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Molecular Brain Research. 14 (1992) 136-138 © 1992 Elsevier Science Pubhshers B V All rights reserved 0169-328X/92/$05 00

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Short Communications

Nerve growth factor precursors in the rat thyroid and hippocampus Eleni Dicou INSERM U 298. CHRU, Angets (Francel

(Accepted 21 January 1991) Key words Nerve growth factor precursor, Immunopreclpltatiom Antl-peptide serum, Thyroid, Hlppocampus

A nerve growth factor (NGF) precursor form of about 24 kDa was identified in homogenates ot rat thyroid and hippocampus by lmmunoprecipltatlon using three sera raised against a synthetic peptlde that reproduces the sequence -71 to -46 of the proNGF molecule Besides this species, a 31 kDa protein, as well as a cleavage product of 12 kDa were also ~mmunoprecipltated in both tissues by one of these sera Nerve growth factor ( N G F ) is a neurotrophic protein involved in the maintenance and d e v e l o p m e n t of the peripheral and central nervous system 16"18. While extensive studies were centered on the expression and localization of the factor, little is known about the in vivo existing N G F precursor forms. The nucleotide sequence derived from the mouse submandibular gland ( M S G ) 13 x7 contained two in-frame initiator methionine codons (Fig. 1). Initiation at the first A U G would result in a 34 k D a protein and at the second A U G in a 28 k D a precursor form. In the long precursor the initiator methlonine is about 70 amino acids upstream from the signal peptidelike sequence while in the short precursor the hydrophobic region is at the N-terminus. Four N G F m R N A transcripts differing at the N-terminus were characterized as products of alternative splicing and/or imtiation from ind e p e n d e n t p r o m o t e r s s'14 The two major transcripts differ by alternative splicing of an N-terminal exon. The long transcript was found to be p r e d o m i n a n t in the M S G while in the peripheral organs and brain regions tested the short transcript was p r e d o m i n a n t A f t e r expression of these two transcripts in m a m m a l i a n cell lines using appropriate recombinant vaccinia virus vectors and by i m m u n o p r e c l p i t a u o n with a n t i - N G F antibodies a 35 k D a glycosylated precursor form was observed in both cases and was tdenttfied as a cleavage product downstreams from the signal p e p t i d e 9 Antibodies were raised m rabbits against synthetic peptides that reproduce sequences of the p r o N G F molecule with the aim to use them in immunohistochemical studies for the localization of cellular sites of N G F synthesis as well as for the biochemical characterizatxon of the N G F precursor forms In particular, antibodies

against a 26 a m m o acid long synthetic peptide N4 that reproduces the sequence -71 to - 4 6 of the p r o N G F were used in immunohistochemlcal studies m a variety of tissues. In the M S G , by immunofluorescence techniques, p r o N G F - l i k e immunoreactwity with affinity chromatography purified anti-N4 antibodies was detected in the basal part of the granular cells forming the convoluted tubules, which are known to be the site of N G F synthesis4. In situ hybridization experiments with N G F c D N A probes showed colocahzation of the N G F m R N A m the basal part of these cells, which lends support that the observed immunoreactlvlty is closely related to a biosynthetic product. N G F m R N A s derived from an SP6 recombinant vector after translation in a rabbit reticulocyte lysate gave rise to two species of about 35 and 28 kDa, thus providing evidence that the translational apparatus can recognize both A U G s 5 This was also shown by in vitro translation of hybrid selected N G F m R N A s from the M S G 6. Both species were xmmunopreclpitated with anti-N4 antibodies 5 It was also shown that processing of the in vitro synthesized N G F precursor by digestion with an arginine esteropeptidase, the 7 subumt of the 7S N G F present m the M S G , liberated the N-terminal peptide but not the f l N G F probably due to abnormal folding of the in vitro precursor. The N-terminal peptlde was identified by immunoprecipitation with an antiserum directed against the N2 peptide, which corresponds to the sequence -163 to -139 of the p r e p r o N G F 5 In the rat brain, p r o N G F - h k e lmmunoreact~vity with anti-N4 antibodies was locahzed in defined areas m the cortex, hippocampal formation, striatum, thalamus/hypothalamus and in several other regions aS. The cell types in the hippocampus, cortex and septum displaying anti-

Correspondence E Dicou, INSERM U298, CHRU, F-49033 Angers Cede× 01, France

137 0

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I--

AUG

AUG

N2 r77z,-~



i N4

0

i

_

0

i

A

3"

rH////J

N3V'TA Fig 1 Schematic representat]on of the NGF transcript The thin hne indicates untranslated regions and the box translated regions The cross

hatched box is the NGF peptlde, stippled box is the putative signal pepUde. Heavy bars indicate proteolytic cleavage sites (shown with arrows), ~ denotes potential glycosylation sites. Epltopes reproduced by the synthetic peptldes are shown by separate hatched boxes.

peptide immunoreactivity were identified to be n e u r o n a l

ronal populations. No anti-N4 immunoreactivity was as-

cells by several approaches, such as double-staining experiments with a n t i - G F A P antibodies and by the use of

sociated with glial cells. This is in agreement with in situ hybridization studies which also indicated n e u r o n a l localization of N G F m R N A in the hippocampus 1"12'19. In

wheat germ agglutinin apohorseradish peroxidase coupled to colloidal gold to retrogradely label selective neu-

the rat retina, however, proNGF-like immunoreactivity with anti-N4 antibodies was observed not only in neuronal components (retinal ganglion cells) but also in MOiler cells which are glial-like cells 3. ProNGF-like immunoreactivity with anti-N4 antibodies was also observed in epithelial secretory cells in the guinea pig prostate and in germ cells in the rat testis and colocalized with NGF-like immunoreactivity1~. In situ

"= 31k -'~ 2 4 k

hybridization experiments also localized N G F m R N A in germ cells of the rat testis 2. A 30-31 kDa protein was previously immunoprecipitated with a serum directed against the N2 peptide in rat thyroid extracts 6. The objective of the present study was to use sera directed against the N4 peptide and characterize the proteins that would crossreact with them not only in the rat thyroid but also in the hippocampus,

-'9=12k

1

2

3

4

~31k "~ 29k -~= 2 4 k

5

Fig 2 Autoradlograms of SDS-polyacrylamIde gels separating proteins from labeled thyroid extracts immunopreclpltated with three different anti-N4 sera The sera used were' lane 1, preimmune, lane 2, N4B, lane 3, N4C: lane 4, N4B premcubated with 40 pg of N4 peptIde overnight at 4°C, lane 5, N4F Methods thyroids were &ssected from male Wlstar rats (250 g) and homogerazed in 0 1 M borate buffer pH 8.5 containing 1 mM phenylmethylsulfonyl fluoride, 1 mM tosyl-L-lysinechloromethyl ketone, 25 ~g/ml aprotmln, 20/tg/ml leupeptxn and 20 pg/ml pepstatin After centrlfugatlon at 12,000 × g for 15 rain, a volume corresponding to about 100 pg of the clear supernatant was labeled with 50/~CI of 3ss-labeled reagent (t-butoxycarbonyl-L-[35S]methlonlne-N-hydroxysuccimidyl ester, Amersham) for 1 h on Ice and the reaction stopped with 100 pl of 0 2 M glycme Immunoprecipitations were performed with 10 pl of each serum and protein A-Sepharose as previously described5 Immunopreopltates were eluted with sample electrophoresis buffer and fractlonated on 18% SDS-polyacrylamlde gels The dry gels were exposed to XAR films for 2 months Molecular weight markers are ovalbumln, 46,000, chymotrypslnogen A, 25,000, and cytochrome c, 12,000

~

1

2

3

4

5

~= 12k

6

Fig 3. Immunoprecxpltated proteins from labeled hippocampal extracts by various anti-N4 sera after separation in SDS-polyacrylamlde gels The sera used were lanes 1 and 3, prelmmune, lane 2, N4C, lane 4, N4B, lane 5, N4B preincubated with 40 /tg of N4 peptlde, lane 6, N4E Hippocampal extracts were labeled as described m legend of Fig 2. Exposure was for 2 months

138 which contains the highest NGF

levels m t h e b r a i n ~°.

age p r o d u c t . A n a l t e r n a t i v e e x p l a n a t i o n is t h a t t h e 12

T h e N 4 B a n t i b o d i e s w e r e e m p l o y e d in t h e i m m u n o h i s -

k D a p r o t e i n is n o t a s t a b l e c l e a v a g e p r o d u c t

tochemical studies described above, the N4C and N4F

s t u d y , t h e 31 k D a p r o t e i n i m m u n o p r e c l p l t a t e d w i t h t h e

In this

sera were obtained more recently. 3sS-Labeled homoge-

N 4 F s e r u m , c o m i g r a t e d w i t h t h e 31 k D a p r o t e m , l m m u -

nates from rat thyroid and hippocampus were immuno-

nopreclpitated with the N2 serum (results not shown)

precipitated with these sera and the immunoprecipitates

a n d m a y c o r r e s p o n d to t h e l o n g p r e c u r s o r . A n o t h e r pos-

w e r e a n a l y z e d in S D S - p o l y a c r y l a m i d e

sibility is t h a t t h e 31 k D a p r o t e i n m a y b e a g l y c o s y l a t e d

gels. A 24 k D a

p r o t e i n in e x t r a c t s of t h e t h y r o i d (Fig. 2) as well as of

f o r m of t h e 24 k D a p r o t e i n T h e 29 k D a species d e t e c t e d

t h e h l p p o c a m p u s (Fig. 3) c r o s s r e a c t e d w i t h all t h r e e s e r a

in t h e r a t h l p p o c a m p u s m i g h t d i f f e r f r o m t h e 31 k D a in

a n d p r o b a b l y c o r r e s p o n d s to t h e s h o r t p r e c u r s o r f o r m

glycosylation content

a f t e r c l e a v a g e o f t h e signal p e p t i d e . T h e s e r u m N 4 F im-

w e r e r e p r o d u c e d in t h r e e i n d e p e n d e n t e x p e r i m e n t s .

m u n o p r e c i p i t a t e d b e s i d e s t h e 24 k D a , t w o o t h e r p r o t e i n s

R e s u l t s s h o w n in Figs. 2 a n d 3

In s u m m a r y , e v i d e n c e is p r e s e n t e d i m p l i c a t i n g a 24

o f a b o u t 31 k D a a n d 12 k D a in t h y r o i d e x t r a c t s a n d 31,

k D a p r o t e i n in t h e r a t t h y r o i d a n d h l p p o c a m p u s

as a

29 a n d 12 k D a in h i p p o c a m p a l e x t r a c t s . T h e 12 k D a

s t a b l e in vivo N G F p r e c u r s o r f o r m , since it c r o s s r e a c t e d

protein most probably corresponds to the cleavage prod-

with t h r e e d i f f e r e n t a n t i - N 4 a n t i b o d i e s , it was a b s e n t in

uct g e n e r a t e d f r o m t h e 24 k D a p r e c u r s o r a f t e r r e m o v a l

~ m m u n o p r e c ~ p i t a t e s w i t h p r e ~ m m u n e s e r a a n d was c o m -

of t h e f l - N G F p e p t i d e . I n s o m e i m m u n o p r e c i p i t a t i o n ex-

peted with the homologous peptide.

p e r i m e n t s N 4 B a n d N 4 C s e r a also i m m u n o p r e c i p i t a t e d t h e 12 k D a p r o t e i n b u t n o t c o n s i s t e n t l y . It was p r e v i o u s l y s h o w n t h a t a n t i - p e p t i d e s e r a display a g r e a t e r affinity for a c h i m e r i c p r e p r o N G F

p r o t e i n t h a n for t h e i r

h o m o l o g o u s p e p t i d e 7, w h i c h m a y e x p l a i n t h e v a r i a b l e efficiency of t h e s e t h r e e s e r a t o d e t e c t t h e 12 k D a cleav-

1 Ayer-Leh~vre, C , Olson, L., Ebendal, T , Selger, A and Persson, H Expression of the 3-nerve growth factor gene m hlppocampal neurons, Sctence, 240 (1988) 1339-1341 2 Ayer-Leh~vre, C , Olson, L , Ebendal, T., et al , Nerve growth factor mRNA and protein m the testis and epldldymls of mouse and rat, Proc Natl A c a d Sct USA, 85 (1988) 2628-2632 3 Chakrabartx, S., Slma, A A F , Lee, J , Brachet, P and Dlcou, E , Nerve growth factor (NGF), proNGF and NGF receptorlike lmmunoreactivlty m BB rat retina, Brain Res , 523 (1990) 11-15 4 Dlcou, E., Lee, J. and Brachet, P , Colocahzatlon of the nerve growth factor precursor protein and mRNA an the mouse submandibular gland, Neurosci L e t t , 85 (1988) 19-23 5 Dlcou, E , Interaction of antibodies to syntheuc peptldes of proNGF with m vitro synthesized NGF precursors, F E B S L e t t , 255 (1989) 215-218 6 Dlcou, E., Lee, J. and Brachet P., Synthesis of nerve growth factor mRNA and precursor protein m the thyroid and parathyroid glands of the rat, Proc Natl A c a d Sct USA, 83 (1986) 7084-7088 7 Dlcou, E., Houlgatte, R , Lee, J and von W~lcken-Bergmann, B , Synthesis of chimeric mouse nerve growth factor precursor and human/3-nerve growth factor in Escherwhta colt immunological properties, J Neurosct Res , 22 (1989) 13-19 8 Edwards, R H , Selby, M J and Rutter, W J , Differential RNA sphcmg pre&cts two distract nerve growth factor precursors, Nature, 319 (1986) 784-787 9 Edwards, R H , Selby, M J,, Mobley, W C , et al , Processing and secretion of nerve growth factor: expression m mammahan cells with a vacclma virus vector, Mol Cell Blol , 8 (1988) 24562464 10 Korshlng, S , Auburger, G , Heuman R , Scott, J and Thoenen, H , Levels of nerve growth factor and its mRNA m the

The author thanks V Nern6re for techmcal assistance and P Brachet for comments on the manuscript Th~s work was supported by grants from ARTP (Assocmtton pour la Recherche sur les Tumeurs de la Prostate) and ARC (Association pour la Recherche sur le Cancer)

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Nerve growth factor precursors in the rat thyroid and hippocampus.

A nerve growth factor (NGF) precursor form of about 24 kDa was identified in homogenates of rat thyroid and hippocampus by immunoprecipitation using t...
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