOLK inhibits the RboP transfer activities of both FKTN activity responsible for the formation of Dol-P. A and FKRP, suggesting that CDP-glycerol inhibits combined N-glycosylation and O-mannosylation de- the synthesis of the functional O-Man glycan of ficiency has been observed in patients with a ,-DG by preventing the further elongation of the predominant presentation of dilated cardiomyopathy glycan chain. A remaining question is whether the 50) due to DOLK mutations has been observed. amount of CDP-glycerol is causative factor for GMPPB (GDP-mannose pyrophosphorylase B) cat- uncharacterized ,-dystroglycanopathy. alyzes the synthesis of GDP-Man from GTP and Glycosylation of Notch receptors regulates Man-1-P. GDP-Man is required for the synthesis ligand-induced Notch signaling in the cell-to-cell of Dol-P-Man. An O-mannosylation deficiency was communication system that is required for cell-fate 57) observed, but no evidence of abnormal N-glycosyla- decisions during development. Aberrant activation tion has been found in patients carrying mutant or inactivation of the Notch signaling pathway leads 51) GMPPB. Dol-P-Man is synthesized from Dol-P to human diseases, including many different cancers. and GDP-Man by the DPM (dolichol-phosphate- Notch receptors are known to be modulated by mannose) synthase complex, which comprises three unusual O-glycans: O-Fuc, O-Glc, and O-GlcNAc. subunits: DPM1, DPM2, and DPM3. Mutations in A notable glycan is O-Glc, the synthesis of which each component result in dystroglycanopathy-type is catalyzed by protein O-glucosyltransferase1 muscular dystrophy, but defects in N-glycosylation (POGLUT1). POGLUT1 is an enzyme that trans- 52)–54) levels differed. Because the DPM1, DPM2, fers Glc from UDP-Glc to a distinct Ser residue in DPM3, DOLK, and GMPPB proteins are all the epidermal growth factor (EGF)-like repeat. involved in the synthesis of Dol-P-Man, the amount Importantly, POGLUT1 is essential for Notch of available Dol-P-Man affects the O-mannosylation receptor function. Recently, a missense mutation of ,-DG. (Asp233Glu) in POGLUT1 was identified in a Rbo5P in a mammalian glycan component is patient showing an impairment in muscle develop- very unique and is incorporated by the sequential ment and hypoglycosylation of muscle ,-DG, but not actions by FKTN and FKRP using CDP-Rbo, as in fibroblasts. A detailed analysis revealed a clearly described above. However, the CDP-Rbo biosyn- different disorder from secondary ,-dystroglycanop- thetic pathway was unknown in mammals. In athy, suggesting that a pathomechanism for this form bacteria, this synthetic pathway has already been of muscular dystrophy is Notch signaling-dependent 58) elucidated. CDP-Rbo is synthesized from CTP and loss of satellite cells. Further studies are necessary No. 1] Mammalian O-mannosyl glycans 47 (α-Dystroglycanopathy) (α-Dystroglycanopathy + Sugars) (Normal) Sugars Laminin α-DG Basal lamina Membrane β β-DG Dystrophin Cytoplasm F-actin Muscle instability, destruction and necrosis Prevention of muscle instability Fig. 3. ,-Dystroglycanopathies caused by a defect in the glycosylation of ,-DG and possible new glycotherapeutic strategies for these diseases. ,-DG is an extracellular peripheral membrane glycoprotein that is anchored to the cell membrane by binding to a transmembrane glycoprotein, O-DG. ,-DG-O-DG is thought to stabilize the plasma membrane by acting as an axis through which the extracellular matrix is tightly linked to the cytoskeleton. In addition, the cytoplasmic domain of O-DG interacts with dystrophin, which subsequently binds to the F-actin cytoskeleton. ,-DG is heavily glycosylated, and its sugars play a role in binding to laminin. Abnormal glycosylation of ,-DG causes ,-dystroglycanopathies (see Table 1). New glycotherapeutic strategies for ,- dystroglycanopathies that induce the interaction laminin and ,-DG include glycan supplementation. ,-DG, ,-dystroglycan; O- DG, O-dystroglycan. to determine the detailed mechanism using data novel structure in mammals. However, the findings from other patients. Similar to O-Glc, O-Fuc and O- produce additional questions. For example, are any GlcNAc modifications also occur at specific positions other protein(s) modified with the core M3 glycan? within an EGF repeat if the appropriate O-glyco- Do proteins other than ,-DG contain the Rbo5P 59) sylation consensus sequence is present, suggesting modification? What is the biological meaning of a mechanism for fine-tuning the Notch signaling Rbo5P in mammalian glycosylation? How is Rbo5P pathway by O-glycan that is probably related to synthesized and degraded in mammals? Additionally, muscle formation. further studies are needed to clarify the distribution The transporters of nucleotide-sugars into the of O-Man glycans in various tissues and to examine Golgi are critical for the glycosylation of glycoconju- their changes during development and in response to gates, and mutations in the transporters may cause a pathological conditions. These questions should be group of genetic disorders named congenital disorders addressed in the future. A major challenge will be to of glycosylation. A missense mutation (Gln101His) in integrate the forthcoming structural, cell biological, one of these transporters, SLC35A1, was identified and genetic information to understand how ,-DG in a patient with intellectual disability, seizures, O-mannosylation contributes to muscular dystrophy ataxia, macrothrombocytopenia, and bleeding dia- and brain development. thesis, and the mutation displayed a reduction in The hypoglycosylation of ,-DG in muscle 60) the sialylation of N- and O-glycosylated glycans. substantially reduces its affinity for a number of Of note, SLC35A1-deficient cells showed a lack of extracellular ligands. Based on this finding, the ,-DG O-mannosylation with a concomitant reduc- defective glycosylation of ,-DG is reasonably ex- 61) tion in sialylation. The results indicate a role for plained to cause muscle cell degeneration and SLC35A1 in ,-DG O-mannosylation that is distinct abnormal brain structures in patients with ,- from sialic acid metabolism. In addition, SLC35A1- dystroglycanopathies. In other words, interference deficient patients present a combined disorder of with the glycosylation of ,-DG may lead to a ,-DG O-mannosylation and sialylation, which is a combination of muscle and brain phenotypes in novel variant of the tertiary ,-dystroglycanopathies. patients with these diseases. Because ,-DG hypo- glycosylation is a common feature of ,-dystroglyca- Future directions nopathies, ,-DG may be a potential target of new The O-Man glycan moiety including Rbo5P is a glycotherapeutic strategies for these diseases (Fig. 3). 48 T. ENDO [Vol. 95, ology 24, 314–324. The formation of the interaction between laminin 9) Yaji, S., Manya, H., Nakagawa, N., Takematsu, H., and ,-DG induced by glycan supplementation may Endo, T., Kannagi, R. et al. (2015) Major glycan improve the symptoms of ,-dystroglycanopathy in structure underlying expression of the Lewis X patients who carry mutations in any enzymes of the epitope in the developing brain is O-mannose- glycan processing pathway. linked glycans on phosphacan/RPTPO. Glycobiol- ogy 25, 376–385. Muscular dystrophy is a group of genetic 10) Manya, H., Yamaguchi, Y., Kanagawa, M., disorders characterized by progressive muscle weak- Kobayashi, K., Tajiri, M., Akasaka-Manya, K. ness and degeneration. Unfortunately, an effective et al. (2016) The muscular dystrophy gene treatment for the disease is not available. I hope our TMEM5 encodes a ribitol O1,4-xylosyltransferase findings will provide new opportunities for the required for the functional glycosylation of dystro- glycan. J. Biol. Chem. 291, 24618–24627. development of treatments for these diseases in the 11) Braulke, T. and Bonifacino, J.S. (2009) Sorting of future. lysosomal proteins. Biochim. Biophys. Acta 1793, 605–614. 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(2009) Deficiency of Dol-P-Man synthase subunit DPM3 bridges the congenital disorders of glycosylation (Received Oct. 5, 2018; accepted Nov. 5, 2018) with the dystroglycanopathies. Am. J. Hum. No. 1] Mammalian O-mannosyl glycans 51 Profile Tamao Endo was born in Asahi-city, Chiba in 1954, and graduated from the Faculty of Pharmaceutical Sciences, The University of Tokyo in 1977 and obtained his Ph.D. at the same institution in 1982. He was a postdoctoral fellow at the Baylor College of Medicine, and a research associate in the Institute of Medical Science, The University of Tokyo. Since 1994, he has been the head of Department of Molecular Glycobiology, Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology (TMIG). He has served as the vice-director of TMIG since 2012. He served as the president of The Japanese Society of Carbohydrate Research (JSCR) and does so currently for the Japan Consortium for Glycobiology and Glycotechnology (JCGG). He is a member of the Science Council of Japan. He is now focusing on glycobiology in aging and diseases. He was honored to receive the Tokyo Metropolitan Governor’s Award (2002), The Pharmaceutical Society of Japan Award for Divisional Scientific Promotion (2003), The Asahi Award (2007), and The Japan Academy Prize (2017).

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Proceedings of the Japan Academy. Series B, Physical and Biological SciencesPubmed Central

Published: Jan 11, 2019

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