Developmental Brain Research, 21 (1985) 73-81 73 Elsevier

BRD 50229

Glycoprotein Composition and Turnover in Subcellular Fractions from

the Cerebral Cortex of Normal and Reeler Mutant Mice

SE,,~N MURPHY and JOHN RUDGE*

Brain Research Group, Biology Department, Open University, Milton Keynes, MK7 6AA (U. K.)

(Accepted January 21st, 1985)

Key words: glycoprotein - - concanavalin A - - fucose - - subcellular fraction - - cortex - - reeler - - development - - mouse

Twenty-day-old reeler and normal mice were either injected intraventricularly with radiolabelled fucose before subcellular frac- tionation of the cerebral cortex followed by SDS-PAGE, or gels of such fractions were overlaid with [125I]concanavalin A (ConA). While there were no differences in polypeptide profiles of normal and reeler subcellular fractions there were marked differences in the abundance of particular ConA-binding glycoproteins and in fucose incorporation into particular glycoproteins, especially in the synap- tic plasma membrane (SPM) and 100,000 g soluble fractions. In addition there was a significantly lower binding (> 50%) of quinuclidi- nyl benzilate to reeler microsomal and SPM fractions as compared with normal. The differences in glycoprotein expression may be pertinent to anatomical observations of abnormal interactions between neurons and glial fibres during development of the reeler cere- brum.

INTRODUCTION

The array of glycosylated proteins that neural cells

express on their surfaces are thought to mediate

events such as adhesion, migration and recognition

during the development of the brain. Some of these

glycoproteins have been identified and character- ised, for example D2 (refs. 11, 20), N-CAM 31 and

BSP-2 (ref. 21), a family of related surface mole-

cules, and L1 (ref. 9) and THY-1 (ref. 36). At the

synaptic level, the array of glycoproteins is very di- verseS; their distribution in synaptic junctions17, ls,2s

and association with postsynaptic densities 15 have been mapped, and their appearance 14,2s.3° and mod-

ification during the development of the synapse have been reported6,10.

In earlier studies we described developmentally

related changes in the polypeptide compositions of neuronally and glially enriched cell populations and

of subcellular fractions from the rat cerebral cortex 2, and reported changes in concanavalin A (ConA) binding glycoproteins (CABG) in subcellular frac-.

tions from the developing rat cerebral cortex 30. In an

attempt to relate changes in specific glycoproteins to

particular developmental processes we have investi-

gated C A B G composition and glycoprotein turnover

in the reeler mutant cerebrum as compared with nor-

mal mice. The reeler carries an autosomal recessive single-

gene mutation and homozygous individuals have a

dystonic posture, action t remor and a reeling ataxic gait 33. Not only is the cerebellum 4 affected but there

are widespread abnormalities in the cerebrum 13,

characterized by malposition of neurons 3, glial cell

defects 12 and inverted or even absent synaptic termi- nations23.

In the present study we describe fucose incorpora-

tion into siaioglycoproteins in vivo and also the C A B G profiles of various fractions isolated from the

cerebral cortex in 20-day-old normal as compared

with reeler mutant mice. While there are no glyco-

proteins unique to normal or to reeler, there are dif- ferences in the abundance of particular CABGs and in the turnover of glycoproteins.

* Present address: Department of Biology, School of Medicine, The University of California at San Diego, La Jolla, CA 92093, U.S.A.

Correspondence: S. Murphy, Brain Research Group, Biology Department, Open University, Milton Keynes, MK7 6AA, U.K.

0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

74

TABLE I

Protein distribution within subcellular fractions from the cere- bral cortex of 20-day-old normal and reeler mutant mice

Subcellular fractions prepared as described in ref. 30. Values in mg protein/cerebral cortex and represent mean _+ S.E.M. of 4 animals. Figures in parentheses denote recovery as % homo- genate.

Fraction Normal Reeler

Homogenate 39.52 + 1.49 (100) 44.73 + 1.42 (100) Nuclei pellet (P1) 9.01 + 0.64 (23) 7.67 + 0.50 (17) Light membrane ($2) 15.42 +_ 1.02 (39) 16.24 +__ 2.70 (36) Myelin 1.18 _+ 0.22 (3) 0.97 + 0.24 (2) SPM 1.85 + 0.24 (5) 1.32 + 0.16 (3) Mitochondria 3.69 + 0.22 (9) 3.28 _+ 0.11 (7)

MATERIALS AND METHODS

Animals Reeler mutants were mainta ined on a C57 BL/6

background with normal mice coming from mutant- free strains of the same genetic background. Homozy-

gous reelers on this background general ly survive for

up to one month. Reelers were identif ied first by

their p ronounced ataxia and identif ication conf i rmed

pos tmor tem by the presence of an immature cerebel-

lum.

In vivo fucose labelling Normal and mutant mice, 20 days old, were in-

jected with e i ther L-5,6-[3H]fucose (71 Ci/mmol) or

L-[t4C]fucose (50 mCi/mmol) , purchased from

Amersham Internat ional (U.K. ) . Each animal re-

ceived 500/~Ci (4 -6 ~1) into the third ventricle via a

Hamil ton syringe, under light halothane anaesthesia.

Animals were allowed to recover for 18 h and then

killed and their cerebral cortices removed. For

double- label l ing exper iments , reeler mice were in-

jected with [3H]fucose and normal mice with [14C]fu-

cose. In these exper iments the specific activity of the

[3H]fucose was reduced to 50 mCi/mmol by the addi-

tion of unlabel led fucose.

Subcellular fractionation and quinuclidinyl benzilate assay

After single fucose labelling pooled normal or reel-

er cerebral cortices were homogenized and centri-

fuged at 100,000 g to produce soluble and part iculate

fractions. Af te r double fucose labelling, pooled reel-

er and normal homogenates were combined. Subcel-

lular fractions for polypept ide , fucose and ConA

analysis were prepared as described previously30 and

reeler and normal samples were run in parallel . DL-

[3H]quinuclidinyl benzilate (QNB, Amersham Inter-

national) binding to various fractions was de te rmined

using a previously described method 16. Protein was

de termined by the method of Lowry et al, 22.

SDS P A G E , gel slicing and [1251]ConA labelling o f

gels

SDS P A G E (7.5% gels) 2 and [125I]ConA labelling

and scanning of slab gels30 were per formed as report-

ed earlier. Reeler and normal comparisons were

made on the same gel. In the fucose exper iments ,

stained gel tracks were frozen on dry ice, sliced into

2-mm blocks, solubilised in 5% Protosol and Econo- fluor and counted at ambient t empera ture in the [3H]

and [laC} channels of a Beckman LS 7500 scintillation

counter.

RESULTS

Subcellular fractionation and Q N B binding

The recovery of protein in various fractions from

the cerebral cortex was similar in both normal and

TABLE II

Binding of [3H]QNB to subceUular fractions from cerebral cortex of normal and reeler mutant mice

Subcellular fractions prepared as described in ref. 30 and QNB assay performed as in ref. 16. Values in fmol × mg protein ~ represent mean + S.E.M. of n = 4 animals. Figures in parentheses denote level of binding as % homogenate. Probability determined using Stu- dent's t-test; n.s., not significant.

Fraction Normal (N) Reeler (R) R/N P

Homogenate 516 + 50 (100) 485 + 35 (100) 0.94 n.s. Light membrane ($2) 830 + 150 (161) 400 + 49 (82) 0.48 < 0.05 SPM 958 + 97 (185) 386 _+ 31 (79) 0.40 < 0.(102

reeler mice (Table I). However , the level of specific QNB binding to $2 and synaptic plasma membrane

(SPM) fractions (Table II) indicated either that the

fractions prepared were different in composition or

75

that muscarinic cholinergic receptors are reduced in

number or expressed later in development in reeler

compared with normal cerebrum. The level of bind-

ing was consistently lower in reeler SPM (40% of

z.

A m

| z

NORMAL HOMOGENATE I 531 i! 44

i52ii

~32 ,

I!

i j i

I I

N O R M A L S P M

l I

,I

53

.102 5 8 1 n t 4 4

I I I I I 1

|

| I I I l 2

s. ~. 8.

REELER HOMOGENATE

71

I I 1 I 1

REELER SPM

I I I I I s.

!:

I I L I 8

I I

Fig. 1. Densitometric scans of polypeptides in homogenates and SPM fractions from the cerebral cortex of 20-day-old normal and reel- er mutant mice. Polypeptides were resolved by SDS-PAGE (7.5% gel) and stained with Coomassie blue. Direction of migration is from left to right and polypeptides are assigned a mol. wt. (x 10 -3) as determined by a Beckman DU8 spectrophotometer.

76

a b 1 2 3 4 5 6 1 2 3 4 5 6

Fig. 2. [1251]ConA labelling of glycoproteins in homogenates, SPM and mitochondrial fractions from the cerebral cortex of 20-day-old normal and reeler mutant mice. 2a is the [lzsI]ConA autoradiogram of the gel shown in 2b. Polypeptides were resolved by SDS-PAGE (7.5% gel) and [125I]ConA binding performed as described in the text. Tracks 1, 3, 5: normal mouse homogenate, SPM and mitochon- dria. Tracks 2, 4, 6: reeler mouse homogenate, SPM and mitochondria. Position of mol. wt. markers (x 10 3) shown adjacent to track 6.

normal) and $2 (48% of normal) fractions. There was

no enrichment of specific binding of QNB in SPM or

$2 over that in homogenate in reeler mice, whereas in

normal mice enrichments of 1.61 ($2) and 1.85 (SPM)

x that in the homogenate were found.

Glycoprotein composition of subcellular fractions There were no polypeptide differences (as re-

vealed by staining with Coomassie blue) between normal and reeler homogenate, Pi, S> mitochondria

and SPM fractions, suggesting that although absolute purity of individual fractions is unknown, the frac- tions isolated are essentially the same from reeler and normal. However, scanning representative tracks revealed differences in the relative amounts of particular polypeptides (Fig. 1 shows a typical exper- iment). In homogenates, a polypeptide of mol. wt.

62,000 dalton is elevated in normal compared with

reeler, while polypeptides of 88,000 and 58,000 dal-

ton are more prominent in reeler. In SPM fractions the doublet at 227,000 and polypeptides of 102,000,

58,000 and 32,000 dalton are elevated in reeler com-

pared with normal. The autoradiogram in Fig. 2a shows the pattern of

[125I]ConA labelling of polypeptides in representa- tive homogenate and SPM fractions resolved by SDS P A G E (Fig. 2b). Scanning the tracks of the autora- diogram reveals (Fig. 3) the relative abundance of particular CABGs. In the homogenate, a C A B G of 241,000 dalton is enriched in normal as compared with reeler, while CABGs of 119,000 and 75,000 dal- ton are less abundant. In SPM there appears to be less ConA labelling in the reeler compared with nor- mal over the mol. wt. range 119,000-142,000 dalton

77

while there is higher labelling over the mol. wt, range

75,000-86,000 dalton in the reeler.

Fucose incorporation in vivo Single-label experiments (using [3H]fucose as a

precursor) and double-label experiments ([3H]/[14C])

revealed differences in incorporation into fuco-

sylated glycoproteins in various fractions of the cere-

bral cortex from normal and reeler mice, after reso- lution by SDS P A G E and gel slicing (Figs. 4 and 5).

Single-label experiments revealed a number of prom-

inent fucosylated proteins (Fig. 4a) and these

, ¢ - -

| e , J -

|

=.

! p j -

t~

NORMAL HOMOGENATE

74

2,1, I 1,2

1 1 [ I J J I I

N O R M A L S P M

174

142 / l ~ 119

I l l ~ 8 6 4 3 ~/'II 'v 1 ' s

I

e~

!_

o J -

| 1 d

REELER HOMOGENATE

I 1 2 4 2 6 5 /i , / , J;i ~ : 1 1 9 I t~ I r

J I J i 1 I ]

R

R E E L E R S P M

II 142 ~ 119 8 6

2 5

I J 1 l J I l J I J I J i J I I 1 1 I I

Fig. 3, Densitometric scans of homogenate and SPM tracks shown in Fig. 2a. Direction of migration is from left to right. CABGs are assigned a mol. wt. (x 10 -3) as determined by a Beckman DU8 computing spectrophotometer.

78

C- ,< 0

£

>. I--

l-.- 0

0

,<

0

"1- °

4 b

3

2

!

HOMOGENATE

,(44

~ 15 ft f t 7t ',

/

- - - NORMAL

- - REELER

Jl V Ir II

~¢J I i Ii

25

I J I I I

200 150 100 50 25

MOLECULAR WEIGHT × 10 .3

lOOK × g S U P E R N A T A N T

40 115

1751 I ~

d

6 8

.(44 tt /

A 55 II

/ I I

I 5 3 i I

i; II 1

- - - - - NORMAL

REELER

4o

~ 25,(

'\ I,'/', t,/,'/I

! k' Jl V,-.v

200 150 100 50

M O L E C U L A R WEIGHT X 10 -3

i

25

lOOK× g PARTICULATE

>

(J ,< 0

.< tic

4Ic 3 1115

It

I I 175

/', I105 236 /^,~ I I / tq

I IV]110211 L t,~

44

- - -- NORMAL

,, REELER

I 140

25

I I I 1 /

200 150 100 50 2 s

MOLECULAR WEIGHT × 103

Fig. 4. [3H]Fucose incorporation into polypeptides of normal and reeler cerebral cortex resolved by SDS PAGE. a: homo- genate, b: 100,000 g soluble and c: 100,000 g particulate frac- tions. Tracks from the gel (7.5%) were sliced at 2-mm intervals and counted. The solid line represents incorporation of [3H]fu- cose into reeler and the dotted line incorporation into normal. The total radioactivity in the track was estimated and each frac- tion expressed as a percentage of the total. Numbers above the peaks indicate mol. wt. ( × 10-~).

showed fraction-specific enrichment, especially in re-

lation to the soluble (Fig. 4b) and particulate frac-

tions (Fig. 4c). The homogenate showed an enrich-

ment in the normal, as compared with reeler, of fuco-

sylated proteins with mol. wts. 236,000, 115,000 and

44,000 dalton, while the reverse was true for mol.

wts. 174,000, 95,000 and 75,000 dalton. In the case of

those polypeptides with mol. wts. 236,000 and

115,000 dalton, the difference was most marked in

the particulate fraction. The soluble fraction showed

a very different profile with polypeptides of 44,000

and 53,000 dalton being much reduced; however, the

44,000-dalton polypeptide was markedly enriched in

normal. In this fraction from reeler, polypeptides

with mol. wts. of 240,000, 75,000 and 68,000 dalton

were much more highly labelled than corresponding

bands in the normal (Fig. 4b).

Using double-labelling procedures, the differences

in incorporation of fucose into subcellular fractions

are even more marked (Fig. 5). In the SPM fraction from reeler there is an increase in fucose incorpora-

tion in polypeptides of mol. wts. 115,000, 95,000,

75,000 and 44,000 dalton but a lower incorporation

into 240,000-, 64,000-, 36,000- and 25,000-dalton

polypeptides. Reversing the label did not affect the

incorporation comparisons.

S Y N A P T I C P L A S M A

M E M B R A N E

- - M E A N

5 - - - - - N O R M A L

R E E L E R 75

>

< 3 95

O 115 64 , /t 25 t'~ 48 1 ~ t

pqt I

~36 I I-- 240

I I 1 I 215 200 150 100 5 0

M O L E C U L A R W E I G H T × 10 -3

Fig. 5. Fucose incorporation into polypeptides in reeler and normal SPM. Homogenates were combined before subcellular fractionation and SDS-PAGE (7.5%). Tracks were sliced at 2-mm intervals and counted in both [3HI and [14C] channels. The solid line represents [3H]fucose incorporation into reeler glycoproteins whereas the dotted line represents [14C]fucose incorporated into normal. The total radioactivity in the track was estimated and each fraction is expressed as a percentage of the total. The [3H]/[14C] ratio for each fraction is shown at the top of the figure. Numbers above the peaks indicate mol. wt. (X 10-3).

79

DISCUSSION

Subcellular fractionation of the reeler cerebral

cortex provided protein yields that were not signifi-

cantly different from normal mice. However, the

binding of QNB, a specific muscarinic cholinergic re- ceptor antagonist which has been used as a marker

for SPM 16,3°, was much reduced in reeler SPM and

crude microsomal ($2) fractions. That this may not be

indicative of a difference in purity of the two frac-

tions, but is evidence of a genuine underexpression of

cholinergic receptors in the reeler, is suggested by

the following observations. First, yields of protein

were the same in the reeler and normal subfractiona-

tion procedure. Second, the level of QNB binding in

reeler was lower in both the $2 and SPM fractions but not different from normal in myelin and mitochondri-

al fractions from the gradient. Last, the polypeptide

and CABG profiles of SPM fractions in normal and

reeler are identical in terms of the range of molecular

species present.

Both East and Dutton 8 and Singer et al. 34, using

crude membrane preparations, have reported a 50%

reduction in QNB binding in the 20-day-old reeler

cerebellum. Singer et al. 34 did not find a reduction in

QNB binding to crude membrane fractions from the reeler parietal cortex, similar to our findings for reel-

er cortical homogenates. The marked loss of QNB

binding to reeler SPM fractions that we have found

might suggest a delay in the appearance or differen-

tiation of a discrete population of neurons in the reel- er cerebral cortex.

While there were no polypeptide differences be-

tween normal and reeler cortex, an observation sup-

porting that of Mikoshiba et ai. 23, there were differ-

ences in the abundance of particular polypeptide spe-

cies in different fractions and more precisely in the

abundance of particular CABG in SPM fractions.

The main differences appear to be within two mol.

wt. ranges. The 119,000-142,000 dalton range of

CABG appears to be enriched in normal SPM while the 75-86,000 dalton range is more prominent in reeler SPM. As ConA binds specifically to a-glucose,

a-N-acetyi glucosamine and a-mannose residues, such differences in CABG may result either from an increase or a decrease in a particular glycoprotein, or

from a posttranslational modification of a particular glycoprotein that is developmentally determined and

80

therefore different in reeler and normal mice at this

age. The C A B G of mol. wt. 25,000, prominent in

both normal and reeler SPM, is p robably THY-1, a

glycoprotein known to be associated with the synap-

tic regions of rodent and chicken brain1,29.30. The

complete lack of C A B G between 25,000 and 43,000

dalton is common to both mouse and rat SPM 30.

Fucose is not significantly catabol ized in brain and

is incorpora ted only to a very small extent into glyco-

lipids 27. Fucose, therefore , part icularly in the

double- label l ing exper iments , provides a good indi-

cation of differential incorporat ion into, and turn-

over of the s ialoglycoproteins in reeler and normal

mice. As Quarles and Brady 27 have shown, the high-

est incorporat ion is into glycoprotein species in the

mol. wt. range of 40-70,000 dalton. In the reeler

100,000 g soluble fraction two glycoproteins of 240,-

000 and 68,000 are prominent in terms of their rate of

fucose incorporat ion. The 240,000 dal ton glycopro-

tein is similar to that described by others 7.27 who

noted that there was extensive incorporat ion in

young as compared with older animals. The double-

labelling comparisons of reeler and normal SPM re-

vealed that a fucosylated glycoprotein of similar mol.

wt. is also present in this fraction. However , the rate

of incorporat ion of fucose into this glycoprotein is

very much lower in reeler than in normal. Similarly,

fucose incorporat ion into a 25,000 dal ton glycopro-

rein in the SPM is much reduced in reeler as com-

pared with normal. THY-1, which is a C A B G of the

same mol. wt., has an N-l inked carbohydra te struc-

ture containing a fucose residue l inked to N-acetyl

glucosamine 29-36. If this 25,000 dal ton glycoprotein,

revealed both by ConA binding and by fucose incor-

porat ion, is THY-1 then our results suggest that

while THY-1 is present in reeler SPM, its turnover is

much less rapid than in normal cerebrum. Al t e red

THY-1 expression may well be associated with the

observed disturbances in cell-to-cell adhesiveness in reeler brain 26.

While changes in the neural circuitry in the reeler

cerebral cortex appear slight compared with the cere-

bellum, nevertheless there are gross al terat ions in

cell posit ion 13 and, as shown here, differences in the

abundance of part icular glycoprotein species. There

have been reports of al tered glycoprotein expression

on the surfaces of cells in mutant mouse cerebel lum

and cerebrum19,24 and of differences in the express-

ion of enzymes such as neuraminidase35 and

galactosyltransferase 32. Pinto-Lord et al. 26 suggest

that the difference in the reeler cortex as compared

with normal is that the migratory ascent of neurons

along radial glial fibres is blocked by postmigratory

neurons that have an unusually extensive contact

with these glial processes. The problem may lie

either with the neuronal or the glial surface 13 and

may reflect an incomplete or delayed develophaental

t ransformation of key glycoproteins. Ghandour et

al.~2 have repor ted abnormali t ies in part icular glial-

specific proteins in the reeler cerebel lum but as yet

there has been no extensive investigation in this mu-

tant of the macromolecules on the surfaces of neu-

rons and glial cells. The differences we report here,

namely in C A B G abundance and the turnover of gly-

coproteins in part icular fractions, may reflect the al-

tered expression of key cell surface glycoproteins in

the reeler mutant.

ACKNOWLEDGEMENTS

We would like to thank Christine Morrow for tech-

nical assistance, Dawn Sadler and Steve Waiters for

maintaining the reeler mutant colony and members

of the Brain Research Group for helpful discussions.

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