Evidence for the presence of major peripheral myelin glycoprotein P0 in mammalian spinal cord and a change of its glycosylation state during aging

Yuji Sato, Mika Kimura, Chie Yasuda, Yasuko Nakano1, Motowo Tomita1, Akira Kobata2 and Tamao Endo3

Department of Glycobiology and 2Director's Office, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan and 1Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-0064, Japan

Received on September 3, 1998; revised on January 4, 1999; accepted on January 8, 1999

Glycoproteins, which react with Lens culinaris agglutinin, in the membrane preparation of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis. In contrast to the samples from brain, which showed similar staining patterns in the two different age groups, the glycoprotein patterns of spinal cords showed marked differences by the age of donors. The most prominent evidence is that a glycoprotein with an apparent molecular weight of 30 kDa (gp30) was detected in the aged rats, but not in the young adult rats. Based on the amino acid sequence data around the glycosylation site, the gp30 was identified as P0, which is a member of immunoglobulin superfamily and a major structural component of mammalian peripheral nerve myelin. This is the first report indicating that P0, which has been considered as a peripheral nerve-specific glycoprotein, occurs also in the spinal cord of mammals. In addition, nonglycosylated P0 molecule could be detected in the spinal cord of young adult rats by anti-P0 polyclonal antibody. These results indicate that the glycosylation state of the P0 molecule in the spinal cord changes during aging.

Key words: aging/glycosylation change/P0/spinal cord

Introduction

Proteins produced by eukaryotic cells are frequently modified posttranslationally by addition of glycans. The glycan moieties of the glycoproteins play important roles not only in modulating the properties of protein molecules, such as stability and activity, but also work as the key elements of various molecular recognition processes found in development and aging (Rademacher et al., 1988; Kobata, 1992; Varki, 1993). In the development of the brain, nervous system glycans have been implicated as important mediators of adhesive interactions among neural cells (Schachner and Martini, 1995). For example, polysialic acid (PSA) is a developmentally regulated glycan composed of a linear homopolymer of [alpha]-2,8-linked sialic acid residues (Rutishauser and Landmesser, 1996). The neural cell adhesion molecule, NCAM, mainly carries PSA, and shows striking spatiotemporal specificity of its expression patterns. The length of this large, negatively charged glycan modulates the adhesive properties of NCAM, and thus perhaps regulates neurite outgrowth and cellular mobility (Rutishauser and Landmesser, 1996). These evidences suggest that the structure of the glycan is very important in the developing nervous system.

Although many studies testify to the importance of the structural changes of glycans during development (Varki, 1993), limited data are available concerning the alteration of glycans during aging. Among them, the most well-known is that loss of sialic acid on red cell membrane proteins, probably resulting in eventual turnover of the red cells (Cohen et al., 1976; Seaman et al., 1977; Schauer, 1982). Another example is that we and others showed previously that the galactose content of human serum IgG decreases with age (Parekh et al., 1988; Tsuchiya et al., 1993). Since degalactosylated human IgG binds less effectively to C1q and Fc-receptors than does galactosylated IgG (Tsuchiya et al., 1989), this alteration may partly explain the phenomenon of immunodeficiency observed in aged people. A change of chondroitin sulfate and keratan sulfate of proteoglycan in cartilage has also been reported (Mathews and Glagov, 1966; Roughley et al., 1981; Barry et al., 1995), but the physiological roles of these changes remain to be clarified in detail. Because the biosynthesis of glycans is not controlled by the interaction of a template and depends on the concerted action of glycosyltransferases and glycosidases in endoplasmic reticulum and Golgi apparatus, the structures of glycans are much less rigid than those of proteins and nucleic acids. Therefore, the structures of glycans can be easily altered by physiological conditions of the cells. Accordingly, age-related alterations of the glycans of various glycoproteins are relevant to the understanding of pathological conditions found in aged individuals.

As the first step toward such a study, we used lectins to find out if any changes occur in the glycoproteins of brain during aging. Our results indicated that the glycoprotein P0, which has been considered to occur strictly in peripheral nerve, is also detected in the spinal cord of rat. This is the first report giving evidence that P0 is present in the mammalian spinal cord. In addition, we found that the glycosylation pattern of the spinal cord P0 changes during aging.

Results

Difference in Lens culinaris agglutinin (LCA)-staining patterns between the glycoproteins in spinal cords of young adult and aged rats

Brains of a young adult (9-week-old) and an aged (29-month-old) rats were separated into white matter, gray matter, basal ganglia, hippocampus, and cerebellum. The membrane fractions of these five brain portions and of spinal cords were prepared as described in Materials and methods. After SDS-PAGE, proteins of the six membrane preparations were transferred on PVDF membranes and stained with Lens culinaris agglutinin (LCA). It is well known that the lectin binds specifically to the following glycan structures (Debry et al., 1981; Kornfeld et al., 1981) :

Each brain portion from a young adult rat and an aged rat showed very similar staining patterns with a heavily stained band at 80 kDa (unpublished observations). In contrast, the staining patterns of spinal cords from a young adult rat and an aged rat were quite different (Figure 1). Reactivity of LCA with a 30 kDa band was observed only in the membrane of spinal cord of an aged rat (Figure 1, lane 2), and no such band was detected in the spinal cord of a young adult rat (Figure 1, lane 1). Accordingly, the glycoprotein was named gp30.


Figure 1. Western blot analysis of the membrane glycoproteins of rat spinal cord using Lens culinaris agglutinin. The membrane fractions of spinal cord from a 9-week-old rat (lane 1) and from a 29-month-old rat (lane 2) were analyzed by SDS-PAGE using a 15% gel. After electrophoresis, the proteins were transferred on a PVDF membrane, and then stained with Lens culinaris agglutinin (LCA) as described in Materials and methods.


Scheme 1.


Figure 2. High-performance liquid chromatography elution profile of the proteolytic peptides of gp30, and detection of LCA-reactive fraction with ELISA. Digests of gp30 were prepared and separated using Wacopak as described in Materials and methods. The binding activity of each fraction with LCA was also determined as described already. Solid line indicates reversed-phase HPLC elution profile of digested gp30, with absorbance detected at 215 nm. Dotted line indicates the concentration of acetonitrile. Solid bar indicates the LCA reactivity of each fraction indicated by the absorbance at 492 nm. The peptide fraction indicated by an arrow was subjected to amino acid sequencing.

Age-related appearance of gp30 in the spinal cord

In order to find out whether the presence or absence of gp30 is the phenomenon of individual difference, analyses were made on the spinal cord membrane fractions from seven additional young adult and eleven aged rats. The band of gp30 was not detected in the young adult rat samples, but was detected in eight of eleven aged rat samples (data not shown). In order to elucidate whether this interesting phenomenon is age-related or not, we analyzed the spinal cord membrane fractions prepared from ten each of 6-month-old, 12-month-old, and 24-month-old rats. The band of gp30 was detected in three 6-month-old and 12-month-old and six 24-month-old rat samples. Therefore, it was concluded that the expression of LCA-positive gp30 in the rat spinal cord is an age-related phenomenon.

Characterization of gp30

To characterize gp30 in further detail, the partial amino acid sequence of gp30 was determined. The peptide mixture obtained from gp30 digested with lysylendopeptidase was fractionated by reversed-phase HPLC (Figure 2). The reactivity of each fraction with LCA was determined by ELISA. Specific reactivity with LCA was observed in the fraction that eluted at 35 min (arrow, in Figure 2). It is noteworthy that LCA reactivity of a fraction that eluted at 60 min was observed even in the case of PVDF membrane pieces containing no protein bands, indicating that this could be due to nonspecific binding. The amino-terminal amino acid sequence of this glycopeptide was determined as XGSIVIHNLD. A database search of protein sequence revealed that the partial amino acid sequence of gp30 was completely identical with 110(G) - 118(D) of rat P0 (Lemke and Axel, 1985), as shown in Figure 3. Since rat P0 contains a single glycosylation site [122(N)], recovery of the glycopeptide [109(D) - 130(K)] is expected as the lysylendopeptidase digestion product with LCA reactivity. In fact, the first nine amino acids were identical to the predicted sequence as described above, except for the N-terminal amino acid, 109(D), which could not be determined. Based on these results, gp30 was identified to P0.


Figure 3. Comparison of the amino acid sequence of rat P0 and amino-terminal amino acid sequence of the LCA-reactive gp30 peptide. The sequence of rat P0 has been taken from Lemke and Axel (1985). The one-letter amino acid notation is used. X indicates that it could not be identified. The position number of the residue at the right end of each line corresponds to that of rat P0 (Lemke and Axel, 1985).

Presence of P0 in the spinal cord of young adult rats

An interesting fact is that gp30(P0) was detected by LCA staining only in the spinal cords of aged rats, but not in those of young adult rats. Two possibilities can be considered to explain this phenomenon. One is that the glycan structure of young adult P0 is different from that of aged rats, i.e., the latter carries a glycan structure that can be recognized by LCA as shown above, and the former does not. If this is the case, it may indicate that the glycan structure of mammalian spinal cord P0 changes during aging. The other possibility is that the P0 molecule is expressed only in aged rats. In order to find out which of these possibilities is correct, we prepared a specific polyclonal antibody against the C-terminal sequence of rat P0 as described in Materials and methods. When we probed Western blots of the membrane proteins of spinal cord and sciatic nerve with the anti-P0 polyclonal antibody, reactivity of the antibody with a 30 kDa band was observed in sciatic nerve (lane 1 in Figure 4), and in spinal cords of both 9-week (lane 2 in Figure 4) and 29-month-old rats (lane 3 in Figure 4). The reactivity was inhibited completely by the synthetic peptide that was used for immunization but not by unrelated peptides (data not shown). These results indicated that P0 does exist in the membrane of spinal cord of young adult rats, and suggested that its glycosylation state may have changed during aging. It should be noted that neither the membrane fractions of the cerebrum nor those of cerebellum was stained with the anti-P0 polyclonal antibody at all (lane 4 and lane 5 in Figure 4, respectively). When Northern blot analysis of poly(A)+ RNA samples from the spinal cords of young adult and aged rats was performed, a single transcript of P0 (2.3 kilobases) from both samples was detected using the probes for N-terminal and central portions of rat P0, nucleotide sequences 1-199 and 265-466, respectively (data not shown). These results also suggested that P0 is produced in the spinal cords of both young adult and aged rats.

Presence of non-glycosylated P0 in spinal cord of young adult rats

In order to determine the presence or absence of glycan moiety on P0 of the spinal cord of young adult rats, we performed the glycoprotein analysis by using periodate oxidation methods as described on Materials and methods. Although the P0 molecules of the spinal cord of aged rats and of sciatic nerve were shown to contain the glycans, the P0 in the spinal cord of young adult rats did not (Figure 5). These results clearly indicated that the P0 of the spinal cord of young adult rats exists as a nonglycosylated form and that the P0 molecules in the spinal cord of aged rats and in sciatic nerve contained the glycans. In addition, the above observations suggested that the P0 molecule in the spinal cord is gradually glycosylated during the aging process.


Figure 4. Western blot analysis of the membrane proteins of rat cerebrum, cerebellum, spinal cord and sciatic nerve using anti-P0 polyclonal antibody. The membrane fractions of rat sciatic nerve (lane 1), and the spinal cords of 9-week (lane 2), 29-month-old (lane 3), 29-month-old cerebrum (lane 4) and cerebellum (lane 5) were analyzed by SDS-PAGE using a linear gradient gel (10-20%) as described in Materials and methods. After electrophoresis, the proteins were transferred on a PVDF membrane, and then stained with the anti-P0 polyclonal antibody as described in Materials and methods.


Figure 5. Detection of glycans of the membrane proteins of spinal cord and sciatic nerve. The membrane fractions of rat sciatic nerve (lane 1), and the spinal cords of 9-week (lane 2) and 29-month-old (lane 3) rats were subjected to SDS-PAGE using 15% acrylamide gel as described in Materials and methods. After electrophoresis, the proteins were transferred on a PVDF membrane, and then subjected to glycoprotein analysis as described in Materials and methods.

Discussion

P0 is a member of immunoglobulin superfamily and is the major structural component of mammalian peripheral nerve myelin. Targeted disruption of the P0 gene in the mouse induced hypomyelination, which was characterized by a failure in the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and continued integrity of associated axons (Giese et al., 1992). Furthermore, it was found that several human P0 gene mutations cause genetic neural disorders with severe clinical symptoms such as Charcot-Marie-Tooth, Dejerine-Sottas syndrome, and congenital hypomyelination (Hayasaka et al., 1993a,b; Warner et al., 1996). These data emphasized the crucial role of P0 in the function of the peripheral nerve (Choe, 1996). It also has been believed that P0 is not present in tissues of the central nervous system (CNS), including the brain and spinal cord (Trapp et al., 1979; Ishaque et al., 1980; Uyemura et al., 1992). Therefore, the finding of the occurrence of P0 in the spinal cord of rat is noteworthy. The reason why P0 was not previously detected in the spinal cord is not clear. Since the final conclusions in previous studies (Trapp et al., 1979; Ishaque et al., 1980) were made on the basis of immunohistochemical data, the epitope specificity of their antibody may underlie this discrepancy. Another possibility that should be considered is stage-specific differences; we used older rats (i.e., 9-week-old) than examined in the previous study (7-day-old rats).

It was previously reported that the glycan moiety of P0 plays a very important role in cell-cell adhesion via homophilic binding, because the P0 peptide fragment containing glycan inhibited cell adhesion to a greater extent than the corresponding peptide without glycan (Yazaki et al., 1992) and the nonglycosylated P0 produced by site-directed mutagenesis did not show homophilic adhesion (Filbin and Tennekoon, 1993). Therefore, the appearance of glycosylated P0 during aging may be relevant to the understanding of the biological and functional significance of P0 in the spinal cord. Although the age-dependent mechanism by which P0 becomes glycosylated remains to be elucidated, several possibilities could be considered. The first possibility is that the activity of a dolichyldiphosphoryl oligosaccharide:polypeptide oligosaccharyltransferase, which resides in the endoplasmic membranes and is responsible for the initiation of N-glycosylation of protein (Silberstein and Gilmore, 1996), might increase in aged spinal cord. The second is that the activity of an uncharacterized "peptide: N-glycanase," which could cleave the glycan moieties from glycoproteins and change them to non-glycosylated proteins, might decrease in aged spinal cord. Recently, such a site-specific glycosylation-deglycosylation modification of peptide portion has been reported in ovalbumin produced in hen oviduct (Suzuki et al., 1997), although the biological meanings of this interesting phenomenon await further elucidation. The third possibility is that membrane localization of P0 may change during aging, resulting in production of glycosylated species of P0 due to altered accessibility to glycan processing enzymes, including "peptide: N-glycanase."

Phylogenetic studies revealed that central as well as peripheral myelin of some fishes contains P0-like glycoproteins (Takei et al., 1993). In the case of amphibians, adult axolotl and African clawed toad contain P0-like glycoproteins in their CNS (Takei and Uyemura, 1993). Interestingly, whereas interspecific variations in glycosylation patterns are widely found (Kobata, 1992; Varki, 1993), the P0[prime]s of mammals, fish and amphibians are all stained with LCA (Uyemura et al., 1992; Takei et al., 1993; Yazaki et al., 1994), indicating that their glycan moieties are phylogenetically conserved. The LCA-reactive glycan possibly may be required to express the functional roles of P0. Information as to the structure of the sugar moiety of glycosylated P0 is limited. It was recently reported that bovine peripheral nerve P0 has a bisected glycan containing an HNK-1 epitope composed of glucuronic acid and sulfate (Voshol et al., 1996). Therefore, more detailed structural study will be required to understand precisely the functional roles of the glycan moiety of P0.

P0 shows neurite-promoting activity (Takei and Uyemura, 1993), and it is therefore possibly involved in the regeneration of the nervous system. Actually, P0 is highly expressed in regenerable tissues such as CNS in lower vertebrates and peripheral nerves in mammals. Whether appearance of glycosylated P0 in the spinal cord during aging is of functional significance, e.g. remyelination, remains to be clarified. Occurrence of age-dependent alteration in the glycan moiety of peripheral nerve P0 was reported by Brunden (1992), although details of structural changes of the sugar chain have not been elucidated, suggesting that the glycan structure might be regulated by alterations in physiological conditions.

A comment is needed for the fact why there was no-shift in apparent molecular mass (Mr) of P0 with aging by SDS-PAGE (lanes 2 and 3 in Figure 4). Although it is not clear the reason at this stage, it is noteworthy that glycosylated P0[prime]s (lanes 1 and lane 3) showed broadening and tailing of the band, but nonglycosylated P0 (lane 2) did not. We cannot completely deny additional posttranslational-modifications, which affect on the migration of P0, to glycosylation during aging.

To our knowledge, this may be the first documented case where a protein is nonglycosylated in early development and then becomes glycosylated later in the animal's life. Elucidation of the background of this phenomenon will provide many useful data not only for finding out the functional roles of glycan, but also for the understanding of the molecular events which occur during aging.

Materials and methods

Chemicals and lectins

Biotin-labeled Lens culinaris agglutinin (LCA) was purchased from Seikagaku Corporation (Tokyo, Japan). Vectastain ABC kit and biotin-conjugated goat anti-rabbit IgG were purchased from Vector laboratories (Burlingame, CA). The reversed-phase column, Wacopak, and lysylendopeptidase of Achromobacter lyticus were obtained from Wako Pure Chemical Industries (Osaka, Japan). Glycoprotein detection kit and enhanced chemiluminescence (ECL) system were purchased from Amersham Pharmacia Biotech (Buckinghamshire, England). Rat P0 carboxy terminal polypeptide (CLYAMLDHSRSTKAASEKKSKGLGESRKDKK) was kindly provided by Dr. Toshiyuki Inazu (Noguchi Institute, Tokyo). Rabbit anti-P0 C-terminal polypeptide polyclonal antibody was obtained by immunization with the polypeptide conjugated with Keyhole lympets hemocyanin. All other reagents were of the highest quality available.

Preparation of membrane fractions from rat brain

The cerebrum, cerebellum, spinal cord, and sciatic nerve were isolated. Each sample, thus obtained from 9-week and 29-month old female Fisher rats, was homogenized with 10 ml/g wet tissue of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA and 250 mM sucrose buffer (SET buffer) in a Potter homogenizer. The homogenates were centrifuged at 100,000 × g for 1 h at 4°C, and the pellets obtained were suspended in SET buffer. Protein was determined by the method of Lowry et al. (1951).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis

SDS-PAGE was performed on a 15% slab gel or a linear gradient gel (10-20%, Bio-Rad Laboratories, Richmond, CA) as described by Laemmli (1970). Samples of 15 µg proteins were applied on each lane. In the case of sciatic nerve, 2.5 µg of the sample was used. After electrophoresis, the proteins were transferred on a polyvinylidene difluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). Western blot analysis using lectins was performed as follows. After blocking with 3% bovine serum albumin (BSA) in 10 mM Tris-HCl, pH 7.4, containing 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2 (TBS) for 30 min at room temperature, the membranes were incubated with biotin-conjugated lectins each in TBS containing 0.05% Tween 20 and 1% BSA (TTBS) for 1 h at room temperature. The PVDF membranes were then incubated with avidin-conjugated horseradish peroxidase (HRP) for 1 h at room temperature, and finally with the peroxidase substrate, 3,3[prime]-diaminobenzamide tetrahydrochloride, to detect lectin-reactive glycoproteins.

Western blot analysis using anti-P0 polyclonal antibody was performed as follows. After blocking with 3% BSA in phosphate-buffered saline (PBS) for 30 min at room temperature, the membranes were incubated with anti-P0 polyclonal antibody in PBS containing 0.05% Tween 20 and 1% BSA (TPBS) for 1 h at room temperature. The membranes were then incubated with biotin-conjugated goat anti-rabbit IgG in TPBS for 1 h at room temperature. The PVDF membranes were incubated with avidin-conjugated HRP in TPBS for 1 h at room temperature, and finally with the peroxidase substrate, 3,3[prime]-diaminobenzamide tetrahydrochloride, to detect the antibody-reactive proteins.

Amino acid sequence analysis

Spinal cord membrane fractions were electrophoresed and transferred on PVDF membrane as described above by using 15% gels. The edge of the PVDF membrane was cut and stained with LCA to detect the location of gp30. The area corresponding to gp30 was then cut out and used for the preparation of peptide fragments as described previously (Nakano et al., 1996). After rinsing briefly with water, the PVDF membrane was incubated with polyvinylpyrrolidine, with an Mr of 40 kDa (PVP-40) for 30 min at room temperature. The excess PVP-40 was removed by extensive washing with water, and the PVDF membrane was treated with lysylendopeptidase in 50 mM Tris-HCl, pH 8.0, at 37°C for 18 h. After digestion, the incubation medium was collected, and the remaining digested peptides were further eluted from the PVDF membrane with 40% acetonitrile with 0.1% trifluoroacetic acid (TFA). All eluates were combined, and the proteolytic fragments in the pooled buffer were fractionated by high-performance liquid chromatography (HPLC) on a reversed-phase column equilibrated in 5% acetonitrile and 0.1% TFA, using a linear gradient of acetonitrile (5-70%).

The reactivity of each fraction with LCA was detected by enzyme-linked immunosorbent assay (ELISA) as follows. Each obtained fraction was coated into microtiter wells, and the wells were blocked with 3% BSA in TBS. Biotin-conjugated LCA was then added to the wells and incubated for 2 h at room temperature. The bound lectin was detected using avidin-conjugated HRP in TTBS. O-Phenylenediamine dihydrochloride was used as a substrate, and absorbance at 492 nm was measured.

The N-terminal amino acid sequence of the peptide was analyzed using a Perkin Elmer Applied Biosystems 473A Protein Sequencer.

Detection of glycoproteins

After electrophoresis and transfer of spinal cord membrane fractions and sciatic nerve on PVDF membrane as described above, the presence or absence of glycan moiety on blotted proteins was examined by a glycoprotein detection kit according to the manufacturer's recommendation. The PVDF membranes were incubated with 10 mM sodium metaperiodate in 100 mM sodium acetate buffer, pH 5.5, for 20 min at room temperature in the dark. The PVDF membranes were then incubated with 0.125 mM biotin hydrazide in 100 mM sodium acetate buffer, pH 5.5, for 1 h at room temperature. After blocking the membrane with the blocking agent in PBS for 1 h at room temperature, the PVDF membranes were incubated with streptavidin-conjugated HRP in PBS for 30 min at room temperature, and finally with the peroxidase substrate, ECL detection reagent, to detect glycoproteins.

Acknowledgments

We are grateful to Dr. Hiroaki Asou, Department of Neurobiology of our institute, for helpful discussions, Dr. Toshiyuki Inazu, Noguchi Institute, for kindly providing the C-terminal portion of rat P0, and Drs. Masafumi Tsujimoto, Hideki Adachi, and Akira Hattori, Riken, for technical assistance with the Northern analysis. This work was supported in part by Research Grant (8A-2) for Nervous and Mental Disorders from the Ministry of Health and Welfare, the Grant-in-Aids for Scientific Research (09558083, 09670167, 10771313) from the Ministry of Education, Science, Sports and Culture of Japan, the New Energy and Industrial Technology Development Organization (NEDO), and The Research Foundation for Pharmaceutical Sciences.

Abbreviations

BSA, bovine serum albumin; CNS, central nervous system; ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; LCA, Lens culinaris agglutinin; NCAM, neural cell adhesion molecule; PSA, polysialic acid; PVDF, polyvinylidene difluoride; PVP-40, polyvinylpyrrolidine; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SET buffer, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA and 250 mM sucrose buffer; TBS, 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2; TTBS, TBS containing 0.05% Tween 20 and 1% BSA; TFA, trifluoroacetic acid.

References

Barry ,F.P., Rosenberg,L.C., Gaw,J.U., Gaw,J.U., Koob,T.J. and Neame,P.J. (1995) N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature and immature bovine cartilage. J. Biol. Chem., 270, 20516-20524. MEDLINE Abstract

Brunden ,K.R. (1992) Age-dependent changes in the oligosaccharide structure of the major myelin glycoprotein P0. J. Neurochem., 58, 1659-1666. MEDLINE Abstract

Choe ,S. (1996) Packing of myelin protein P0. Neuron, 17, 363-365. MEDLINE Abstract

Cohen ,N.S., Ekholm,J.E., Luthra,M.G. and Hanahan D.J. (1976) Biochemical characterization of density-separated human erythrocytes. Biochim. Biophys Acta, 419, 229-242. MEDLINE Abstract

Debry ,H., Decout,D., Strecker,G., Spik,G. and Montreuil,J. (1981) Specificity of twelve lectins towards oligosaccharides and glycopeptides related to N-glycosylproteins. Eur. J. Biochem., 117, 41-45. MEDLINE Abstract

Filbin ,M.T. and Tennekoon,G.I. (1993) Homophilic adhesion of the myelin P0 protein requires glycosylation of both molecules in the homophilic pair. J. Cell. Biol., 122, 451-459. MEDLINE Abstract

Giese ,K.P., Martini,R., Lemke,G., Soriano,P. and Schachner,M. (1992) Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules and degeneration of myelin and axons. Cell, 71, 565-576. MEDLINE Abstract

Hayasaka ,K., Himoro,M., Wang,Y., Takata,M., Minoshima,S., Shimizu,N., Miura,M., Uyemura,K. and Takada,G. (1993a) Structure and chromosomal localization of the gene encoding the human myelin protein zero (MPZ). Genomics, 17, 755-758. MEDLINE Abstract

Hayasaka ,K., Himoro,M., Sato,W., Takada,G., Uyemura,K., Shimizu,N., Bird,T.D., Conneally,P.M. and Chance,P.F. (1993b) Charcot-Marie-Tooth neuropathy type 1B is associated with mutation of the myelin P0 gene. Nature Genet., 5, 31-34. MEDLINE Abstract

Ishaque ,A., Room,M.W., Szymaska,I., Kowalski,S. and Eylar,E.H. (1980) The P0 glycoprotein of peripheral nerve myelin. Can. J. Biochem., 58, 913-921. MEDLINE Abstract

Kobata ,A. (1992) Structures and functions of the sugar chains of glycoproteins. Eur. J. Biochem., 209, 483-501. MEDLINE Abstract

Kornfeld ,K., Reitman,M.L. and Kornfeld,R. (1981) The carbohydrate-binding specificity of pea and lentil lectins: fucose is an important determinant. J. Biol. Chem., 256, 6633-6640. MEDLINE Abstract

Laemmli ,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. MEDLINE Abstract

Lemke ,G. and Axel,R. (1985) Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin. Cell, 40, 501-508. MEDLINE Abstract

Lowry ,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.

Mathews ,M.B. and Glagov,S. (1966) Acid mucopolysaccharide patterns in aging human cartilage. J. Clin. Invest., 45, 1103-1111. MEDLINE Abstract

Nakano ,Y., Tobe,T., Miura,N,C., Mazda,T. and Tomita,M. (1996) Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. (Tokyo), 120, 803-812. MEDLINE Abstract

Parekh ,R., Roit,I., Isenberg,D., Dwek,R. and Rademacher,T. (1988) Age-related galactosylation of the N-linked oligosaccharides of human serum IgG. J. Exp. Med., 167, 1731-1736. MEDLINE Abstract

Rademacher ,T.W., Parekh,R.B. and Dwek,R.A. (1988) Glycobiology. Annu. Rev. Biochem., 57, 785-838. MEDLINE Abstract

Roughley ,P.J., White,R.J. and Santer,V. (1981) Comparison of proteoglycans extracted from high and low weight-bearing human articular cartilage, with particular reference to sialic acid content. J. Biol. Chem., 256, 12699-12704. MEDLINE Abstract

Rutishauser ,M. and Landmesser,L. (1996) Polysialic acid on the surface of axons regulates patterns of normal and activity-dependent innervation. Trends Neurosci., 19, 422-427. MEDLINE Abstract

Schachner ,M. and Martini,R. (1995) Glycans and the modulation of neural-recognition molecule function. Trends Neurosci., 18, 183-191. MEDLINE Abstract

Schauer ,R. (1982) Chemistry, metabolism and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem., 40, 131-234. MEDLINE Abstract

Seaman ,G.V.F., Knox,R.J., Nordt,F.J. and Regan,D.H. (1977) Red cell aging. I. Surface charge density and sialic acid content of density-fractionated human erythrocytes. Blood, 50, 1001-1011. MEDLINE Abstract

Silberstein ,S. and Gilmore,R. (1996) Biochemistry, molecular biology and genetics of the oligosaccharyltransferase. FASEB J., 10, 849-858. MEDLINE Abstract

Suzuki ,T., Kitajima,K., Emori,Y., Inoue,Y. and Inoue,S. (1997) Site-specific de-N-glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N-glycanase as a quality control system for newly synthesized proteins. Proc. Natl Acad. Sci. USA, 94, 6244-6249. MEDLINE Abstract

Takei ,K., Kitamura,K., Banno,K. and Uyemura,K. (1993) Major glycoproteins in carp CNS myelin: homology to P0 protein with HNK-1/L2 carbohydrate epitope. Neurochem. Int., 23, 239-248. MEDLINE Abstract

Takei ,K. and Uyemura,K. (1993) Expression of a P0-like glycoprotein in central nervous system myelin of amphibians (Ambystoma mexicanus, Xenopus laevis and Rana catesbeiana). Comp. Biochem. Physiol., 106B, 873-882.

Trapp ,B.D., McIntyre,L.J., Quarles,R.H., Strenberger,N.H. and Webster,H.deF. (1979) Immunocytochemical localization of rat peripheral nervous system myelin protein: P2 protein is not a component of all peripheral nervous system myelin sheaths. Proc. Natl Acad. Sci. USA, 76, 3552-3556. MEDLINE Abstract

Tsuchiya ,N., Endo,T., Matsuta,K., Yoshinoya,S., Aikawa,T., Kosuge,E., Takeuchi,F., Miyamoto,T. and Kobata,A. (1989) Effects of galactose depletion from oligosaccharide chains on immunological activities of human IgG. J. Rheumatol., 16, 285-290. MEDLINE Abstract

Tsuchiya ,N., Endo,T., Matsuta,K., Yoshinoya,S., Takeuchi,F., Nagano,Y., Shiota,M., Furukawa,K., Kochibe,N., Ito,K. and Kobata,A. (1993) Detection of glycosylation abnormality in rheumatoid IgG using N-acetylglucosamine-specific Psathyrella velutina lectin. J. Immunol., 151, 1137-1146. MEDLINE Abstract

Uyemura ,K., Kitamura,K. and Miura,M. (1992) Structure and molecular biology of P0 protein. In Martenson,E. (ed.), Myelin: Biology and Chemistry. CRC Press, Boca Raton, FL, pp. 481-508.

Varki ,A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97-130. MEDLINE Abstract

Voshol ,H., van Zuylen,C.W.E., Orberger,G., Vliegenthart,J.F.G. and Schachner,M. (1996) Structure of the HNK-1 carbohydrate epitope on bovine peripheral myelin glycoprotein P0. J. Biol. Chem., 271, 22957-22960. MEDLINE Abstract

Warner ,L.E., Hilz,M.J., Appel,S.H., Killian,J.M., Kolodny,E.H., Karpati,G., Carpenter,S., Watters,G.V., Wheeler,C., Witt,D., Bodell,A., Nelis,E., Van Broeckhoven,C. and Lupski,J.R. (1996) Clinical phenotypes of different MPZ(P0) mutations may include Charcot-Marie-Tooth type 1B, Dejerine-Sottas and congenital hypomyelination. Neuron, 17, 451-460. MEDLINE Abstract

Yazaki ,T., Miura,M., Asou,H., Kitamura,K., Toya,S. and Uyemura,K. (1992) Glycopeptide of P0 protein inhibits homophilic cell adhesion: competition assay with transformants and peptides. FEBS Lett., 307, 361-366. MEDLINE Abstract

Yazaki ,T., Miura,M., Asou,H., Toya,S. and Uyemura,K. (1994) Peripheral myelin P0 protein mediates neurite outgrowth of cortical neurons in vitro and axonal regeneration in vivo. Neurosci. Lett., 176, 13-16. MEDLINE Abstract


3To whom correspondence should be addressed at: Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan


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