Identification of glycosaminoglycans in nerve terminals

Journal of the Neurological Sciences, 1979, 41 : 261-269 © Elsevier/North-HollandBiomedicalPress

261

IDENTIFICATION OF GLYCOSAMINOGLYCANS IN NERVE TERMINALS

C. J. BRANFORD WHITE Department of Biology, Oxford Polytechnic, Headington, Oxford OX3 0BP (Great Britain)

(Received 7 August, 1978) (Accepted 4 December, 1978)

SUMMARY Synaptosomes were prepared using sucrose and Ficoll density-gradient-centrifugation procedures. Extraction of lipid-free material from both preparations with guanidinium chloride together with papain treatment released hexuronic-containing fractions. The glycosaminoglycan fractions (C1, C2, and Ca) were isolated after ionexchange chromatography. Chemical studies identified fraction Ct as a chondroitin sulphate proteoglycan whereas fractions Cz and C3 were characterised as hyaluronic acid and heparan sulphate. The relative yields of glycosaminoglycans (as compared with brain mitochondria) strongly suggest that these macromolecules form an integral component of nerve endings. The possible role of glycosaminoglycans in synaptosomes is also discussed.

INTRODUCTION Glycosaminoglycans are high-molecular weight polymers containing a hexosamine-hexuronic acid repeating unit. The disaccharide units are built up on a protein backbone and the glycosyl transferases involved in the biosynthesis of these polymers have been found in brain (Brandt et al. 1975). Subcellular studies have shown relatively high levels of glycosaminoglycans in the microsome fraction with low amounts in synaptosomes (Margolis et al. 1975). It has been proposed that glycosaminoglycans might be present in synaptic vesicles, but there is little chemical evidence for the occurrence of these macromolecules in nerve endings (Pycock et al. 1975). In this paper the characterisation of glycosaminoglycans from synaptosomes prepared using sucrose and Ficoll gradients is reported, and their possible functions in nerve endings are discussed. MATERIALS AND METHODS Sephadex G-25, Sepharose 4B and Ficoll were purchased from Pharmacia, DE-

262 52 cellulose was bought from Whatman. Bovine serum albumin, chondroitin sulphate. hyaluronic acid, papain and chondroitinase ABC were obtained from Sigma. Heparan sulphate standard was isolated from whole brain tissue (Branford White and Hardy 1977). All other reagents were supplied by British Drug Houses. Fresh adult ovine brains from a local abattoir were transported to the laboratory on ice within an hour after death. Brains were freed from meninges, serous membranes and other adhering tissue. Cortex slices were homogenised in 0.32 M sucrose adjusted to pH 6.4 (15 vol) with 7 strokes of a Teflon-glass homogeniser (speed of 800 rev/min clearance 0.25 mm; Heaton and Bachelard 1973). The synaptosomes were either isolated by the sucrose discontinuous procedure outlined by Gray and Whittaker (1962), or by a modified method of Cotman and Matthews (1971). In this modified procedure, after removal of the nuclear pellet at low speed (1,000 x g: 15 min) the crude mitochondrial fraction was isolated (12,000 x g: 20 min). This preparation was resuspended in 0.32 M sucrose and further centrifuged (17,000 × g: 30 min). The resultant pellet was suspended in sucrose (5 ml) and centrifuged on a discontinuous gradient containing 7.5 % and 13 ~o (22 ml) Ficoll in 0.32 M sucrose (80,000 × g: 60 min). After centrifugation the uppermost interfacial layer (mainly myelin) was discarded and the residue was retained as whole brain mitochondrial fraction (fraction M). The interfacial layer above the 13% Ficoll (synaptosomes) was aspirated and washed once with sucrose (65,000 × g: 20 min). The synaptosomes were then subjected to osmotic shock by dialysis against water at 4 °C and clarified by centrifugation (35,000 x g: 20 min). The soluble fraction was collected and lyophilised. The residue was defatted by extraction with chloroformmethanol (Branford White et al. 1976), washed with acetone and dried in vacuo. This fraction was designated fraction A, and its subsequent treatment is outlined in Fig. 1. Fraction C (Table 1) was extracted with 0.4 M citrate buffer pH 5.0 and the supernatant chromatographed on a DEAE-cellulose column gradient (0-1 M NaCI) in 0.1 M citrate buffer, pH 4.5. Fractions were assayed for protein and hexuronic

Fraction A (1) Suspend in 4 M guanidinium chloride in 0.05 M Tris-HCI pH 7.5 f o r 4 8 h at 25°C (2) Centrifuge 35 0 0 0 × g

I

I Pel let

Supernatant (I) Dialyse against water (2) Centrifuge as before

Fraction B

I Pellet

Supernatant Precipitate by addition of 3 vol. ethanOl and I vol. ethanol saturated w i t h KAc.

Fraction Fraction D discard (he×uronic acid-free)

Fig. 1. Extraction of synaptosomal fraction A.

263 TABLE 1 DISTRIBUTION OF GLYCOSAMINOGLYCANS IN SUBFRACTIONS PREPARED FROM SYNAPTOSOMES Subfraction

Method of preparation Sucrose-prepared

A B C

Ficoll-prepared

dry weight (mg)

hexuronic acid (,ug/mg)

dry weight (mg)

hexuronic acid (,ug/mg)

100 27.6 50.2

3.1 0.55 5.0

100 18.5 71

6.0 trace 7.2

acid. The major fraction was precipitated by the addition of 3 vols. of ethanol and 1 vol. of ethanol saturated with potassium acetate. The portion of fraction C which remained after citrate buffer extraction was hydrolysed with papain (E.C. 3.4.22.2) for 20 hr at 55 °C. The hydrolysate was chromatographed on a DEAE-cellulose column. Glycosaminoglycans associated with brain mitochondria were also isolated by treating fraction M with papain followed by ion-exchange chromatography as described above. Amino sugars were quantitatively identified using a Jeol JLC 6AH amino acid analyser. Samples were hydrolysed under nitrogen in sealed tubes with 6 M HC1 at 100 °C for 6 hr. Acid was removed by rotary film evaporation at 30 °C. N-sulphated hexosamines were determined by the nitrous acid oxidation procedure (Lagunoff and Warren 1962) and hexuronic acid by the carbazole reaction (Bitter and Muir 1964). Protein concentrations were determined either from amino acid analysis values after samples had been hydrolysed with 6 M HC1 at 105 °C for 20 hr, or by the micro-biuret method of Itzhaki and Gill (1964). Sulphate was estimated by the colorimetric procedure described by Antonopoulos (1962). Anticoagulant activity was obtained by the protamine sulphate titration assay of Sharp and Eggleton (1963). Glycosaminoglycans were degraded with nitrous acid (Cifonelli 1968); after incubating samples for 5 hr at 25 °C the neutralised products were chromatographed on Sephadex G-25. Chondroitinase ABC (E.C. 4.2.2.4) digestion was carried out as described by Yamagata et al. (1968). Electrophoresis of all fractions was performed on cellulose acetate (Wessler 1971). RESULTS

Glycosaminoglycans in synaptosomes The hexuronic acid material of synaptosomes was found exclusively in the particulate fraction. Ninety-three per cent of hexuronic acid in fraction A derived from sucrose prepared synaptosomes was solubilised after extraction with guanidinium chloride (Fraction C). In contrast all the hexuronic acid in synaptosomes prepared on Ficoll was soluble and this fraction contained twice as much hexuronic acid material (Table 1).

264 C1

30

/.,

J 1 ! l

"~ 2 0 3-

~ /

' t

/ /~ ;

0

E 10

<

/ ......°.---"°'

0

,....---"

20

,i~ /

.°°.--

NaCtM ....",1.0

~ ...........

2"\

-- 7':

40

.

\

0.5

60

80

100

V o t u m e (mr)

Fig. 2. DEAE-cellulose chromatography of citrate buffer-extracted fraction C. Fractions were assayed for hexuronic acid (0) and protein (;q). As described in Methods, fraction C was extracted with 0.4 M citrate p H 5.0. Chromatography on DEAE-cellulose of the extract gave a single band which was eluted at 0.5 M NaCI (Fraction C1; Fig. 2). The homogeneity of this fraction was confirmed by the elution from a previously calibrated Sepharose 4B column of a single peak with an apparent distribution coefficient (Kav) of 0.32 (Laurent and Killander 1964). On chemical analysis fraction C1 was found to contain 19.2% protein, with aspartic acid, glutamic acid, serine and glycirte contributing 65 % of the total amino acids. The carbohydrate moiety contained equimolar proportions of hexuronic acid, hexosamine and sulphate. The amino sugar was identified as galactosamine (Table 2). These results indicate that fraction Ct consists of chondroitin sulphate. This conclusion is also supported by the finding that fraction Ct was degraded by chondroitinase ABC, yielding a protein containing fraction and unsaturated oligosaccharides which were separated by gel filtration on Sephadex G-25. Furthermore, the free glycosaminoglycan chains migrated as a monodispersed species similar to a chondroitin sulphate standard (Fig. 5). Treatment of this fraction with 0.5 M N a O H (Branford White and Hudson 1977) showed a reduction in serine content with no apparent loss ofthreonine. This result indicates that the glycosaminoglycan is linked to protein through specific serine residues. TABLE 2 Fraction

C1 Cz Ca M1 M~

Hexosamine

Galactosamine Glucosamine Glucosamine Galactosamine Glucosamine

Hexuronic acid/hexosamine (molar ratio)

Sulphate/hexosamine (molar ratio)

%

Su a

Fi ~

Su

Fi

Su

Fi

0.9 1.0 0.9 ---

1.0 1.1 1.0 0.9 1.1

1.1 -1.7 ---

1.0 -1.7 0.9 1.6

41 22 37 ---

36 23 41 67 33

a Su and Fi correspond to sucrose and Ficoll prepared material, respectively.

265

C3

3o

~". [

'~ 20

M NaCI .. 1.0

t

C2

~

..~ . . . . . .

g E lo <

, .

0

" -

0.5

--'''''

mJ.-m 8'0

410 ~

'W"n

120 0

Volume (mr)

Fig. 3. DEAE-cellulose chromatography of papain-treated fraction C remaining after citrate buffer extraction. Fractions were assayed for hexuronic acid (11). The residue from fraction C which was insoluble in citrate buffer was digested with papain. Fractionation on DEAE-cellulose resulted in the elution of two peaks which both contained hexuronic acid and hexosamine (Fig. 3). The first peak was resolved at 0.32 M NaC1 (fraction C2) and contained glucosamine as the sole amino sugar, and hexuronic acid in equimolar proportions. No sulphate groups were detected in this fraction. The second band which was eluted at 0.7 M NaC1 (fraction C3) also contained equal portions of glucosamine and hexuronic acid, but in contrast to C2 this fraction contained about 2 moles of sulphate per hexuronic acid (Table 2). N-sulphated hexosamine residues react with nitrous acid to form 2,5-anhydro-D-mannose (Cifonelli 1968). When Ca was treated with nitrous acid between 60 % and 65 % of the he×osamine residues were desulphated. In addition an anticoagulant activity of 12 I.U./mg was found for this fraction compared to 160 I.U./mg for a commercial heparin sample. These results suggest that C2 is hyaluronic acid and C3 is heparan sulphate. This conclusion is also supported by electrophoresis against glycosaminoglycan standards (Fig. 5).

Glycosaminoglycans in mitochondria The hexuronic acid content of glycosaminoglycans of the mitochondria fraction was 1.1/~g per mg lipid-free dry weight. Two glycosaminoglycans fractions (M1 and ml

~'"=

30

I ; /

ol

320

........

fi < o

"

..... r I

go

M NaCt

~, l 1

m2.... ....

\

?, ,

L8.0/ V o l u m e (m[)

d

....

-'11"°

t

o.5

X :

120

Fig. 4. DEAE-cellulose chromatography of papain-treated lipid-free brain mitochondria material. The column was eluted with increasing concentration of NaC1 in 0.1 M citrate buffer pH 4.5 at a flow rate of 30 ml/h. Fractions were assayed for hexuronic acid (11).

266

Fig. 5. Electrophoretic profile of glycosaminoglycansfrom synaptosomes and mitochondria (see text). Hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were run as standards. Glycosaminoglycans were identified by staining with Alcian blue. M2) were isolated after papain treatment of the lipid-free materials and elution on DEAE-cellulose (Fig. 4). Fractions M1 and M2 contributed 0.7 #g and 0 , 4 / t g of hexuronic acid per mg lipid-free dried weight, respectively. M1 was identified as chondroitin sulphate because it contained galactosamine, hexuronic acid, and sulphate in equimolar proportions. This fraction was also susceptible to chondroitinase ABC degradation and migrated with a chondroitin sulphate standard on electrophoresis. Furthermore, from chemical analysis data (Table 1) and electrophoretic mobility (Fig. 5) it would appear that fraction M2 is heparan sulphate. DISCUSSION The glycosaminoglycan fractions characterised during this study do not appear to have been degraded during isolation. A hexuronic acid :hexosamine molar ratio of approximately one was found for all fractions which is in keeping with the view that the polysaccharides are made up of repeating disaccharide units.

267 Using whole ovine brain extracts a chondroitin sulphate proteoglycan has been recently identified in this laboratory (Branford White and Hudson 1977). This macromolecule consists of a multi-chain complex in which the potentially reducing ends of the glycosaminoglycan chains are attached to a common protein core. From the analytical data and gel filtration studies it would seem that fraction C1 is a similar proteoglycan. Partial degradation of heparan sulphate from a variety of sources, other than brain tissue, using heparitinase have yielded a number of disaccharide forms including N-acetylated, and N- and O-sulphated glucosamine derivatives (Silva et al. 1976). Moreover, a 2,6-disulphated glucosamine-containing fraction similar to those found in heparin was also identified by these workers. The proportion and location of partially or fully sulphated regions in heparan sulphate vary and appear to be tissue specific (Linker and Hovingh 1973). The limited amount of C3 in synaptosomes meant that I was unable to study further the distribution of N-acetylated/N-sulphated glucosamine residues in this fraction. The high sulphur content (sulphate :glucosamine molar ratio of 1.7:1), supports the idea of a block-like structure in which 85 ~ of the repeating disaccharide units are N-/O-sulphated. The high proportion of disulphated glucosamine residues may account for the anticoagulant activity exhibited by this glycosaminoglycan. Using an agarose gel electrophoresis system Dietrich et al. (1976) have demonstrated the presence of chondroitin sulphate and heparan sulphate in rat brain mitochondria. As a result of homogenisation the "pinched off" nerve endings have been shown to contain mitochondria (Gray and Whittaker 1962). In this present study 3 times more glycosaminoglycan was found in synaptosomes than in mitochondria. Furthermore, the distribution of chondroitin sulphate and heparan sulphate in the synaptosome and mitochondria preparations differed. This evidence suggests that a large proportion of the glycosaminoglycans in nerve endings occur in association with other structures and are not entirely localised in synaptosomal mitochondria. Margolis and Margolis (1977) have questioned the occurrence of glycosaminoglycansin nerve endings. Using sucrose-prepared synaptosomes there is a likelihood of contamination from fragments of intracellular membrane material together with neuronal and glial plasma membranes (microsomal fraction) which sediment with the crude mitochondrial pellet from which the synaptosome fraction is prepared. Subcellular studies have demonstrated that about 35 ~ of the glycosaminoglycan in rat brain is found in the microsomal fraction (Margolis et al. 1975). However, it is generally accepted that reduction in contaminating membrane material occurs in nerve enditlgs prepared using Ficoll-sucrose gradients. This has been substantiated using specific enzyme markers and electron microscopy (Morgan et al. 1971 ; Gurd et al. 1974; Langley and Kennedy 1977). It can be calculated that if the microsomal content of the synaptosomes fraction is 15~ (Cotman and Matthews 1971) then the proportion of glycosaminoglycan attributed to this preparation would be 5 ~. In this present study I have been able to chemically identify glycosaminoglycans using both methods of preparation, and it should also be noted that although the sucrose-prepared synaptosomes contained lower concentrations of hexuronic acid, the proportions of glycos-

268 aminoglycans in both preparations showed a marked similarity (see Table 2). The slight variation in chondroitin sulphate and heparan sulphate content (fractions (_j and C3) could perhaps have arisen from the microsomal impurities discussed earlier. Although the biological role of glycosaminoglycans is still unclear, electrophysiological studies involving intraventricular injections of calcium and hyaluronidase have suggested that they may be involved with neurotransmission (Wang and Adey 1969). In addition it has been proposed that these polymers may be linked with the possible storage, stabilisation and release of transmitter amines. The exchange of cations, notably calcium, could occur on fusion of the vesicles with the pre-synaptic membrane, and with the subsequent release of the transmitter into the synaptic cleft (Uvnas 1973). Supporting this theory Pycock et al. (1975) found a similar distribution of labelled transmitter and aSS-containing material after treating rats with radioactive noradrenaline and sulphate. Glycosaminoglycans in synaptosomes may also stabilise acetylchotinesterase. The enzyme isolated from the electric organ of Electrophorus electricus exists in a number of distinct molecular forms (Bon et al. 1976). Aggregation of the enzyme complexes is thought to be due to a non-protein polymer interacting with the positively charged groups of a tail protein. Using an in vitro system between 80-100 ~,', aggregation of acetylocholinesterase occurred in the presence of hyaluronic acid, chondroitin sulphate and heparan sulphate. These findings have led to the suggestion that in the synaptic cleft acetylcholinesterase may be immobilised through high affinity, ionic strength-dependent interactions involving the tail protein with glycosaminoglycans (Bon et al. 1978). Morphological studies have demonstrated both the presence of glycosaminoglycans surrounding nerve cells and the possibility of their existence in the synaptic cleft (Pease 1966; Rambourg and Leblond 1967). During this present investigation the solubilisation of glycosaminoglycans was found to require a dissociative solvent system and enzymic treatment. Therefore, it is conceivable that these molecules may be found in association with membrane-like components, which could influence the binding and possible transport of certain cations during neural transmission. In order to study this further, the distribution of glycosaminoglycans in synaptic membranes is now being investigated. ACKNOWLEDGEMENTS I am grateful to Mr T. Jones for his technical assistance and to Mr D. R. A. Mobbs, Mrs I. Webber and Mrs M. Sanders in assisting in the preparation of this manuscript.

REFERENCES

Antonopoulos, C. (1962) A modificationfor the determinationof sulphate in mucopolysaccharides by the benzidine method, Acta chem. scand., 16: 1521-1522. Bitter, T. and H. M. Muir(1962) A modifieduronicacidcarbazolereaction,Analyt. Biochem., 4: 330334.

269 Bon, S., J. Cartaud and J, Massouli6 (1978) The dependance of acetylcholinesterase, aggregation at low ionic strength upon a polyanionic component, Europ. J. Biochem., 85: 1-14. Bon, S., M. Huet, M. Lemonnier, F. Rieger and J. Massouli6 (1976) Molecular forms of Electrophorus acetylcholinesterase, Eur op. J. Biochem. , 68: 523-530. Brandt, A. E., J. J. Distler and G. W. Jourdain (1975) Biosynthesis of chondroitin sulphate proteoglycan, J. biol. Chem., 250: 3996-4006. Branford White, C. J. and J. F. Hardy (l 977) The presence of a heparan sulphate proteoglycan in ovine brain, FEBS Lett., 78: 229-232. Branford White, C. J. and M. Hudson (1977) Characterisation of a chondroitin sulphate proteoglycan from ovine brain, J. Neurochem., 28: 58•-588. Branford White, C. J., M. Hudson and K. Howells (1976) Evidence for the existence o f a proteoglycan in sheep brain, Biochem. Soe. Trans., 4: 1136-1138. Cifonelli, J. A. (1968) Reaction of heparitin sulphate with nitrous acid, Carbohyd. Res., 8: 233-242. Cotman, (7. W. and D. A. Mat thews (1971) Synaptic plasma membranes from rat brain synaptosomes - - Isolation and partial characterisation, Bioehim. biophys. Aeta (Amst.), 249: 380-394. Dietrich, C. P., L. O. Sampaio and O. M. S. Toledo (1976) Characteristic distribution of sulphated mucopolysaccharides in different tissues in their respective mitochondria, Biochem. biophys. Res. Commun., 71 : 1-10. Gray, E. G. and V. P. Whittaker (1962) The isolation of nerve endings from brain - - An electronmicroscope study of cell fragments derived from homogenisation and centrifugation, J. Anat. (Lond.), 96: 79-88. Gurd, J. W., L. R. Jones, H. R. Mahler and W. J. Moore (1974) Isolation and partial characterisation of rat brain synaptic plasma membranes, J. Neurochem., 22: 281--290. Heaton, G. M. and H. S. Bachelard (1973) The kinetic properties of hexose transport into synaptosomes from guinea pig cerebral cortex, J. Neurochem., 21 : 1099-1108. ltzhaki, R. F. and D. M. Gill (1964) A micro-biuret method for estimating protein, Anal. Biochem., 9 : 401-410. Lagunoff, D. and G. Warren (1962) Determination of 2-deoxy-2-sulfoaminohexose content of mucopolysaccharides, Arch. Biochem. Biophys., 99: 396-400. Langley, K. O. and P. Kennedy (1977) The metabolism of nerve terminal glycoproteins in the rat brain, Brain Res., 130: 109-120. Laurent, T. C. and J. Killander (1964) A theory of gel filtration and its experimental verification, J. Chromatog., 14: 317-330. Linker, A. and P. Hovingh (1973) The heparitin sulphates (heparan sulphates), Carbohyd. Res., 29: 41-62. Margolis, R. U. and R. K. Margolis (1977) Metabolism and function of glycoproteins and glycosaminoglycans in nervous tissue, Int. J. Biochem., 8: 85-91. Margolis, R. K., R. U. Margolis, C. Preti and D. Lai (1975) Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain, Biochemistry, 14: 4797-4804. Morgan, I. G., L. S. Wolfe, P. Mandl and G. Gombos (1971) Isolation of plasma membranes from rat brain, Biochim. biophys. Acta (Amst.), 241 : 737-751. Pease, D. C. (1966) Polysaccharides associated with the exterior surface of epithelial cells: kidney, intestine, brain, J. Ultrastruct. Res., 15: 558-588. Pycock, C., E. Blaschke, U. Bergqvist and B. Uvnas (1975) An attempt to explain nervous transmitter release as due to nerve impulse-induced cation exchange, Acta physiol, scand., 87: 168-175. Rambourg, A. and C. P. Leblond (1967) Electron microscope observations on the carbohydrate-rich cell present at the surface of cells in the rat, J. Cell BioL, 32: 27-53. Sharp, A. A. and M. J. Eggleton (1963) Haematology and the extracorporeal circulation, J. clin. Path., 16: 551-557. Silva, M. E., C. P. Dietrich and H. B. Nader (1976) On the structure of hepartin sulphates, Biochim. biophys. Acta (Amst.), 437: 129-141. Uvnas, B. (1973) An attempt to explain nervous transmitter release as due to nerve impulse-induced cation exchange, ActaphysioL scand., 87: 168-175. Wang, H. H. and S. R. Adey (1969) Effects of calcium and hyaluronidase on cerebral electrical impedance, Exp. NeuroL, 25: 70-84. Wessler, E. (1971) Electrophoresis of acidic glycosaminoglycans in hydrochloric acid, Anal. Bioehem., 41 : 67-69. Yamagata, J., J. Saito, O. Habuchi and S. Suzuki (1968) The purification of bacterial chondroitinase and chondrosulphatases, J. bioL Chem., 243: 1523-1535.