Glycoprotein biosynthesis in the developing rat brain I. Microsomal galactosyltranferase utilizing endogenous and exogenous protein acceptors

390

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26644

G L Y C O P R O T E I N B I O S Y N T H E S I S IN T H E D E V E L O P I N G RAT B R A I N I. MICROSOMAL G A L A C T O S Y L T R A N S F E R A S E U T I L I Z I N G ENDOGENOUS AND E X O G E N O U S P R O T E I N ACCEPTORS

G. K. "W. NO AND E. R A G H U P A T H Y

Brain-Behavior Research Center, Sonoma Stale Hospital, Eldridge, Calif. 9543 z ( U . S . A . ) ( Receiv ed Ap ril i 4 t h , 1971)

SUMMARY

I. The enzyme UDP-galactose: glycoprotein galactosyltransferase, which transfers galactose from UDP-galactose to both endogenous protein acceptors and a defined exogenous acceptor (desialized, degalactosylated fetuin) has been characterized from rat brain microsomes. The enzyme requires Mn ~+ for maximal activity and has a p H optimum of 6.3. Co s÷ can partially replace Mn=+; other bivalent cations had no effect. The sulfhydryl nature of the enzyme is indicated by the observations that the activity is inhibited by p-chloromercuriphenylsulfonate and that the inhibition can be prevented by the addition of dithiothreitol. The galactose transfer activity is higher in the smooth than in the rough lnicrosomes and is present in cortex, cerebellum, brain stem, hypothalamus and hippocampus. The cortex has the highest activity,. 2. The transfer of galactose from UDP-galactose to endogenous acceptors is very low in the neonatal rat brain, but increases steadily during development. The activity in the adult brain is about 6 times as that found in the brains of the newborn rats. in contrast to its limited ability to transfer -~4C]galactose to endogenous acceptors, the enzyme preparation from the neonatal rat brain transfers considerable amounts of galactose to added desialized, degalactosylated fetuin. This activity towards the exogenous protein acceptors increases slightly during development, the activity of the mature brain being only" about 1.5 times that of the infant brain. These results lend support to the view that a specific glycoprotein(s) appear in brain during ontogenesis.

INTRODUCTION

A great number of cellular proteins have been shown to be associated with polysaccharides. These glycoproteins have diverse functions and presumably possess specific biological significance. Abnormalities of metabolism of these conjugated Abbreviation used : HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic Biochim. Biophys. Acta, 244 (I971) 396-409

acid.

BRAIN GLYCOPROTEIN SYNTHESIS

397

proteins have been associated with a number of clinical disorders (e.g. cystic fibrosis, amyloidosis). Studies on the structures and biosynthesis of these carbohydrate-containing macromolecules are extensive and include several species and a variety of tissues. Nervous tissues contain an abundance of glycoproteins 1,2 which are associated with the membranous elements in synaptosomes, microsomes and glial processes. While the functions of these proteins are not precisely understood, it is clear from the work of GESNER AND GINSBERGa and of CRANDALLAND BROCK4 that they may be involved in the mediation of intercellular recognition. BARONDES AND DUTTON5 have recently demonstrated a rapid turnover of soluble nerve ending glycoproteins and emphasized the possible importance of glycoprotein metabolism in the regulation of nerve-ending function. The experiments of BOGOCH6 on the alteration in the levels of certain glyeoproteins during training also underscore the potential importance of these molecules. In our laboratory we have undertaken a systematic study of the pathways by which glycoproteins are synthesized in the central nervous system with the ultimate view of establishing the role of brain glycoproteins in the coding of experiential information. The present report deals with some properties of a microsomal enzyme which catalyzes the transfer of galactose from UDP-I14CJgalactose to both endogenous and exogenous protein acceptors. It is shown that the enzyme is preferentially located in the smooth microsomal fraction. Evidence is also presented which indicates that while enzyme activity is high in brain tissues from neonatal rats, the acceptor capacity of the microsomal protein fractions from brains of mature rats is higher than that of the fractions from neonatal rats. EXPERIMENTAL

Materials Uniformly labeled UDP-E14Clgalactose (298 mC/mmole) was purchased from New England Nuclear Corporation, Boston, Mass. a-Amylase, purified from hog pancreas was a product of Sigma Chemical Co., St. Louis, Mo. Fetuin, dithiotreitol and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) were obtained from Calbiochem, Los Angeles, Calif.

Preparation of subcellular fractions Unless otherwise stated, brains from adult male Sprague-Dawley rats were used. The animals were lightly anesthetized with ether and then killed by decapitation. The brains were removed and homogenized with 5 vol. of a buffer (pH 7.5); containing 25 ° mM sucrose and 40 mM Tris, in a Potter-Elvehjem homogenizer equipped with a teflon pestle. The homogenate obtained after 12 up and down strokes was spun at 12000 × g for 15 min and the pellet was discarded. The supernatant was centrifuged at iooooo × g for 60 rain and the pelleted microsomes were washed twice with the buffer. In some experiments, the microsonles were further fractionated into smooth and rough mierosomes according to the method of DALLNERv. The 12000 × g supernatant was adjusted with I M CsCI to a final CsC1 concentration of 15 raM. 6 ml of the mixture was layered over 6 ml of 1.3 M sucrose in 15 mM CsC1 and centrifuged at 145o00 × g for 3.5 h. The pellet, consisting of rough microsomes, was washed with Biochim. Biophys. Acta, 244 (1971) 396-409

39~;

t;. K. W. KO, E. RAGHUPATHY

the homogenizing medium before use. The clear upper phase was removed b y aspiration and discarded. The entire fluffy interface at the gradient b o u n d a r y was collected, diluted with cold water and centrifuged at 145 ooo × g for I h to sediment the smooth microsomes. The sediment was washed twice with the honlogenizing nledium. In experiments in which synaptosomes and mitoehondria were isolated, the brains were homogenized in IO vol. of 0.32 M sucrose and the homogenate was spun at 800 × g for IO min. The pellet was washed twice with 0.32 M sucrose and the washings were combined with the original 8o0 × g supernatant. The combined supernatant was spun at 14500 × g for IO min and the supernatant was saved for preparation of microsomes (the total yield of the microsomes prepared b y this procedure was higher than t h a t obtained by the previous procedure, but the specific activities of the nlierosomal fractions were the same). The 14500 × g pellet was resuspended in o.32 M sucrose (3 ml/g of original tissue) and was layered on ficoll gradients according to the procedure of KUROKAWA et al. 8. The gradients were centrifuged at 18825 y. g for 15 rain. The mitochondrial pellet and the interface consisting primarily of synaptosomes were suspended in 0.32 M sucrose and spun at 14500 × g for 15 rain. 1"he pellets were rinsed with the cold sucrose solution once and then frozen at 9 °° until use.

Assav of galactose transferase activity The galaetose transferase activity of the various brain fractions was assayed by measuring the transfer of [14C]galactose from UDP-[14Cjgalactose to protein acceptors insoluble in IO~"o tlichloroacetic acid. The incubation mixture contained the following, in a final volume of 0.25 ml: o.336 nmole UDP-D-I l*C]galactose, (approximately I , lO 5 counts/rain), 61riM MnCI:, 3 mM dithiothreitol, 60 mM H E P E S buffer (pH 6.3) and I mg of protein. When the Triton-solubilized enzyme was used, I mg of desialized, degalactosylated fetuin was also included in the above incubation mixture. After incubation at 37 ° (the incubation periods are specified in the tables and figures), the mixture was chilled in ice and the following additions were made: 50 nmoles U D P galactose, o.I ml 1.5°/0 deoxycholate in 60 mM H E P E S buffer (pH 6.3), and 5o0 Iooo units of a-amylase. The entire mixture was again incubated at 37 ° for 2 h. The latter incubation served to destroy all the radioactivity associated with glycogen. After incubation, 2 ml of ice-cold IO°.'o ttichloroacetic acid were added to the mixture and the precipitated protein pellet was washed successively with 2 ml aliquots of the trichloroacetic acid solution, acetone, chloroform-methanol (2 : I, b y vol.), and ether. The washed protein residue was dissolved in 0.5 ml of formic acid and assayed for radioactivity as described elsewhere ~. For each sample, the "zero time" value was subtracted. In general, this value was 25-4 ° counts/rain. Since the brain tissue contains substantial amounts of lipids, the possibility existed t h a t under the above washing conditions, lipids were adsorbed to the trichloroaeetic acid-denatured proteins and were hence not efficiently removed b y the organic solvents. This possibility was examined b y terminating some reactions b y the addition of chloroform-methanol (2:1, v/v) to the incubation mixture and then washing the insoluble proteins with trichloroaeetie acid and the other solvents as described above. Both the procedures gave identical results.

Triton extraction of microsomal enzyme The microsomal pellet was suspended in the Tris-sucrose buffer solution (I Inl/g Biochim. Biophys. Acta, 244 (1971) 396-4o 9

BRAIN GLYCOPROTEINSYNTHESIS

399

of original weight of brain taken). The suspension was treated with an equal volume of 0.5% (v/v) Triton X - I o o for 3 ° rain at 4 °. The mixture was spun at 144000 × g for 12o rain and the supernatant removed. The resulting pellet was extracted once more with Triton X - I o o and this extract was pooled with the first. The combined extracts were dialyzed against cold distilled water for 24 h with at least 3 changes. The dialyzed solution was lyophilized and the d r y material was stored for later assay of enzyme activity.

A cceptor preparation Endogenous acceptors. Washed microsomes prepared from brains or rats of different ages were suspended in buffer medium and placed in a boiling water bath for 3 rain ; the mixture was re-homogenized gently and then used as source of endogenous acceptors for galactose transferase. Desialized, degalactosylated fetuin. Sialic acid and galactose were removed from native fetuin essentially according to the procedure of SPIRO TM. Fetuin was treated with 0.05 M H2SO, at 80 ° for 60 rain and the solution after neutralization was dialyzed successively against o.i M NaC1 and distilled water. The desialized fetuin was next treated with o.oi M sodium metaperiodate in 0.05 M sodium acetate buffer p H 4.5 in the dark at 0-4 ° for 16 h. The oxidation reaction was stopped b y the addition of an excess of ethylene glycol. The entire mixture was dialyzed against o.i M NaC1 and distilled water. The dialyzed solution was then treated with an equal volume of o. I M NaBH~ in sodium borate buffer p H 8.0 and the reduction was carried out at 4 ° for 12 h. Tile p H of the mixture was then adjusted to 5.0 with acetic acid and the mixture was dialyzed against o.i M NaC1 and distilled water. The solution was then made 25 mM with respect to H~S04 and heated at 9 °o for 2 h. The solution was neutralized and then dialyzed exhaustively against o.I M NaC1 and distilled water and the dialyzed solution was lyophilized.

Identification of 14C-labeled compound incorporated In order to identify the sugar t h a t was added to the acceptor, the assays were carried out in the usual manner with the exception t h a t the incubation mixture was scaled up b y a factor of IO. The precipitated protein was washed as described in an earlier section, and then hydrolyzed with 4 M HC1 at IOO° for 4 h. The hydrolysate was centrifuged to remove insoluble material and the supernatant was dried in a v a c u u m dessicator. The dried sample was taken up in a small volume of ethanol and chromatographed on W h a t m a n No. I paper in the following solvent systems: (I) n - b u t a n o l acetic acid-water (5 : 2 : I, b y vol.) and (2) pyridine-ethyl acetate water (5 : 18 : 6, by vol.). I d e n t i t y of the sugar was established b y comparison with authentic galactose marker. Protein in the fractions was determined b y the biuret procedure of GORNALL

et al.lk RESULTS Preliminary studies indicated t h a t exclusion of Mg 2+ in the homogenizing medium increased the subsequent galactose transferring activity of the brain fractions. Consequently, in all our experiments, Mg2+-free medium was employed for homogeniz-

Biochim. Biophys. Acta, 244 (1971) 396-409

400

G.K.W.

K O , E. R A G H U P A T H Y

ation. The results presented in Table I show that among the different subcellular fractions of rat brain which were studied, microsomes had the highest capacity to transfer [i~C]galactose from UDP-[14C]galactose to endogenous protein acceptors. The other fractions showed little or no activity. It should, however, be pointed out, that since the various subcellular fractions contain both the enzyme and the acceptor proteins, the observed differences in the activity of the various fractions could be the result of differences in enzyme activity, acceptor capacity, or both. In studies which follow microsomes were used as the source of enzyme.

Characteristics of the galactose transfer system in microsomes Endogenous system. Some properties of the microsomal enzyme system for the TABLE

1

TRANSFER OF L14CJGALACTOSE FROM I~Dp-~I4C]OALACTOSE TO ENDOGFNOUS PROTEINS BY SUBCELLULAR FRACTIONS OF RAT BRAIN S u b c e l l u l a r f r a c t i o n s w e r e o b t a i n e d f r o m b r a i n s of 6 2 - d a y - o l d r a t s , as d e s c r i b e d in EXPERIMENTAL. I n c u b a t i o n m i x t u r e c o n t a i n e d 6 m M MnCI~, 3 m M d i t h i o t h r e i t o l , 6 o m M H E P E S b u f f e r ( p H 6.3), o . 3 3 6 n m o l e U D P - F 1 4 C ] g a l a c t o s e ( o . i ,aC; i . o 1. 5 • i o ~ c o u n t s / m i n ) , a n d I m g p r o t e i n , all i n a f i n a l v o l u m e of o . 2 5 m l . T h e m i x t u r e w a s i n c u b a t e d f o r i h a t 37 °. E a c h v a l u e r e p r e s e n t s t h e m e a n ± S . D . of 3 o b s e r v a t i o n s .

Fraction

Protein (mg/g of tissue)

[laC]Galactose transferred (counts~rain per mg protein)

Honlogenate Synaptosonles Mitochondria Microsomes IOOOOO × g s u p e r n a t a n t (dialyzed)

125 4 .2 2.5 9.7 26.0

229 44 55 3519 240

TABLE

~: ~= ± ± ±

16 0.7 0.5 1.9 6..5

+ 20 Jz 4 • 7 ± 14o ~_ 42

1[

CHARACTERISTICS OF THE GALACTOSE TRANSFER SYSTEM IN RAT BRAIN MICROSONIES E x p t . I a n d I [ : m i c r o s o i x l e s p r e p a r e d f r o m b r a i n s of 3 5 - d a y - o l d r a t s w e r e u s e d . E x p t . I H : m i c r o s o m e s w e r e f r o m b r a i n s of 2 5 - d a y - o l d r a t s . Incubation systems : E x p t . I : t h e s a m e as d e s c r i b e d in l e g e n d t o T a b l e I e x c e p t M n 2+ w a s o m i t t e d . W h e r e i n d i c a t e d 6 m M m e t a l i o n s w e r e a d d e d . E x p t . I [ : t h e s a m e a s i n T a b l e I. E x p t . I I [ : a s d e s c r i b e d i n T a b l e I e x c e p t t h a t d i t h i o t h r i t o l w a s o m i t t e d . W h e r e i n d i c a t e d 1. 5 m M p - c h l o r o m e r c u r i p h e n y l s u l f o n a t e and/or 3 mM dithiothreitol was added.

Expt. No.

A dclition

[14C]Galactose transferred to protein (counts/rain)

% Of control

I

None M n 2+ C a ~+ C o 2+ Ba=+ Cs + M g =+

850 3275 870 2285 935 870 lO5O

ioo 385 IO2 269 I io lO2 124

II

None DSG-fetuin*

3275 6175

IOO 189

2515 2840 260

IOO I 13 lO

331o

132

II[

(2 rag)

None Dithiothreitol p-Chloromercuriphenylsulfonate p-Chloromercuriphenylsulfonate + dithiothreitol

* Desialized, degalactosylated

fetuin.

Biochim. Biophys. Mcta, 2 4 4 (1971) 3 9 6 - 4 0 9

401

BRAIN GLYCOPROTEIN SYNTHESIS

transfer of galactose from UDP-galactose to protein acceptors are shown in Table II. Significant transfer of galactose to trichloroacetic acid insoluble proteins took place even in the absence of any added acceptor proteins, presumably to incompletely glycosylated endogenous proteins. The enzyme showed a strict requirement for Mn 2+, which could not be replaced b y Mg 2+. Among the other ions tested, only Co ~÷ was partially able to replace Mn2+; it was about 70% as active as Mn 2+. Ba 2+, Ca 2+ and Cs+ were ineffective. The inclusion of 2 mg of desialized, degalactosylated fetuin in the incubation medium resulted in a substantial increase in the radioactivity recovered in the protein indicating that the microsomal enzyme is also capable of glycosylating exogenous protein. Native fetuin was not galactosylated by the microsomal enzyme. Hydrolysis of the ~4C-labeled protein at IOO° for 4 h in 4 M HC1 released over 9o% of the radioactivity, all of which cochromatographed with carrier galactose in the solvent systems described in EXPERIMENTAL. The sulfhydryl nature of the enzyme is shown by the results of Expt. I I I in Table II. Although dithiothreitol at 3 mM concentrations increased the galactose transfer to endogenous proteins by only 13%, addition of 1. 5 mM p-chloromercuriphenylsulfonate resulted in an almost complete inhibition of the transfer reaction. The presence of dithiothreitol in the incubation system, however, prevented the inhibition due to p-chloromercuriphenylsulfonate. The transfer of E~4Clgalactose from UDP-[14Clgalactose to endogenous acceptors was proportional to microsome concentration (Fig. I) and had a p H optimum of 6.3 (Fig. 2). The data in Fig. 3 illustrate the time course of transfer of ~4C]galactose to endogenous acceptors by microsomes. The reaction proceeded rapidly during the 9500 3500

~E

750C

~X

cx

8 ¸

£_ 0

3ooc

.£

E m g

xJ

55oc

O

25OC

\.

0

350C

o 2OOC _s

7 150C

IO

I I 10 20 Microsomal protein (mg)

I 30

L 5.0

I

6D

I

I

70

80

pH

Fig. i. E f f e c t of m i c r o s o m e c o n c e n t r a t i o n on [14C]galactose t r a n s f e r t o e n d o g e n o u s p r o t e i n s . Inc u b a t i o n c o n d i t i o n s w e r e as in T a b l e I. Microsomes w e r e p r e p a r e d f r o m 35-da y-ol d r a t bra i ns . B l a n k v a l u e ( w i t h o u t microsomes) w a s 22 c o u n t s / m i n . Fig. 2. T h e influence of p H on t h e t r a n s f e r of E14C]galactose from U D P - [ x i C ] g a l a c t o s e b y b r a i n m i c r o s o m e s p r e p a r e d from 3 3 - d a y - o l d r a t s to e n d o g e n o u s a c c e pt ors . I n c u b a t i o n c o n d i t i o n s w e re t h e s a m e as in T a b l e I a n d w i t h H E P E S buffers of t h e p H i n d i c a t e d .

Biochim. Biophys. Acta, 244 (1971) 3 9 6 - 4 0 9

402

G. K. W. KO, E. RAGHUPATHY 9000

4OOO

8000 E O_ E

30oo

E *d

/

m

7000 //

6000

5000

2000

/J 0

~ 4000

/ o

~ C 3oo(

~ooo

u 200(

o;

I

60

I

120

111-.-2

180 "240

Time(rain)

0

0

I

1

[

2

I

3

r

4

I

5

DSG-fetuin added (nag)

Fig. 3. Effect of length of incubation time on the transfer of [14C]galactose to endogenous acceptors by brain microsomes prepared from 35-day-old rats. Incubation conditions were as described in Table I. Fig. 4- The influence of desialized degalactosylated fetuin (DSG-fetuin) concentration on the transfer of []4C]galactose to DSG-fetuin by brain microsomes prepared from 55-day-old rats. Incubations were carried out under conditions described for Expt. II in Table II. initial 3 ° min a n d the reaction was essentially complete within the first hour. The transfer of galactose to an exogenous acceptor, n a m e l y desialized, d e g a l a c t o s y l a t e d fetuin as a function of desialyzed, d e g a l a c t o s y l a t e d fetuin c o n c e n t r a t i o n is expressed in Fig. 4. The a c t i v i t y was linear u p to a desialized, d e g a l a c t o s y l a t e d fetuin concent r a t i o n of 3 rag/0.25 ml of i n c u b a t i o n m e d i u m .

Triton-solubilized enzyme system. The t r a n s f e r of [14C~galactose from U D P [14C~galactose into endogenous acceptors b y the Triton-solubilized e n z y m e was g e n e r a l l y less t h a n a t h i r d of t h a t o b t a i n e d w i t h the microsomes. However, a d d e d desialized, d e g a l a c t o s y l a t e d fetuin was g a l a c t o s y l a t e d far more efficiently b y the Triton-solubilized system. T h e general p r o p e r t i e s of the Triton-solubilized s y s t e m (with respect to p H , m e t a l ion r e q u i r e m e n t , t i m e course, etc). were essentially similar to those described a b o v e for the endogenous system. Studies on the sugar a n d s u g a r nucleotide specificity in the t r a n s f e r r e a c t i o n c a t a l y z e d b y the t r i t o n e x t r a c t showed t h a t little or no i n c o r p o r a t i o n of label into p r o t e i n (both endogenous as well as a d d e d desialized, d e g a l a c t o s y l a t e d fetuin) was o b s e r v e d w i t h ~C-labeled UDP-glucose, U D P - N - a c e t y l g l u c o s a m i n e , glucose, galactose or glucosamine. A ~-ecent r e p o r t from this l a b o r a t o r y ~2 dealt with the transfer of galactose from U D P - g a l a c t o s e to lipid acceptors b y r a t b r a i n m i c r o s o m a l fractions. Since the enz y m e s t u d i e d in the p r e s e n t work also resides in the m i c r o s o m a l fraction, t h e specificity of the T r i t o n - e x t r a c t e d e n z y m e was s t u d i e d using desialized, d e g a l a c t o s y l a t e d fetuin as a p r o t e i n a c c e p t m a n d h y d r o x y f a t t y acid c e r a m i d e as a lipid acceptor. The results p r e s e n t e d in T a b l e n I clearly show t h a t while the e n z y m e g a l a c t o s y l a t e d desialized, d e g a l a c t o s y l a t e d fetuin, no transfer of galactose to t h e lipid acceptor t o o k place. Biochim. Biophys. Acta, 244 (1971) 396-409

403

BRAIN GLYCOPROTEIN SYNTHESIS TABLE III LACK OF TRANSFER OF [14C]GALACTOSE FROM CERAMIDE BY TRITON-SOLUBILIZED ENZYME

UDP-[X4C]GALACTOSE

TO

HYDROXY

FATTY

ACID

Triton-solubilized enzyme (I m g protein; p r e p a r e d f r o m brains of 6o-day-old rats) was incubated with I mg of desialized, degalactosylated fetuin (DSG-fetuin) a n d UDP-[t4C]galactose u n d e r conditions described in Table I or w i t h 0.25 mg h y d r o x y f a t t y acid ceramide dispersed on 25 mg of celite u n d e r conditions described in ref. 12. I n c u b a t i o n time, 60 rain.

A cceptor

[x~C]Galactose transferred (counts/rain)

DSG-fetuin H y d r o x y f a t t y acid ceramide

12 8o0 4°

Galactose transfer by rough and smooth microsomal fractions of rat brain The results of experiments presented in the preceding sections clearly show that rat brain microsomal fractions are capable of catalyzing the enzymatic transfer of labeled galactose to endogenous as well as added protein acceptors. In attempts to localize the enzyme within submicrosomal fractions of the rat brain, brain microsomes were fractionated into rough and smooth fractions as described in EXPERIMENTALand the capacity of these subfractions to transfer galactose from UDP-galactose to endogenous proteins was investigated. I t can be seen from the results presented in Table IV that galactosyltransferase activity is higher in the smooth microsomal fraction, the ratio of smooth to rough being 3.5 : I. TABLE IV DISTRIBUTION RAT BRAIN

OF GALACTOSE

TRANSFERASE

ACTIVITIES

IN SMOOTH AND ROUGH

MICROSOMES

FROM

R o u g h and s m o o t h microsomes were p r e p a r e d from i8-day-old r a t brains b y the procedure described in text. I n c u b a t i o n conditions are as described in Table I. E a c h value is the m e a n ± S.D. of 3 observations.

Fraction

Protein (mg/g of tissue)

[14C]Galacgose transferred (counts/min per rng protein)

R o u g h microsomes S m o o t h microsomes Total microsomes

1.43 :~ 0.32 2-64 -~ 0.49 lO.5O ± 1.82

lO9O -4- 13o 3820 -~ 375 215o ± 17 °

These results are compatible with those of MOLNARet al. 13 who have shown that in the case of liver, smooth microsomes incorporate galactose 3-4 times faster than the rough microsomes. A similar preferential localization of thyroidal galactosyltransferase enzyme in the smooth microsomal fraction has been demonstrated by BOUCHILLOUX et al. 14.

Other studies on the distribution of galactose transfer activity in different regions of the rat brain showed that although the transfer activity was highest in the cortex, it was distributed in all the regions studied.

Changes in microsomal galactose transfer activity during brain development The galactose transfer activity in the microsomes of rat brain exhibits a definite developmental increase during the early life of the animal (Fig. 5). The activity, which was low in the first week of life, increased rapidly at the beginning of the second week and reached the levels observed in mature animals b y the end of the fourth week. Biochim. Biophys. Acta, 244 (1971) 396-4o 9

404

G.K.W.

KO, E . R A G H U P A T H Y

30oo

2500

E

j ....

c

2000

E

,/ / /

g

1500 /

1000

/

J

/

5ooi

,/ o"

%

10

,¢

I I 15 20 Age (days)

25

6O

Fig. 5. Transfer of [~C]galactose from UDP-~4C]galactose to endogenous acceptors by brain microsomes obtained from rats of different age groups. Each point represents the nlean ± S.D. obtained from 6 animals. Conditions of incubations were as in Table I. A l t h o u g h these d a t a d e m o n s t r a t e d t h a t the c a p a c i t y to transfer galactose from U D P - g a l a c t o s e to endogenousproteins increases during brain d e v e l o p m e n t , t h e y do not indicate w h e t h e r this increase is due to an increase in t h e e n z y m e a c t i v i t y itself, or an increase in the acceptor c a p a c i t y of the proteins. In e x p e r i m e n t s to clarify this question the e n z y m e a c t i v i t y of microsomal fractions of brains o b t a i n e d from rats of different age groups was assayed against the exogenous acceptor, desialized, degalactos y l a t e d fetuin. T h e results of this e x p e r i m e n t (Fig. 6) show t h a t considerable transfer of galactose to desialized, d e g a l a c t o s y l a t e d fetuin took place in the microsomes of n e o n a t a l rat brains. W i t h increasing age, there was an increase in the galactosylation of a d d e d fetuin. P e a k transfer a c t i v i t y was observed in brains of 22-day-old

Endogenous ~ protein LJ

E

~ DSG~j fetuin

5o00

o

4ooo

V b

3000

N 2000

M -~

lOOO 0,

3

15 22 Age (days)

28'

60

Fig. 6. The transfer of [t4C]galactose from UDP-[t4C]galactose to desialized, degalactosylated fetuin (DSG-fetuin) by brain microsomes obtained from rats of different age groups. The value represent the mean 4- S.D. of 3 experiments using at least 3 rats in each age group. Conditions of incubation were as in Expt. II, Table II. Biochim. Biophys. Acta, 244 (1971) 396-4o9

405

BRAIN GLYCOPROTEIN SYNTHESIS

rats and remained relatively constant thereafter. In 3 separate experiments, it was observed that 5o-7o°/0 of this peak activity was present in the brains of newborn rats ( 1 - 3 day old). These results strongly suggest that substantial galactosyltransferase activity is present in the neonatal rat brain, but raises to a steady value by the third week of life. However, since the developmental pattern of galactosylation of desialized, degalactosylated fetuin did not follow that observed with endogenous protein acceptors, it m a y also be concluded that the acceptor capacity of endogenous protein increases markedly during development of brain. Additional support for this hypothesis comes from the results of another type of experiment in which brain microsomes from 3-day-old rats were assayed for galactose transfer activity against heat-treated microsomal preparations from brains of 3-, ii-, and 6o-day-old rats. If our postulate were correct, then one should observe greater stimulation with the addition of acceptors from the mature than from the neonatal brain. As can be seen from the results presented in Table V, there was a slight stimulation (28%) when heat-treated microsomes from 3-day-old rat brains TABLE V ACCEPTOR CAPACITIES OF HEAT-TREATED MICROSOMAL FRACTIONS FROM BRAINS OF 3% I I - AND 60-DAY-OLD RATS Microsomes (i rag) p r e p a r e d from b r a i n s of 3 - d a y - o l d r a t s were i n c u b a t e d w i t h 6 mM MnCI~, 3 mM d i t h i o t h r e i t o l , 60 niM H E P E S buffer (pH 6.3) a n d 0.336 n m o l e of U D P - p 4 C ] g a l a c t o s e a t 37 ° for I h. F i n a l v o l u m e was 0.35 ml. H e a t - t r e a t e d f r a c t i o n s w e re i n c l u d e d as i n d i c a t e d . Ma, M n a n d M60 r e p r e s e n t p r o t e i n s of m i c r o s o m a l p r e p a r a t i o n s of b r a i n s of 3-, I I- a n d 6 o - d a y - o l d rats, re s pe c t i vely. H e a t t r e a t m e n t of s a m p l e s w a s b y i m m e r s i n g t h e p r o t e i n s u s p e n s i o n s in a b o i l i n g w a t e r b a t h for 3 rain. E a c h v a l u e is t h e m e a n -z S.D. of 4 e x p e r i m e n t s .

Addition

p4C]Galactose transferred (counts /min )

None Heat-treated Ma Heat-treated Mn H e a t - t r e a t e d M60

lO55 135o 1535 1735

± ± ± ±

54 79 66 145

°/o Increase

28 46 65

were used as acceptors. With heat-treated acceptor proteins from ii-day-old rat brain microsomes, the increase in galactose transfer was 46% while with the acceptors from 6o-day-old rats, the increase was 65~o. Similar results were obtained in experiments in which the acceptor capacity of microsomal proteins of young and adult rat brains were compared using the Tritonsolubilized galactose transferase enzyme. The results presented in Table VI show that the transfer of galactose from UDP-galactose to endogenous acceptors of adult brain is clearly greater than to the acceptors from young brain, regardless of whether the Triton extract was prepared from brains of adult or young rats (lO63 v s . 837 in Expt. I and lO6O v s . 91o in Expt. II). In other experiments, the heat-treated microsomes from brains of 4- and 6o-day-old rats were tested over a wide range of concentration (0.25-3 mg protein/o.35 ml of incubation medium) for their capacities to accept [14C]galactose. At every concentration, the transfer of galactose was lower with the microsomes from the brains of 4-day-old rats than with those from the 6o-day-old rats. Biochim. Biophys. deta, 244 (1971) 396-4o9

406

G . K . W . KO, E. RAGHUPATHY

TABLE VI TRANSFER AND

OF GALACTOSE

ADULT

RATS

AND

GALACTOSYLTRANSFBRASE

TO H E A T - T R E A T E D TO

DESIALIZED,

MICROSOMAL

PREPARATIONS

DEGALACTOSYLATED

FROM RATS OF TWO DIFFERENT

FETUIN

FROM BY

BRAINS

OF

YOUNG

TRITON-SOLUBILIZED

AGE GROUPS

Microsomal galactosyltransferase was extracted with Triton X-Ioo as described in EXPERIMENTAL, from brains of 16- and 5o-day-old rats. Ma and M~0 represent proteins obtained from naicrosomal preparations from brains of 4- and 6o-day-old rats, respectively. These preparations were immersed for 3 min in a boiling water bath to obtain the heat-treated sanlples. The enzyme (i mg protein) was incubated with and without the heat-treated acceptors (i mg) under conditions described in Table V. Incubation period, 3° rain. Each value is the mean 2~ S.D. of 4 experiments. Expt. No.

Age of rats from which Triton-solubilized enzyme was prepared (days)

Addition

I

16

None Heat-treated Heat-treated DSG-fetuin* None Heat-treated Heat-treated DSG-fetuin*

lI

*

50

[nC]Galactose transferred (counts~rain)

M~ M60

M4 M60

493 ~- 18 837 ± 22 lO63 ± 43 795o ~ 355 5oo ± 27 91o ± 17 lO6O± 63 9072 ± 42o

Desialized, degalactosylated fetuin

DISCUSSION

Studies on i n vitro biosynthesis of glycoproteins generally fall into two categories: (i) those which utilize an endogenous acceptor system, a n d (2) those in which a well-defined exogenous acceptor is used. O'BRIEN et aI.15,16 have pointed out, t h a t the enzyme acceptor system in a particulate fraction represents a highly efficient organization of enzyme a n d s u b s t r a t e which eliminates the need for r a n d o m complex form a r i o n which occurs with the soluble systems. T h e y have also p o i n t e d out t h a t such systems are generally more efficient t h a n the wholly soluble ones. The endogenous acceptor systems, furthermore, are valuable for studies in the sequence of events leading to the formation of complete glycoproteins within cells. On the other hand, the assay of glycosyltransferases utilizing endogenous acceptors can yield misleading results insofar as (a) the system m a y contain several glyeosyl transferases t h a t utilize the same sugar nucleotide b u t different acceptors a n d (b) the endogenous acceptor a n d n o t the enzyme m a y be rate-limiting17. For such studies on the n a t u r e of the enzyme itself or on its aceeptor specificity it is essential t h a t well-defined exogenous acceptor substrates be used. I n the present s t u d y we have described the properties of the cerebral microsomal system which transfers [14C]galaetose to endogenous proteins a n d have further established t h a t these properties are also shared b y the T r i t o n solubilized enzyme which efficiently utilizes added desialized, degalactosylated fetuin as the acceptor. The cerebral galactose transferase has some properties in common with similar transferases reported in other tissues. Thus it requires Mn 2+ as an activator, a p r o p e r t y shared b y galactosyltransferases in r e t i n a 15 a n d t h y r o i d 18. The enzyme a c t i v i t y was primarily localized in the smooth endoplasmic reticulum, like the hexose transferases in liver 19 a n d thyroid 14. The p H o p t i m u m is 6.3, similar to t h a t of the retinal galaetosyltransfer enzyme 15. Bioehim. Biophys. Acta, 244 (1971) 396-4o9

BRAIN GLYCOPROTEIN SYNTHESIS

407

McGuIRE et al. 2° have also studied the distribution of galactosyltransferase activity in several tissues of the rat, including brain. Their results show that brain particulate fractions contain galactosyltransferase activity and that the supernatant fraction does not possess the enzyme activity. The particulate enzyme studied by these authors also required Mn ~+ for m a x i m u m activity but Co 2÷ showed slight or no activity. In our experiments, Co ~+ was 70% as active as Mn 2÷. It is difficult to compare their results with those presented here, since their particulate preparation cousisted of all materials sedimenting between 121 × g and 39000 × g and thus perhaps contained heavy microsolnes too. A recent paper from this laboratory ~ has dealt with the galactosyl transferase of rat brain microsomes which catalyzes the transfer of galactose from UDP-galactose to lipid acceptors such as hydroxy f a t t y acid ceramide and sphingosine. In view of the fact that the UDP-galactose:glycoprotein galactosyltransferase described in this paper is also located in the microsomal fraction, it became necessary to distinguish between the two activities. The enzyme system reported here clearly differs from the UDP-galactose:ceramide galactosyltransferase in the following respects: (a) it requires Mn 2÷ for full activity while the enzyme which transfers galactose to ceramide is not dependent on bivalent ions for optimal activity ~1 and (b) the enzyme activity is present in the brains of postnatal rats while the lipid galactose transferase activity has been reported not to appear until the i o t h day of postnatal age 1~. Furthermore, while the Triton-solubilized enzyme efficiently uses fetuin deprived of its carbohydrate content as acceptor (but not native fetuin), it does not transfer galactose to the lipid acceptor (Table III). The preferential localization of the enzyme in the smooth microsomal subfractions rather in the rough suggests that the galactose transfer takes place within the microsomal membranes devoid of ribosomes and hence presumably at sites distal to those for the assembly of polypeptides. Support for this conclusion also stems from our observation (not presented in this paper) that isolated ribosomes which are highly active in protein synthesis do not possess galactose transferase activity. The observations described above are consistent with the multisite hypothesis of glycoprotein biosynthesis 2~,~3. The hypothesis suggests that carbohydrate attachment to the polypeptide chain m a y be initiated during the growth of the polypeptide on the ribosomes. Additional carbohydrates are acquired while the protein is migrating in the channels of the endoplasmic reticulum. The demonstration of the presence of galactose transferase activity, albeit low, in the rough endoplasmic reticulum strongly supports this view. The biosynthesis of glycoproteins involves two fundamental steps. The first step requires the formation of the backbone polypeptide chain (which subsequently becomes the acceptor molecule for the monosaccharides) via the classical pathways of protein biosynthesis. The second involves the stepwise addition of the various carbohydrate moieties to the preformed polypeptide chains - a sequence of reactions in which the different glycosyltransferases participate. The results of the present study suggest that these two steps m a y be subject to independent alterations during brain development. Thus, when the galactosyltransferase assay was carried out with the endogenous system in the absence of an added acceptoI protein, the incorporation was very low in the neonatal rat brain, but increased 4-6 fold as the rat grows older. However, when the assay was done in the presence of an exogenous acceptor, desialiBiochim. Biophys. Acta, 244 (1971) 396-4o9

40~

G . K . W . KO, E. RAGHUPATHY

zed, degalactosylated fetuin, the immature rat brain (3 day old) possessed considerable hexose transfer activity. With increasing age, this increase in the transfer to exogenous protein during the age period 3-6o days was usually less than 2 fold. These results therefore suggest that during maturation of brain, the increase that is observed in the acceptor capacity of the endogenous protein is far greater than the increase in the activity of the galactosyltransferase itself. It is not possible to establish from the data presented in this paper the precise mechanism by which the increase in the acceptor capacity of endogenous protein is brought about during the development of the brain. The following possibilities should, however, be considered: (I) A specific acceptor protein (or proteins) appears during the development of the brain (2) the young brain does possess some glycoproteins, but the capacity of these to accept additional carbohydrates changes during development, presumably due to the action of specific glycosidases and (3) the microsomal preparation from the young rat brain contains some factor or factors which inhibit the transfer reaction. Studies on the dissociation of the enzyme from endogenous protein acceptors as well as on the isolation and characterization of the purified acceptor proteins which will aid in distinguishing between these possibilities, are now in progress in this laboratory. At least two groups of workers have described the appearance of a specific glycoprotein in brain during myelination. WARECKA AND BAUER24 and WARECKA25 reported that a brain-specific glycoprotein appears in white matter during ontogenesis and that the appearance was correlated with the differentiation of the glia during myelination. A sinfilar accumulation of glycoprotein in the glial cells during myelination was also reported by BENETATO et al.26. These observations are entirely consistent with our results on the developmental patterns of the acceptor capacity of endogenous proteins and the activity of the enzyme, UDP-galactose:glycoprotein galactosyltransferase. The recent study of KELLEHER AND SMITH2v on serum glycoprotein synthesis by the fetal rat provides an example of yet another tissue in which the synthesis of the polypeptide and carbohydrate components of glycoproteins might be regulated independently during development. On the basis of in vivo studies these authors have concluded that the lack of detectable amounts of certain adult rat serum proteins in the fetal rat is not due to an inability to incorporate carbohydrates into serum glycoproteins. They further propose that the fetal rat lacks some specific synthetic mechanism for these proteins which then must become operative later in development. ACKNOWLEDGMENTS

This study was supported by a grant from National Institutes of Health HD01823 and 05317 . The technical assistance of Mrs. Lois Mowat is gratefully acknowledged. REFERENCES I E. G. BRUNNGRABER, V. AGUILAR AND A. ARO, Arch. Biochem. Biophys., 129 (1969) 131. 2 S. ]~OGOCH, P. C. BELVAL, W. H. SWEET, W. SACKS AND G. I~ORSH, Protides of Biological Fluids, Elsev ier, A m s t e r d a m , 1968, p. 129. 3 ]3. M. GESNER AND V. GINSBURG, Proc. Natl. Acad. Sci, U.S., 52 (1964) 750.

Biochim. Biophys. Acta, 244 (1971) 396-409

BRAIN GLYCOPROTEIN SYNTHESIS 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

409

M. A. CRANDALL AND T. D. BROCK, Science, 161 (1968) 473S. H. BARONDES AND G. R. DUTTON, J. Neurobiol., I (1969) 9. S. BOGOCH, The Biochemistry of Memory, O x f o r d Press, Oxford, 1968, p. 185. G. DALLNER, Acta Pathol. Microbiol. Scand. suppl., 166, i (1963). M. KUROKAWA, T. SAKAMOTO AND M. KATO, Biochem. J., 97 (1965) 833. E. RAGHUPATHY, N. A. PETERSON AND C. M. McKEAN, Biochem. Pharmacol., 19 (197 o) 993. R. G. SPIRO, J. Biol. Chem., 239 (1964) 567 • A. G. GORNALL, C. S. BARDAWILL AND 1V[.M. DAVID, J. Biol. Chem., 177 (1949) 751. S. N. SHAH, J. Neurochem., 18 (1971) 395J. MOLNAR, M. TETAS AND H. CHAO, Biochem. Biophys. Res. Commun., 37 (1969) 684S. BOUCHILLOUX, M. FERRAND, J. GREGOIRE AND O. CHABAUD, Biochem. Biophys. R°s. Commun., 37 (1969) 538. P. J. O'BRIEN AND C. G. MUELLENBERG, Biochim. Biophys. Acta, 158 (1968) 139. P. J. O'BRIEN AND C. G. MUELLENBERG, Biochim. Biophys. Acta, 167 (1968) 268. H. SCHACHTER, I. JABBAL, R. L. HUDGIN, L. PINTERIC, E. J. McGuIRE AND S. t{OSEMAN, J. Biol. Chem., 245 (197 o) lO9O. R. G. SPIRO AND M. J. SPIRO, J. Biol. Chem., 243 (1968) 6529. R. R. WAGNER AND M. A. CYNKIN, Arch. Biochem. Biophys., 129 (1969) 242. E. J. McGUIRE, G. VV. JOURDIAN, D. IV[. CARLSON AND S. }{OSEMAN, J. Biol. Chem., 240 (1965) 4II2. P. MORELL AND lXT.S. RADIN, Biochemistry, 8 (1969) 5o6. J. MOLNAR, G. B. ROBINSON AND R. J. WINZLER, J. Biol. Chem., 240 (1965) 1882. C. R. LAWFORD AND H. SCHACHTER, J. Biol. Chem., 241 (1966) 54o8. K. WARECKA AND H. J. BAUER, Deut. Z. Nervenheilkd., 194 (1968) 66. K. WARECKA, J. Neurochem., 17 (197 o) 829. G. BENETATO, ]~. GABRIELESCU, L. PARTENI, A. BORDEIANU AND I. HORUS, Fiziol. Norm. Patol. Bucharest, 7 (1961) 73V. C. KELLEHER AND C. J. SMITH, Biochim. Biophys. Acta, 2Ol (197 o) 76.

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