Review. Structures and functions of the sugar chains of glycoproteins

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1 Eur. J. Biochem. 209, (1 992) (c> FEBS 1992 Review Structures and functions of the sugar chains of glycoproteins Akira KOBATA Departmcnt of Biochemistry, Institute of Medical Science, University of Tokyo, Japan (Rcccived June 25,1992) - EJB Most proteins within living organisms contain sugar chains. Recent advancements in cell biology have revealed that many of these sugar chains play important roles as signals for cell-surface recognition phenomena in multi-cellular organisms. In order to elucidate the biological information included in the sugar chains and link them with biology, a novel scientific field called glycobiology has been established. This review will give an outline of the analytical techniques for the structural study of the sugar chains of glycoproteins, the structural characteristics of the sugar chains and the biosynthetic mechanism to produce such characteristics. Based on this knowledge, functional aspects of the sugar chains of glycohormones and of those in the immune system will be described to help others understand this new scientific field. Many proteins produced by mammalian cells contain covalently linked sugar chains and are called glycoproteins. However, because of the difficulty of studying the structure of the sugar chain moieties of glycoproteins, their functional aspects have been ignored during the long history of protein research. In contrast to nucleic acids and proteins, many different sugar chains can be formed by using a small number of monosaccharide units. Let us consider the smallest unit of chains: A-B. In the case of protein, only one structure is made when A and B are assigned to, for example, alanine and leucine. In the case of a sugar chain, however, many isomeric structures are formed. Suppose that A and B were assigned to N-acetylglucosamine and mannose, respectively. As shown in Fig. 1, N-acetylglucosamine can be linked at the four hydroxyl groups of mannose: C-2, C-, C-4 and C-. Therefore, four isomeric structures can be formed. Since two anomeric link- HO 0- NHCOCH, Fig. 1. Construction of sugar chains. Correspondence to A. Kobata, Department of Biochemistry, University of Tokyo, 4--1 Shirokanedai, Minato-ku, Tokyo, Japan 108 FUX: Ahhreviutions. Subscript OT indicates NaBH4-reduced oligosaccharides; cndo H, endo-fl-n-acetylglucosaminidase H ; ConA, concanavalin A; Fuc, I-fucose; NeuSAc, N-acetylneuraminic acid; Sia, sialic acid; GnT, N-acetylglucosaminyltransferase; IFN, interferon; CHO, Chincse hamster ovary; hcg, human chorionic gonadotropin; EPO, crythropoietin ages exist for the N-acetylglucosaminyl linkage, the possible isomeric structures of GlcNAc-Man is thus considered to be eight. Furthermore, we should consider the possibility that the N-acetylglucosamine residue can exist in the furanose form as well as the pyranose form shown in Fig. 1, so that 1 isomeric structures are possible for the disaccharide. When the number ofidentical units increases to three, four etc., only one structure can be formed in the case of protein. In contrast, the number of isomeric sugar chains increases by geometrical progression, because branching can occur in sugar chains larger than disaccharide. In addition to the structural multiplicity, many glycoproteins contain more than one sugar chain in one molecule. Even in the case of a glycoprotein with only one sugar chain, microheterogeneity [l] of sugar chain structure occurs widely. This is because the absence of a template in the biosynthetic machinery of sugar chains affords the chance of incomplete sugar chain formation. Therefore, each sugar chain must be separated for its structural study. Development of cell biology in 1970 s revealed the possibility that the sugar chains of glycoproteins play an important role as the signal of cell-to-cell recognition. Elucidation of this exciting phenomenon on a molecular basis, however, had to await the establishnient of several new sensitive methods to analyze the structures of the sugar chains of glycoproteins. Methods to analyze the structures of sugar chains of glycoproteins Background to the development ojnew sensitive methods Human milk contains various oligosaccharides with the GalPl-4Glc group at their reducing termini. These oligosaccharides are considered to be formed by the concerted action of the glycosyltransferases responsible for formation of the

2 484 4OC ' A /I 401 :I ol;;mil,.l,-.---~ TUBE NUMBER Fig. 2. Fingerprints of human milk oligosaccharides. The fraction numbcrs obtained by Sephadex G-25 column chromatography of human milk oligosaccharide are plotted against the distance of migration by paper chromatography using ethyl acetate/pyridine/acetic acid/water (5 : 5: 1 : ) as solvent. Black spots represent oligosaccharides visualized by alkaline-agno, reagent, hatched ones those obtained by both alkaline-agno, reagent and thiobarbituric acid reagent. Three typical patterns are shown in A -C. L, lactose; 2'-FL, 2-fucosyllactose; - FL, -fucosyllactose; LD, lactodifucotetraose; LNT, lacto-n-tetraose LNF-I, lacto-n-fucopentaose I; LNF-11, lacto-n-fucopentaose 11; LNF II1,lacto-N-fucopentaose 111; LND-I, lacto-n-difucohexaose I; LDN-11, lacto-n-difucohexaose 11; N-1, lacto-n-hexaose; N-2, fucosyllacto-n-hexaoses; N-, difucosyllacto-n-hexaoses; N-4, trifucosyllacto-n-hexaoses; '-SL, '-sialyllactose; '-SL, '-sialyllactose; LST-a, LS-tetrasaccharide a; LST-b, LS-tetrasaccharide b; LST-c, LS-tetrasaccharide c; DLN, disialyllacto-n-tetraose; S-5, sialyllacto-n-hexaoses; S-, fucosylsialyllacto-n-hexaoses. sugar chain structures on the Gal/l+4GlcNAc group, which is widely distributed in the sugar chains of glycoproteins and glycolipids. By combining gel permeation and paper chromatography, we established a method to fingerprint all these oligosaccharides [2]. Application of this method for the 4 t Le*i Fkal J analysis of milk oligosaccharide samples obtained from individual donors revealed that they can be divided into three groups. The first group includes approximately 80% of the population and contains all oligosaccharides (Fig. 2A). The second group has an oligosaccharide pattern in which 2'- fucosyllactose, lacto-difucotetraose, lacto-n-fucopentaose I and lacto-n-difucohexaose I are missing (Fig. 2B). Approximately 15% of the population are included in this group. The third group includes the remaining 5% of the population, which lacks lacto-n-fucopentaose 11,lacto-N-difucohexaose I and lacto-n-difucohexaose I1 (Fig. ZC). It soon became evident that the first group includes all individuals with blood type Le(a+b+), the second group thdse with blood type Le(a+b-) and third group those with Le(a-b-). Based on the evidence that the four oligosaccharides missing in the milk of donors with blood type Le(a+b-) contain the +2Gal group in common, it was concluded that these women lack the al,2-fucosyltransferase catalyzing the following reaction : GDP-Fuc + Gal/l-+GlcNAc Gal/l-+GlcNAc--- + GDP Since the three oligosaccharides missing in the milk of donors with blood type Le(a-b-) contain the -+4GlcNAc group in common, these donors must lack the a1,4- fucosyltransferase responsible for the following reaction : GDP-FUC + GAP1 +GlcNAc Gal/1+GlcNAc--- + GDP These data lead to the elucidation of the biosynthetic scheme of the human ABO and Lewis blood group determinants as shown in Fig. []. Furthermore, labelling of the milk ohgosaccharides by reduction with NaBH4 [4] and their sequencing by exoglycosidase digestion [5-71 were developed as new sensitive techniques for the structural study of oligosaccharides. Based on the success of the milk oligosaccharide study, I considered that the techniques could. be effectively used for the study of the sugar chains of glycoproteins if an appropriate method to release them quantitatively as oligosaccharides were established. Release of the sugar chains of glycoproteins as oligosaccharides The sugar chains of glycoproteins can be classified into two groups. One contains an N-acetylgalactosamine residue at its reducing terminal, which is then linked to the hydroxyl group ofeither a serine or a threonine residue ofa polypeptide and is called mucin type or 0-linked sugar chain. The other, which is called asparagine-linked or N-linked sugar chain, contains an N-acetylglucosamine residue at its reducing terminal and is linked to the amide group of an asparagine residue of a polypeptide. 0-linked sugar chains can be released as oligosaccharide alcohols by heating in an alkaline solution in the presence of NaBH4 (/-elimination) [8]. By using NaBH4 for this reaction, H-labeled oligosaccharides can be obtained [9]. However, the requirement of a high concentration of NaBH4 (usually 1 M) to protect the released oligosaccharide from a 'peeling' reaction, made this method impractical because quite large amounts of Hz were produced as a waste product. Recently, Amano and Kobata [lo] opened a way to label quantitatively

3 485 Galpl+GbNAcpl UDP-GalNAc A-enzyme UDP GalPl+GlcNAcPl GalPl+GlcNAcpl T (H antigen) GDP-Fuc / 7 (Lea antigen) \ GalNAcal +GalB1 +GlcNAcBl ' T (A antigen) Fig.. Biosynthetic pathways of ABO and Lewis blood group determinants. Gala1 +Galp1+GlcNAcpl T ( antigen) +GlcNAcPl T (Leb antigen) Table 1. Common structures of the susceptible glycopeptides for endo-/?-n-acetylglucosaminidases. Dotted lines indicate the positions of hydrolysis, and R represents either hydrogen or sugars. Endo-p-N-acetyl-glucosaminidase Structures of substance References D and C, H R R+4Manal+Manfil +4GlcNAc/1+4GlcNAc-rAsn I R R 'R Manal+Manal \ 1 1 R~4Man~1+4GlcNAc~1+4GlcNAc+Asn I R 7 R [ CII Manal-Manal \ I R+4Manpl+4GlcNAc/1 -+4GlcNAc+Asn I R-+2Manal 7' the oligosaccharide alcohols obtained from glycoproteins by /&elimination. This method consists of removing acyl groups from N-acylated amino sugar residues of oligosaccharide alcohols by hydrazinolysis and labeling them by re-n-acetylation with I4C- or 'H-labeled acetic anhydride. Application of this method for the analysis of the 0-linked sugar chains of various glycoproteins has indicated that it is quite effective [ll Since no useful method to release N-linked sugar chains as oligosaccharides was available until 1970, an exhaustive pronase digestion to convert glycoproteins into glycopeptides had been widely used for the study of these sugar chains. However, this method is not satisfactory for the fractionation of N-linked sugar chains because pronase digestion is usually incomplete and the heterogeneities of the peptide moieties and of the sugar chains made it difficult to obtain pure samples of glycopeptides. In early 1970's, several endo-p-n-acetylglucosaminidases, which hydrolytically cleave the N, N'-diacetylchitobiose moieties at the reducing termini of N-linked sugar chains were found in the culture fluid of various microorganisms [ By carefully investigating the action of these enzymes on various glycopeptides, the substrate specificities of these enzymes were elucidated as summarized in Table 1. Endo-P-N-acetylglucosaminidases D and H were effectively used to determine the sugar chain structures of hen egg albumin [1, 18, 20, 211. Endo-P-N-acetylglucosaminidase H (endo H) was also widely used to elucidate the structures of

4 48 various high-mannose-type [22] and hybrid-type sugar chains, which will be described later. However, none of the enzymes listed in Table 1 could release oligosaccharides from ovomucoid or from glycopeptides containing complex-type sugar chains, which will also be described later. By looking for a method to release quantitatively these endo-p-nacetylglucosaminidase-resistant N-linked sugar chains as oligosaccharides, we finally reached a chemical method : hydrazinolysis. Hydrazinolysis was originally used by Matsushima and Fujii [2] for the study of glycoconjugates and applied by Yoshizawa et al. [24] for the study of the sugar chains of N- linked sugar chains. Bayard and Montreuil [25] reported that the N-linked sugar chains of several glycoproteins can be quantitatively released as oligosaccharides by heating them at 100 C for 0 h. Although the method was successfully used for the the study of sugar chains of subcomponent Clq of the first component of human complement [2], we soon noticed that messy results were obtained when studying other glycoproteins. This was because the hydrazin derivatives of released oligosaccharides cannot be easily converted to free oligosaccharides like glycosylamine in an aqueous solution and side reactions such as isomerization and degradation of the derivatives are easily induced. By this time, we knew that all of the N-linked sugar chains of hen egg albumin can be released as oligosaccharides by digestion with endo H [2]. By using the oligosaccharide pattern of this glycoprotein obtained with endo H as a standard, we carefully investigated the condition of hydrazinolysis, and established a method to release quantitatively all N-linked sugar chains as oligosaccharides [27]. Since then, this chemical method has served as a standard method to investigate the N-linked sugar chains of many glycoproteins. Since hydrazinolysis can release all N-linked sugar chains even from cells and tissues if only the samples are thoroughly dried, it has recently been used effectively for the study of the changes of sugar patterns induced by differentiation [28, 291 or transformation of cells [0]. After that, glycopeptidases (N-glycanases), which cleave the GlcNAch Asn linkage, were found and purified from many biological sources [1-1. Since these enzymes release all N-linked sugar chains of glycoproteins without destroying the polypeptide moieties, they can be used effectively for the study of the functional aspects of N-linked sugar chains. Care must be taken, however, to limit the application of these enzymes because they can have substrate specificities, including the steric hindrance of the polypeptide portion. Since the oligosaccharides released by the various methods so far described all contain an N-acetylglucosamine residue at their reducing termini, they can be converted quantitatively to H-labeled oligosaccharide alcohols by reduction with NaBH4. Converting them to fluorescent [4] or ultravioletabsorbing derivatives [5] may also be useful for their detection. Fractionation of the oligosaccharides Before starting a structural study of the oligosaccharides, they must be fractionated by appropriate methods. Oligosaccharides with a charged group, such as sialic acids, phosphate and sulfate, can be fractionated by paper electrophoresis and ion-exchange column chromatography. Gel-permeation chromatography [,7] and HPLC [8] are also widely used. In addition to these, affinity chromatography using immobilized lectin columns has been used as a unique and very effective tool to fractionate oligosaccharides. Since the method CHzOH Fig. 4. The structure recognized by ConA. 0- seems likely to form the basis of an automated sugar pattern analyzer in the future, I will describe it in a little more detail. Concanavalin A (ConA) binds specifically to the structure shown in Fig.4 [9]. Therefore, Glcalh, Manalh and GlcNAcal -+ residues, which are located at the non-reducing termini of sugar chains, and -2Glcal+ and -2Manalresidues within sugar chains can bind to this lectin. By investigating the behavior of various oligosaccharides and glycopeptides in a ConA-Sepharose column, Ogata et al. [40] reached an important conclusion that the presence of two binding residues is required for a sugar chain to be reiained on the column. Based on this finding, affinity chromatography using a ConA-Sepharose column was developed as an effective method to fractionate and analyze the structures of N-linked sugar chains of glycoproteins. In brief, monoantennary and biantennary complex-type sugar chains (described later), which contain two binding residues, bind to a ConA- Sepharose column and are eluted with a solution of 5 mm methyl a-glucopyranoside, while high-mannose-type sugar chains, which contain many binding residues, bind to the column and are eluted only with 200 mm methyl a-mannopyranoside. In contrast, triantennary complex-type sugar chains, which contain only one binding residue, pass through the column without interaction. Introduqtion of a ConA-Sepharose column to the field of glycoprotein research has attracted the attention of many researchers and the behavior of N-linked oligosaccharides in various immobilized lectin columns has been investigated. In Table 2, the binding specificities of- the immobilized lectin columns useful for the fractionation of N-linked sugar chains are summarized. For detailed information of these lectin columns, please consult with our recent review [41]. Since the behavior of an oligosaccharide is mainly determined by the structural requirement in Table 2 and is not affected by additional sugar chain moieties, a mixture of many oligosaccharides can be separated into each component by choosing a series of appropriate immobilized lectin columns. Therefore, combination of the serial lectin column chromatography with hydrazinolysis affords a simple and very sensitive method for analyzing the sugar patterns of glycoproteins. Monosaccharide sequence analysis of an oligosaccharide Sequential exoglycosidase digestion has been widely used for the determination of the monosaccharide arrangement of an oligosaccharide. Since exoglycosidases hydrolytically cleave a particular monosaccharide from the non-reducing terminal of an oligosaccharide, the radioactivity incorporated at the reducing terminal of an oligosaccharide remains in the oligosaccharide portion until it is completely digested to [H]N-acetylglucosaminitol. Exoglycosidases have two kind of specificities : the glycon specificity and the aglycon specificity. The glycon specificity is directed at the structure of the

5 Table 2. Binding specificities of immobilized lectin columns. Lectin Structure necessary for binding Reference Concanavalin A R+2Manal \ ManPl -PR R+2Mancll 7 Manal+2Mancll +R Phytohemagglutinin E4 strong binding (retarded at 20 C and 2 C) & Fuccll R+Galj1+4GlcNAc~I +2Manal 1 GlcN Acj 1 + 4Manfi 1 + 4GlcNAcP 1 + 4GlcN AcoT. RL 4 P Mancll 2 R+4GlcNAcfl1 7 weak binding (retarded at 2 C only) + Fuccll R+GalP1+4GlcNAcjI +2Mancll \ 1 Manfll-+4GlcNAcfll +4GlcNAcoT I7 Duturu strumonium agglutinin strong binding (bound) R+Gal~1+4GlcNAcfll \ Mancll + R 2 R+Gal~1+4GlcNAc~I 7 R+GalpZ +4GlcNAc~l+Gal~l+4GlcNAc~1 +R weak binding (retarded) R+Galfll -+4Gl~NAcjl \ 4 Mancll +R 2 R+Gal~I+4GlcNAcfl1 7 Aleuriu uuruntiu lectin Fucccl 1 R+4GlcNAcoT Allomyrinn dichotomu lectin NeuSAca2+Galfl1-4GlcNAcfll +R monosaccharide residue to be released from the sugar chain; linkages. The specificity directed to the monosaccharide is it also includes the anomeric configuration of the monosac- also strict in most cases. For example, a-fucosidases and charide. This latter specificity is very high and none of the a-mannosidases can only hydrolyze a-fucosidic and a- enzymes so far reported hydrolyzes both a- and 0-glycosidic mannosidic linkages, respectively. However, some exoglycosi-

6 488 B I Table. Structures resistant to diplococcal j-n-acetylhexosaminidase digestion. Structure GlcNAcPl+4Manal+ GlcNAcPl+Manal+ I ELUTION VOLME (ml) Fig. 5. Sequential exoglycosidase digestion of a radioactive dodecasaccharide fraction obtained by hydrazinolysis of a glycopeptide fraction purified from the urine of a patient with fucosidosis. The radioactive oligosaccharides at each digestion step were analyzed by Bio-Gel P- 4 column chromatography. The black arrows indicate the elution positions of glucose oligomers (numbers indicate the glucose units), and the white arrow indicates the the elution position of authentic Mancll +(Man~l+)Man~l+4GlcNAc~l+4(Fuccll -+)N-acetylglucosaminitol. (A) The dodecasaccharide fraction; (B) the dodecasaccharide fraction incubated with almond a-fucosidase I; (C) the combined peaks in after incubation with diplococcal p-galactosidase; (D) peak c after incubation with diplococcal b-nacelylhexosaminidase. dases show lower specificities in this category. For example, all glycosidases which cleave P-N-acetylglucosaminyl linkages also hydrolyze P- N-acetylgalactosaminyl linkages. Therefore, these enzymes are called P-N-acetylhexosaminidases. Because exoglycosidases cleave only monosaccharide residues which are located at the non-reducing terminal, they can be used as effective reagents to sequence the sugar chains. For example, sugar chains with a Galfll-+4GlcNAcPI --t 2Manal+Manfll+ sequence at their non-reducing termini are hydrolyzed only by sequential digestion with P- galactosidase, P- N-acetylhexosaminidase and a-mannosidase. Susceptibility of the sugar chain to each exoglycosidase digestion can be examined by gel-permeation chromatography, as shown in Fig. 5. The data in Fig. 5 also indicate an advantage claimed by this method : the structure of each oligosaccharide can be determined by using a mixture of oligosaccharides. Before explaining the data in Fig. 5, aglycon specificity of exoglycosidase must be described. The aglycon specificity is directed at the structures of the sugar chains to which the monosaccharide to be hydrolyzed is linked. For example, the a-fucosidase purified from Bacillus fulminans cleaves the +2Gal linkage only [4]. a-fucosidase I purified from almond emulsin hydrolyzes the -+GlcNAc and the +4GlcNAc linkages but not other a-fucosyl linkages [47]. In contrast, the enzyme from Charonia lampas cleaves all a-fucosyl linkages including synthetic substrates such as p- nitrophenyl a-l-fucopyranoside [48]. The P-galactosidase purified from the culture medium of Diplococcus pneumoniae cleaves the Gal11-4GlcNAc linkage, but not the Gal~l-tGlcNAc or the GalPl-+GlcNAc linkages [49]. In contrast, P-galactosidase purified from Streptococcus 4 K cleaves all the Gal -+GlcNAc linkages [50]. GlcNAc,CIl \ Mancll + 2 GlcNAcPl 7 GlcNAc,81+2Mancll \ GlcNAc/1+4Manpl + Mancll 7 Diplococcal P-N-acetylhexosaminidase also shows a unique aglycon specificity [ Basically, the enzyme cleaves only the GlcNAcfll-+2Man linkage. However, the enzyme cannot cleave the GlcNAcPl+2Man linkage in the oligosaccharides as listed in Table. With this aglycon specificity, the enzyme is used as an effective reagent to analyze the antennary structures of various complex-type sugar chains. With this knowledge in mind, I would like to come back to the data in Fig. 5. In Fig. 5A, the elution pattern of radioactive dodecasaccharide obtained by hydrazinolysis of a glycopeptide isolated from the urine of a fucosidosis patient [52] is shown. Incubation of this fraction with almond emulsin a- fucosidase I resulted in a partial degradation of the fraction (Fig. 5B). The difference of the eluting position of the two peaks was approximately one glucose unit. Since one a-fucosyl residue in the Galb1- or 4(+4 or )GlcNAc group behaves as half a glucose unit [,7], the result indicated that two such fucosyl residues were removed from approximately 50% of the oligosaccharide mixture. When the mixture of the two peaks in Fig. 5B was incuba ted with diplococcal P- galactosidase, two galactose residues were removed from peak b, while peak a remained unchanged (Fig. 5C). It must be added that the Galfll - or 4(+4 or )GlcNAc group cannot be degraded by any fl-gdlactosidase so far reported. After incubation with diplococcal P-N-acetylhexosaminidase, the radioactive peak c was converted to Manalj (Manal-+)Man~l+4GlcNAc~1+4( +)GlcNAco, (subscript OT indicates NaBH4-reduced), releasing two N-acetylglucosamine residues (Fig. 5D). The results of sequential exoglycosidase digestion revealed that approximately 50% of the oligosaccharides in the original fraction had the structure: Gal~l+4(+)GlcNAcfl1-+ 2Manal-+[Gal~1+4(+)GlcN AcPI -2Manal+]- Man~l-+4GlcNAc~1 -+4( +)GlcNAco,. When peak a in Fig. 5C was incubated with B.,fulminans a-fucosidase, two fucose residues were removed from all of the components in this fraction (data not shown). The decasaccharide gave exactly the same degradation patterns as in the case of peak b. Therefore, the remainder of the original fraction has the structure: -+ 2GalP1 -+ 4GlcNAcP1 + 2Manal -+ (-+2Gal~1-+4G lcnac~l-+2manal+)man~l+ 4GlcNAcPI +4( +)GlcNAcor. As another example of aglycon specificity useful for the structural study of N-linked sugar chains, I would like to

7 ~ I 489 introduce the specificities of various a-mannosidases. Jack bean a-mannosidase cleaves the Manal+2Man and the ManaljMan linkages at almost the same rate, but the hydrolysis rate of the Manal+Man linkage is about one-fifteenth of that of the Manal42Man linkage [1]. Accordingly, this enzyme can be used at high concentration to cleave all a-mannosyl residues included in N-linked sugar chains. At low concentration, it can be used to discriminate the Mana1-Man linkage from the other two a-mannosyl linkages [1]. Two a-mannosidases with important aglycon specificities were found in Aspergillus saitoi. a-mannosidase I cleaves the Manal+2Man linkage but not the Manal +Man and the Manal-Man linkages [5]. Therefore, this enzyme is useful for assigning a series of high-mannose-type sugar chains as will be discussed later. a-mannosidase I1 from A. saitoi shows another interesting aglycon specificity. It cleaves an a-mannosyl residue from an R-,Manal+B(Manal- )Manpl -+group but not from a Manal +(R+Manal+ )Manfil +group (where R represents a sugar) [54]. Accordingly, the enzyme is used to assign a particular outer chain on the two a-mannosyl arms of complex-type sugar chains. Several other analytical techniques, such as methylation analysis and Smith degradation, are essential for the structural study of oligosaccharides. Interested readers should consult our recent review [55]. GalPl-14GlcNAcpl,, 1 Asn Gal~1-14GlcNAcPl GalPl+4Gl~NAcpl ~l I Manal% Manal, Fig.. The subgroups of N-linked sugar chains. (1) Complex-type sugar chains; (2) high-mannose-type sugar chains; () hybrid-type sugar chains. Structures within the solid line is the pentasaccharide structure common to all N-linked sugar chains. The structure enclosed with a dotted line is the common heptasaccharide of high-mannose-type sugar chains. Structures outside the solid line can vary in their sugar chains. 1 Structural rules included in N-linked sugar chains and the biosynthetic mechanism to produce them Suhgroups of N-linked sugar chains The establishment of a series of analytical methods as described in the previous section enabled us to study the structure of N-linked sugar chains accurately. Accumulation of the structural data revealed that N-linked sugar chains conform to more structural rules than 0-linked sugar chains. All N-linked sugar chains contain a pentasaccharide : Manal+(Manal+)Man~1+4GlcNAc~1+4GlcNAc as a common core, which will be called trimannosyl core in the following part of this review. According to the structures and the location of the extra sugar residues added to the trimannosyl core, N-linked sugar chains are further classified into three subgroups (Fig. ) [5]. Sugar chains classified as complex type, contain no other mannose residue other than the trimannosyl core. Outer chains with an N-acetylglucosamine residue at their reducing termini are linked to the two a-mannosyl residues of the trimannosyl core. These outer chains are composed of N- acetylglucosamine, galactose, fucose, sialic acids, N- acetylgalactosamine and sulfate. The presence or absence of an a-fucosyl residue linked to the C position of the proximal N-acetylglucosamine residue and the P-N-acetylglucosamine residue linked to the C-4 position of the P-mannosyl residue of the trimannosyl core (bisecting GlcNAc) contribute the structural variation of the complex-type sugar chains. High-mannose-type sugar chains contain only a-mannosyl residues in addition to the trimannosyl core. A heptasaccharide with two branching structures: Manal + (Manal+)Manal+ (Manal+)Man/1+4GlcNAcPl+ 4GlcNAc is commonly included in this type of sugar chain, as shown by the dotted line in Fig.. Variation is formed in these sugar chains by the numbers and the locations of up to four Manal+2 residues linked to the three non-reducing terminal a-mannosyl residues of the common heptasaccharide. The third group is called hybrid type because the oligosaccharides have the structural features of both high-mannosetype and complex-type sugar chains. One or two a-mannosyl residues are linked to the Manal- arm of the trimannosyl core as in the case of the high-mannose-type, and the outer chains found in complex-type sugar chains are linked to the Manal+ arm of the core of this group. The presence or absence of the a-fucosyl residue and the bisecting GlcNAc linked to the trimannosyl core also produce structural variations in the sugar chains of this subgroup. Among the three subgroups of N-linked sugar chains, the complex type has the largest structural variation. This variation is due mainly to two structural factors. As shown in Fig. 7, from one to five outer chains are linked to the trimannosyl core by different linkages, resulting in formation of mono-, bi-, tri-, tetra- and penta-antennary sugar chains. Two isomeric triantennary sugar chains containing either the GlcNAc~l+4(GlcNAc~1-2)Manal- group or the GlcNAcPl+(GlcNAc/?1-+2)Manal+ group are found. These isomeric sugar chains are called 2,4-branched and 2,- branched triantennary sugar chains, respectively. Various structures are found for the outer chain moieties of complextype sugar chains. In Fig. 8, structures of some of the representative outer chains are listed. Combination of the antennary and the various outer chains will form a large number of different complex-type sugar chains. In contrast to N-linked sugar chains, 0-linked sugar chains have fewer structural rules. So Far, these sugar chains can be categorized into at least four groups according to their core structures (Fig. 9). In addition, 0-linked sugar chains with the GlcNAcPl -GalNAc core and the GalNAcPl+GalNAc core are found in a limited number of glycoproteins. Biosynthesis of the sugar chains of glycoproteins 0-linked sugar chains are formed by stepwise addition of monosaccharides to the Ser and Thr residues of polypeptides from nucleotide sugars. In contrast, N-linked sugar chains are

8 490 1) Monoantennary GalPl+GlcNAcPl+ 2) Biantennary ) Triantennary Manal -'Manpl -4R GI~NA~DI + 2 ~ ~ ~ ~ 1 " ~ GlcNAcPl +2Manalb ManPl-+4R GlcNAcPl -+2Manalr a) 2,4-branched GlcNAcPl -12Manalb GlcNAcPl Manpl-i4R V:Manalr GlcNAcPl-) b) 2.-branched GlcNAcPlV planal GlcNAcPln YManpl +4R GlcNAcPI +2Manalp 4) Tetraantennary SiaaP 1 Siaa2-iGal~l-iGlcNAc~1+ +PGalpl+GlcNAc~l+ f+2gal~l+glcnac~l -1 4 T SiaaL+Galjl-iGlcNkc~l+ 4 T GalP1-14GlcNAcPI + Siaa2+()Gal~l-i4GlcNAc~l -i -+2GalPl -14GlcNAcPI + f-i2gal~i-i4glcnac~l T Siaa2-tGal~l-t4GlcNAc~I -i f 5) Pentaantennary GlcNAcP1, GlcNAcPl +anal, GICNAC~I+ GIcNAcPI,~ '!Manpl+4R 2Mana17 GlcNAcPl 2 R = GlcNAcPI +4GlcNAc-+Asn Fig. 7. Branching of complex-type sugar chains. Gala1 +Gal~1-14GlcNAc~I -i SO4-4GalNAc~l-i4GlcNAc~l+ Fig. 8. Various outer-chain structures found in complex-type sugar chains. Core 1 GalPl-i4Gl~NAc~l,~ r_._.. -_.._ --._. Galp1 +GlcNAcp1+ /Galpl-1GalNAc+Ser (Thr) A Galp1+GlcNAcp1~ L.._..._. Core 2 formed by a series of complex pathways including lipid-linked intermediates [571. First, GlcNAc-1-P is transferred from UDP-GlcNAc to a polyisoprenol monophosphate: dolichyl phosphate (Dol-P). The N-acetylglucosamine residue of the GlcNAc-P-P-Do1 is the starting point of N-linked sugar chains. To this N-acetylglucosamine residue, another N- acetylglucosamine and five mannose residues are transferred from UDP-GlcNAc and GDP-Man, respectively. The lipidbound heptasaccharide, which is enclosed by a line in Fig. 10, is converted to G~c~-M~~~-(G~cNAc)~-P-P-Do~ by the further addition of four a-mannosyl residues from Dol-P-Man and three a-glucosyl residues from Dol-P-Glc. The tetradecasaccharide of the lipid derivative is then transferred en bloc to the asparagine residue of the polypeptide chain, which is translated in the rough endoplasmic reticulum by the catalytic action of a dolichyldiphosphoryl oligosaccharide : polypeptide oligosaccharyltransferase residing in the endoplasmic membrane. Only the asparagine residue in the sequence of Asn- Xaa-Ser/Thr, where Xaa can be any amino acid other than proline, is glycosylated. This is because the oligosaccharyltransferase recognizes the ring structure of the polypeptide shown in Fig. 11. By investigating the transfer of the tetradecasaccharide from G~c~-M~~~-(G~cNAc)~-P-P- Do1 to various tripeptides in vitro, Ronin et al. [58] found that Asn-Xaa-Cys can be glycosylated equally as well as Asn-Xaa- core Core 4 Gal01 +4Gl~NAc~l,~ r _ Galp14 4 jglcnacp1 +dgalnac+ser (Thr) -2 Galpl+4GlcNAcpl~ L.._ Fig. 9. Four types of core structures found in 0-linked sugar chains. The cores are enclosed by dotted lines. Ser/Thr. However, very few of the tripeptide sequences in natural glycoproteins are actually glycosylated [59]. This is probably because the SH group of the cysteine residue quickly forms an S-S linkage with another cysteine residue and cannot contribute to the ring formation shown in Fig. 11. The completely translated polypeptide with the tetradeca- saccharide is then transported to the Golgi apparatus (Fig. 10). During this transport, three a-glucosyl residues and at least one Manal-+2 residue are removed by the action

9 49 1 Manal +PManal. Manal Manal +PManal F M a n p l +4GlcNAcp1+4GlcNAc-P-P-Do~ GIcal+?Glcal +Glcul+ IManal +PManal+2 Manal rr Dol-P-P Asn (peptide) Glc,.Manp.GlcNAc2+Asn (peptide) Glucose 4 Mannose Manal imanal Manal!Manpl-14R Manal UDP-GlCNAc UDP Manal ;Manal Manal EManpl+4R GlcNAcPl+PManal UDP-GlcNAc II GlcNAcpl Manal L %4anal 4 Manal M ;Manpl+4R IV GlcNAcP1 +ZManal UDP GlcNAcPl 5. Manal Manal+JManal 4 imanpl+4r GlcNAcPl+PManal +4R T* GlcNAc!1+2Manal UDP-GlcNAC UDP V 111 Manal :Manpl+4R GlcNAcPl+PManal Fig. 10. Processing in the biosynthesis of N-linked sugar chains. R = GlcNAc/1+4GlcNAc+(Asn-Xaa-Ser or Thr)protein. Fig. 11. The ring structure formed by Asn-Xaa-Ser (or Thr) Ser, R, = H; Thr,!A1 = CH. B is the basic residue of the oligosaccharyltransferase. of two a-glucosidases and an a-mannosidase residing in the membrane of the endoplasmic reticulum. After being translocated to the cis-golgi, the N-linked sugar chain of the polypeptide is converted to Man,-(GlcNAc)z by the action of Golgi a-mannosidase I, which removes all Mana+2 residues from the sugar chain. A series of high-mannose-type sugar chains is now considered as the intermediary product of this trimming process. When the glycoprotein is translocated to the medial Golgi, an N-acetylglucosamine residue is added at the C2 position of the Mand + arm of the trimannosyl core portion by the action of N-acetylglucosaminyltransferase I (GnT-I) [0]. Addition of this N-acetylglucosaniine residue changes the steric arrangement of the two a-mannosyl residues linked to the Manal- arm, so that they can be removed by Golgi a-mannosidase 11 [1]. These are the entire features of the processing pathway to form monoantennary complex-type sugar chains. The action of another N-acetylglucosaminyltransferase (GnT-111) to the processing intermediates I and I1 in Fig. 10 converts them to bisected sugar chains IV and V, respectively. The a-mannosyl residues of these bisected sugar chains cannot be removed by Golgi a-mannosidase I1 [2]. Hybrid-type sugar chains are produced from these deadlocked intermediates together with the intermediates I and 11. The a-fucosyltransferase responsible for formation of the fucosylated trimannosyl core needs the presence of the GlcNAcpl-t2 residue on the Manal+ arm []. This specificity explains the evidence that no fucosylated high-mannosetype sugar chain is detected in glycoproteins. When a GlcNAcPl42 residue is galactosylated, the addition of a fucose residue does not take place. The presence of a bisecting GlcNAc also prevents the core fucosylation [4]. Therefore, the core fucosylation is controlled by the competitive action of these transferases for the common acceptor sugar chain. Starting from monoantennary sugar chains, a series of complex-type sugar chains is formed by the action of various N-acetylglucosaminyltransferases (GnTs in Fig. 12). The first product is the biantennary sugar chain produced by the action of GnT-I1 [0]. This biantennary sugar chain is converted to 2,4-branched and 2,-branched triantennary sugar chains by the action of GnT-IV [5] and GnT-V [], respectively. By the concerted action of GnT-IV and GnT-V, a tetraantennary sugar chain is formed. All these complex-type sugar chains are bisected by the action of GnT-111. Each p-nacetylglucosamine residue is further elongated by the action of various glycosyltransferases as depicted in Fig. 1. A bisecting GlcNAc residue cannot be the acceptor of any of these glycosyltransferases and remains unchanged during these maturation processes. Glycosyltransferases catalyzing these reactions have strict specificities for donor nucleotide sugars and acceptor sugar

10 492 GlcNAcPl GlcNACPl\ s. panal 4 GlcNAcPlfl zmanp1+4r GlcNAcPl +PManal fl t GnT-Ill GnT-I PManal, panpl+4r GlcNAcPl +PManal I fl GnT-II - GlcNAcPl +PManai \ panpl+4r GlcNAcPl +PManal fl GlcNAcPl L GnT-I11 GlcNAcPl+2Manal 4 \$Aanpl+4R GlcNAcPI -tpmanal H GlcNAcPI GlcNAcPl,4 \imanpl+4r 2Manal GlcNAcPI - GIcNAcPI N GlcNAcPl +PManal GnT-Ill GlcNAcPl +PManal 4 2Manal GIcNAcPI\~ zmanpl+4r GlcNAcP1 fl ;Manf1+4R 2Manal GlcNAcPl+ZManal fl GlcNAc PI fl GnT-IV \ GnT-V \ J I GlCNACP1 x GlcNAcPl, lcnacp1 2Manal L GnT-Ill 2Manal, 4-0. GlcNAcPlfl zmanpl+4r GlcNAcP1,4 GlcNAcPlfl :Manp I ++ R GlCNAcPl \4,Manal -Manal GICNACPI ~ GICNACPIH~ Fig. 12. Maturation of N-linked sugar chains: formation of branching structures of complex-type sugar chains. R ist the same as in Fig. 10. GnT, N-acetyl-glucosamin yltransferase. / / GlcNAcDl-tR \ Galpl-tGlcNAcpl-tR - i 4 Galpl -1GlcNAcpI-tR Neu5Aca2 1 \ Neu5Aca2+Galpl -tglcnacpl-tr - Galpl-t4GlcNAcpl-tR Galpl+4GlcNAcpI -tr I? t GlcNA~pl-tGalp1+4GlcNAcp1 +R Galp1+4GlcNAcp1+Galp1-t4GlcNAcp1-tR Fig. 1. Maturation of N-linked sugar chains: formation of various outer chains. R represents the trimannosyl cores. chain structures. Under the cellular conditions where enough nucleotide sugar pools are available, levels and acceptor specificities of glycosyltransferases are of primary importance for determining the final structure of an outer-chain moiety of a complex-type sugar chain. For example, an a2,- sialyltransferase purified from rat liver can transfer sialic acid to the GalBl+4GlcNAc group producing the Siaa2-+ Galfl1+4GlcNAc group, but not the GalBl+GlcNAc group [7. An a2,-sialyltransferase purified from the same source can transfer sialic acid to both the Galj1+4GlcNAc and the GalB1+GlcNAc groups [7]. Therefore, an oligosaccharide structure can be recognized by a glycosyltransferase or by some glycosyltransferases. In the latter case, distinct glycosyl linkages are formed in a different ratio as a result of competition between the glycosyltransferases. Competition also occurs between glycosyltransferases which add different monosaccharides to a common acceptor sugar chain. A typical example is the case of sialylation and fucosylation of the GalPl+4GlcNAc group widely found in the complex-type sugar chains. An a2,-sialyltransferase purified from bovine colostrum and an a1,-fucosyltransferase from human milk can produce the Si&2-,GalB1+4GlcNAc and the Gal01 -+4(+)GlcNAc groups by acting on asialotransferrin [49], respectively. However, fucosylated asialotransferrin cannot serve as a substrate for the a2,- sialyltransferase. Similarly, prior sialylation of the glycoprotein prevents the subsequent fucosylation by the d,- fucosy!transferase. Therefore, the ratio of sialylated and fucosylated outer chains must be determined by the ratio of the sialyltransferase and the fucosyltransferase activities expressed in a cell. The Siaa2+Gal~1+(Fucul-+4)GlcNAc group, called sialyl-lea, was found to be the antigenic determinant of a monoclonal antibody 19-9 directed to gastrointestinal and pancreatic cancer cells [8]. Together with cancer, the antigenic determinant occurs in fetal tissues [9], normal adult pancreas [70], salivary mucins [71] and seminal fluid [72] of normal Le(a+b-) and Le(a+b+) individuals. An enzymatic study using the microsome fraction of SW111 cell line and human milk oligosaccharides revealed that the a2,-sialylation precedes fucosylation of the penultimate N-acetylglucosamine residue [7]. An analogous situation has been observed in the synthesis of sialyl X antigenic determinant, Siaa2+ GalP1+4( -+)GlcNAc [74]. Accordingly, these fucosyltransferases have wider substrate specificities than human milk fucosyltransferase. Species- and organ-specific glycosylation The enzymes responsible for the processing of N-linked sugar chains are included in all mammalian cells. However, the expression of the glycosyltransferases related to the maturation of the sugar chains is quite different between animal species and between the organs of one animal. The most clear-cut evidence for the species-specific and organ-specific glycosylation was provided by the comparative study of the

11 49 Kidney rat and bovine mouse () L +4GlcNAc L GlcNAcpl 1 1 Galp1+4GlcNAcp1hPManal x 4 ManpI +4GlcNAcp1-4GlcNAc Galpl+4GlcNAcpl-12Manal f human f GlcNAcpl Liver rat and human mouse (Galpi +4GlcNAcpI +) I I 1 PManal 4 $anal ~Manpl+4GlcNAcpl+4GlcNAc i Sia~Galpl+4GlcNAcpI ~ PManal., (fsia~galpl+4glcnacpi Manpl +4GicNAcp1 +4GlcNAc \ )Manal* Sia*Galpl+4GlcNA~pl*~ Siaa2 1 Siaa2+Galpl+GlcNAcpI hpmana1 K panpi +4GlcNAcp1+4GlcNAc Sian2+Galpl+GlcNAopl h2manal f SiaaP Fig. 14. Major sugar chain structures of y-glutamyltranspeptidases from the kidney and the liver of various mammals. sugar chains of y-glutamyltranspeptidase (y-gtp). This enzyme widely occurs in all InamnYdlS as a membrane-integrated glycoprotein of epithelial cells of various organs. Only N- linked sugar chains are included in these enzymes. In Fig. 14, structures of the major sugar chains of y-gtps purified from the kidneys and the livers of various mammals are summarized [ Both kidney and liver enzymes of mouse contain biantennary sugar chains. However, the structures of the outer chains of the two enzymes are totally different. Furthermore, the sugar chains of the kidney enzyme contain bisecting GlcNAc and core fucose, which are not found in the sugar chain of the liver enzyme. Therefore, organ-specific differences exist in the sugar chains of mouse y-gtps. Structural differences are also found in the sugar chains of kidney and liver enzymes of other mammals. Comparison of the sugar chains of kidney enzymes or liver enzymes of different animals indicates that species-specific glycosylation is also found to occur in these enzymes. Interesting and important evidence is the fact that bisecting GlcNAc is detected in the sugar chains of all kidney enzymes but not in those of liver enzymes. Not only liver y-gtps but all glycoproteins produced in the liver, so far studied, have non-bisected N-linked sugar chains. Therefore, expression of GnT-I11 must be suppressed in all mammalian cells during their differentiation to hepatocytes. In contrast, the enzyme is strongly expressed in the kidney cells. Another good example of the species-specific glycosylation is the expression of the Galal +Gal group in the sugar chains. That this disaccharide group occurs as the non-reducing terminus of the sugar chains of glycoproteins and glycolipids produced by the cells of New World monkeys and non-primate mammals, but not by human and Old World monkey cells, I was confirmed by Galili et al. [81] by using antibodies directed to the disaccharide structure. This evolutionarily regulated expression of the disaccharide group has been shown to correlate inversely with the presence of natural antibodies against the Galal +Gal epitope. Organ-specific glycosylation was also found widely in other glycoproteins. It has been demonstrated, by using various immobilized lectin columns, that the sugar chains of human ribonucleases are different between organs [82]. Human placental glycoproteins contain only the Neu5Aca2-+Gal group [8-81 in contrast to the human serum glycoproteins, which dominantly contain the Neu5Aca2+Gal group or both the NeuSAca2+Gal and the NeuSAca2+Gal groups. The presence of species- and organ-specific differences in the glycosylation of proteins will create many problems in the production of glycoproteins by recombinant techniques. Actually, a comparative study of the N-linked sugar chains of natural human interferon-pl (IFN-P1) and three recombinant IFN-Pls produced by different mammalian cell lines transfected with the gene coding for human IFN-P1 revealed that they all contain different sets of N-linked sugar chains [87]. More than 80% of the sugar chains of natural IFN-fll have a biantennary structure. The remainder is composed of 2,4- branched (10%) and 2,-branched (8%) triantennary sugar chains. Recombinant IFN-Pl produced by Chinese hamster ovary (CHO) cells contains sugar chains mostly similar to those of the natural counterpart. However, it contains an increased amount of 2,-branched triantennary sugar chains (1%) with a slight decrease of biantennary sugar chains and the complete absence of S4-branched triantennary sugar chains. In addition to the increase of 2,-branched triantennary sugar chains, the appearance of a small amount of tetraantennary sugar chains (4-5%) was found in other recombinant IFN-Pls produced by mouse epithelial cells derived from breast carcinoma (C127) and by human lung carcinoma cells (PC8). 2,4-Branched triantennary sugar chains do not occur In PC8-cell-derived IFN-Pl as in the case of CHOcell-derived IFN-bl. Host-cell-dependent differences in glycosylation were observed not only in the antennary structures, but in the structures of outer chains and the trimahnosyl core. Natural and CHO-cell-derived IFN-P1 s were quite similar in that none of the sugar chains are bisected and contain only the Siaa2+ Gal group. in contrast, sialic acids in the sugar chains of C127- cell-derived IFN-P1 exclusively occur as the Siaa2+Gal group, and the Galal 4Gal group was extensively expressed. The most remarkable feature found in the sugar chains of PC8-cell-derived IFN-P1 is the presence of a bisected trimannosyl core, which is expressed in a fifth of the sugar chains. Organ-specific and species-specific expression of glycosyltransferases must be the enzymatic basis of the different sugar patterns of these recombinant IFN-PIS. Another factor, transformational change in the expression of glycosyltransferase, should also be considered as the background of this phenomenon, because all host cell lines used for the production of recombinant IFN-Pls are tumor cells. The phenomenon of altered glycosylation of proteins is widely found in various tumors Functional roles of the sugar chains of glycoproteins Accumulation of information on the structural characteristics of the sugar chains of glycoproteins has enabled us to consider their functional roles on a molecular basis. In this section, I will introduce only some of the topics.

12 494 A A- 1 I NeuAcaZ+Gal1+4GlcNAc1 *2Manalk A- 2 NeuAca2+Gall+4GlcNAc~1+ZManal CManB1+4GlcNAcf1+4GlcNAcOH N- 1 B c Galgl+4GlcNAc~l+ZManal Galgl+4GlcNAc1*2Manal ~an~l+4glcnacb1+4gl~naco,, Ga11+4GlcNAcf1+2Manal~ +4GlcNAc1+4,GlcNAcoH NeuAca2*Ga11*4GlcNa~~~+ZManal~~~~~~ A- NeuAca2+Gal~1+4GlcNAc~1+2Manal %Man B GlcNAc B 1 + 4G1cNAco, NeuAcaZ*GalB1+4Gl~NAc~l+2Mana~~ N-2 Gal 1 *4GlcNAC 1 *2Manul\ A- 4 +4GlcNAc B1 +4GlcNAcOH Gal 1 *4GlcNAc 1 +2ManalAManB1 GalBl+4GlcNAcBl+2Man~l~,Man 1 +4GlcNAC 1 *4GlCNAcOH NeuAca2+Gal~l+4GlcNAc1*2Manal A- 5 N- Manak,ManB1+4GlcNAcf1+4GlcNAc0H NeuAca2+GalB1+4GlcNAc~1+ZManal Maria% +4GlcNAcB 1 +4GlcNAcOH Gal 1-4GlcNAcB 1 +2ManalAManB1 Fig. 15. Structures of the sugar chains of hcg purified from the urine of pregnant women (A) and their desialylation products (B). N-linked sugar chains of glycohormones Four glycohormones are found in mammals. Three of them are produced by the anterior pituitary gland. They are luteinizing hormone and follicle-stimulating hormone produced by gonadotrophs [91, 921 and thyroid-stimulating hormone produced by thyrotrophs [9] of the gland. Only chorionic gonadotropin (CG) is produced by trophoblasts of the placenta [94]. All these four glycohormones are heterodimers consisting of a and p subunits. Studies of the amino acid sequences of the two subunits of the four glycohormones revealed that the a subunits have an identical structure within one animal species [95, 91. Because of this, it has been believed, without proof, that the hormones bind specifically to their target cells by their p subunit and inject their a subunit into the cells by endocytosis. However, structural information of their sugar moieties obtained recently has changed this well accepted concept of the action mechanism of glycohormones. Sugar chain structures were first elucidated on human CG (hcg). The two subunits of hcg contain two N-linked sugar chains [97, 981. The p subunit contains four 0-linked sugar chains in addition [98]. Endo et al. [8] investigated the structures of oligosaccharides released by hydrazinolysis from hcg purified from pooled urine of healthy pregnant women and found that five acidic N-linked sugar chains, shown in Fig. 15A, are included in hcg. A little later, Kessler et al. [99] reported that oligosaccharide A-1 is included in hcg. Detection of the five acidic complex-type sugar chains in hcg could be considered as a typical case of microheterogeneity. Namely, A-2-5 in Fig. 15A could be incomplete biosynthetic products of A-1. However, a study of the N-linked sugar chains of a and p subunits revealed that this is not the case. Desialylation of the five acidic oligosaccharides in Fig. 15A results in the production of the three neutral oligosaccharides shown in Fig. 15B. A comparative study of the desialylated oligosaccharide fractions from the two subunits revealed that the a subunit contains N-2 and N- in 1 : 1 molar ratio but no N-1, while the / subunit contains an equal amount of N-1 and N-2 but no N- [loo]. This result indicated that the two N- linked sugar chains of the a subunit are never fucosylated and one of them remains in the monoantennary state. In contrast, both N-linked sugar chains of the subunit mature to biantennary sugar chains but one of them is not fucosylated, while the other is mostly fucosylated. Therefore, even the smallest N- should not be considered as an incomplete biosynthetic product but is a final product at a particular site of the hcg molecule. The specific distribution of different sugar chains in the two subunits of hcg suggested that the steric structure of the polypeptide portion of hcg controls the maturation of its sugar moieties. A recent study of the N-linked sugar chains of the free a subunit supports this notion. Together with hcg, a very small amount of free a subunit is detected in the urine of pregnant women. In spite of having the same amino acid sequence as the a subunit of hcg, free a subunit cannot associate with the p subunit of hcg. In order to elucidate the molecular basis of this phenomenon, Kawano et al. [loll investigated the sugar chain of the free a subunit. Interesting evidence has been obtained that the subunit contains only one N-linked sugar chain. The structure of the sugar chain is a mixture of sialylated N-l and N-2. Based on this evidence, the biosynthetic mechanism to form the N-linked sugar chains of hcg can be estimated as shown in Fig. 1. Both a and p subunits of hcg produced in the rough endoplasmic reticulum of the trophoblasts of placenta have two tetradecasaccharides : Gl~,-Man~-(GlcNAc)~, and the sugar chains are soon converted to Man9-(GlcNAc);? by the action of a-glucosidases as described previously. During this early process, a small amount of a subunit accepts only one N-linked sugar chain, probably because the folding of its polypeptide moiety inhibits it from accepting another tetradecasaccharide from the dolichyl derivative. The occurrence of such steric inhibition of N-glycosylation by folding the polypeptide moiety was suggested from a structural study of ovalbumin. This glycoprotein contains two potential glycosylation sites Asn292- Leu-Thr and Asn1l-Leu-Ser in its polypeptide [102], but the

13 495 kgu. U'S 5?G' % Fig. 1. Maturation of the N-linked sugar chains of hcg and of free a subunit. S, sialic acid; G, galactose; M, mannose; F, fucose; GN, N- acct ylglucosamine. latter site is never glycosylated [10]. However, denatured ovalbumin can be further N-glycosylated by an in vitro system. Therefore, a non-glycosylated Asn 1 1-Leu-Ser group might be buried within the folded peptide before the tetradecasaccharide is transferred from the dolichyl derivative. An a subunit with two high-mannose-type sugar chains will associate with a p subunit which also has two high-mannose-type sugar chains. The four high-mannose-type sugar chains of the heterodimer will be processed to the oligosaccharide I11 in Fig. 10 until the dimer reaches the medial Golgi. The maturation of the four N-linked sugar chains of hcg will then be controlled by the steric effect of the two subunits. By this means, one N-linked sugar chain of an a subunit remains in the monoantennary state, while the other is converted to a biantennary sugar chain. Because of this effect, fucose is not added to the two N-linked sugar chains. In contrast, the control effect will allow the two N-linked sugar chains of a p subunit to become biantennary sugar chains, but prohibit one sugar chain from being fucosylated. The a subunit with one N-linked sugar chain cannot combine with a p subunit and therefore reaches the Golgi as a free a subunit. Since the maturation of its N-linked sugar chain is not controlled by the steric effect of the / subunit, it will grow to an oligosaccharide N-1 by the complete action of the glycosylation machinery of trophoblasts. The first data indicating the importance of the sugar chains of hcg for the expression of its hormonal action was presented by Moyle et al. [104]. They showed that removal of the sugar chains of hcg by sequential exoglycosidase digestion gradually reduced the ability of the hormone to stimulate the production of camp and testosterone by rat Leydig cells. Having been stimulated by this report, investigations of the effect of deglycosylation by either enzymatic or chemical means on the biological activity of hcg were reported by several research groups [lo As summarized in Fable 4, removal of sialic acid from hcg enhances the binding Table 4. Comparison of biological properties of hcg and its derivatives [104, 108, hcg Receptor-binding Stimulation of camp affinity production Intact Desialylated Deglycos ylated of the hormone to the receptor on the surface of target cells, but its hormonal activity is depressed to 50%. Removal of the remaining part of its sugar chains further enhances the binding of the hormone to the receptor up to twice that of the sialylated hormone, but deprives it completely of its hormonal activity. Based on this interesting evidence, we first investigated the role of sialic acid. As shown in Fig. 15A, all sialic acid residues of the N-linked sugar chains of hcg occur solely as the NeuSAca2+Gal group. This is probably because no a2,- sialyltransferase is expressed in the placenta. The exclusive occurrence of the NeuSAca2+Gal linkage raises the question of whether the linkage is important for the functional role of the sialic acid residues of hcg. In order to answer this question, isomeric hcg containing the NeuSAca2 + Gal group was prepared from desialylated hcg, by incubating it with commercial Ga1Pl-j 4GlcNAc : a2+ sialyltransferase and CMP-NeuSAc, and its hormonal activity in vitro was investigated [110]. As summarized in Fig. 17, addition of the NeuSAca2- residues to the desialylated hcg completely restored its hormonal activity to the level of natural hcg. This result indicated that the sialic acid linkage of hcg is not important although the presence of sialic acid residues is essential for the full expression of the hormonal activity of hcg.

14 49 Fig. 17. Doselresponse curves for the activation of adenyl cyclase in MA-10 cells on the addition of the control hcgs (0), desialylated hcg (0 in A), and the isomeric hcg (0 in B). Experiments were performed in triplicate and the data are presented as mean values standard errors. In thc absence of hcg, no CAMP was detected. As introduced in our previous reviews [88, 111, 1121, altered glycosylation is found in the hcg samples purified from the urine of patients with choriocarcinoma and invasive mole. Measurement of the hormonal activities of these hcg samples revealed that more highly sialylated samples show higher hormonal activity on a mass basis [11]. Furthermore, the hormonal activities of the desialylated samples revealed that the hcgs with more altered N-linked sugar chains show less hormonal activity, indicating that the structures of the neutral oligosaccharide portion are also important for the expression of full hormonal activity. Studies by Matzuk and Boime [114, 1151, using the site-directed mutagenesis of the two potential glycosylation sites in each subunit of hcg, revealed that the oligosaccharide at each site may have different functions with respect to the stability, assembly and biological activity of the hcg molecule. The function of the N-linked oligosaccharides of hcg may be expressed in either a direct or indirect manner. The report of Keutmann et al. [11] that a conformational change is induced in deglycosylated hcg may indicate that the oligosaccharides of hcg contribute to form the correct and restricted conformation of the hormone. Another possibility is that the sugar chains of hcg interact directly with some molecule, which is either a part of the hcg receptor or another molecule on the surface of target cells, simultaneously with the binding of the polypeptide moiety to its receptor. This dual binding might be essential for the expression of the hormonal action. This possibility is supported by the report of Calvo and Ryan [117], which indicated that the glycopeptides obtained from hcg by exhaustive pronase digestion specifically inhibit the activation of adenylate cyclase in target cells by the action of hcg. Recently, we have also found that sialylated complextype oligosaccharides show an efficient inhibition of hcg action (unpublished data). The interaction between the sugar chains and the suspected cell surface molecule might be extremely weak, because we have failed to detect any membrane proteins which bind to the oligosaccharides released from hcg by hydrazinolysis (unpublished data). Following hcg, the structures of N-linked sugar chains of human luteinizing hormone, human thyroid-stimulating hormone and human follicle-stimulating hormone have been elucidated [ These data indicated that the sugar chains of the four glycohormones are quite different. In view of the evidence obtained from the study of hcg so far discussed, the sugar chains of other glycohormones might also be essential for the expression of their biological activity. The data also indicated that the M subunit of the four glycohormones should no longer be considered the same. Functional role of N-linked sugar chains in immunology Recent developments in immunology indicated that cellular and humoral immunological systems are controlled by a complicated network connecting the interaction of immunocompetent cells. Many facts indicating the important roles of N-linked sugar chains in the construction of this network have been reported. Various kinds of blood cells are found within a closed blood-vessel system. Among them, lymphocytes leave the system at a capillary, enter a lymph vessel and return to a blood vessel through the thoracic duct [121]. This recirculation is considered to be essential for the immune functions of lymphoid cells. The passage of lymphocytes from blood to lymph is made by their specific interactions with endothelial cells lining the lymph node post-capillary venules [122]. Triggered by this interaction, lymphocytes are engulfed by endothelial cells and forced out. In 194, Gesner and Ginsburg [12] showed that the sugar chains of membrane glycoconjugates of lymphocytes play a role as the passport for passage through the endothelial cells. A recent sophisticated study using the techniques of cell biology, however, indicated that the luminal surface of high endothelial venules contains glycoproteins which are recognized by the lymphocyte homing receptor [124]. This homing receptor, which is currently called L-selectin, is found to be a calcium-dependent lectin [125]. Together with E-selectin and P-selectin, which are also related to endothelial cell leukocyte adhesion, L-selectin is grouped in the selectin family of cell adhesion proteins. E-selectin does not occur in endothelial cells but is formed on the surface of these cells by stimulation with various inflammatory mediators and induces the attachment of various leukocytes [12, P-selectin is stored in the Weibel-Palade bodies of endothelial cells [128] and the a granules of platelets [129], and elicited to the surface of the cells by stimuli such as histamine and thrombin. The ligand for L-selectin on the surface of endothelial cells is a complex

15 497 sugar chain with fucose, sialic acid and sulfate residues [10]. In view of the recent finding that sialylated X [Neu5Aca2-+Gal~1-+4(+)GlcNAc] and sialylated Lea [Neu5Aca2+Gal~1+(Fuccrl+4)GlcNAc] are possible candidates for ligand in E-selectin [11-141, elucidation of the exact structure of the ligand for L-selectin will lead to great progress in the molecular understanding of cellular immunity. Mutual interactions of immuno-competent cells are mediated by cell-surface glycoproteins encoded by genes within the major histocompatibility complex. Hart demonstrated that pretreatment of allogeneic stimulator cells with tunicamycin prevents their induction of the blastogenic response by thymic lymphocytes [15]. Since the mixed lymphocyte reaction has been considered as a model for cell-to-cell interactions which regulate various kinds of immune responses, this evidence indicates that the N-linked sugar chains on the surface of stimulator cells play an essential role in the cell-to-cell interactions involved in the regulation of immune responses. In addition to the direct interaction of cells, several glycoproteins secreted from immuno-competent cells work as soluble mediators. There is no evidence indicating that the sugar moieties of these glycoproteins play important roles in their biological functions. However, recent finding that interleukins 1 and 2 have lectin activities indicate that sugar-receptor interaction may also be included in their actions [1, 171. Immunoglobulin G (IgG) plays a major role in humoral immunity. This glycoprotein is unique among serum glycoproteins because it contains more than 0 different N-linked sugar chains [18]. This extremely high multiplicity is considered to be due to the fact that human individuals have a series of B-cell clones equipped with different sets of glycosyltransferases. Despite this complex nature, the ratio of each oligosaccharide of IgG samples purified from whole human sera is almost identical, indicating that the ratio of B- cell clones synthesizing IgG with different sugar chains in an individual is rather constant [19]. Recent success in degalactosylating IgG by Streptococcus 4 K P-galactosidase digestion has enabled us to compare the function of this glycoprotein before and after degalactosylation [140]. Interestingly, the degalactosylated human IgG binds less effectively to the subcomponent Clq of the first component of human complement and the Fc receptor. No decrease in binding to protein A was observed in the degalactosylated IgG. This result indicated that the function of IgG molecules can be modified by different degrees of maturation of their sugar moieties. Fractionation of IgG glycoforms and a comparative study of the function of each glycoform may open novel aspects of IgG research. The knowledge will no doubt be useful for the effective development of monoclonal antibodies for passive immunization. Use of recombinant glycoproteins,fbr the study of the,functional roles of sugar chains As introduced in the case of hcg, studying the effects of sugar chain removal by enzymatic or chemical means on the biological activities of glycoproteins is one of the strategies used to investigate the functional roles of sugar chains. For the effective use of this method, however, careful purification of the exoglycosidases to remove any contaminating proteases are essential. Each step of the trimming of the sugar chains is laborious and time-consuming. This is one of the major reasons for the slow pace in elucidating the function of sugar chains of glycoproteins. Recent developments in gene technology have opened a door to handle substantial amounts of bioactive glycoproteins, which occur in very minute amounts in the animal body. Since these recombinant glycoproteins are produced by various cells of different species by transfecting them with the aimed gene, different sets of sugar chains can be formed on the same polypeptide. This situation was exemplified by a comparative study of the interferon-pl samples produced by different mammalian cell lines transfected with, the gene coding for human interferon-pl [87]. A comparative study of the biological activities of these glycoproteins with different sets of sugar chains is now expected to be a new approach to elucidate the function of the sugar chains of a glycoprotein. As an example of such a study, recent work on recombinant human erythropoietin (EPO) will be introduced below. EPO is a hemopoietic hormone specific to cells of erythroid lineage [141]. Human EPO consists of 15 or 1 amino acids and contains three N-linked sugar chains and one 0-linked sugar chain. Total sugar chains amount to 40% of the molecular mass of this glycohormone. Although desialylated EPO was more active than the sialylated hormone, as measured by the bioassay system in vitro [142], it showed no in vivo hormonal activity [14]. Hence the functional role of the sugar chains of EPO have been attracting the attention of many investigators. Successful cloning of the structural gene of human EPO in 1985 opened a way to obtain a large amount of recombinant EPO [144, Sasaki et al. [14] and Takeuchi et al. [147] independently analyzed the structures of the N- linked sugar chains of recombinant EPO produced by Chinese hamster ovary (CHO) cells and of natural EPO purified from the urine of patients with aplastic anaemia. As summarized in Table 5, no qualitative difference was detected in the sugar chains of the two EPO samples, except that the sugar chain of the recombinant EPO contains only the NeuSAca2+Gal group while natural EPO contains both the Neu5Aca2-+Gal and the NeuSAca2+Gal groups. This structural similarity suggested the usefulness of the recombinant EPO as a drug to treat anaemia. During the course of studies on the effective production of EPO in recombinant CHO cell lines, a cell line, B8-00, was found to produce an EPO preparation with extremely low in vivo hormonal activity. Study of its sugar chains revealed that the EPO is enriched in biantennary sugar chains, in contrast to natural and highly active recombinant EPO in which tetraantennary sugar chains were major constituents [148] (Table 5). A comparative study of the sugar patterns and the in vivo activities of several preparations of recombinant EPO revealed that the activity was proportional to the ratio of tetraantennary to biantennary sugar chains [148]. The low in vivo activity of the EPO obtained from B8-00 cells was not due to a defect in the activation of the biological response at the receptor level, since its in vitro activity was considerably higher than that of natural EPO. Similar but more dramatic changes can be expected for the recombinant EPO preparations obtained by using cell lines of different species or of different organs in which more extensively altered glycosylation might be induced. Concluding remarks Illustration of a glycoprotein molecule by computer graphics shows that a sugar moiety occupies a large space, indicating the importance of the sugar chains in considering the function of glycoproteins. Evidence introduced in this

16 ~~ Table 5. Structures of the N-linked sugar chains of recombinant (r) EPO and natural EPO. R = GlcNAcPl+4(+)GlcNAc; R = GlcNAc/ll+4GlcNAc. Natural EPO has a2+ and a2+ linkages at the position marked by a single asterisk. The locations of N- acetyllactosamine rcpeat in the sugar chains of natural EPO have not been determined but are indicated by **. Structure Amount in repo with urinary EP with R R R R mo1/100 mol * (NeuSAccll+)1-2 GalPl+4GlcNAcPI +2Manal \ ManPl+4R/R \ Galfll+4GlcNAcPI +2Mancll 7 CalPl+4GlcNAc~1 +2Manal \ * Gal~l+4GlcNAc~I \ ManPl-+4R/R (NeuSA~cl2--+)~-~ 4 Mancll 7 2 GalPl+4GlcNAcPI * (NeuSAca2- )1 - Mancll \ 2 ManPl-t4R/R GalPl+4GlcNAc~l+2Manal 7 (NeuSAca2+?), -4 GalPl+4GIcNAc~I \ Mancll \ 2 GalPl+4GlcNAc~l 7, ManBl+4R/R Gal~l+4GlcNAc~l \ 4 7 Manal 2 ( Gal~l+4GlcNAc~l Gal~l+4GlcNAcP1 \ * GalPl+4GlcNAcPI + Manal \ * I 2, Gal~l+4GlcNAc~I 7 Man11 +4R/R Gal/ll+4GlcNAc~I (NeuSAc~2+)~ Mancll 2 Gal/1+4GlcNAcfiI 7 * (Neu5Accl2+), -2 GalPl +GlcNAcPI 2Gal/l1+4GlcNAc/1 \ Mancll \ 2 GalPl +GlcNAcfil +~GalP1+4GlcNAc~I 7 ManPl+4R/R GalPl +4GlcNAcpl \ 4 7 Mancll 2 Gal/1+4GlcNAc~I

17 499 review indicates that a study of the sugar moiety is essential for the sound development of gene technology and protein engineering. Based on an idea for elucidating the biological information included in the sugar chains of glycoconjugates, a novel scientific field called 'glycobiology' has recently been established. Such knowledge will be effectively used for the development of biotechnology in the future. The research of structures and functions of the sugar moieties of glycoproteins is one of the important pillars of glycobiology. Only a few of the possible topics for the functional study of the sugar chains of glycoproteins are introduced in this review. However, the topics are widely related to many other biological recognition phenomena that control life from fertilization through to aging of multicellular organisms. Because the biosynthesis of sugar chains is not controlled by the interpolation of a template, the structures of sugar chains are less rigid than those of proteins or nucleic acids. This means that sugar chains can be altered by the physiological condition of the cells. As shown by studying the glycoproteinsproduced by many tumor cells [88-90, 1491 and several diseases [150], such alterations may be the cause of various diseases. Accumulation of more such information will develop another sugarchain-related research field : glycopathology. As introduced here, many important techniques for the structural analysis of the sugar chains of glycoproteins have been established today. However, many problems still remain unsolved for the popular use of these techniques. One of the big problems is the lack of quality control of the lectins and glycosidases, which are essential for the fractionation and structural analysis of sugar chains. Most of these reagents are now commercially available. 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