Abstract: Many glycosidases, which work as useful reagents for the studies of structures andfunctions of free and conjugated oligosaccharides, have been found and thoroughly purified. Theseenzymes are classified into exo- and endoglycosidases by their glycon specificities. Their usefulnessand limits as reagents are explained in detail in this review.
Endoglycosidases, which were originally found in the culture fluid of bacteria and in theextracts of plants, are now widely found in the mammals including humans. The physiological rolesof these enzymes are discussed in relation to the oligosaccharides accumulated in the urine ofpatients with exoglycosidase deficiencies. Furthermore, PNGase is found to play important roles inthe ER-associated degradation pathway of glycoproteins.
Recent studies of the glycosidases in Bifidobacteria have revealed that GNB/LNB pathway,which uniquely exist in this bacteria, works for the expression of Bifidus factor activity of humanmilk oligosaccharides, an important topic in the baby nutrition. This interesting field will beintroduced in detail in one section of this article.
Enzymes, which hydrolyze sugar chains arecalled glycosidases. The topics related to glycosidasesare very wide. For example, various glycosidasesobtained from microorganisms, and recombinantglycosidases are used as important industrial re-agents to produce monosaccharides from varioushomoglycans. Furthermore, applications of glycosi-dases as important industrial reagents in the fieldsof food, pharmaceutical, and paper manufacturingindustries are well known.
In this review article, however, I would like tolimit the subjects to the glycosidases, which havebeen used as useful reagents for the structural studies
and functional studies of sugar chains. In addition,the physiological roles of these enzymes in theproducing cells will also be discussed.
1. Glycosidases used as reagents for thestructural studies of sugar chains
1-1. Exoglycosidases. Exoglycosidases are theenzymes, which release particular monosaccharidesfrom non-reducing termini of oligosaccharides andthe sugar chains of glycoproteins and glycolipids. Thegeneral structure of ,-L-fucopyranoside is shownin Fig. 1. Exoglycosidases, which hydrolyze thiscompound into L-fucose and ROH, are called ,-L-fucosidases. In this article, discrimination of D- andL- of monosaccharides are ignored, and the enzymeswill be simply named ,-fucosidases.
,-Fucosidases work on ,-fucopyranosides, butnot on its anomeric isomers: O-fucopyranosides, andon the other glycosides having different monosac-charides.
As described so far, exoglycosidases usually showstrong substrate specificities to both monosaccharidemoiety and its anomeric structure. Therefore, theyare named by the monosaccharide and its anomeric
*1 Scientific advisor, The Noguchi Institute, Tokyo, Japan.† Correspondence should be addressed: A. Kobata, The
Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan(e-mail: [email protected]).
structure, which they hydrolyze, like ,-fucosidase, orO-galactosidase etc. This substrate specificity is calledglycon specificity. By using the substrate specific-ities of exoglycosidases, we can easily determine themonosaccharide located at the non-reducing terminiof a sugar chain including its anomeric configuration.
Furthermore, monosaccharide sequence of asugar chain can be easily determined, by sequentialexoglycosidases digestion, since exoglycosidases re-lease monosaccharides only from the non-reducingtermini of a sugar chain.
For example, the sugar chains, which have thetetrasaccharide group: GalO1-4GlcNAcO1-2Man,1-3ManO1—at their non-reducing termini, can bedegraded to the ManO1—group, only when theyare sequentially digested with O-galactosidase, O-N-acetylglucosaminidase and ,-mannosidase, by releas-ing one mole each of galactose, N-acetylglucosamineand mannose.
How many monosaccharides are released ateach step of exoglycosidase digestion can easily bedetermined by using Bio-Gel P-4 column chromatog-raphy.1)
As an example, analytical data of a biantennarycomplex-type sugar chain obtained from humanserum transferrin by hydrazinolysis followed byNaB3H4 reduction are shown in Fig. 2.
1-2. Glycon and aglycon specificities ofexoglycosidases. Glycon specificities of someexoglycosidases are not so strict as others. Forexample, the O-glucosidase, purified from Almondemulsin, hydrolyzes not only O-glucopyranosyl link-age but also O-galactopyranosyl linkage.2) Many O-N-acetylglucosaminidases hydrolyze O-N-acetylgalacto-saminyl linkages also, and are called O-N-acetylhex-osaminidases. Namely, these enzymes do not recog-nize the epimeric difference at the C-4 position ofthe substrates. However, enzymes, which show suchwide glycon specificities, are rather rare, and mostexoglycosidases hydrolyze only one monosaccharidelinkage. Now, I would like to describe about the
aglycon specificity of glycosidases. This specificityis related to the structure of R in Fig. 1.
Many ,-fucosidases were found from varioussources. Among them, the enzymes purified from thehepatopancreas of marine gastropods can hydrolyzeall ,-fucopyranosyl derivatives having various Rgroups.3) In contrast, the ,-fucosidase purified fromthe culture fluid of Bacillus fulminans hydrolyzes theFuc,1-2Gal group but no other disaccharides havingFuc,1- residue at their non-reducing termini.4) Two,-fucosidases were purified from Almond emulsion.5)
Among them, ,-fucosidase I hydrolyzes the ,-fucosylresidues of the GalO1-3(Fuc,1-4)GlcNAc group andthe GalO1-4(Fuc,1-3)GlcNAc group, but cannothydrolyze other ,-fucosyl linkages.6) Accordingly,not only the number of ,-fucosyl residues at the non-reducing termini of a sugar chain, but their bindingsites in the sugar chain can be determined by usingthe three ,-fucosidases. Quite recently, Sakuramaet al.7) elucidated the structural basis of differences inthe aglycon specificities of two ,-fucosidases purifiedfrom Bacteroides thetaiotaomicron. Hopefully, thiswill open the door to elucidate the structural basisof differences of aglycon specificities of other ,-fucosidases in the future.
Similar aglycon specificities were also found inother exoglycosidases, and effectively used for thestructural analysis of sugar chains. For example,many O-galactosidases hydrolyze all disaccharidescontaining GalO1- residue at their non-reducingtermini, but cannot remove the galactose residue ofthe GalO1-3(Fuc,1-4)GlcNAc group and the GalO1-4(Fuc,1-3)GlcNAc group. O-Galactosidase purifiedfrom the culture fluid of Diplococcal pneumoniae canhydrolyze the GalO1-4GlcNAc group but not theGalO1-2GlcNAc, the GalO1-3GlcNAc and the GalO1-6GlcNAc groups.8)
A more complicated result was obtained by thestudy of O-galactosidase purified from Jack bean.The enzyme hydrolyzes the GalO1-4GlcNAc group50 times faster than the GalO1-3GlcNAc group atlow enzyme concentration. However, at high enzymeconcentration, the enzyme hydrolyzes both groupsat almost the same speed.9)
Many ,-mannosidases hydrolyze all ,-mannosyllinkages. However, the enzyme purified fromAspergillus saitoi hydrolyzes the Man,1-2Man grouponly, and cannot hydrolyze the Man,1-3Man and theMan,1-6Man groups.10) Accordingly, this enzymehas been effectively used to characterize the highmannose-type sugar chains, which contain largeamount of the Man,1-2Man groups. It was also
O
HO
OH
GLYCON AGLYCON
HOCH3
O R
Fig. 1. General structure of ,-L-fucopyranoside.
A. KOBATA [Vol. 89,98
known that the ,-mannosidase purified from Jackbean hydrolyzes the Man,1-6Man group and theMan,1-2Man group at almost the same rate, buthydrolyzes the Man,1-3Man group at only 1/15speed.
Different aglycon specificities were also foundin the sialidases purified from various sources.The enzymes purified from Clostridium perfringensand from Arthrobacter ureafaciens hydrolyzesthe Neu5Ac,2-3Gal, the Neu5Ac,2-6Gal and theNeu5Ac,2-6GalNAc groups at almost the samerates. In contrast, the enzyme purified from Vibriocholera hydrolyzes these three groups at the rates of11:4:1, and the enzyme from influenza virus cleavesonly the Neu5Ac,2-3Gal group (Takasaki, S. andKobata, A. unpublished data).
The most prominent aglycon specificity wasfound by the study of O-N-acetylhexosaminidase
purified from Diplococcus pneumoniae,11) and usedfor the structural studies of complex-type and hybrid-type sugar chains. This enzyme hydrolytically cleavesthe GlcNAcO1-2Man, the GlcNAcO1-3Man and theGlcNAcO1-6Gal groups, but cannot hydrolyse theGlcNAcO1- residues shown in bold letters in Fig. 3.Namely, bisecting GlcNAc is resistant to the enzy-matic digestion, and also inhibits the hydrolysis of theGlcNAcO1-2 residue linked to the Man,1-6 arm.Furthermore, the GlcNAcO1-6Man group is resistantto the enzyme digestion and also inhibits thehydrolysis of the GlcNAcO1-2Man group on the samemannose residue. As described already, more delicatestructural study of the sugar chains can be performedby using the aglycon specificities of exoglycosidases.
As an example of such study, structural studyof a mixture of two octasaccharides12) obtained fromhuman milk will be introduced.
2Gal
2GlcNAc
2Man
Man
GlcNAc
300 400 500
Elution Volume (ml)
Rad
ioac
tivi
ty
Fig. 2. Sequential exoglycosidases digestion analysis of the tritium labeled biantennary nonasaccharide obtained from human serumtransferrin by hydrazinolysis. In the right figure, radioactive oligosaccharide at each step of exoglycosidase digestion was analyzed byBio-Gel P-4 column chromatography. Arrows at the top of the figure are the elution positions of glucose oligomers (numbers indicatethose of glucose units). O-Gal’ase, O-galactosidase; O-HexNAc’ase, O-N-acetylhexosaminidase; ,-Man’ase, ,-mannosidase; O-Man’ase,O-mannosidase.
Glycosidases as tools and their physiological rolesNo. 3] 99
By combining the sequential exoglycosidasedigestion and methylation analysis, this octasacchar-ides fraction was found to be a mixture of oligosac-charides shown in Fig. 4A. When this oligosaccha-rides mixture was incubated with Almond ,-fucosidase I, only the fucose residues close to thenon-reducing termini were removed and the GalO1-3(4)GlcNAcO1- portions were exposed. When themixture of these heptasaccharides was digested witha mixture of diplococcal O-galactosidase and Jackbean O-N-acetylhexosaminidase, approximately 30%of the oligosaccharides were converted to pentasac-charides and remaining 70% stayed as heptasaccha-rides. When the mixture of heptasaccharides wasdigested with a mixture of Jack bean O-galactosidaseand Jack bean O-N-acetylhexosaminidase, all oligo-saccharides were converted to a mixture of penta-saccharides. When this pentasaccharides mixture wasdigested with Almond ,-fucosidase, it was convertedto tetrasaccharide. This tetrasaccharide was com-pletely converted to GlcNAcO1-3GalO1-4Glc bydigestion with diplococcal O-galactosidase. Becausediplococcal O-galactosidase cleaves the GalO1-4GlcNAc group but not the GalO1-3GlcNAc group,the tetrasaccharides fraction is concluded to be apure lacto-N-neotetraose. Based on this information,the original octasaccharides fraction was found tobe a mixture of the octasaccharides constructed byaddition of the GalO1-3(Fuc,1-4)GlcNAcO1- groupand the GalO1-4(Fuc,1-3)GlcNAcO1- group atthe C-3 position of the non-reducing terminalgalactose of lacto-N-fucopentaose III: GalO1-
4(Fuc,1-3)GlcNAcO1-3GalO1-4Glc in 7 to 3 molarratio.
Usually, exoglycosidase activities were measuredby using p-nitrophenyl glycosides as substrates.However, some of the enzymes purified by using theartificial substrates are not useful for the structuralstudies of the sugar chains because they do not workwell on the natural sugar chains. Furthermore, manyexoglycosidases, which have high aglycon specificitiesand very useful for the structural study of sugarchains, cannot be detected because they do nothydrolyze p-nitrophenylglycosides. Therefore, onemust be careful on such evidences in searching for anew useful exoglycosidases.
1-3. Endoglycosidases. As described already,exoglycosidases hydrolytically cleave the monosac-charide residues located at the non-reducing terminiof sugar chains. Many enzymes, which hydrolyzeproteoglycans and various homoglycans, were knownto hydrolyze the inner part of the sugar chains.Namely the glycon specificities of these enzymes arenot directed to monosaccharides but to larger partsof sugar chains.
Since early 1970th, such group of enzymes,which work on the N-linked and O-linked sugarchains of glycoproteins, have been found andeffectively used for the structural studies of sugarchains of complex carbohydrates. Since these en-zymes cleave the inner part of sugar chains, they aregenerically called endoglycosidases. In Table 1, endo-glycosidases that work on N-linked sugar chains andO-linked sugar chains are summarized. In order touse these enzymes as the effective reagents for thestructural studies of the sugar chains of glycoproteins,their exact substrate specificities must be elucidated.
The detailed substrate specificities of Endo D,Endo H, Endo CII, diplococcal endo-,-N-acetylga-lactosaminidase, and four endo-O-galactosidases fromdifferent origins have been elucidated as summarizedin Table 2 and Table 3. Namely, these enzymes haveglycon specificities recognizing from disaccharide topentasaccharide. It was also found that Endo CI hasthe same substrate specificity as Endo D.16)
1-3-1. Endo-O-N-acetylglucosaminidases and theirapplication to structural study of glycopeptides. Becauseof its unique substrate specificity, Endo H could re-lease all high mannose-type sugar chains and hybrid-type sugar chains, and used as a very effective re-agent for the structural studies of these sugar chains.Since none of the four endo-O-N-acetylglucosamini-dases could act on the complex-type sugar chains,which are the largest population of the N-linked
Fig. 3. The GlcNAcO1- residues (bold letters), which areresistant to diplococcal O-N-acetyl hexosaminidase digestion.
A. KOBATA [Vol. 89,100
sugar chains, structural studies of these sugar chainswere mainly performed by using hydrazinolysis30) atthe early stages of N-linked sugar chains research.
Many endo-O-N-acetylglucosaminidases werefound from various sources later.31),32) However,they all have similar substrate specificities, and noneof them acts on the complex-type sugar chains. Aninteresting enzyme called Endo A was purified fromArthrobacter protophormiae.26) This enzyme, whichis induced by adding glycopeptides containing highmannose-type sugar chains in the culture medium,shows the same substrate specificity as Endo CII,and shows strong transglycosylation activity togetherwith hydrolytic activity.33) The unique character ofthis enzyme will open the gate of production ofneoglycoproteins as will be discussed at the end ofthis review article.
In 1982, Elder and Alexander found a new endo-O-N-acetylglucosaminidase in the culture fluid ofFlavobacterium meningosepticum, and named itEndo F.24) Interesting evidence is that this enzymecleaves the complex-type sugar chains together withthe high mannose-type sugar chains. However, theusefulness of the enzyme was not established becausedetailed substrate specificity was not investigated.
In 1990, Kadowaki et al. found an endo-O-N-acetylglucosaminidase, which acts on the complex-type sugar chains, in Mucor hiemalis and named itEndo M.25) They then comparatively investigated indetail the substrate specificities of Endo M and EndoF.34) By using DNS (5-dimethylaminonaphthalene-1-sulfonyl) derivatives of Asn-oligosaccharides as sub-strates, they found that both enzymes showed thesame activities to Asn-oligosaccharides obtained from
A
B
Fig. 4. Structural elucidation of an octa-saccharides mixture obtained from human milk. O-Gal’ase, O-galactosidase; O-HexNAc’ase,O-N-acetylhexosaminidase.
Glycosidases as tools and their physiological rolesNo. 3] 101
ovalbumin, indicating that both enzymes act on thehigh mannose-type and the hybrid-type sugar chainsalmost at the same rates. In contrast, Endo M acts onthe biantennary complex-type sugar chains muchfaster than Endo F. Furthermore, Endo M can acton the triantennary complex-type sugar chains, butEndo F cannot act on them at all. Accordingly, EndoM is much more useful for studying the complex-typesugar chains than Endo F.
1-3-2. Endo-,-N-acetylgalactosaminidase. In1972, Huang and Aminoff reported that the crudeexoglycosidase mixture obtained from the culturefluid of Clostridium perfringens releases a disaccha-ride: GalO1-3GalNAc from pig submaxillary mucin.35)
However, this enzyme had not been purified, and itsspecificity was not elucidated. A similar enzyme wasfound in the culture fluid of Diplococcus pneumoniae,and was purified 180 fold by using a humanerythrocyte glycoprotein, glycophorin, containingthe [3H]GalO1-3GalNAc residues, as substrate.21)
This enzyme releases the GalO1-3GalNAc groups,which are linked to either serine or threonine residuesof glycoproteins. However, R,1-Ser and Thr groups,in which R represent the Fuc,1-2GalO1-3GalNAc,
the GalO1-3(NeuGly,2-6)GalNAc and the Fuc,1-2GalO1-3(NeuGly,2-6)GalNAc groups, do not workas substrate of the enzyme indicating that substitu-tion of any hydroxyl groups of the GalO1-3GalNAcgroup by other sugars abolished the substrateactivity of the disaccharide to the enzyme.
The enzyme was effectively used to determinethe structure of the serine and threonine-linked sugarchains of a glycopeptide released from bovine plasmahigh-molecular-weight kininogen by the action ofplasma kallikrein.36)
1-3-3. Endo-O-galactosidases and their applicationto the studies of the sugar chains of complex carbohy-drates. A very interesting endoglycosidase was foundin the culture fluid of Diplococcus pneumoniae.23)
This enzyme releases the GalNAc,1-3(Fuc,1-2)Galgroup and the Gal,1-3(Fuc,1-2)Gal group fromblood type A and B substances, respectively, but doesnot act on blood type H substance. Since the enzymecleaves O-galactosyl linkages, it was classified asan endo-O-galactosidase. The enzyme also acts onoligosaccharides with blood type A and B determi-nants. After 1000-fold purification, action of theenzyme on various oligosaccharides was investigated,
Table 1. Endoglycosidases acting on the sugar chains of complex carbohydrates
Names Sources References
Endo-O-N-acetylglucosaminidase
D (Endo D) Diplococcus pneumoniae 13
H (Endo H) Streptomyces griseus 14
L (Endo L) Streptomyces plicatus 15
CI and CII (Endo CI and CII) Clostridium perfringens 16
F (Endo F) Flavobacterium meningosepticum 24
M (Endo M) Mucor hiemalis 25
A (Endo A) Arthrobacter protophormiae 26
Fig 17, 18
Mammalian organs 19
Hen oviduct 20
Endo-,-N-acetylgalactosaminidase
Diplococcus pneumoniae 21, 22
Endo-O-galactosidase
Diplococcus pneumoniae 23
Escherichia freundii 27
C Clostridium perfringens 28
Endo-O-GalGnGa Clostridium perfringens 29
Peptide:N-glycanase (PNGase)
Almond emulsin 50
Flavobacterium meningosepticum 51
Animal tissues 56, 57
A. KOBATA [Vol. 89,102
and finally concluded that the enzyme has thesubstrate specificity shown in Table 3-2. Severalexoglycosidases, which destroy the activities of bloodgroup antigens, were found and had been called bloodgroup-destroying enzymes.37)–41) However, these en-
zymes were either ,-galactosidases or ,-N-acetylga-lactosaminidases, which also hydrolyze other ,-galactosyl or ,-N-acetylgalactosaminyl linkages thanblood group determinants. In contrast, the diplococ-cal endo-O-galactosidase hydrolyzes only blood group
Table 2. The substrate specificities of Endo D,53),54) Endo H,53) and Endo CII.55) R represent H, monosaccharides, or sugar chains
Endo enzymes Structures of substrates
D NH-C-CH2-CH
=
O
C=O
NHO
CH2OH
NHAc
OCH2OR
NHAcO
OCH2OH
OCH2OR
O O
OH HO
OHHO
OH
RO
HO
HO
NH-C-CH2-CH
=
O
C=O
NHO
CH2OH
NHAc
OCH2OH
NHAc
OCH2
O ORO
ORRO
OO
CH2OH
OCH2OR
HO
HOHO
OH OHOH
HO
CII
RO
O
NH-C-CH2-CH
=
O
C=O
NHO
CH2OH
NHAc
OCH2OH
NHAc
OCH2
O ORO
ROO
OCH2OH
OCH2OR
OO
CH2OH OH OHHO
HO
OHHO
OHHO
HO
Table 3. The substrate specificities of 1, endo-,-N-acetylgalactosaminidase;21) 2, Diplococcal endo-O-galactosidase;23) 3, Escherichiafreundii endo-O-galactosidase;27),45) 4, endo-O-galactosidase C;28) and 5, endo-O-GalGnGa.29) R1 represents either –OH, or –NHAc, R2
represents either H, or SO4, and R3 represents either –H or –CH3. R represents either H, O-galactosyl residue or sugar chains withO-galactosyl residue at their reducing termini
Enzymes Structures of substrates Enzymes Structures of substrares
1 CH-CHC=O
NHR3
CH2OHO
O
OH
HO
O
CH2OHO
NHAc
HO
OH3
O
CH2OR2O
ORRO
O OH
R1
CH2OHO
CH2OHO
OH
HOO
NHAc
2 OO
CH3
CH2OHO
CH2OHO
O
R1
O
CH2OHO
HO
HO
OH OH
HO
HO
OH
R1
O
4 O
CH2OHO
OH
HO
OH
CH2OHO
CH2OHO
OH
HO
OHO
R1
O
5
CH2OHO
CH2OHO
OH
OHO
R1
O
OHOH
CH2OHO
NHAc
HOO
Glycosidases as tools and their physiological rolesNo. 3] 103
A and B determinants and can be called a true bloodgroup-destroying enzyme.
The enzyme was successfully used to character-ize the ABO blood group-active glycoproteins ofhuman erythrocyte membrane,42) and to characterizethe ABO blood group determinants with branchedcore.43) Another endo-O-galactosidase with quitedifferent specificity was found in the culture fluidof Escherichia freundii by Kitamikado and Ueno.44)
This enzyme releases the Neu5Ac-Gal-GlcNAc(SO4)-Gal group from purified keratosulfate preparations.Fukuda and Matsumura studied the action spectrumof this enzyme on various glycoproteins and oligosac-charides, and found that the enzyme releases theGlcNAcO1-3Gal and the GlcNAc(6-SO4)O1-3Galgroups from a mucin isolated from pig, and glucosefrom GalO1-3GlcNAcO1-3GalO1-4Glc.27) The exactsubstrate specificity of this enzyme was elucidated bystudying its action on various glycolipids.45) Theenzyme showed no action on the glycosphingolipidsof globo and ganglio series. However, it cleaves allthe glycolipid with the R-GlcNAcO1-3GalO1-4Glc(or GlcNAc-) groups in which R represents eitherhydrogen or sugars. Further substitution of thegalactose residue in the essential trisaccharide groupby additional sugars greatly reduces the susceptibil-ity of the sugar chains to the enzyme. Anotherinteresting characteristic of this enzyme is that thepresence of a sialyl residue in the R moiety enhancesthe hydrolyzability of the sugar chains. This is veryunusual because sialyl substitution of sugar chainsusually reduces their susceptibility to most of theexo- and endoglycosidases. Possibly, the endo-O-galactosidase has cationic peptide group close to thecatalytic polypeptide moiety.
In 1987, Muramatsu’s group reported the findingof a novel endo-O-galactosidase, which releases theGal,1-3Gal group from the carbohydrate moietiesof complex carbohydrates, in the culture fluid ofClostridium perfringens, and named it as endo-O-galactosidase C.28) After cloning the enzyme gene,46)
they performed a series of studies to apply the genefor producing the Gal,1-3Gal group free mice as amodel to produce the animal suitable for nonprimate-to-primate xenotranplantation.47)–49)
In 2001, Ashida et al. found another endo-O-galactosidase, which releases the GlcNAc,1-4Galgroup from glycans expressed in the gastric glandmucous cell-type mucin, as a contaminant ofcommercial Clostridium perfringens sialidase. Theypurified the enzyme in electrophoretically homoge-neous form from the culture supernatant of Clostri-
dium perfringens, and named it as GlcNAc,1-4Gal-releasing endo-O-galactosidase (Endo-O-GalGnGa).29)
1-3-4. Peptide:N-glycanase. Peptide:N-glycanase(PNGase), another group of endoglycosidase actingon the N-linked sugar chains, cleaves the amide bondof the GlcNAcO1-Asn group located at the linkageregion of N-linked sugar chains to the polypeptideportion as shown in Fig. 5. The enzyme was firstextracted and purified from Almond emulsion byTakahashi in 1977.50) Similar enzyme was later foundin the culture fluid of Flavobacterium meningosepti-cum by Plummer et al.,51) and has been used for thestudies of structures and functions of N-linked sugarchains of glycoproteins.
2. Endoglycosidases found in the animal kingdomand their physiological functions
In 1974, Nishigaki et al. found an endo-O-N-acetylglucosaminidase in the organs of mammals.19)
Following this, Tarentino and Maley20) found similarenzyme in hen oviduct, indicating that endo-O-N-acetylglucosaminidases occur widely in the animalkingdom. In 1989, DeGasperi et al.52) found twoendo-O-N-acetylglucosaminidases in human kidneyand named them E-O-GNase 1 and 2. E-O-GNase 1has similar substrate specificity as Endo H, cleavingeffectively glycopeptides containing the high man-nose-type sugar chains but not those containing thecomplex-type sugar chains. Furthermore, acetylationor dansylation of the Asn residue of the substratesdoes not affect susceptibility to the enzyme. Since theGlcNAcO1-4GlcNAc moieties of Man,1-6(Man,1-
NH-C-CH2-CH
=
O
C=O
NHO
CH2OR
NHAc
O OH
=
O
C=O
NH
HOC-CH2-CH+O
CH2OR
NHAc
O OH
OH+NH3
Fig. 5. Mechanism of the release of N-linked sugar chains byPNGase action. R represents either H or Fuc,1-. Release ofammonia from glycosylamine occurs spontaneously.
A. KOBATA [Vol. 89,104
3)Man,1-6(Man,1-2Man,1-3)ManO1-4GlcNAcO1-4GlcNAc and ManO1-4GlcNAcO1-4GlcNAc were alsohydrolyzed by the enzyme, Asn residue is not strictlyrequired for the enzyme activity.
On the contrary, E-O-GNase 2 does not act onthe Asn-linked oligosaccharides at all, but hydrolyti-cally cleaves the GlcNAcO1-4GlcNAc moiety of thehigh mannose-type oligosaccharides and the com-plex-type oligosaccharides containing the N,NB-diace-tylchitobiose at their reducing termini. Among theseenzymes, E-O-GNase 1 is considered to be the sameas that we found in the rat organs,19) and probablydistributes widely in the mammalian organs. E-O-GNase 2 showed the same characteristics as thedi-N-acetylchitobiase reported by Kuranda andAronson.110) As to PNGase, Inoue’s group foundthat this enzyme widely occurs in animal tissues.56),57)
Tadashi Suzuki, who was one of the young scientistsin Inoue’s group, recently elucidated that PNGases inanimal and plant kingdoms are playing importantroles in the ER-associated degradation (ERAD)pathway.58)–60)
All N-linked sugar chains are added as thetetradecasaccharide: Glc3·Man9·GlcNAc2 to the Asnresidue constructing the Asn-X-Thr (or Ser) group ofthe polypeptide chain being translated in the roughendoplasmic reticulum, and then trimmed to theheptasaccharide: Man5GlcNAc2 until the polypeptidereaches to the Golgi. After addition of an N-acetylglucosamine residue to the hexasaccharide,the formed octasaccharide is further trimmed itstwo ,-mannosyl residues to form the hexasaccharide:Man,1-6(GlcNAcO1-2Man,1-3)ManO1-4GlcNAcO1-4GlcNAc by the Golgi resident ,-mannosidase. Thehexasaccharide is then converted to a series ofcomplex-type and hybrid-type sugar chains in Golgi.During this pathway, the N-linked sugar chain ofnascent glycoprotein is converted to GlcMan9Glc-NAc2 by the action of ,-glucosidases I and II, whilethe protein still exist in the endoplasmic reticulum.This dodecasaccharide plays important role as aligand of two chaperons: Calnexin and Calreticulin,which retain the polypeptide within ER for properfolding. When the protein happens to be miss-foldedby some reason, the protein will be released from ERto cytoplasm and degraded by proteasome. At thisstage, PNGase, which occurs in the cytoplasm, playsan important role in removing bulky N-linked sugarchains from a miss-folded nascent protein and helpsits entry into proteasome. The oligosaccharides, thusreleased from miss-folded nascent protein, have theGlcNAcO1-4GlcNAc group at their reducing termini.
As shown in Fig. 6C, one N-acetylglucosamineresidue is removed from these oligosaccharides byE-O-GNases 1 and 2 found by DeGasperi et al.52)
Action of E-O-GNase 2 is indispensable here, becauselarge amount of complex-type sugar chains contain-ing the GlcNAcO1-4GlcNAc group at their reducingtermini must be handled by this pathway as willbe described in the story of glycosidase deficiencieslater.
The oligosaccharides are then brought intolysosomes after being partially hydrolyzed by cyto-plasmic ,-mannosidase, and completely hydrolyzedinto monosaccharides by the action of lysosomalexoglycosidases.
As described so far, PNGase is considered toplay a main role in the removal of N-linked sugarchains in the catabolism of glycoproteins. However,there is one important problem we must have in ourmind. As described already, there occurs endo-O-N-acetylglucosaminidase (which correspond to E-O-GNase 1 of human kidney) in the cytoplasm ofcells of animal organs. If this enzyme acts onthe glycoproteins within the animal body, variousproteins with N-acetylglucosamine residues on theirAsn residues would be produced as shown in Fig. 6B.However, PNGase cannot release these N-acetylglu-cosamine residues.61) Therefore, biological actionsof such modified glycoproteins are the interestingtargets for investigation.
Very useful data for considering the metabolismof the N-linked sugar chains of glycoproteins areobtained by the structural studies of oligosaccharidesexcreted in the urine of patients with glycosidasedeficiency. Among the hereditary metabolic diseases,those induce the abnormal metabolic disorder ofcomplex carbohydrates are generically called glyco-sidase-deficiencies.
A series of these diseases are genetically sufferinga loss of one of the exoglycosidases. Among thediseases, GM1-gangliosidosis lacking O-galactosidase,Sandhoff disease lacking O-N-acetylhexosaminidase,mannosidosis lacking ,-mannosidase and fucosidosislacking ,-fucosidase have been found. Since degra-dation of a sugar chains by exoglycosidase proceedfrom non-reducing termini, lack of an exoglycosidasestops the degradation at the monosaccharide residue,which should be removed by the missing exoglyco-sidase and accumulate in the tissues.
In the case of glycolipid metabolism, suchaccumulating materials were precisely detected, andthe abnormal schemes were clearly elucidated asdescribed in the review of Seyama and Yamakawa.62)
Glycosidases as tools and their physiological rolesNo. 3] 105
In contrast, the accumulating materials originatedfrom glycoproteins were not found, and it wasconsidered that the exoglycosidases working for themetabolism of the sugar chains of glycolipids and ofglycoproteins might be different, and there was atrend to call the diseases as sphingoglycolipidosis.
We were interested in the preliminary findingthat large amount of oligosaccharides are excreted inthe urine of patients of exoglycosidase deficiencies,and performed a systematic research of the structuresof oligosaccharides accumulated in the urine samplesof various exoglycosidase-deficiency patients incollaboration with the Departments of Pediatricsof Osaka University, Kumamoto University andHokkaido University. It was found that the urinesamples of patients with mannosidosis, and those ofpatients with GM1-gangliosidosis contain the oligo-saccharides shown in Fig. 7.63)–65) The structuralcharacteristics of these oligosaccharides are that theycontain monosaccharides, which should be removedby the missing exoglycosidases, at their non-reducingtermini, and contain the ManO1-4GlcNAc group attheir reducing-termini. These characteristics can beeasily explained if we estimate the N-linked sugar
chains of glycoproteins are released from polypep-tides by endo-O-N-acetylglucosaminidase, and thenthe sequential digestion from non-reducing termini isstopped at the missing exoglycosidase. The structuresof oligosaccharides in the urine samples of GM1-gangliosidosis patients and mannosidosis patients(Fig. 7) indicated that human endo-O-N-acetylgluco-saminidase hydrolyzes both the high mannose-typesugar chains and the complex-type sugar chains.These results indicated that the oligosaccharides inFig. 7 are mainly produced by pathway C shown inFig. 6.
Further information as to the human endo-O-N-acetylglucosaminidase was obtained from the struc-tural study of the incomplete degradation products ofN-linked sugar chains of glycoproteins accumulatingin the urine samples of the fucosidosis patients.66),67)
In contrast to the urine samples of other patientswith exoglycosidase-deficiency, the urine sample ofpatients with fucosidosis contain large amounts ofAsn-oligosaccharides (Fig. 8B) together with smallamounts of oligosaccharides (Fig. 8A). A note-worthy evidence is that all these Asn-oligosaccharidescontain the Fuc,1-6(3)GlcNAc-Asn groups. This
Asp
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B CE-β -GNase 1
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E-β -GNase 1 andE-β -GNase 2
Fig. 6. Metabolic pathway of the N-linked sugar chains of a glycoprotein (A) by the actions of PNGase, E-O-GNase 1 and 2. A highmannose-type undeca-saccharide: Man,1-2Man,1-6(Man,1-2Man,1-3)Man,1-6(Man,1-2Man,1-2Man,1-3)ManO1-4GlcNAcO1-4GlcNAc ( indicates GlcNAc and indicates Man) is drawn as the sugar moieties of the glycoprotein in this figure. However, otherportions of the undeca-saccharide than the core penta-saccharide: Man,1-6(Man,1-3)ManO1-4GlcNAcO1-GlcNAc can be changed toform other high mannose-type, hybrid-type and complex-type sugar chains in pathway C. Only high mannose-type and hybrid-typesugar chains can be included in pathway B. Oligosaccharide with asterisk will be transported to lysosomes after removal of a part ofmannose residues by the action of cytoplasmic ,-mannosidase, and completely hydrolyzed by lysosomal exoglycosidases.
A. KOBATA [Vol. 89,106
evidence indicated that human endo-O-N-acetylglu-cosaminidase cannot act on the N-linked sugar chainscontaining a fucose residue linked to the proximal N-acetylglucosamine residue. In order to confirm thisestimation, we used the homogenates of skin fibro-blasts obtained from fucosidosis patients as enzymesources of endo-O-N-acetylglucosaminidase, and in-vestigated their action to various Asn-oligosaccha-rides. By this study, it was confirmed that both highmannose-type sugar chains and complex-type sugarchains can be cleaved by the enzyme, but none ofthe oligosaccharides containing a fucose linked to theproximal N-acetylglucosamine residue can be cleavedby the enzyme.68) These results indicated that humanskin fibroblasts contain endo-O-N-acetylglucosamini-dase with wider glycon-specificity than E-O-GNase 1.However, the evidence that the complex-type sugarchains without fucose residue was hydrolyzed atmuch slower rate: 1/10 of the high mannose-typesugar chains, must be taken into account.
Furthermore, it was also reported by Inoue’sgroup that the glycopeptides containing the Fuc,1-6(3)GlcNAc-Asn groups cannot be hydrolyzed byPNGase.61) Therefore, the above results could beinterpreted that it was obtained by the pathwayFig. 6C not by Fig. 6B. If it is, the evidence that thecomplex-type sugar chains work as substrate can bewell explained.
Because N-linked sugar chains with the Fuc,1-3GlcNAc-Asn group do not exist in human body,occurrence of such sugar chains in the urine offucosidosis patients may be originated from otherliving organisms taken as foods.69)
3. Role of glycosidases of intestinal bacteriafor the expression of Bifidus factor activity
of human milk oligosaccharides
As already described, many exo- and endogly-cosidases were found in the culture fluid and cells ofvarious bacteria and used as very effective reagents to
A B
Fig. 7. Structures of oligosaccharides excreted in the urine of GM1-gangliosidosis patients (A), and mannosidosis patients (B).
Glycosidases as tools and their physiological rolesNo. 3] 107
study the structures and functions of oligosaccharidesand the sugar chains of complex carbohydrates.
Recently, these enzymes were found to playimportant roles in relation to the Bifidus factor, animportant topic in the baby nutrition.
It has been known that Lactobacillus bifidusbecomes a predominant intestinal flora of babies fedwith human milk. This bacterium digests lactose, andproduces large amount of lactic acid and acetic acid.The acidic condition in the intestine of babiessuppresses the growth of many other microorgan-isms, and may protect babies from harmful intestinalinfection.70)
Schönfeld found a growth factor of Lactobacillusbifidus var. pennsylvanicus in the whey of humanmilk, and named it Bifidus factor.71) In collabo-ration with György, Kuhn started a series of
systematic studies to elucidate the chemical entityof Bifidus factor, and found many oligosaccharides inhuman milk.72)–74)
The problem of the chemical entity of Bifidusfactor had been considered to be solved, whenLactobacillus bifidus var. pennsylvanicus was foundto request N-acetylglucosamine as a growth factorand only the milk oligosaccharides containing theGlcNAc residue were effective as Bifidus factor.However, the project of Bifidus factor is developingnow to a new point of view by the finding that humanmilk oligosaccharides show a unique growth activityworking specifically for various Bifidus strains as willbe described below.
Most human milk oligosaccharides are consid-ered as soluble fibers, because they are not degradedby exoglycosidases in the digestive tract of suckling
B
A
Fig. 8. Structures of oligosaccharides (A) and Asn-oligosaccharides (B) excreted in the urine of fucosidosis patients.
A. KOBATA [Vol. 89,108
babies and reach to colon intact75)–79). Accordingly,these oligosaccharides must first be digested to theirmonosaccharide constituents in order to be used byintestinal flora as nutrients. However, it is not so easytask for the bacteria settling in colon, because mostof the O-galactosidases produced by the intestinalbacteria hydrolyze readily the GalO1-4GlcNAc groupbut do not strongly act on the GalO1-3GlcNAc group.Some of the enzymes do not work on the GalO1-3GlcNAc group at all as described for the diplococcalO-galactosidase. As already described in my recentreview,80) human milk contains more than hundredsof oligosaccharides, which are constructed fromthe thirteen core oligosaccharides summarized in
Table 4. As underlined in the table, the GalO1-3GlcNAc groups are amply located at the non-reducing termini of human milk oligosaccharides.Therefore, many human milk oligosaccharides arenot digested by bacterial O-galactosidase. Accordingto the recently published review,81) occurrence ofoligosaccharides enriched in the GalO1-3GlcNAcgroup is a unique phenomenon of human milk amongthe milk of mammals.
In 1993, Sano et al. found in the culture fluidof Streptomyces sp. 142 an endoglycosidase, whichcleaves the GalO1-3GlcNAc group from the non-reducing termini of sugar chains, and named itlacto-N-biosidase.82) Recently, Ando et al. reported
Table 4. Core structures found in human milk oligosaccharides. The GalO1-3GlcNAc groups, located at the non-reducing termini of theoligosaccharides, are underlined
Names Structures References
Lactose
Lacto-N-tetraose (LNT) 87
Lacto-N-neotetraose (LNnT) 88
Lacto-N-hexaose 89
Lacto-N-neohexaose 90
para-Lacto-N-hexaose 91
para-Lacto-N-neohexaose 91
Lacto-N-octaose 92
Lacto-N-neooctaose 92
iso-Lacto-N-octaose 93
para-Lacto-N-octaose 94
Lacto-N-decaose 95
Lacto-N-neodecaose 96
Glycosidases as tools and their physiological rolesNo. 3] 109
that this enzyme is widely distributed in variousBifidus strains, but not in Clostridia, Bacteroidesand Lactobacilli.83) While Wada et al. found atransporter protein in the plasma membrane ofBifidus strains, which takes up the disaccharidereleased from sugar chains by lacto-N-biosidaseaction.84) Together with the GalO1-3GlcNAc group,this transporter also binds to the GalO1-3GalNAcgroup, which is released from mucin by the action ofendo-,-N-acetylgalactosaminidase.21) Accordingly, itwas named galacto-N-biose/lacto-N-biose I-bindingprotein (GL-BP).
In 1999, Derensy-Dron et al. found in theultrasonicate of Bifidobacterium bifidum DSM20082 a phosphorylase, which phosphorylates GalO1-3GlcNAc or GalO1-3GalNAc to form Gal,1-PO4
and GlcNAc or GalNAc. They partially purified theenzyme, and named it as O-1,3-galactosyl-N-acetyl-hexosamine phosphorylase.85) Kitaoka et al. purifiedthis enzyme and cloned its gene from Bifidobacteriumlongum JCM1217.86) Furthermore, they suggestedthat the enzyme plays an important role in themetabolism of the GalO1-3GlcNAc group releasedfrom human milk oligosaccharides by lacto-N-bio-sidase. Namely, collaboration of three proteins: extra-cellular lacto-N-biosidase, GL-BP in the plasmamembrane and intra-cellular O-1,3-galactosyl-N-ace-tylhexosamine phosphorylase, can effectively metab-olize human milk oligosaccharides containing theGalO1-3GlcNAc group at their non-reducing termini,even if no O-galactosidase cleaving the disaccharidegroup is available. Kitaoka further found a series ofgenes of enzymes responsible for the metabolism ofall monosaccharides constructing human milk oligo-saccharides in the operon containing the gene ofO-1,3-galactosyl-N-acetylhexosamine phosphorylase,and presented the whole scheme of the metabolicpathway of human milk oligosaccharides.97)–99)
A series of these research results confirmed thatBifidobacteria are equipped with a unique mechanismcalled GNB/LNB pathway,99) and effectivelyutilizes oligosaccharides having the GalO1-3GlcNAcgroup at their non-reducing termini. Accordingly,human milk oligosaccharides, which are enrichedin the GalO1-3GlcNAc group at their non-reducingtermini, works as specific nutrients for Bifidobacteria.
Presence or absence of fucosyl residues con-structing the Fuc,1-2Gal, the Fuc,1-3GlcNAc, theFuc,1-4GlcNAc and the Fuc,1-3Glc groups, and ofsialic acid residues constructing the Neu5Ac,2-3Gal,the Neu5Ac,2-6Gal, and the Neu5Ac,2-6GlcNAcgroups were the major source to produce over one
hundreds oligosaccharides from the thirteen coreoligosaccharides in Table 4.80)
Quite recently, Asakuma et al. selected fourtypical Bifidus strains: B. bifidum JCM1254, B.longum subsp. infantis JCM1222, B. longum subsp.longum JCM1217 and B. breve JCM1192, andcultured them in a medium containing 1% humanmilk oligosaccharides as carbohydrate source.JCM1254, and JCM1222 quickly grew in themedium, while JCM1217, and JCM1192 grew veryslowly.100) They then analyzed the time coursealteration of the sugar components in the spentmedia by using 2-anthranilic acid labeling methodfollowed by HPLC analysis. In the 1L of originalmedium, 0.45 g of lactose, 2.88 g of monofucosyllac-toses, 1.11 g of lacto-N-tetraose (LNT), 0.32 g oflacto-N-neotetraose (LNnT), 2.45 g of a mixture ofmono-fucosyl LNT and LNnT, 2.65 g of di-fucosylLNT and LNnT, and few percent of other sugars weredetected.
Time course studies of spent media revealedthat in the case of two strains growing quickly, alloligosaccharides are used up before the growth ofbacteria reach to maximum, and large amounts ofthe monosaccharides: fucose, galactose and glucose,quickly appeared and then vanished.
In contrast, in the case of two strains growingvery slowly, only LNT disappeared but no degrada-tion is observed in other oligosaccharides.
Assay of exoglycosidases, revealed that the twoslowly growing strains completely lack ,-fucosidaseswhich hydrolyze the Fuc,1-2Gal group and theGalO1-4/3(Fuc,1-3/4)GlcNAc groups, while largeamounts of such ,-fucosidases were detected inthe two quickly growing strains. O-Galactosidase,O-N-acetylhexosaminidase and GL-BP were detectedin all four strains. However, lacto-N-biosidase wasdetected in JCM1254, and JCM1217, but not inJCM1222 and JCM1192. These enzymatic studiesrevealed that the two slowly growing strains couldnot use the fucosylated oligosaccharides at all,because of the lack of ,-fucosidases, and JCM1217rely on LNT only by GNB/LNB pathway. Althoughhuman milk contains LNnT in about 1/4 amount ofLNT, the two slowly growing strains could not usethe tetrasaccharide. This evidence may indicate thatthe O-galactosidase in the slowly growing strains actson lactose but not on the GalO1-4GlcNAc group. Incontrast, the quickly growing strains can use LNnTalso, indicating that they contain another O-galacto-sidase acting specifically on the GalO1-4GlcNAcgroup, which will be described later in this section.
A. KOBATA [Vol. 89,110
Anyway, the data presented here clearly indi-cated that the major oligosaccharides in human milk,which have the GalO1-3GlcNAc group in their non-reducing termini, are selectively used by Bifidusstrains. Probably because of this mechanism,Clostridia, Bacteroides and Lactobacilli, which donot have lacto-N-biosidase, cannot use human milkoligosaccharides and cannot grow in the colon ofbreast-fed baby.
Absence of lacto-N-biosidase in B. longumsubsp. infantis JCM1222 and B. breve JCM1192indicated that these strains couldn’t use the GNB/LNB pathway. Therefore, the fact that these strainscan digest LNT was a mystery. Recently, Yoshidaet al. solved this mystery by finding that B. longumsubsp. infantis takes up directly LNT, and digest itby intra-cellular lacto-N-tetraose O-1,3-galactosidase,produced by Blon_2016 gene.101) Another interestingevidence is that this strain contains another O-galactosidase made by Blon_2334 gene, and thisenzyme acts on the GalO1-4GlcNAc group andlactose. Namely, this bacteria is equipped with twokinds of O-galactosidases working specifically tothe GalO1-3GlcNAc group and the GalO1-4GlcNAcgroup. These two enzymes now started to be foundin other bifidobacteria. Although the metabolism ofLNT in B. breve JCM1192 has not been investigated,similar mechanism as JCM1222 may work in thisstrain also.
Concluding remarks
Studies of exo- and endoglycosidases, which havebeen utilized as useful reagents for the structuralstudies of complex carbohydrates, are recentlyexpanding to their physiological function in theliving organisms producing the enzymes.
Two topics: metabolism of the sugar moieties ofglycoproteins within human body, and specificdigestion of human milk oligosaccharides by Bifidusstrains are introduced in this review. Nothing isknown about the physiological functions of proteinscontaining the GlcNAc-Asn residues produced by thepathway of Fig. 6B. However, in view of the variousimportant physiological functions found in the caseof proteins containing the GlcNAc-Ser (Thr) resi-dues,102) the role of GlcNAc-Asn residues may beelucidated as a new field of glycobiology in the future.
As shown in the case of Bifidobacteria, glyco-sidases produced by bacteria are playing importantroles for their growth. These facts indicated that abacterium could acquire a greater adaptation to thecircumstances by obtaining a new glycosidase.
Furthermore, a glycosidase, with peculiar sub-strate specificity, may be useful for the bacterialgrowth. For example, diplococcal endo-O-galactosi-dase, listed in Table 1, shows a very interestingspecificity cleaving the antigenic determinant trisac-charides from the human blood group A and Bsubstances.23) Since similar enzyme was recentlyfound in Clostridium perfringens ATCC 10543 byAnderson et al.,103) this unique enzyme may widelydistribute in many bacteria. Blood group A and Bantigenic determinants are distributed at the non-reducing termini of the sugar chains of mucins, whichare covering the surface of epithelial cells of humanrespiratory tract, and digestive tract. These mucinswork as the barrier to protect epithelial cells frombacterial invasion. Accordingly, bacteria must de-stroy this barrier in order to start infection. However,it is not an easy task for a bacterium, because at leastfour glycosidases: ,-N-acetylgalactosaminidase, ,-galactosidase, ,-fucosidase and O-galactosidase, arenecessary in order to simply remove the A and Bblood group determinants from the sugar chainsof mucins. Accordingly, the endo-O-galactosidase,which can remove the two trisaccharides at once,is a very useful weapon for a bacterium.
Similarly, endo-,-N-acetylgalactosaminidase,21)
which can remove all of the core disaccharide:GalO1-3GalNAc at once may work as a useful weaponfor an invading bacterium.
Because of the limited space, I did not describeabout the reverse reaction of glycosidases for thesynthesis of oligosaccharides as the topic of glyco-sidases. Oligosaccharide synthesis has been tradition-ally performed by using the methods of organicchemistry. However, this approach has a seriousdrawback: it is essential to use a vicious cycle ofprotection-deprotection steps, which often result in adramatic decrease in reaction yields. By the develop-ment of molecular biology, use of glycosyltransferasesfor the synthesis of oligosaccharides has quicklybeen developed. Though this new technique is veryeffective because one can synthesize an aimedoligosaccharide effectively, it requires expensive sugarnucleotides and has a drawback of using fragileenzymes. Therefore, this method is not practical tosynthesize oligosaccharides in industrial scale.
Under such circumstances, oligosaccharide syn-thesis catalyzed by the reversed reaction of exogly-cosidases has been developed recently. As alreadydescribed, exoglycosidases show various aglyconspecificities. Since these specificities will be reflectedin the reverse reactions also, a particular disaccharide
Glycosidases as tools and their physiological rolesNo. 3] 111
can be synthesized by selecting a specific exoglyco-sidase. Furthermore, synthetic method of glycopro-teins containing N-linked sugar chains by usingthe reversed reaction of Endo M by Yamamotoet al.104) has tremendously been developed in recentyears,105),106) and it is now possible to obtain even aglycoprotein, which has no micro-heterogeneity in itssugar moieties, and opening a gate for the compara-tive study of a glycoprotein with different N-linkedsugar chains. I would like to present two representa-tive reviews107),108) for the readers who are interestedin this newly developing field.
Quite recently, even site-directed mutants ofglycosidases, in which hydrolytic activities are sup-pressed, were developed and collectively termedglycosynthases. In addition to these developmentsin the enzyme side, very effective donors likeoligosaccharide oxazoline109) were also developed.
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(Received Nov. 29, 2012; accepted Jan. 10, 2013)
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Profile
Akira Kobata was born in 1933. Graduated and received Ph.D. from the Universityof Tokyo. Elucidated the biosynthetic pathway of ABO and Lewis blood groupdeterminants in Victor Ginsburg’s laboratory in NIH (1967–1971). In 1971, he becamethe Professor of the First Department of Biochemistry, Kobe University School ofMedicine. From 1982 to 1993, he served as the Professor and Chairman of Department ofBiochemistry, Institute of Medical Science, the University of Tokyo. During these 22years, he developed a series of reliable and sensitive methods for the structural study ofthe N-linked sugar chains, and investigated functions and pathology of the sugar chainsof glycoproteins. Studies on glycosidases, which are introduced in this review, wereperformed for the purpose of developing enzymatic reagents for the structural studies ofthe sugar chains of glycoproteins.
He was awarded the Prize for the Promotion of Young Scientists for 1963 from the Pharmaceutical Society ofJapan, Science and Technology Prize for 1985 from Toray Science Foundation, PSJ Award for 1992 from thePharmaceutical Society of Japan, Claude S. Hudson Award for 1992 from American Chemical Society, and also the1992 Japan Academy Prize. He was a Fogarty Scholar-in-Residence in NIH (1985–1987), Auckland FoundationVisiting Professor in New Zealand in 1988, and also served as the Director of Institute of Medical Science (1990–1992).
In 1993, he was appointed as the Director of Tokyo Metropolitan Institute of Gerontology, and became aProfessor Emeritus of the University of Tokyo. In this last carrier as a scientist, he developed a new glycobiologyarea in the field of aging research. From 2000, he has been the Director Emeritus of Tokyo Metropolitan Institute ofGerontology, and served as the advisor of Seikagaku Kogyo Co., LTD. until 2003. Currently, he is the scientificadvisor and a member of the board of directors of the Noguchi Institute, a non-profit institution established for thestudy of carbohydrate chemistry in Japan.
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