Glycobiology in the field of aging research—introduction to glycogerontology

Biochimie 85 (2003) 13–24 www.elsevier.com/locate/biochi

Glycobiology in the field of aging research—introduction to glycogerontology > Akira Kobata * Tokyo Metropolitan Institute of Gerontology, 5-18-2 Tsurumaki, Tamashi, Tokyo 206-0034, Japan Received 23 September 2002; accepted 24 December 2002

Abstract In populations where demographies are shifting towards increased average age, the importance of gerontology is also increasing. The main purpose of gerontology is to elucidate the mechanisms of deterioration, which occur in various parts of the human body through aging, and use this knowledge to improve quality of life among the elderly. By the elucidation of the human genome, a revolutionary development is expected to occur in the field of medical science in the near future. Many important genes related to the aging processes of various organs have already been found and are expected to be useful in the future development of geriatric medicine. However, most of the proteins produced by the human body contain sugar chains, whose importance as biosignals for multi-cellular organisms was revealed by the recent development of the new field of glycobiology. Since sugar chains are formed as secondary gene products by the concerted action of glycosyltransferases, the structures of sugar chains are less strictly regulated than proteins. Accordingly, most of the biosignals associated with sugar chains are not essential for the maintenance of life itself, but are necessary to maintain the ordered social life of cells constructing multi-cellular organisms. Hence, investigation of structural changes of sugar chains that is caused by aging is expected to produce quite a lot of useful information pertaining to the elucidation of diseases induced by aging. This review will summarize our current knowledge of such changes found in the sugar chains of glycoconjugates resulting from the aging process. © 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. Keywords: Glycobiology; Proteoglycan; Glycolipid; Glycoprotein; Gerontology

1. Introduction As many proteins produced by mammalian cells contain covalently linked sugar chains, these proteins are known as glycoproteins. As in the case of proteins and nucleic acids, sugar chains within glycoproteins are constructed by joining units called monosaccharides. However, sugar chains have a

Abbreviations: Glc, glucose; Man, mannose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; GlcN, glucosamine; Fuc, L-fucose; Neu5Ac, N-acetylneuraminic acid; Cer, ceramide; CS-4, chondroitin-4-sulfate; CS-6, chondroitin-6-sulfate; KS, keratan sulfate; HA, hyaluronan; HABR, hyaluronan binding region; AD, Alzheimer’s disease; Ab, b-amyloid peptide; APP, amyloid precursor protein; PHF, paired helical filament; SPM, synaptic plasma membrane; IgG, immunoglobulin G; LCA, Lens culinaris agglutinin; WGA, wheat germ agglutinin; HME, hereditary multiple exostosis. > Dedicated to late Professor André Verbert, in memory of his excellent activity and leadership in the field of glycobiology and his warm friendship. * Corresponding author. E-mail address: [email protected] (A. Kobata).

characteristic feature, which is not found in proteins or nucleic acids i.e. the structural multiplicity formed by a limited number of units. For example, the disaccharide, ManGal, can form 16 isomeric structures, while only one structure can be made in the case of a dipeptide or a dinucleotide. In the cases of proteins and nucleic acids, when their number of units increases to three, or four, etc., only one structure can be formed because they are linear constructs. However, in the case of sugar chains, the number of isomers increases by geometrical progression, due to the branching that can be formed in the sugar chains larger than a disaccharide. Developments in cell biology in the 1960s suggested that sugar chains of glycoproteins and other glycoconjugates play important roles as the initiators of various cellular recognition signals, which are essential for the maintenance of the ordered social life of each cell within a multi-cellular organism. This presumption was substantiated for the first time in 1970 by the finding of the hepatic galactose binding receptor, which is responsible for the clearance of glycoproteins in the blood stream [1]. Since then, many lectins, which play roles

© 2003 Éditions scientifiques et médicales Elsevier SAS and Société française de biochimie et biologie moléculaire. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 3 0 0 - 9 0 8 4 ( 0 3 ) 0 0 0 0 3 - 8

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as the receptors of the sugar chains, were found in the animal kingdom [2]. Developments in gene technology in the past two decades have accelerated the study of the functional roles of the sugar chains of glycoproteins. With use of this technology, we can obtain substantial amounts of bioactive proteins, which may be useful for the development of new drugs but occur only in trace amounts in the animal body. However, many proteins produced by animal cells occur as glycoproteins. Since bacteria such as E. coli lack glycosylation machinery, recombinant proteins produced by using such hosts lack sugar chains. Since many of these non-glycosylated proteins do not express the expected biological activities, functional roles of the sugar chains have been attracting the interest of molecular biologists. In order to obtain recombinant proteins containing sugar chains, cultured animal cell lines are now used instead of bacteria. However, as evidenced by the comparative study of the sugar chains of c-glutamyltranspeptidases, which were purified from kidneys and livers of various mammals, both organ- and species-specific differences occur in the sugar chains of glycoproteins [3]. In addition, altered glycosylation phenomenon which is observed in the malignant cells [4], is also reflected in the sugar chains of recombinant glycoproteins because many of the cell lines used are, to some extent, malignant cells. Accordingly, structures of sugar chains of recombinant glycoproteins could vary according to the host cells used, despite having the same polypeptide structure [5]. This affords us a new approach to elucidate the function of the sugar chains of glycoproteins by investigating the comparative biological activities and sugar chain structures of recombinant glycoproteins [6]. The accumulated data obtained by the progress of studies of the sugar chains of glycoconjugates indicated that elucidation of the information included in sugar chains as well as those in nucleic acids and proteins is essential for the future development of biology. Based on such a trend in the bioscience world, a new life science field called glycobiology was launched in the mid 1980s [7]. Since the biosynthesis of the sugar chains is not controlled by the intervention of a template, structures of the sugar chains are much less rigidly defined than those of proteins and nucleic acids. This means that the sugar chains can be easily altered by the physiological conditions of the cells. Accordingly, age-related alteration of the sugar chains of various glycoconjugates could be an important element in solving various pathological problems found in elderly individuals. However, the number of papers reporting studies of glycoconjugates in relation to aging of animals is still quite limited. In this review, I would like to introduce readers to some prominent reports, and discuss prospects related to this new field of life science, which might be called glycogerontology.

Fig. 1. Structures of the basic disaccharide units of glycosaminoglycans. Except for HA, many minor modifications were found to occur in glycosaminoglycans.

2. Changes in the molar ratio of proteoglycans in mammalian tissues through aging The study of glycoconjugates as it relates to aging was introduced via the field of proteoglycans, which are essential elements for the construction of tissues. At the end of the 1960s, Iwata [8] investigated sugar compositions of proteoglycan preparations, extracted from the rib cartilage of 43 humans ranging in age from 10 weeks fetus to 79 years, and found that values change according to age namely, chondroitin (Fig. 1), which constitutes 10–20% of the whole glycosaminoglycan chains in the fetus, gradually decreases and almost disappear at least by 10 years of age. The amounts of chondroitin-4-sulfate (CS-4) and chondroitin-6-sulfate (CS-6), shown in Fig. 1, are approximately 40% at the fetal stage. Amounts of CS-6 remain almost constant during the remaining part of life, while amounts of CS-4 decrease rather quickly after 10 years of age. Alteration of sugar chains of proteoglycan through aging was investigated in more detail by Mathews and Glagov [9]. They found that the proteoglycan preparation of human rib cartilage at the fetal stage was mainly comprised of CS-4, CS-6, and chondroitin in the approximate molar ratio of 4:4:2. Only chondroitin disappears by the birth stage. After birth, CS-4 starts to decrease markedly and reaches 5% of the

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total glycosaminoglycan by the age of 40, while the amount of CS-6 did not change noticeably through aging. On the other hand, keratan sulfate (KS) in Fig. 1, which could not be detected at birth, linearly increases and reaches 60% of total glycosaminoglycan by the age of 40. The phenomenon in which the amount of CS-4 decreases through aging was also found in the articular cartilage of the knee by Greiling and Stuelsatz [10]. They reported that the amount of CS-4 in the cartilage decreases after birth at a rate of approximately 2 µg/g of cartilage in a year until 85 years, while the amount of CS-6 increases at the rate of 0.5 µg/year until 70 years, and then starts decreasing. There are several reports indicating that CS-4 decreases and CS-6 increases in the proteoglycan preparations obtained from the cartilages of other organs, and the ratio of KS to CS-4 and CS-6 increases with the onset of aging [11,12]. Therefore, the change found by the study of human rib cartilage is considered to occur widely in the cartilages of various portions of mammalian bodies. However, the physiological meaning of this change remains unknown. In view of recent development in studies on the functions of the sugar moieties of proteoglycans, we may be able to expect that the meaning of age-related change of proteoglycans will be elucidated in the near future. Although in much lower amounts, hyaluronan (HA), shown in Fig. 1, also occurs in the cartilage tissues. Holmes et al. [13] reported that the HA content in human articular cartilage increases from 0.5 to 2.5 µg/mg wet weight of the cartilage between the age of 2.5 and 86 years. Interestingly, the chain size of the HA decreases from a molecular weight of 2.0 × 106 to 3.0 × 105 over the same age range. However, no age-related change was observed in the size of newly synthesized HA, as determined by metabolic labeling with 3H-glucosamine, indicating that the age-related modification of the HA chain may take place in the extracellular matrix. The mechanism of such extracellular degradation is an important target for future investigation. Many proteoglycans of different molecular constructs have been reported to occur. Among them, aggrecan is the most widely distributed one in cartilages. The aminoterminal region of the core protein of aggrecan, which is called the G1 domain, forms an hyaluronan binding region (HABR). Aggrecan is so named because it forms an aggregate by binding to HA through HABR, in concerted action of the link protein. In the HABR of aggrecan, there is a marked increase in the level of N- and O-linked KS during aging [14]. Therefore, such a structural alteration may also be an important factor in causing tissue to age. Under the chemical conditions to dissociate the HAproteoglycan aggregates in the cartilages, proteoglycan monomers could be analyzed for various tissues. An agerelated reduction of the mean molecular size of proteoglycan monomers was observed by such a study [15]. A comparative study of the glycosaminoglycan chains of such proteoglycan mixtures revealed that the amount of chondroitin sulfates decreases, while the amount of KS increases through aging [16,17].

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Table 1 Major gangliosides found in the brain

The smaller proteoglycan monomers, which increase in the tissue through aging, appear to be bound to an increased number of HA chains with reduced chain length, and form an elevated number of smaller aggregates in the aged cartilages. For the readers who are interested in this field, I will list some reviews [18–22] published in more recent years. 3. Changes in the sugar chain pattern of glycolipids through aging Acidic glycosphingolipids, which contain sialic acids, are named gangliosides. Since they are abundant in the brain, their structures have been elucidated for the first time by using the samples obtained from the brain as summarized in Table 1. Since the brain is an organ related to dementia, which is one of the most important diseases in aging research because it severely deteriorates the patient’s quality of life, alteration of the sugar chains of gangliosides was considered to be an important target in gerontology. The following are representative publications picked up from a limited number of papers.

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In 1989, Svennerholm et al. [23] reported that the amount of total gangliosides in the frontal lobe of human brain increases prominently at birth, and remains at the same level until the age of 100. When the amount of each ganglioside was investigated, that of GM1 sharply increases at birth and remains at the same level during the entire life. The amounts of GD1b and GT1b increase until 5 years of age, and remain at the same level afterwards. In the case of GD1a, the amount increases up to the age of 5, and then starts to decrease. Alongside this pioneering report, many conflicting data were reported. There were many papers showing that ganglioside content decreases in human and small animal brains [24,25] in advanced age. The age-related decrease of gangliosides may be related to the functional decline of the brain-nervous system of elderly persons. Based on such an idea, the effect of ganglioside supplements has been investigated by many research groups. It was reported that administration of GM1 to senescent rats prevents decrease of their neurochemical parameters [26]. Systemic administration of GM1 increases choline acetyltransferase activity in the brains of aged rats [27]. It was also found that GM1 can alleviate genetic- and lesion-induced memory deficits [28], improve spatial learning and memory [29], restore in part, the neuropeptide deficits in the ventral homs [29], and restore abnormal responses to acute thermal and mechanical stimuli in aged rats [30]. Administering GM1 was also reported to show a limited ability to improve age-associated motor deficits of rats [31,32]. Another interesting aspect of GM1 is the finding of GM1-bound amyloid b-peptide (Ab) in the brains of early pathological stages of Alzheimer’s disease (AD), which may work as a seed for amyloid fibril formation [33,34]. I will come back to the topic of Ab for the pathogenesis of AD, later in this review. The fluidity of synaptic membranes in rat brains decreases through aging. However, the ratio of cholesterol/ phospholipids, which is widely accepted to correlate with membrane fluidity, does not change in the synaptic membrane through aging. Based on evidence that the amount of total gangliosides in synapses decreases via aging, it was proposed that such a decrease of gangliosides is the main cause of the decrease in fluidity of the synaptic membrane [35]. Actually, addition of ganglioside preparation, obtained from the brain of young rats, prominently increased the fluidity of the membrane of the synaptosomes which were obtained from the brains of old rats. It was further proposed that the decrease of fluidity of the synaptosome membrane, induced by the decrease of gangliosides through aging, declines mutual fusion of membranes. The decline may deteriorate the signal transduction by inhibiting the emission of acetylcholine [36]. An important paper, which contradicted the well accepted concept that gangliosides decrease through aging, was reported by Waki et al. [37]. They developed a sensitive new method to investigate gangliosides, and used it for the exquisite analysis of age-related change of gangliosides in the mouse brain. They found that synaptic plasma membrane (SPM) preparations contain approximately three

Fig. 2. Structures of C-series gangliosides. R = H, GT3; GalNAcb1, GT2; Galb1-3GalNAcb1, GT1c; Neu5Aca2-3Galb1-3GalNAcb1, GQ1c; Neu5Aca2-8Neu5Aca2-3Galb1-3GalNAcb1, GP1c.

times more gangliosides than cerebral cortices and synaptosomes on protein bases. When the ganglioside contents were investigated in the SPM preparations obtained from mice of different ages, it was found that the ganglioside level decreases until 6 months of age, and remains constant thereafter. When the amount of each ganglioside in SPM was investigated, it was found that those of GM1, GD1b, GT1a, GT1b and GQ1b, remained constant from the age of 1-30 months. In contrast, the amount of GD1a sharply decreases from the age of 1-6 months, and then remains at the same level afterwards. Therefore, the level of each ganglioside in SPM remains constant throughout adulthood and senescence. Based on these data, Waki et al. [37] determined that the results reporting the age-related decrease of brain gangliosides may imply the diminished density of synapses rather than compositional changes in SPM. Therefore, it was suggested that the effects found by the experiments of ganglioside supplement should be considered to be pharmacological, and are different from the physiological roles played by intrinsic gangliosides in the plasma membrane. In this respect, the findings that extrinsic gangliosides activate the voltage-dependent calcium channels in synaptic membranes [38], and induce the release of neurotransmitters [39] are important. Gangliosides can be classified into three structural groups: A-series, B-series, and C-series by the number of sialic acids, linked at the C-3 position of the galactose residue of the Galb1-4Glc group. The C-series gangliosides (Fig. 2) are expressed in fetal brains, but not in the adult brains. Successful development of monoclonal antibodies, which specifically recognize C-series gangliosides, opened a way for the investigation of the distribution of this series of gangliosides in the brain. An interesting finding is that the C-series gangliosides are detected in the dystrophic neurites of senile plaques, neurofibrillary tangles, and neurophil thread in the cerebral cortex of the brains of AD patients [40]. All these structures are considered to be pathological hallmarks of AD. Together with the C-series gangliosides, microtubuleassociated protein 5, which is also detected only in fetal brains, is detected in the AD brains. Therefore, expression of the fetal antigens seems to be one of the characteristics of AD brains. Possibly, regeneration or sprouting of neurons is occurring in association with the re-induction of gene expres-

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Fig. 3. The largest desialylated N-linked sugar chain detected in human serum IgG. The presence or absence of the underlined monosaccharides produces the multiplicity of the sugar chains.

sion, which is characteristic of brains at early stages of development. Recently, a series of monoclonal antibodies, each of which strictly recognizes a specific ganglioside, was developed [41]. By using these antibodies, Tai et al. [42] found that each ganglioside is distributed in a very strict manner in different parts of the brain. Investigation of the change in the distribution of each ganglioside in the brain through aging will, therefore, better elucidate brain aging, and may solve the problem of age-related change in the amount of gangliosides under debate. Other than the brain, age-related changes of gangliosides in the rat liver [43] and the human lens [44] were reported. Detection of neolacto-series gangliosides, containing the sialyl-LeX determinant in the human lens, is noteworthy. Recently, age-related changes of the ganglioside composition in human lens have been speculated to lead to the initiation and progression of cataract [45].

4. Changes in the sugar chain structures of IgG through aging Although many studies were reported to show the importance of the structural changes of sugar chains of glycoproteins during development [46], limited data are available on the changes in the sugar chains that take place during aging.

The first reliable data were obtained by the studies of the sugar chains of human serum immunoglobulin G (IgG). IgG is a glycoprotein composed of two types of polypeptide chains named heavy (H) and light (L) chains with a stoichiometry of H2L2. Among the approximately 2.8 mol of the N-linked sugar chains, detected in 1 mol of IgG samples purified from human sera, 2.0 mol are located in the Asn297 residues of the two H chains, and the remainder are linked to the variable regions of both H and L chains. A structural study of all of the sugar chains of human serum IgG revealed that several unique characteristics are included in this glycoprotein [47]. The largest sugar chain is the biantennary complex-type shown in Fig. 3 in its desialylated form. Only 25% of the sugar chains are sialylated. This is very unusual because the N-linked sugar chains of other human serum glycoproteins are highly sialylated. Another characteristic feature of the IgG sugar chains is the occurrence of extremely high microheterogeneity, which is produced by the presence or absence of the two galactoses, the bisecting N-acetylglucosamine and the fucose as underlined in the structure in Fig. 3. Therefore, human IgG is a mixture of a large number of glycoforms. In spite of this extremely high multiplicity, the molar ratio of each oligosaccharide included in the IgG samples obtained from the sera of healthy individuals is quite constant. As an example, the analytical data of three normal IgG samples are shown at the top in Table 2.

Table 2 Analytical data of the sugar chains of normal and myeloma IgG samples IgG Molecule Subclass Normal IgG Normal IgG Normal IgG Yot Kyo Han Ogo Tom Ike Saw Rov Dom Til Jir Gab Heb

– – – IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG2 IgG2 IgG3 IgG3 IgG4

Percent molar ratio Fractions N A1 75.7 18.0 75.9 18.3 75.2 18.7 75.2 23.0 77.6 21.4 76.6 21.1 83.7 15.7 88.1 11.9 54.3 42.2 57.6 33.7 81.6 17.7 65.8 26.5 89.9 9.4 88.8 11.2 89.3 10.7 77.1 18.9

A2 6.3 5.8 6.1 1.8 1.0 2.3 0.5 0 3.5 8.7 0.7 7.7 0.7 0 0 4.0

Percent of total oligosaccharides Bisected Fucosylated 18.2 86.4 18.4 84.5 18.1 86.2 7.8 75.2 14.3 82.6 23.3 83.4 28.8 85.8 18.2 88.8 10.2 85.8 29.8 86.6 18.1 73.3 58.4 92.2 5.7 92.3 9.3 81.2 8.9 77.5 10.3 82.3

Number of N-linked sugar chains mol/mol IgG 2.6 2.8 2.9 2.1 1.9 2.2 2.0 1.8 2.1 4.9 1.9 4.1 1.8 2.8 2.9 1.9

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Fig. 4. Proposed structures of the oligosaccharides released from three glycosylated Bence Jones proteins: Wh k, Sm k and Nei k.

In all the cases, the percent molar ratios of the neutral (fraction N), monosialyl (fraction A1) and disialyl (fraction A2) oligosaccharides were close to 76:18:6. The percent molar ratio of the bisected sugar chains was 18, and the percent molar ratios of fucosylated ones were 85–86. In contrast, the percent molar ratios of the oligosaccharides released from 13 myeloma IgG samples are not constant [48,49]. The fraction N ranged from 54% to 90%, fraction A1 ranged from 9% to 42%, and fraction A2 ranged from 0% to 9%. Oligosaccharides with bisecting N-acetylglucosamine were 6-58%. The values for fucosylated oligosaccharides were fairly constant, but also showed some variation. As listed in Table 2, most IgG myeloma proteins contain two N-linked sugar chains in one molecule. However, four IgG myeloma proteins contain larger amounts of sugar chains, indicating that variation also occurs in the number of sugar chains. The structural and numerical variations of the carbohydrate moieties of the monoclonal IgG samples are not correlated with their subclasses, which are also shown in Table 2. As already described, the extra oligosaccharides in the four myeloma IgGs are linked at the variable regions of the H and L chains. Indeed, some of the Bence-Jones proteins, free L chains detected in the sera of myeloma patients, are glycosylated. In contrast to the sugar chains of IgG, the oligosaccharides released from three glycosylated Bence-Jones protein samples are highly sialylated [50]. After desialylation, all of them were found to be almost single components. A comparative study of the structures of the oligosaccharides revealed that the three Bence-Jones proteins contain different sugar chains as shown in Fig. 4.

A comprehensive explanation for these analytical data is that the B cells are a mixture of clones, which are equipped with different sets and ratios of glycosyltransferases. The constancy in the ratio of each oligosaccharide of IgG, purified from the sera of healthy individuals, indicates that the ratio of B cells with different sets of glycosyltransferases is fairly constant in healthy individuals. The extremely high heterogeneity found in the sugar chains of myeloma IgGs might also be produced because the two H chains of IgG are coupled before their processed sugar chains start to mature in the Golgi apparatus. Steric effect of the coupled two H chains could inhibit the complete maturation of the sugar chains linked to the Asn297 residues. Although most of the galactose residues of the human serum IgG occur as non-reducing termini, it is difficult to remove them by the action of various b-galactosidases. This is probably because the galactose residues are buried in the polypeptide moiety of the Fc portion of IgG molecule [51]. So far, only Streptococcus 6646K b-galactosidase can successfully remove the galactose residues of IgG [52]. It was found that the degalactosylated human IgG binds less effectively to C1q and Fc-receptor. However, no change in its binding to polyclonal rheumatoid factor (IgM-type) and protein A was observed in the degalactosylated IgG. These results indicated that the sugar moiety, particularly the galactose residues of IgG are important for the expression of its functions in the Fc portion. An important observation is that the galactose content of human serum IgG decreases via aging [53,54]. This evidence may indicate that the population of B cells with less b-galactosyltransferase increases through

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aging. Hence, the alteration induced in the sugar chains of IgG can partly explain the phenomenon of immunodeficiency observed in aged individuals. 5. Alteration of the N-linked sugar chains of the glycoproteins in the brain through aging For the purpose of investigating the alteration of N-linked sugar chains of the glycoproteins in the brain through aging, Sato et al. [55] comparatively analyzed the glycoproteins in the soluble fractions and the membrane preparations of various portions of brains and spinal cords, obtained from 9 week-old rats and 29 month-old rats. After sodium dodecylsulfate-polyacrylamide gel electrophoresis, the proteins were transferred on a polyvinylidene difluoride membrane, and the glycoproteins on the membrane were reacted with biotin-conjugated lectins, which were selected to detect representative groups of N-linked sugar chains, followed by avidin-conjugated horseradish peroxidase. Detection of the bound peroxidase, by using 3,3’-diaminobenzamide tetrahydrochloride as a substrate, revealed that many age-related alterations occur in the glycoproteins of various portions of the central nervous system. The most prominent alteration was detected in the membrane fraction of spinal cords by staining with Lens culinaris agglutinin (LCA). A band, which moved to the front of the gel, was detected in the membrane preparations obtained from aged rats, but not in those of young adult rats. In the soluble fractions of white matter, basal ganglia, and spinal cord, glycoproteins with an apparent molecular weight of 123 and 115 kDa, as detected by wheat germ agglutinin (WGA), increased in the aged rats. In the membrane fractions of white matter, basal ganglia, hippocampus, cerebellum, and spinal cords, a glycoprotein with a molecular weight of 115 kDa, which was positively stained by WGA, increased in aged rats. In contrast, a Maackia amurensis agglutinin-positive glycoprotein with a molecular weight of 115 kDa, which is detected only in the membrane fraction of cerebellum, decreased in the aged rats. Glycoproteins with molecular weights of 133 and 125 kDa, detected by staining with LCA in the membrane fractions of white matter and basal ganglia, also decreased in the aged rats. The exact molecular weight of the LCA-positive glycoprotein, detected only in the membrane fraction of the spinal cord of aged rats, was determined to be 30 kDa by SDSPAGE using a higher concentration of gel, and was named gp30 [56]. Further investigation using a larger number of rats confirmed that gp30 is detected in the spinal cord membrane fractions of aged rats, but not in those of young adult rats. In order to determine the structure of gp30, it was digested by lysylendopeptidase and fractionated by reverse-phase HPLC. A glycopeptide fraction, which positively reacted with LCA, was collected and sequenced. The N-terminal amino acid could not be identified, but the following sequence of GSIVIHNLD was obtained for the N-terminal portion of the glycopeptide. A data-base search revealed that

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it was completely identical to the 110(G)–118(D) of rat P0 [57]. Since rat P0 contains a single N-glycosylation site at Asn122, the recovery of 109(D)–130(K) is expected as the glycosylated product of lysylendopeptidase digestion. Based on these results, gp30 was identified as P0. P0 is a member of the immunoglobulin superfamily and has been known as a major structural component of mammalian peripheral nervous myelin. Targeted disruption of the structural gene of P0 in mouse induced hypermyelination, which was characterized by a failure in the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons [58]. Furthermore, it was reported that several mutations in the gene of human P0 cause genetic neural disorders [59,60]. Therefore, a crucial role of P0 in the function of the peripheral nerve was expected [61]. It also had long been believed that P0 is not present in the central nervous system [62–64]. Therefore, detection of P0 in the rat spinal cord became the target of dispute. This is probably because the previous studies were never performed on aged rats. With respect to the absence of P0 in the spinal cord of young adult rats, two possibilities were considered. One is that the sugar chain structure of young adult P0 is different from that of aged rats, and cannot interact with LCA. The other possibility is that the P0 molecule is expressed only in the spinal cord of aged animals. In order to find out which alternative is the correct one, a polyclonal antibody which specifically recognizes the C-terminal sequence of rat P0 was prepared. Western blot analyses of the membrane proteins of spinal cords and sciatic nerves by using this antibody revealed that P0 was detected in the spinal cords as well as sciatic nerves of both aged and young adult rats [56]. Therefore, P0 does exist in the spinal cord membrane of young adult rats. For further confirmation of the occurrence of P0 in the spinal cord of young adult rats, the occurrence of a single transcript of the gene (2.3 kilobases) was investigated. By northern blot analysis using the probes for N-terminal (1–199) and central portions (265–466) of nucleotide sequences of rat P0 gene, such a transcript was detected in both poly(A)+RNA samples obtained from the spinal cords of aged and young adult rats [56]. The commercial product named ‘‘glycoprotein detection kit’’, provided by Amersham Pharmacia Biotech Co., facilitates the detection of glycoproteins on a polyvinylidene difluoride membrane. On investigating with the aid of this kit, it was revealed that the P0 in the spinal cord of aged rats and sciatic nerve contain sugar chains, while the P0 of the spinal cord of young adult rats does not [56]. Therefore, the absence of sugar chains in the P0 in the spinal cords of young adult rats caused its negative reaction with LCA. A cell line, which shows strong homophilic adhesion, was obtained by P0 cDNA transfection into C6 glioma cells. The addition of glycopeptide fragment obtained from P0 strongly inhibited the adhesion of cells. In contrast, the corresponding peptide without sugar chain showed much lower inhibition

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Fig. 5. Production of Ab species from APP. CHOs represent N-linked sugar chains.

[65], indicating that the sugar moiety of P0 plays a very important role in homotypic cell adhesion. It was also found that a non-glycosylated P0, obtained by site-directed mutagenesis, was not involved in homotypic adhesion [66]. Therefore, the biological meaning of the occurrence of nonglycosylated P0 in the spinal cord of young adult rats is an interesting target to be elucidated in a future study. Elucidation of the mechanism, that starts the glycosylation of spinal cord P0 through aging, is also an interesting target for future study. In view of the presence of other glycoproteins containing N-linked sugar chains in the spinal cords of young adult rats, the mechanism should be working only for P0 or proteins of limited number. In any event, elucidation of the background of this interesting phenomenon will surely provide ample useful data not only for finding out the functional roles of the sugar chains, but also for understanding the molecular events that occur during aging.

6. Future prospects As already discussed, dementia is one of the most important targets of aging research. By microhistological studies of the brains of patients with AD, occurrence of senile plaques and neurofibrillary tangles together with the extensive loss of neuronal cells were found to be highly correlated events associated with this disease. It was found that Ab is the major component of senile plaques [67]. A structural study of Ab and subsequent cloning of the gene, which includes the code of the amino acid sequence of Ab, revealed that the sequence is included in a membrane glycoprotein called amyloid precursor protein (APP) [68]. Since a large amount of APP is produced in the brains of healthy individuals at almost the same level as in those of AD patients, elucidation of the

mechanism to induce an abnormal cleavage of APP, leading to the production of Ab, was considered the key in this line of study. Although AD is predominantly a disease occurring late in life, there are families in which AD is inherited as an autosomal dominant disorder in mid-life. By searching the genes of familial ADs, presenilin 1 gene and presenilin 2 gene were picked up in addition to the gene of APP as causative genes of early onset ADs [69,70]. Both presenilins are multiple-transmembrane spanning proteins [71]. Currently, it is proposed as a scenario that APP is first cleaved at its b-site by b-secretase [72–75], forms a complex with presenilins, and is cleaved at the c-site by the protease postulated to reside in presenilins [75,76] (Fig. 5). The c-site cleavage generates a variety of Ab species: predominantly a 40-amino acid peptide, Ab1-40, with a smaller amount of a 42-amino acid peptide, Ab1-42. The latter peptide is more prone to form amyloid deposits. Mutations in all three genes, which were detected by the studies of familial ADs, alter the processing of APP to produce more Ab1-42. Although two potential N-glycosylation sites were detected in the extracellular portion of APP, which is close to the N-terminal portion of the Ab fragment as shown in Fig. 5 [68], and moreover, the structural study of the sugar chains of recombinant human APP was reported [77], no investigation has been made on the effect of the sugar chains on the abnormal cleavage. In view of the case of P0 described already, studies of the structures of the sugar chains of APP and their age-related alteration may result in another new aspect of production of Ab1-42 from APP. The major fibrous components of neurofibrillary tangles are paired helical filaments (PHFs). PHFs are composed of a microtubule-associated protein, called tau, in an abnormally hyperphosphorylated state [78,79]. The majority of the PHF-

A. Kobata / Biochimie 85 (2003) 13–24

tau polymerization property is induced by the abnormal phosphorylation at serine 262 in the human protein [79]. In order to find out that hyperphosphorylation of tau is actually the main cause of AD, tau kinase was thoroughly investigated [80]. However, the enhancement of tau kinase has so far not positively correlated with AD. Hart’s group found that the tau in the brains of normal animals is extensively modified by O-N-acetylglucosaminylation (O-GlcNAc) instead of phosphorylation [81]. Therefore, it is possible that the hyperphosphorylation of PHF-tau is the direct result of defective regulation resulting from either increased activity of cytosolic b-N-acetylglucosaminidase [82], or decreased activity of O-GlcNAc transferase [83]. O-GlcNAc residue is also found on the cytoplasmic domain of APP [84]. Therefore, O-N-acetylglucosaminylation may also be involved in the production of Ab1-42 from APP. Hereditary multiple exostosis (HME) is an autosomal dominant disorder characterized by the development of cartilage-capped exostosis in the epiphyseal ends of long bones. Three genetic loci; called EXT1, EXT2 and EXT3 were found to be responsible for the development of HME [85,86]. It was found recently that EXTs encode for the glycosyltransferase required for the synthesis of heparan sulfate (Fig. 1) [87]. On investigating the transgenic expression of the EXT2 gene in developing chondrocytes, it was found that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation [88]. These recent developments in the understanding of bone formation, in which proteoglycans are actively involved, suggested that the age-related alteration of proteoglycans, as already described in a previous section, may be important in solving the mechanism of joint problems of aged people. Recently, we noticed that glycobiology has been making large strides in the field of cellular immunology. Selectins were found by those studies to elucidate the mechanisms which facilitate the recirculation of lymphoid cells from the intravascular compartment to lymph nodes. L-Selectin, which is expressed on the surface of leucocytes, is considered to play an essential role for the homing of peripheral lymphocytes [89]. The structure of its ligand was recently elucidated as sialyl-6-sulfo-Lex [90,91]. P-Selectin, which is expressed on the surfaces of activated platelets, megakaryocytes, and vascular endothelial cells, plays an important role in the early phases of leukocyte recruitment [92], and adherence of activated platelets to neutrophils, monocytes, natural killer cells, and some subsets of T lymphocytes [93]. The ligands of P-selectin are rather complicated. It recognizes both tyrosine sulfate residues and the sialyl-Lex residues on the target cell surface glycoproteins [94,95]. Many intercellular mediators of glycoprotein origin were found to be playing roles in controlling the T-cells and B-cell network. It was determined that the sugar chains work as ligands of these mediators, and initiate their signal transductions [96–99]. Therefore, agerelated alteration of the sugar chains of the surface glycopro-

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teins of immunocompetent cells, in addition to the agerelated alteration of the N-linked sugar chains of IgG described already, may be an important background of the age-related suppression of immunological system in humans. As already discussed, sugar chains can be altered by the physiological conditions of cells. Accordingly, agerelated alteration of sugar chains of various glycoconjugates is likely to be an important target for solving other various pathological problems found in aged individuals. Therefore, gerontology would be a gold mine for glycobiological studies.

Acknowledgements I would like to thank Dr. Susumu Ando and Dr. Tamao Endo of the Tokyo Metropolitan Institute of Gerontology for critically reading this manuscript. Thanks are also due to Mr. Derek Adelman for kindly editing this manuscript.

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