Determination of gangliosides, glycoproteins, and glycosaminoglycans in brain tissue

CLINICA CHIMICA ACTA

DETERMINATION

I59

OF GANGLIOSIDES,

GLYCOSAMINOGLYCANS

IN BRAIN

GLYCOPROTEINS,

ERIC G. BRUNNGRABER,

BARBARA

Research Department, Illinois Chtcago, Ill. 60612 (U.S.A.)

State Psychiatric Institute, r6or

(Received

September

AND

TISSUE

D. BROWN

AND HILDEGARD

HOF*

W. Taylor Stveet,

4. 1970)

SUMMARY

A procedure is described which permits the determination of gangliosides, glycoproteins, and glycosaminoglycans in brain tissue samples. Gangliosides are extracted with chloroform-methanol (2: I and I : 2, v/v). The defatted tissue is treated with papain or pronase to liberate glycopeptides and glycosaminoglycans. The solubilized glycopeptides are separated into non-dialyzable and dialyzable glycopeptide fractions. Glycosaminoglycans are separated from the non-dialyzable glycopeptides by precipitation with cetylpyridinium chloride. Application of the method to normal and pathological samples provided clearcut separations of glycopeptides, glycosaminoglycans, and gangliosides. The following pathological conditions were studied : a case of subacute sclerosing leukoencephalitis, a case of early infantile neurolipidosis with failure of myelination, and a case of galactosemia. Alterations in the carbohydrate moiety of the gray matter glycoproteins was indicated in the three cases studied. Glycosaminoglycan levels were low in galactosemia and in the case of neurolipidosis with a failure of myelination.

Chemical analysis of pathological brain specimens have largely been directed toward a study of the lipid material extracted by organic solvents. In some cases, the defatted protein residue which remains after the extraction has been analyzed for “residual hexosamine” or “residual N-acetylneuraminic acid (NANA)“. It is now clear that the “residual hexosamine” is associated with glycoproteins as well as glycosaminoglycansW. In view of the existence of various metabolic lesions which induce anomalies in the amount and structure of aminosugar-containing ‘constituents of brain tissue, it is desirable to have available a means whereby gangliosides, glycosaminoglycans, and glycoproteins can be evaluated in a given tissue sample. Such an analysis would permit confirmation of diagnosis of such diseases as the gangliosidoses, mucopolysacharidoses, and the glycoproteinoses 3-5. Furthermore, it would be of in-

* Research Illinois.

Fellow

on--Training

Grant

PHS-MH

8396 of the

University

of Illinois,

Chicago,

Clin. Chim. Acta, 32 (1971) 159-170

160

BRUNNGRABER

et&.

terest to know the effect of a specific metabolic lesion affecting one of the aminosugar constituents on other constituents. For example, deficiency of p-galactosidase has been implicated as the cause of generalized gangliosidosis. A deficiency of this lysosomal enzyme might be expected to affect the galactose-containing glycoproteins as well. In the present communication, we describe a procedure presently in use in this laboratory which permits an evaluation of the three aminosugar-containing constituents in a given sample of brain tissue. The method has been applied to an examination of a case of subacute sclerosing leukoencephalitis (SSLE) , since there has been a report6 of an elevation of “residual hexosamine” in this disease. A case of early infantile neurolipidosis with failure of myelination and a case of galactosemia were also studied. METHODS

Extraction

of gangliosides

A flow chart of the procedure to be described is provided in Fig. I. Brain tissue samples were homogenized in the Serval Omni-Mixer, Waring Blendor (or equivalent instrumentation) for 3 min with rg volumes of chloroform-methanol (2: I, v/v). The material was centrifuged. The precipitate was homogenized with IO volumes of chloroform-methanol (I :2, v/v) and centrifuged as before. The two supernatants were combined and an appropriate amount. of chloroform was added to yield a final concentration of chloroform-methanol of 2 :I (v/v). The gangliosides were partitioned into the upper phase by the addition of 0.2 volumes of 0.1 M KCl. The lower phase was washed once with theoretical upper phase containing KC1 (chloroform-methanol-0.1 M KCl, 3 :48:47) and once with the theoretical upper phase containing no salt (chloroform-methanol-water, 3 :48:47). The method is that of Suzuki’. The upper phases were combined, concentrated, and dialyzed prior to determination of hexosamine and NANA. The lower phase contains cerebrosides and phospholipids. of the glycopeptides and glycosaminoglycans The defatted tissue residue was dried to remove the solvent, by warming to 37” in an incubator, The dried powder derived from I g of original tissue is suspended by homogenization in 4.5 ml of 0.1 M sodium acetate buffer, pH 5.5, containing 0.005 M EDTA. Sufficient cysteine is added to the mixture so that the concentration of cysteine is 0.005 M. Two times recrystallized papain (Sigma Chemical Co., St. Louis, MO.) is added. For each ml of acetate buffer used, the amount of papain added is 0.175 mg protein. The mixture is incubated at 60” in a shaking bath for 24 h. The suspension is centrifuged. ,The supernatant is set aside and the precipitate is resuspended by means of a hand homogenizer in the sodium acetate buffer and the proteolytic digestion is repeated as before. After centrifugation, the supernatant is combined with the first supernatant. The precipitate is the papain-resistant fraction. Extraction

Separation

of nondialyzable

and dialyzable glycopeptides

The volume of the combined supernatants is reduced to I/S of the original volume by use of a rotary evaporator. The solution is dialyzed for 24 h with three changes of water. The ratio (of dialysand to dialysate is I to Ioo for the first 6 h of dialysis. Dialysis overnight is carried out with a ratio of I to 200. A third period of dialysis is carried out for 6 h with a ratio of I : IOO. The dialysate is concentrated and contains Clin. Chim.

Acta.

32 (1971)

159_170

GANGLIOSIDES

AND GLYCOPROTEINS

x61

IN BRAIN Brain Ttssue m 19 vol./g C-M (2 1, viv)

I-----

SupeL*an* I Precipitate uspend m 3 ml 0.1 M

Combme supernatants. Add chloroform to bring to a C-M concentration of 2: 1 (v/v). Partition into upper phase wttb 0.2 vol 0.1 hi KCI. Wash lower phase with theoretical

sodrum a.cetate, pH 5.5

incubate 24 h wth papam. Centrifuge. Repeat. Combme two

upper phase containing

s”per”a*a”*S

r

I

Upper

I

I Precipitate Giyco~*noglycans nucleic actds

I Dialyzable glycopepttdes fwth some peptide impurttres)

I Free NANA, hexose, hexosamine, peptide and amino acld nnpurlties

I

I

1

Lower phase

Upper phases

Gel hItrattan on Sephadex G50

Non-dialyzable glycopeptldes

Excess cetylpyrldmum chloride fdlscwd)

P&de

lmpunty (discard)

Fig. I. Flow chart of the procedure which separates gangliosides, glycopeptides, and glycosaminoglycans from brain tissue samples.

the dialyzable glycope$tides. The dialysand contains the nondidyzable glycopeptides and ~~~~os~~~~o~~~~uns. Dialysis tubing (Size 20) was obtained from Union Carbide Corp%ation, Food Products kision, 6733 W. 55th Street, Chicago, Ill. 60638, U.S.A. SeparatioPt of the rcondialyzable glycopeptides

from the glycosamtioglycans

The E,,, due of the solution contaiking the, nondialy~ble ~lyco~ptides is determined. To each ml of the solution, I ml of 0.01 M sodium sulfate and I ml of cetylpyridinium chloride solution is added. A freshly prepared cetylpyridinium chlol%a. Ckim. Acta, 32 (1971)

159_170

162

BRUNNGRABERetd.

ride solution at a concentration of 0.034% per E,,, unit was employed. For example, for a solution with an E,,, value of 1x.8, the concentration of the cetylpyridinium chloride reagent was 11.8xo.o34~/~ or 0.4% cetylpyridinium chloride. The mixture was incubated at 30’ in a water bath for 30 min. The precipitate containing the glycosaminoglycans was separated by centrifugation. The supernatant was treated with pentan-r-01 to remove the cetylpyridinium chloride. For each mg of cetylpyridinium chloride calculated to be present in the solution, 0.2 ml of pentan-r-01 was added with stirring at 0’ in an ice bath. The two phases were separated in a separating funnel in the cold room; the upper pentan-r-01 phase was discarded. This step was repeated. The last traces of pentan-r-01 were removed by dialysis or evaporation. The final solution contained the nondialyzable glycopeptides. The precipitate obtained after the addition of cetylpyridinium chloride was dissolved in 2.1 M sodium chloride and treated with pentan-r-01 as above in order to remove the cetylpyridinium chloride. The salt was then removed by dialysis to provide a solution containing the glycosaminoglycans. Partial purification

of the dialyzable glycopeptides

The dialyzable glycopeptides contain salts and amino acids and peptides which have to be removed prior to analysis. This is accomplished by gel filtration on a Sephadex G-15 columns. Four ml samples are applied to a 42 x 2.5 cm column (bed volume of 180 ml). The column is equilibrated and eluted with water. The glycopeptides are not retarded, and are followed by a NANA-containing peak corresponding to free NANA. In order to obtain complete recovery of the glycopeptide sample, the test tubes containing free NANA are analyzed first. The position of free NANA can be determined by a prior standardization of the column. Free NANA is determined by applying the thiobarbituric acid method of Warrens without prior acid hydrolysis. All of the material eluted before the appearance of free NANA in the eluates is combined and concentrated. These are the dialyzable glycopeptides. The contents of test tubes containing free NANA can also be combined and analyzed. Analytical

Methods

The Elson-Morgan reaction as described by Boa+’ was used to analyze all of the fractions for hexosamine. Hydrolysis is performed for 6 h at 100’ in 1.5 M HCl. NANA is determined by the thiobarbituric acid methods, after hydrolysis in 0.1 N sulfuric acid at 80” for 2 h for the gangliosides and I h for the glycopeptides. The values obtained are corrected for destruction of NANA during the hydrolysis by multiplying by 1.3 and 1.1 for gangliosides and glycoproteins respectivelyll. Hexose is determined by the method of Dische et al.*= and FranGois et al.13 Fucose was determined by the method of Dische et ~1.~~. Materials

The defatted powder from cerebral gray tissue of normal 3- and 8-year-old children and the case of SSLE was provided by Dr. Robert Ledeen, who had previously reported on the ganglioside content of these specimens”. Brain samples from the case of early infantile neurolipidosis with failure of myelination (3 yrs-II mos of age)16 and brain tissue from a zg-year-old galactosemic patient were provided by Dr. Catherine Haberland. Neuropathological details on these cases will be published C%n. Chim. Acta, 32

(1971)

15g-170

GANGLIOSIDES

AND GLYCOPROTEINS

163

IN BRAIN

elsewhere. A sample of labeled chondroitinsulfuric R. U. Margolis and R. K. Margolis.

acid was kindly supplied by Drs.

RESULTS AND DISCUSSION

Extraction

of gangliosides

with chloroform-methanol

The ideal extraction procedure is one which clearly separates gangliosides from glycoproteins and glycosaminoglycans. Extraction of normal brain tissue with chloroform-methanol (2: I and I:Z, v/v) as described by Suzuki’ appears to meet these requirements. Suzuki noted that the thin layer chromatogram of the ganglioside preparations provided no evidence., for the presence of NANA-containing material other than gangliosides. Over 95% of the NANA presnt in the sxtract appears in the ganglioside area of the thin layer chromatogram. Glycoproteins contain large amounts of mannose, glucosamine, and fucose. It is of significance that the considerable research devoted to the elucidation of ganglioside structure (see review by Wiegandtle) has failed to uncover the presence of these sugars in chloroform-methanol extracts. The presence of glycoproteins or mucopolysaccharides in chloroformmethanol extracts has been suggested by Svennerholm17 and Balakrishnan and McIlwainls and more recently by Bogoch lB. However, isolation and identification of the non-gangliosidic material has never been reported. These suggestions were made to explain losses of NANA that occurred in subsequent fractionation procedures. Bogoch’P claim was dependent upon his finding that some of his ganglioside preparations had a high value for the hexosamine/NANA ratio. High hexosamine/NANA ratios, however, can be due to the loss of NANA during the more drastic conditions of his isolation procedure. An abnormally high hexosamine/NANA ratio was found in the case characterized by lipidosis and failure of myelination (Table I). This specimen had been preserved in’ formalin, which caused a loss of gangliosidic NANA groups’. The failure to find glucosamine by use of the GardellaopZ1method to distinguish between galactosamine and glucosamine and the failure to find mannose and TABLE

I

N-ACETYLNEURAMINIC SAMPLES

ACID-

AND

HEXOSAMINE-CONTAINING

CONSTITUENIS

RECOVERED

FROM

VARIOUS

BRAIN

All values are expressed in terms of pmoles/g tissue (wet). Cerebral gray matter was analyzed in all cases. Normal 8-yr-old

I. Gangliosides

2. Nondialyzable

glycopeptides 3. Dialyzable glycopeptides 4. Total glycopeptides (sum of 2+3) 5. Papain-resistant fraction 6. Glycosaminoglycans 7. Free NANA

Normal

SSLE 8-yr-old

Lipidosis. with Galactosemia failure myelination 25-yr-old (3 yv-rr mos)

3yr-old

NANA

Hexosamine

NA NA

Hexosamine

NANA

Hexosamine

NANA

Hexosamine

NANA

Hexosamine

2.53 0.72

n.d. I.93

2.19

n.d.

n.d. 0.70

n.d. 2.32

3.00 0.31

3.05 1.33

2.56 0.60

0.77

1.65

0.21

I.03

0.24

I.49

0.23

1.64

0.57

O.gI

0.27

0.40

0.93

2.96

0.87

3.14

0.97

3.96

0.88

2.24

0.87

1.60

0

0.32

0.03

0.23

0.06

0.26

0

0.30

0.07

0.07

o 0.08

0.39 -

0 0.06

0.34 -

o 0.06

0.22

0

O.IO

-

0.12

-

n.d.

-

n.d.

-

0.63

C&n. Chim. Acta,

32 (1971)

1.20

15~I70

BRUNNGRABER

164

et al.

fucose by paper chromatographic methods demonstrated that glycoprotein material was not present in the chloroform-methanol extract. Refluxing of brain tissue, particularly with solvents containing a high concentration of methanol, cannot be recommended. It is known22 that non-gangliosidic material increases as a contaminant with higher methanol concentrations, particularly at methanol concentrations above 500/o. Witting et al. 23 refluxed brain tissue and reported the presence of NANA at the origin of thin layer chromatograms and the possible presence of glucocamine in the extracts. This study is noteworthy since these materials were minor components. Since the hexose in these’preparations consisted solely of galactose and glucose, as determined by gas-liquid chromatography, one must conclude that glycoprotein material was absent. While extraction of normal brain tissue with chloroform-methanol provides extracts freed of glycoprotein material, it cannot be assumed that this would necessarily be the case with pathological material. Thus, Suzuki et aLe4 noted that chloroform-methanol extracts from visceral organs of a case of generalized gangliosidosis contain sialomucopolysaccharides and/or keratan sulfate. Testing for the presence of glucosamine, fucose or mannose in the gangliosidic material or separation of gangliosides from glycoproteins by thin layer chromatographya is a necessary confirmatory test. Brunngraber et al.25 provided evidence that the Suzuki’ extraction procedure removes all, or nearly all, of the gangliosides from brain tissue. The defatted protein residue from rat brain contains considerable amounts of NANA. However, nearly all of this material can be accounted for by the presence of glycoprotein-NANA and free NANA. A small amount (0.08 pmoles/g wet tissue) was found to be present in a papain-resistant fraction. The nature of this material is unknown. In several experiments with human (Table I), bovine 25, and rat brain2s tissue, the NANA content of this fraction was o to 4% of the NANA present in the gangliosidic fraction. Consequently, the Suzuki extraction procedure can be considered to be virtually complete. Some of the more polar gangliosides are not extracted with chloroforn-methano1 (2:1, v/v). If the extraction with chloroform-methanol (1:2, v/v )is omitted, most of the unextracted gangliosides appear in the papain-resistant fraction (Table II). TABLE

II

NANA-CONTAINING

CONSTITUENTSRECOVERED FROM RAT BRAIN

Double extraction (chloroform-methanol

Single extraction (chloroform-methanol

211 and 1:2,

2 :I. v/w) pmoles NA NA /g tissue

pmoles (wet) Gangliosides Glycopeptides Papain-resistant Free NANA

fraction

Total

v/w)

NA NA /g tissue

(wet)

2.22 1.00 0.08 0.11

0.94 0.45 0.11

1.62

3.41

3.12

Papain solubilizes some of the unextracted gangliosides, but these enter the amyl alcohol phase during the last stage of the purification procedure. Consequently, they would not contaminate the glycopeptide fractions. Clin. Chim. Acta, 32 (1971)

159-170

GANGLIOSIDES AND GLYCOPROTEINS

Papain

IN BRAIN

165

digestion

Proteolytic treatment of the defatted tissue residue solubilizes the carbohydrate portion of the glycoproteins as well as the glycosaminoglycans. Variations in pH of the digestion mixture as well as the use of pronase were studied (Table III). All conditions used were equally effective in solubilizing the aminosugar-containing constituents: The source of the free NANA is unknown. The presence of free NANA in TABLE EFFECT

III OF

pH

PROTEOLYSIS

AND

WITH

TEMPERATURE

PAPAIN

AND

ON

RELEASE

OF

GLYCOPEPTIDES

FROM

RAT

BRAIN

DURING

PRONASE

Incubations were carried out in sodium acetate buffer (0.1 M, pH 5.5) and sodium phosphate buffer (0.2 M, pH 6.5 anf 7.5). All values are expressed in terms of pmoles/g (wet) tissue. _ Total glycopeptades Free Papain- and pronaseNANA resistant fvactton NA NA Hexosamine NA NA Papain, pH 5.5, 60”. Papain, pH 6.5, 37” Pronase, pH 7.5, 37’

0.94

1.81

I.01

x.72 1.72

0.9x

0.07 0.03 0.07

0.16 0.15 0.15

brain tissue is to be expected. However, some of the free NANA recovered may have been split from the glycopeptides during the isolation procedure. The use of higher pH’s or lower temperatures during the proteolytic digestion did not eliminate the appearance of free NANA. Dialysis

The glycopeptides fall into three groups of molecular sizes. The large glycopeptides have molecular weights ranging from 4000 to 4650. These will not permeate a dialysis bag even after exhaustive dialysis against running water. All of these will appear in the nondialyzable fraction by the procedure described. The smallest glycopeptides (molecular weights ranging from 1300 to about 3000) are dialyzable, and all of these will appear in the dialyzable glycopeptide fraction by the procedure described. This fraction contains all of the NANA- and fucose-free glycopeptides. A glycopeptide or glycopeptides of intermediate size will appear in the dialyzable and nondialyzable fraction if dialysis is carried out as described. These glycopeptides are of special interest in that they contain a high concentration of fucose and these might be expected to be elevated in amount in cases of fucosidosis39b. If dialysis is prolonged, a greater proportion of these would appear in the dialyzable fraction. The dialysis procedure adopted by us is a compromise dictated by practical considerations. We have sought to keep the time of dialysis and the volume of dialysate which must be concentrated to a minimum in order to expedite the simultaneous handling of several samples. The amount of nondialyzable glycopeptides recovered from rat brain tissue has been reproducible to within 8%. of glycosaminoglycarcs by use of cetylpyridinium chloride The precipitation of glycosaminoglycans by means of cetylpyridinium chloride appears to provide a clearcut separation of glycopeptides from glycosaminoglycans. The precipitate (glycosaminoglycans) does not contain NANA and the supernatant

Precipitation

Cl%. Chim. Acta,

32 (1971)

15g-170

BRUNNGRABER et al.

166

(glycopeptides) does not contain glucuronic acidae.a7. Some low molecular weight hyaluronic acid may remain unprecipitatedae, although the amount present was not detectable by use of the carbazole reaction 28.The nondialyzable glycopeptides contain ester sulfate27922and both glycosaminoglycans and glycoproteins of rat brain become labeled after the intraperitoneal administration of Na, 35S0,.The labeled glycopeptides and chondroitinsulfuric acid were isolated by procedures described and subjected to column electrophoresis (Fig. 2). The mobility of the highly labeled chondroitinsul-

A. Glycopeptides ,, 06 _-

*

~moles NANA/ml cpm/ml

----

- 4000

z-l .;

:‘. : :

5 u x

- 1000 5

B. CSA ‘\ . .

lz ‘.

! 30

‘.

‘\

--.

60 Volumes

90 effluent

120

150

Fig. z. Chromatograms obtained when rat brain nondialyzable glycopeptides and chondroitinsulfuric acid were subjected to column electrophoresis utilizing the LKB apparatus No. 3340. Rats were injected intraperitoneally with carrier-free Na, %O, (6-8 PC/~) and sacrificed after 24 h in order to label the glycopeptides and chondoitinsulfuric acid. Electrophoresis was conducted at 3’ in 0.1 M glycine-NaOH buffer, pH 10.3 at 200 V and 23-27 mA for 55-60 h. The column was eluted with the same buffer and 3-ml fractions were collected. The fractions were analyzed for NANA and radioactivity.

furic acid is greater than that of the glycopeptides. The failure to find a component with a mobility corresponding to that of chondroitinsulfuric acid in the glycopeptide preparation indicates that this glycosaminoglycan was quantitatively precipitated by means of cetylpyridinium chloride. The advantage of the method described is that it provides for the separation and determination of the three major aminosugar-containing constituents of brain tissue. The ganglioside preparation can be analyzed for ganglioside components by thin layer chromatography’. The dialyzable and nondialyzable glycopeptides can be fractionated by column electrophoresis, anion exchange chromatography, or gel filtrationl~2~s. Glycosaminoglycans can be fractionated by fractional precipitation with cetylpyridinium chloride or electrophoresis in order to determine the amount of chondrcitinsulfuric acid, dermatansulfate, heparan, and hyaluronic acid present in the tissuegO-aP.The major impurity in the fraction containing the nondklyzable glycopeptides is a relatively large peptide which can be removed by gel filtrationsa. The dialyzable glycopeptides are also contaminated with peptide impurities; these can be Cl&. Chim. Acta, 32

(1971)

159-170

167

GANGLIOSIDES AND GLYCOPROTEINS IN BRAIN

removed by ion-exchange chromatography or paper chromatography3*. The major impurity in the glycosaminoglycan preparation is nucleic acid. This can be removed by the action of RNAase and DNAases6 or fractional precipitation with cetylpyridiniurn chloride36. Knowing the sensitivity of the analytical methods one is using, it is possible to estimate the amount of tissue required for analysis. Four grams of tissue would suffice to provide the data of Table I. A complete analysis of glycopeptides in the nondialyzable and dialyzable fractions by means of column electrophoresis and gel filtration1~2~8requires 70 g of wet tissue. Approximately 20 g of tissue would suffice to obtain data on the individual components comprising the glycosaminoglycan fraction. The amounts of tissue used can be reduced considerably by the use of microor semimicro-analytical methods. The predominate hexosamine in the nondialyzable glycopeptide fraction is glucosamine. Small amounts of galactosamine are also present. The dialyzable glycopeptides from rat brain have been reported to contain larger amounts of galactosamine*. The estimation was based on the use of the Radhakrishnamurthy and Berenso9 procedure for distinguishing between the two aminosugars. If the GardelP procedure is used a1,the predominate hexosamine appears to be glucosamine. Although this discrepancy can possibly be explained by the presence of mannosamine in the fractions21, positive identification of mannosamine has not yet been accomplished. A recent report33 that mannosamine is not incorporated into liver glycoproteins would appear to reduce the liktlihood that brain glycoproteins contain mannosamine. The use of the Garde11 column to separate glucosamine and mannosamine from galactosamine and the subsequent employment of analytical methods to distinguish between glucosamine and mannosamine a1 has shown that in human brain, the predominant hexosamine in the dialyzable glycopeptide fraction is glucosamine and that mannosamine is absent. In the case of the rat preparation, conflicting data have been obtained depending upon the analytical method used to distinguish between glucosamine and mannosamine. Dialyzable and nondialyzable glycopeptides can be analyzed for hexose and fucose. The results are shown in Table IV for various human and animal brain samples. TABLE

IV

CARnoHYDRATE CoMPosIrroN OF FROM

VARIOUS

BRAIN

THE

DIALYZABLE

AND

NONDIALYZABLE

GLYCOPEPTIDES

DERIVED

SAMPLES

All values are expressed in terms of pmoles/g tissue (wet). __NA NA Fucose

Hexosamine

Hexose

r .93 1.60 2.32

Nondialyzable glycopeptides Normal 8yr-old human cerebral gray SSLE 8-yr-old cerebral gray, human Normal 3-yr-old human cerebral gray Galactosemia, z5-yr-old cerebral gray Rat brain (whole) Bovine brain, cerebral gray

0.63 c.70 0.60 0.64 0.62

0.54 0.4r 0.47 0.29 0.30 0.3r

0.89 1.32

2.16 I.79 I.77 1.85 1.26 1.62

Dialyzable glycopeptides Normal 8-yr-old human cerebral gray SSLE 8-yr-old cerebral gray, human Normal 3-yr-old human cerebral gray Galactosemia. z5-yr-old cerebral gray Rat brain (whole)

0.21 0.24 0.23 0.27 0.24

0.22 0.25 0.27 n.d. 0.13

I .03 I.49 1.64 0.40 0.92

1.05 I .48 I.33 n.d. r .44

0.72

1.2c

Cl&z. Chim. Acta.

32

(1971)

I=jg-170

168

BRUNNGRABERef

al.

The nondialyzable glycopeptides have not presented any problems. On the other hand, some preparations of dialyzable glycopeptides from human and rat brain contain a substance which seriously interferes in the Dische et aLI reaction for hexose and fucase. This substance presents a spectrum similar to that of ribose in the 48-h modification of the cysteine reaction as described by Dische et a1.12. Some of the glycopeptides present in the dialyzable fraction from human and rat brain, unlike the larger glycopeptides, contain glucose as well as mannose and galactose. All of the glycopeptides present in the nondialyzable fraction contain NANA, glucosamine, galactosamine, mannose, galactose, and fucose1~2+~11. However, there is considerable variation in the relative amount of these sugars present in individual glycopeptides which comprise this fraction. For example, the largest glycopeptides in this fraction are rich in NANA (approximately 30-35% of the carbohydrate content) and poor in fucose (2%). The smallest glycopeptides are rich in fucose (approximately II%), but poor in NANA content. The dialyzable glycopeptide fraction contains many minor glycopeptide components, but two types of glycopeptide material predominate. One is a fucose-rich, NANA-poor glycopeptide the composition, molecular weight, and electrophoretic mobility of which corresponds closely to the smallest glycopeptide present in the nondialyzable fraction. The second glycopeptide does no+ contain NANA or fucose, and its hexosamine and hexose content accounts for nearly half of the amount of these sugars present in the dialyzable glycopeptide fraction. It is this glpcopeptide which may be elevated in SSLE (cf. below). Formalin-fixed brains cannot be analyzed by produres described in this communication. Lower values for glycopeptides are obtained and the material contains substances which interfere in the analytical methods. On the other hand, dried powders obtained after extraction of lipids with chloroform-methanol can be stored for at least 3 years if kept cold and dry. Analysis of such material for glycopeptides yielded results similar to those obtained with fresh tissue. Analysis of brain tissue stored frozen for periods up to 6 months have also yielded results similar to those obtained with fresh tissue. A one-hour autolysis (at 37’) of rat brain tissue had no effect on the amount of glycopeptides recovered from the tissue, nor was the carbohydrate composition of the glycopeptide preparation affectedZ5. While the NANA content of the nondialyzable glycopeptide fraction obtained from the gray matter of the normal 3-year-old child (Table I) was equal to that obtained from the 8-year-old, the hexosamine content of this fraction appears to higher in the 3-year-old. The ratio of hexosamine/NANA increased from 3.2 to 3.7 between 3 and 8 years of age. The hexosamine content of the dialyzable glycopeptide fraction was considerably higher in the 3-year-old gray matter. Higher values for “residual hexosamine” in the 3-year-old cerebral cortex had been noted earlier by Cumings et a1.39. The present work indicates that this elevation at the earlier age is due to, an increased level in glycoprotein-hexosamine rather than glycosaminoglycan-hexosamine. An abnormality in the glycopeptides derived from the case of neurolipidosis characterized by a failure of myelinationl6 was indicated by the low NANA and,hexosamine content of the nondialyzable glycopeptide fraction as well as an abnormally low value for the hexosamine/NANA ratio. The dialyzable glycopeptides in this case contained an abnormally high level of NANA and a low value for hexosamine. The galactosemic brain contained considerably less hexosamine in both the dialyzable and nondialyzable glycopeptide fractions. This decrease was accompanied by a slight, ClZn. Chim. ACta, 32

(1971)

15+170

GANGLIOSIDES

AND GLYCOPROTEINS

IN BRAIN

169

probably insignificant, decrease in NANA content of the glycopeptide fractions. The cerebral gray matter of the B-year-old SSLE case showed only a slight reduction in nondialyzable glycopeptide material, and the value for the ratio hexosamine/NANA was normal. There appeared to be a significantly increased amount of hexosamine in the dialyzable glycopeptide fraction, if this value is compared to that obtained for the normal B-year-old. The values for glycosaminoglycan-hexosamine obtained for normal and SSLE brain (Table I) are 0.39 and 0.34 pmoles hexosamine per gram brain respectively. These values are close to those obtained by Suzuk? (0.26 pmoles) for normal adult brain, but lower than the value reported by Singh et a1.3°~32 (0.82). Szabo and RobozEinstein41 and MargoliF obtained values of 0.65 and 0.61 pmoles/g for bovine brain. Glycosaminoglycans were not altered in SSLE, but low values were noted in a case of early infantile neurolipidosis with failure of myelination15 and galactosemia. Cumingsa reported an elevation in residual hexosamine in the cerebral cortex in SSLE. The total hexosamine recovered from the glycoproteins and glycosaminoglycans in our case (3.48 pmoles/g) was close to that obtained for the normal B-yearold brain (3.35 ,umoles/g). However, a significant increase in the hexosamine content of the dialyzable glycopeptide fraction was noted. This increase was compensated for by the decrease in the hexosamine in the nondialyzable glycopeptide fraction. Consequently, the total glycopeptide hexosamine of the SSLE brain (3.14 ymoles/g) was not significantly different from that of the normal brain (2.96 pmoles/g). Since the increase of hexosamine in the dialyzable glycopeptide fraction was not accompanied by an increase in its NANA content, it is suggested that the glycopeptide which contains hexosamine and hexose, but which is devoid of NANA and fucose, is elevated. The fucose-rich NANA-containing glycopeptides present in this fraction are probably not affected. The data suggests that the increase noted by Cumings in this disease may have been due to an elevation of glycoproteins which contain hexosamine and hexose, but lack NANA and fucose. REFERENCES I E. G. BRUNNGRABER, in A. LAJTHA (Ed.), Handbook of Neurochemistry, Vol. I, Plenum Press, New York, 1969, p. 223. 2 E. G. BRUNNGRABER, in A. LAJTHA (Ed.), Protean Metabolism of the Nervous System, Plenum Press, New York, 1970. p. 338. 3 E. G. BRUNNGRABER, J. P&at., 77 (1970) 166. 4 P. A. OCKERMANN, J. Pediat., 77 (1970) 168. 5 P. DURAND, C. BORRONE AND G. DELLA CELLA, J. Pedaat., 75 (Ig6g) 665. 6 J. N. CUMINGS, in A. S. ROSE AND C. M. PEARSON (Eds.). Mechanisms of Demyelination, McGraw H111,Inc., New York, 1963, p. 58. 7 K. SUZUKI, J. Neurochem., IZ (1965) 629. 8 C. DI BENEDETTA, E. G. BRUNNGRABER, G. WHITNEY, B. D. BROWN AND A. ARO. Arch. Biochem. Biophys., 131 (1969) 404. g L. WARREN, J. Biol. Chem., 234 (1959) 1971. IO N. F. BOAS, J. Bzol. Chem.. 204 (1953) 553. II E. G. BRUNNGRABER AND B. D. BROWN, Biochem. J., 103 (1967) 65. 12 Z. DISCHE, L. B. SHETTLES AND M. OSNOS, Arch. Biochem., 22 (1949) 169. 13 C. FRANFOIS, R. D. MARSHALL AND A, NEUBERGER, Biochem. J., 83 (1962) 335. 14 R. LEDEEN, K. SALSMAN AND M. CABRERA. J. Lipid Res,, g (1968) rzg. 15 C. HABERLAND AND E. G. BRUNNGRABER, Arch. Neural., 23 (1970) 481. 16 H. WIEGANDT, Evgeb. Physiol., Biol. Chem. Exptl. Phavmakol., 57 (1966) Igo. r7 L. SVENNERHQLM, J. Neurochem., I (1956) 42. 18 S. BALAKRISHNAN AND H. MCILWAIN, Biochem. J., 81 (196x) 72. Clin. Chim.

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