Tenascin-R (TN-R), originally named J1-160/180, janusin, in rodents (Kruse et al., 1985; Pesheva et al., 1989, 1993) and restrictin in chicken (Rathjen et al., 1991) is a member of the tenascin multigene family of structurally-related, modular glycoproteins of the extracellular matrix (ECM) (Chiquet-Ehrismann, 1995). Molecules of the ECM play a key role in the development, maintenance, and repair of the nervous system (Reichardt and Tomaselli, 1991; Ruoslahti, 1996). The tenascins are multifunctional molecules that can either promote or inhibit cell adhesion and neurite outgrowth in specific assays (Faissner and Kruse, 1990; Morganti et al., 1990; Lochter et al., 1991; Erickson, 1993; Taylor et al., 1993; Lochter and Schachner, 1997). They are prominent in the development of the nervous system, where both adhesive and anti-adhesive interactions between neurons and the ECM are essential in neuronal pattern formation.
TN-R is detected exclusively in the CNS, where it is expressed by oligodendrocytes and subpopulations of neuronal cells (Pesheva et al., 1989; Bartsch et al., 1993; Wintergerst et al., 1993). The protein appears mostly associated with the surface of oligodendrocytes and myelinated axons in the white matter and with perineuronal nets of interneurons and motoneurons in different parts of the mammalian CNS (Pesheva et al., 1989; Bartsch et al., 1993; Angelov et al., 1998). This pattern of TN-R expression suggests a role in the process of CNS myelination, stabilization, and maintenance of nerve fiber tracts and axosomatic synaptic contacts, the latter required for normal connectivity and neuronal survival. TN-R has been shown to be adhesive for oligodendrocytes (sulfatide-mediated; Pesheva et al., 1997) and astrocytes (Pesheva et al., 1989; Morganti et al., 1990) and anti-adhesive for activated microglia (Angelov et al., 1998) and CNS neurons (Pesheva et al., 1989, 1993; Taylor et al., 1993). The latter action is mediated by the interaction of TN-R with the neuronal protein F3/F11/contactin (Pesheva et al., 1993; Xiao et al., 1996, 1997b, 1998), which also acts as a receptor for the 190 kDa isoform of tenascin-C (TN-C) (Zisch et al., 1992). The F3/F11-mediated inhibition of neurite outgrowth by TN-R (and probably TN-C) may also contribute to the marked lack of regeneration of CNS axons following injury. The contrary functions of tenascins localize to different structural domains of the molecule (Erickson, 1993; Schachner et al., 1994; Faissner and Schachner, 1995; Faissner et al., 1996; Norenberg et al., 1996; Fischer et al., 1997).
TN-R is composed of four structural motifs. The N-terminus, unique to the tenascin family, contains a cysteine-rich segment followed by EGF-like repeats (4.5 for TN-R) and eight motifs related to fibronectin type III-like domains. At the C-terminus, TN-R contains a 220 amino acid segment similar to the [beta] and [gamma] chains of fibrinogen, including a Ca2+ binding segment (Norenberg et al., 1996; Carnemolla et al., 1996). Rat, chicken, and human TN-R (Norenberg et al., 1992; Fuss et al., 1993; Carnemolla et al., 1996) show a high degree of sequence homology (typically at least 75%), including conservation of potential sites for N-glyco-sylation (14 in the rat). There are also numerous potential sites for O-glycosylation, the proportions of serine and threonine in several of the fibronectin type III domains indicating, moreover, regions for potential O-linked domains (Wilson et al., 1991). Although tenascins contain in the region of 10-20% by weight of carbohydrate, nearly all structural and functional analyses to date have concentrated exclusively on the polypeptide. The possibility that binding between lecticans (e.g., the chondroitin sulfate proteoglycans versican, brevican) and TN-R may be mediated through a C-type lectin-carbohydrate interaction has been studied. However, the potential role of the glycoconjugate ligands of TN-R (e.g., the HNK-1 carbohydrate, containing a 3-sulfated glucuronic acid; see Schachner and Martini, 1995, for review) remained unclear (Aspberg et al., 1995, 1997).
For an understanding of the details of molecular interactions involving the glycans of TN-R, it is first necessary to describe the nature of the glycosylation of the glycoprotein. The results are presented in this paper for TN-R prepared from adult mouse brain and their potential biological significance is discussed.
The total N- and O- linked glycans, released by hydrazinolysis, were subjected to weak anion-exchange (WAX) HPLC (Figure
Figure 1. WAX-HPLC of the total glycans released from TN-R. Fractions were collected, as shown at the bottom of the figure, into neutrals and pools 1-5. The column void and the elution positions of mono-, di-, tri-, and tetra-sialylated N-linked glycans obtained from fetuin are indicated at the top. Neutral N-glycans
The Bio-Gel P4 profile of neutral N-linked glycans from TN-R, obtained from the WAX HPLC void fraction, is shown in Figure
Figure 2. BioGel P4 chromatogram of the neutral N-linked oligosaccharides from TN-R. Fractions a-f were pooled as shown for further analysis. The numbers at the top indicate the elution positions of glucose oligomers (P4 glucose units, P4GU) from a partial dextran hydrolysate, 2AB-labeled. Due to a slight charge interaction with the matrix, the presence of the 2AB-fluorescent label decreases the glucose unit value of a given oligosaccharide by 2.65 compared with the corresponding nonlabeled alditol. The void volume containing acidic glycans is not shown.
Figure 3. NP-HPLC profiles of neutral N-glycan fractions from TN-R. Fractions shown are pools a, c, d, and e & f from BioGel P4 chromatography (see Figure 2). Numbers adjacent to peaks indicate compound number in Figure 4 (and Table I). Symbols: triangles, fucose; solid squares, GlcNAc; open circles, mannose; open squares, galactose. The numbers at the top of the NP-HPLC profiles indicate the elution positions of glucose oligomers (normal phase glucose units, NPGU) from a partial dextran hydrolysate, 2AB-labeled.
Figure 4. Structures of the neutral N-glycans of TN-R. The compound numbers correspond to those given in Table I and to those peak numbers given in Figure 3. Components in boxes represent the most abundant glycans.
Table I.
Oligomannosidic glycans Man(6-5)GlcNAc2 were the main components of P4 fractions e and f, respectively, and were the only constituents detected from this glycan family. Thus, in contrast to brain tissue, larger oligomannosidic glycans Man(9-7)GlcNAc2 were completely absent from TN-R, as were hybrid structures.
P4 fraction b contained a complex mixture of the more highly processed outer-arm- and core-fucosylated biantennary structures ± bisecting GlcNAc (Figure Acidic glycans
Acidic N- and O- linked glycans were voided on the BioGel P4 column and were separated analytically by NP-HPLC (Figure
Figure 5. NP-HPLC profiles of 2AB-labeled acidic glycans of TN-R. (a) Total acidic glycans. The horizontal bar indicates the elution period for O-linked glycans. (b) WAX pool 3. (c) WAX pool 4. The numbers at the top indicate the elution positions of glucose oligomers (NPGU) from a partial hydrolysate of dextran, 2AB-labeled.
No.3
Fract.4
Mass1
Comp.2
Found
Calc.
H
N
F
1
ef
1377.6
1377.5
5
2
0
2
ef
1540.0
1539.6
6
2
0
3
ef
-
1256.5
3
3
0
4
ef
1402.5
1402.6
3
3
1
5
ef
1460.2
1459.6
3
4
0
6
d,c
1605.4
1605.4
3
4
1
7
c
1605.7
1605.4
3
4
1
8
c
1768.2
1767.7
4
4
1
9
c
1913.9
1913.7
4
4
2
10
b
1784.1
1783.7
5
4
0
11
b
1930.3
1929.7
5
4
1
12
c
1808.7
1808.7
3
5
1
13
b
1971.4
1970.8
4
5
1
14
b
-
2132.8
5
5
1
15
b
-
2116.8
5
5
2
16
a
2149.0
2148.8
6
5
0
Figure 6. RP-HPLC of 2AB-labeled monosaccharides. (a) Profile for a mixture of common standard monosaccharides. Abbreviations: Fuc, fucose; Xyl, xylose; Gal, galactose; Glc, glucose; Man, mannose; Arab, arabinose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine. (b) Profile for the reducing terminal monosaccharide of the component in WAX pool 4. The peak has a retention time corresponding to that of 2AB-labeled GalNAc.
WAX pool 4. This pool also showed homogeneity on NP-HPLC (Figure
The acidic N-glycans, detected in WAX pools 1 and 5 only, were present in a relatively low overall abundance and showed a high degree of structural heterogeneity. More than 90% contained charged groups other than or in addition to NeuAc. On account of the low abundance and complexity of these structures, further analysis was not performed.
This study addresses the characterization of the most abundant glycans expressed on TN-R from adult mouse brain. The glycans comprised neutral N-linked oligosaccharides and O-linked sialylated structures.
N-Linked glycans
The neutral N-linked glycans comprised >80% of the N-linked oligosaccharide pool and consisted mainly of complex biantennary structures with outer-arm and core fucosylation, bisecting GlcNAc residues and arm truncation. The truncated complex biantennary glycans were major components of TN-R and are highlighted as boxed structures in Figure
In contrast to brain tissue (Chen et al., 1998), hybrid structures were not expressed on TN-R. Moreover, oligomannosidic glycans, known to be expressed on several neural glycoproteins (Fahrig et al., 1990; Horstkorte et al., 1993), including synaptic glutamate receptors (Clark et al., 1998), were relatively minor components, consisting only of Man(6-5)GlcNAc2. Thus, the N-glycan processing pathway was directed almost entirely towards the production of complex oligosaccharides only. This suggested that there was an extensive array of processing enzymes in the oligodendrocyte cell and good accessibility to the glycosylation sites on TN-R for the completion of the processing.
TN-R contained one complex glycan, in addition to those described above, which was not identified as a major structure in rat brain tissue (Chen et al., 1998). This component was a (2,4)-branched nonfucosylated triantennary oligosaccharide. The sialylated counterpart of this structure was seen, however, in rat brain (Zamze et al., 1998).
Most of the minor fraction of acidic N-glycans from TN-R possessed anionic charges in addition to NeuAc, with <10% containing NeuAc, either [alpha]2,3- or [alpha]2,6-linked, as their sole charged group. In accordance with this observation, TN-R is known to express the L2/HNK-1 epitope (Kruse et al., 1985; Pesheva et al., 1989), a characteristic carbohydrate structure of many neural glycoproteins based on a terminal 3-sulfated glucuronic acid moiety. It is concluded that those glycans carrying this epitope on TN-R were probably present in minor and heterogeneous fractions.
O-Linked acidic glycans
The most striking observation of the glycosylation of TN-R was the relatively large abundance of O-linked acidic glycans, and particularly the disialylated structure NeuAc[alpha]2-3Gal[beta]1-3(NeuAc[alpha]2-6)GalNAc (WAX pool 4, Figure
WAX pool 3 (Figure
The myelin-associated glycoprotein (MAG) is the major CNS-located member of the family of lectins recognizing sialic acids (Kelm et al., 1994; Probstmeier and Pesheva, 1999). Its specificity of recognition has been demonstrated by Yang et al., (1996) using a variety of gangliosides and related glycosphingo-lipids. An [alpha]2-3-N-acetylneuraminic acid residue on the terminal galactose was necessary for binding and additional sialic acid residues on the other core saccharides contributed significantly. (Additional supporting evidence has been provided recently by Strenge et al., 1998.) The physiological sugar ligands for MAG recognition are believed to be conjugated to protein (Kelm et al., 1994) since the interaction was trypsin-sensitive. Clearly the multivalent display of the disialylated O-linked glycan described above on TN-R would strongly point to this carbohydrate as a potential ligand for MAG. This possibility is accentuated by the fact that MAG is present in the noncompacted myelin loops at the node of Ranvier, where tenascin is accumulating (Bartsch et al., 1992, 1993). The elasticity of the tenascins (Oberhauser et al., 1998) additionally would make this type of molecule ideally suited for an intermediary role in such a process as myelination, substantiating results using oligodendrocytic cells (Jung et al., 1993; Pesheva et al., 1997).
In addition, the present results suggest that sialoadhesin may represent another cellular receptor for TN-R on activated microglia. This I-type lectin, known to favor recognition of sialic acids in [alpha] 2-3 linkage (Crocker et al., 1991; Kelm et al., 1994), is normally absent in the adult CNS. It becomes expressed, however, by a subpopulation of brain microglia/macrophages upon CNS injury (Perry et al., 1992) and is likely to mediate the repellent action of TN-R on activated microglia reported recently, i.e., such interaction may play a role in the proposed function of TN-R in neuronal protection against these cells (Angelov et al., 1998). During neurodegeneration following peripheral nerve axotomy, the downregulation of TN-R in the perineuronal net of motoneurons would (1) result in an impaired structural integrity of this specialized ECM, due to a disruption of the assembly of TN-R with ECM proteins and chondroitin sulfate proteoglycans, such as versican and phosphacan (Celio and Blumcke, 1994; Aspberg et al., 1997; Xiao et al., 1997a; Milev et al., 1998), and, as a consequence, (2) affect neuronal function and/or survival.
Materials
All exoglycosidases were purchased from Oxford GlycoSciences (Abingdon, Oxon, UK) except for Charonia lampas [alpha]-fucosidase which was prepared in the Oxford Glycobiology Institute. Oligosaccharide standards were obtained from Oxford Glyco-Sciences and were fluorescently labeled with 2-AB as described below. Additional standards were prepared by exoglycosidase digestion of A2G0FB with Diplococcus pneumoniae [beta]-hexosaminidase, 0.01 U/ml, to remove selectively the GlcNAc residue linked to the Man [alpha](1-3)-arm, and digestion of A2G0FB with C.lampas [alpha]-fucosidase to remove core fucose. A disialylated tetrasaccharide standard, NeuAc[alpha]2-3Gal[beta]1-3(NeuAc[alpha]2-6)-GalNAc, was obtained from IgA1 (Mattu et al., 1998).
Tenascin-R
TN-R was immunoaffinity purified from adult mouse brains as described previously (Pesheva et al., 1989). After dialysis against 0.1 M ammonium bicarbonate pH 7.0, the sample was lyophilized in preparation for anhydrous hydrazinolysis.
Release and labeling of oligosaccharides
The release of oligosaccharides from TN-R (1-2 mg) by small scale hydrazinolysis was carried out essentially as described previously (Ashford et al., 1987; Parekh et al., 1987) for the optimal release of N- and O-linked glycans (Patel et al., 1993). Oligosaccharides were fluorescently labeled with 2-AB by reductive amination according to the method of Bigge et al., (1995), using an Oxford GlycoSciences Signal labeling kit and following the manufacturer's instructions. Fluorescence was measured at [lambda]em420 nm and [lambda]ex330 nm.
Bio-Gel P4 gel filtration chromatography
The size fractionation of neutral glycans by Bio-Gel P4 gel filtration chromatography in water was performed as described previously (Ashford et al., 1987; Parekh et al., 1987). Neutral glycans are resolved by this method and are separated from acidic glycans which elute in the void volume of the column. Fluorescently labeled neutral oligosaccharides were analyzed on an Oxford GlycoSciences GlycoMap 1000 equipped with an on-line fluorescence monitor.
HPLC
Anion-exchange HPLC was carried out using a Vydac 301VHP575 7.5 × 50 mm WAX column (Hichrom Ltd., Reading, UK) with ammonium formate at pH 9.0 at a flow rate of 1 ml/min (Guile et al., 1994). The following linear stepwise gradient was used: an increase from 0 to 25 mM formate over 12 min, followed by increases to 105 mM over 13 min, 400 mM over 25 min, 500 mM over 5 min, and then held for 5min.
NP-HPLC of neutral 2-AB-labeled N-linked glycans was performed using a polyhydroxyethyl aspartamide column as described previously (Chen et al., 1998). Structures were assigned normal phase glucose unit (NPGU) values in relation to external calibration with a 2-AB-labeled partial hydrolysate of dextran (Guile et al., 1996). NP-HPLC of acidic 2-AB-labeledN- and O- linked glycans was also performed using a poly-hydroxyethyl aspartamide column using conditions described in Guile et al., (1996).
RP-HPLC was performed on a Hypersil BDS C18 column as described previously (Chen et al., 1998) using 50 mM ammonium formate as solvent A and acetonitrile as solvent B. The following gradient conditions were used for the separation of 2-AB-labeled N- and O-linked glycans: an increase of 2-4% B over 25 min, followed by an increase of 4-8% B over 65 min and a further increase of 8-12% B over 5 min. The flow rate was 0.5 ml/min. This was then held at the final conditions for 5 min at 1 ml/min. For the separation of 2-AB-labeled monosaccharide residues: a linear gradient was used of 0-4% B over 50 min, followed by an increase of 4-9% B over 42 min and a further increase of 9-12 % B over 3 min. The flow rate was 0.5 ml/min. The final conditions were held for 5 min at 1 ml/min.
Mass spectrometry
Positive ion matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was performed on selected peaks from the NP-HPLC separations on either a Micromass AutoSpec-QFPD magnetic sector mass spectrometer or a PerSeptive Biosystems Voyager Elite mass spectrometer operated in the reflectron mode with delayed extraction. Samples were prepared by mixing 0.5 µl of the glycan solution, in water, with 3.0 µl (AutoSpec) or 0.5 µl (Voyager) of a saturated solution of 2,5-DHB in acetonitrile, allowing the mixture to crystallize and then recrystallizing it from ethanol. Spectra were acquired on the AutoSpec instrument with the array detector in the high resolution position to provide monoisotopic masses. The mass range was chosen to be appropriate to the compounds to be detected and spectra were accumulated until a satisfactory signal:noise ratio was obtained. During spectral acquisition, the laser spot was moved manually over the target surface to compensate for sample depletion. Spectra on the Voyager instrument were acquired with an accelerating voltage of 20 kV, a pulse decay of 75 ns, and a grid voltage of 70%; 256 laser shots were fired to produce each spectrum.
Exoglycosidase digestions
The digestion of 2-AB-labeled oligosaccharides was carried out in a volume of 20 µl for 18 h at 37°C using the following enzymes: D.pneumoniae [beta]-galactosidase, 0.4 mU/ml in 100 mM sodium acetate pH 5.5; bovine testes [beta]-galactosidase 10 mU/ml in 100 mM citrate-phosphate, pH 4.0; jack bean [beta]-hexosaminidase 10 U/ml in 100 mM citrate-phosphate, pH5.0; Aspergillus saitoi [alpha](1,2)-mannosidase 1 mU/ml in 100 mM sodium acetate, pH5.0; almond meal [alpha]-fucosidase 1 mU/ml in 100 mM sodium acetate, pH 5.0 and Charonia lampas [alpha]-fucosidase 10 mU/ml in 50 mM sodium acetate, pH 4.5 containing 0.15 M sodium chloride. D.pneumoniae [beta]-hexosaminidase was used at either 10 U/ml or, for conditions providing linkage specificity, at 0.01 U/ml in 100 mM citrate-phosphate, pH 6.0 (Yamashita et al., 1992). At the lower concentration, only GlcNAc residues linked [beta] (1-2) to Man are cleaved, with two provisos: (1) that [beta] (1-2) GlcNAc is not cleaved if the Man to which it is attached is also substituted at the C-6 position and (2) that [beta](1-2) GlcNAc linked to the Man [alpha](1-6)-arm of the tri-mannosyl core is not cleaved in the presence of a bisecting GlcNAc.
Jack bean [alpha]-mannosidase was used at either 25 U/ml or 10 U/ml in 100 mM sodium acetate pH 5.0 containing 2 mM Zn2+. At the lower concentration R-Man[alpha]1-6(Man[alpha]1-3)-Man[beta]1-4GlcNAc[beta]1-4GlcNAc but not R-Man[alpha]1-3(Man[alpha]1-6)-Man[beta]1-4GlcNAc[beta]1-4GlcNAc is susceptible where R is not H or Man (Yamashita et al., 1980). Mixed digests of D.pneumoniae [beta]-galactosidase (0.4 U/ml) and almond meal [alpha]-fucosidase (1 mU/ml) were carried out in 100 mM sodium acetate, pH 5.5.
Digestion of acidic glycans with NDV neuraminidase (specificity for [alpha]2-3(8)NeuAc) was performed at 0.2 U/ml in 50 mM sodium acetate, pH 5.5; and Arthrobacter ureafaciens neuraminidase (specificity for [alpha]2-6(3,8)NeuAc) was used at 2 U/ml in either 100 mM ammonium acetate or 50 mM sodium acetate, pH 5.0.
Reactions were terminated and samples deproteinated using 0.45 µm cellulose nitrate Pro-Spin Micro centrifugal filters. After application to the filter, samples were left at room temperature for 60 min. Glycans were then recovered by centrifugation three times with 30 µl 5% acetonitrile.
Monosaccharide analysis
The identity of the terminal reducing monosaccharide of the O-linked glycans was determined (1) by RP-HPLC of the 2-AB-labeled monosaccharide following complete exoglycosidase digestion of the glycan to its reducing monosaccharide unit and (2) by GC of the radiolabeled alditol acetate following reduction of the glycans with 3H-labeled sodium borohydride and acid hydrolysis (Ashford et al., 1987, and references therein).
We are grateful to Brian Matthews for expert assistance withthe hydrazinolysis procedure and GC analysis of radiolabeled monosaccharides and to Iris Bahnmüller for expert help withthe preparation of mouse brain-derived tenascin-R. We alsothank Professor E.M.Southern and Amersham International for access to the PerSeptive Biosystems Voyager Elite MALDI-TOF mass spectrometer. This work was supported by a project grant, no 041916, from the Wellcome Trust (to R.A.D.).
A2G0FB, agalactosylated biantennary N-glycan with core fucose and bisecting N-acetylglucosamine; 2-AB, 2-aminobenzamide; CNS, central nervous system; 2,5-DHB, 2,5-dihydroxy benzoic acid; ECM, extracellular matrix; EGF, epidermal growth factor; GC, gas chromatography; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GU, glucose unit; IgA, immunoglobulin A; MAG, myelin-associated glycoprotein; MALDI-MS, matrix-assisted laser desorption/ionization-mass spectrometry; Man, mannose; NDV, Newcastle disease virus; NeuAc, N-acetyl neuraminic acid; NP, normal phase; RP, reverse phase; TN-C, tenascin-C; TN-R, tenascin-R; WAX, weak anion exchange.
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