Occurrence of a differential expression of the glyceraldehyde-3-phosphate dehydrogenase gene in muscle and liver from euthermic and induced hibernating jerboa (Jaculus orientalis)

(1)

ELSEVIER Gene 181 (1996) 139 145

G E N I E

A N I N T E R N A T I O N A L *,JOURNAL O N O E N E S A N D G E N O M E S

Occurrence of a differential expression of the glyceraldehyde-3-phosphate dehydrogenase gene in muscle and liver from euthermic and induced

hibernating jerboa (Jaculus orientalis)

A. Soukri a, F. Valverde b, N. Hafid a, M.S. Elkebbaj a, A. Serrano b,,

a Laboratoire de Biochimie, Biologie Cellulaire et Mol6culaire, Facult6 des Sciences-Ain Chock, B.P. 5366 Maar~ Casablanca, Morocco

b Instituto de Bioqu#nica Vegetal y Fotosintesis, Consejo Superior de Investigaciones Cientificas y Universidad de Sevilla, Apdo. 1113, 41080 Sevilla, Spain

Received 6 February 1996; revised 3 June 1996; accepted 12 June 1996

Abstract

A cDNA clone which contains the near-complete open reading frame (ORF) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was obtained by screening a muscle cDNA library of jerboa (Jaculus orientalis), a true hibernating rodent, with a PCR-amplified 0.5-kb genomic DNA probe from an internal region of the gene. The 1.l-kb cDNA clone consists of a 927-bp O R F which codifies for 309 aa, about 93% of the original GapC gene encoding the 36-kDa protein, and a 3'-noncoding region of 167 bp. The full-length aa sequence of G A P D H was achieved by sequencing the N-terminal region of the purified protein completing the missing part in the cDNA clone. Both nt and aa sequences exhibit a high degree of homology to other mammalian GAPDHs. The expression of the GapC gene was studied in skeletal muscle and liver of euthermic and hibernating jerboas both on the mRNA level by Northern blot hybridization using the cDNA clone as a probe and on the protein level by Western blot immunodetection using an antibody raised against muscle GAPDH. A clear decrease (about threefold) in the amount of GapC mRNA, a single 1.2-kb transcript, was observed in muscle of hibernating jerboa when compared with the same tissue from the euthermic animal. This mRNA level decrease directly correlates with a reduction in both protein amount and specific activity in crude protein extracts. In contrast, both G A P D H protein and GapC mRNA levels remained unchanged in liver from euthermic and hibernating jerboas although the enzymatic activity was also about threefold lower in the hibernating tissue. These results, together with previous data obtained from protein studies [Soukri et al. (1995) Biochim. Biophys. Acta 1243, 161-168 and (1996) 1292, 177-187] indicate that jerboa G A P D H is regulated by different mechanisms during hibernation in these tissues, that is, at transcriptional level in muscle and at posttranslational level in liver. The reduced G A P D H activity should result in both cases in a decrease of the glycolytic flux that would eventually contribute to the dramatic metabolic depression of this dormant state.

Keywords: cDNA cloning; Glyceraldehyde-3-phosphate dehydrogenase; Transcriptional regulation; Induced hibernation; Jaculus orientalis

1. Introduction

G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ( G A P D H , E C 1.2.1.12) is a cytosolic e n z y m e that plays

* Corresponding author. Tel. +34 5 4557083; Fax +34 5 4620154;

e-mail: aurelio@cica.es

Abbreviations: aa, amino acid(s); bp, base pair(s); GapC, gene (DNA, RNA) encoding GAPDH; GAPDH, D-glyceraldehyde-3-phosphate dehydrogenase; GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

a key role in the glycolytic p a t h w a y a n d has been r e m a r k a b l y conserved d u r i n g evolution, the GapC gene being c o n s e q u e n t l y expressed in all tissues (Fothergill- G i l m o r e a n d Michels, 1993). T h e enzyme, that is c o m - posed of four identical 3 6 - k D a subunits, is responsible for the oxidative p h o s p h o r y l a t i o n of glyceraldehyde- 3 - p h o s p h a t e by N A D + a n d inorganic p h o s p h a t e (Fothergill-Gilmore a n d Michels, 1993).

T h e level of GapC m R N A correlates well with the expression of the e n z y m e in a variety of tissues (Quail a n d Yeoh, 1995). F o r this cause GapC has been widely used as an internal c o n t r o l for transcription a n d N o r t h e r n analysis, being then considered to have a

(2)

140 A. Soukri et al./Gene 181 (1996) 139 145

constitutive expression. For example, the level of rat liver GapC m R N A matches well with that of ribosomal RNA during development (Quail and Yeoh, 1995).

However, several studies suggest that at least in some instances GapC is not constitutively expressed. In this way, insulin markedly increases GapC mRNA in 3T3-F442A adipocytes and H35 hepatoma cell lines (Alexander et al., 1988). On the other hand, the plant Craterostigma plantagineum responds to dehydration and abscissic acid treatment by exhibiting an increase in GapC mRNA due to higher gene transcription (Velasco et al., 1994). Furthermore, increased levels of GapC transcripts have already been observed in other environmental stress conditions in animals and plants, such as iron deficiency in rat liver (Quail and Yeoh, 1995) and anaerobic stress in maize and soybean (Russel and Sachs, 1989; Russel et al., 1990). A decrease in the amount of GapC mRNA was also detected in tobacco plants which exhibit a differential light regulated expression of nuclear genes encoding chloroplast and cytosolic G A P D H s (Shih and Goodman, 1988).

Hibernation is an adaptive strategy that is used by some species of several mammalian orders to conserve energy in cold or inhospitable environments. This strategy helps some, particularly small, mammals to survive cold winter conditions in temperate climates when food and water are scarce yet the demand for metabolic heat generation is high. These hibernating mammals show a dramatically lower body temperature, metabolic and respiratory rates and heart activity during hibernation (Castex and Hooc-Paris, 1987). These changes are pre- cisely controlled and can be reverted only by internally driven mechanisms, suggesting a specific biochemical regulation (Srere et al., 1992). We are interested in the regulation of the GapC gene in jerboa (Jaculus orientalis), a small rodent from desertic areas of Moroccan Highlands that can undergo hibernation (El Hilali and Veillat, 1972). In this paper we describe a cDNA clone which contains most of the predicted open reading frame (ORF) encoding jerboa G A P D H , being the first gene of this rodent cloned so far. The entire aa sequence of the protein has been obtained by a combination of the deduced aa sequence from the cDNA and the N-terminal aa sequence. Using this cDNA clone as a probe in Northern blots, a decrease of about threefold of the GapC mRNA level was found in skeletal muscle of hibernating J. orientalis. However, no difference in the mRNA expression was detected in liver of hibernating and euthermic animals. These results, together with those obtained at the protein level, indicate that a differential transcriptional regulation of the GapC gene should occur in these tissues during the hibernating state.

2. Experimental and discussion

2.1. Isolation of a cDNA clone containing a partial GapC ORF and determination of the full-length aa sequence of jerboa muscle GAPDH

Jerboas (J. orientalis), captured in the sub-desert Moroccan East Highlands and acclimated in a temperature-controlled room (22°C) for about 3-6 months, were forced to hibernate in the laboratory by artificial cooling (Bourhim et al., 1993). Young adult jerboas of both sexes, about 6 months old and weighing 130 150 g, were decapitated and whole liver and leg skeletal muscle were immediately removed and frozen. Tissue samples from four to five individuals were used as starting material for all the experiments. A cDNA library from skeletal muscle of euthermic jerboa constructed in )~ ZAP II vector (Stratagene, La Jolla, CA, USA) was screened with a DNA probe generated by the polymerase chain reaction (PCR). The probe was a DNA fragment of 0.5 kb, the size expected without introns, produced when genomic DNA from euthermic skeletal muscle was used as template (annealing temperature 45°C, 35 cycles).

Degenerate oligonucleotides based on the aa sequence of an internal G A P D H region (residues 153-321) strictly conserved in all species so far studied (Fothergill- Gilmore and Michels, 1993) were used as primers (sense primer 5'-GCC(T)T(A)C(G)C(T)TGC(T)AC G(C)AC G(C)AAC(T)TG-3', antisense primer 5'-CCC(G) CAC(T)TCG(A)TTG(A)TCG(A)TACCA-3'). After agarose gel electrophoresis the PCR-amplified DNA fragment was purified by selective adsorption/desorption on glass beads (Gene Clean, Biol01, La Jolla, CA, USA), cloned into the EcoRI site of pBluescript II SK + vector (Stratagene, La Jolla, CA, USA), and eventually used as a probe to select clones containing the GapC gene from the cDNA library using standard methods (Sambrook et al., 1989). Three positive clones, sizing 0.8 1.1 kb, were found from which the longest one was selected.

Fig. 1A shows the nt sequence of the selected cDNA clone (1094 bp, EMBL/X87226) determined for both strands by the dideoxy chain termination method (Sanger et al., 1977). This sequence contains an ORF (nt 1 927) encoding the near-complete (about 93%) G A P D H protein and a 167-bp 3'-noncoding region which exhibits a consensus poly(A) addition signal sequence (AATAAA) also found in other GapC cDNA clones from mammals (Tso et al., 1985). The 5'-noncoding region of the cDNA and the O R F portion corresponding to the 24 first aa of the protein were missing in this clone. Therefore, the complete aa sequence of jerboa G A P D H (Fig. 1A) was achieved by a combination of the N-terminal aa sequence of the main muscle G A P D H isoform purified from euthermic animals (54 residues, sp/P80534, determined by the Edman method on a 476A Applied Biosystems auto-

(3)

A

M V K V G V N G F G R I G R L V T R A

F N S G K V D I V A I N D P F I D L N

A A A G T C G A T A T T G T G G C C A T C A A T G A C C C C T T C A T C G A C C T G A A C

M V Y M F K Y D S T H G K F K G T V K

A T G G T T T A C A T G T T C A A A T A T G A T T C T A C C C A T G G C A A G T T C A A G G G C A C C G T C A A G

E N G K L V I N G H A I T I F Q E R D

G A G A A C G G G A A G C T G G T G A T C A A T G G G C A C G C C A T C A C T A T C T T C C A G G A G C G C G A C

S K I K W G D A G A E Y V V E S T G V

T C C A A A A T C A A A T G G G G C G A T G C A G G C G C T G A G T A C G T C G T G G A G T C T A C T G G T G T C

T T M E K A G A H L K G G A K R V I I

A C C A C C A T G G A G A A G G C T G G G G C T C A T C T G A A G G G G G G T G C C A A A A G G G T C A T C A T C

A P S R D A P M F V M G V N H E K Y D

G C C C C C T C T C G T G A T G C C C C C A T G T T T G T G A T G G G T G T C A A C C A T G A G A A G T A T G A C

S L K I V S N A S C T T N C L A P L A

A G C C T G A A G A T C G T C A G C A A T G C C T C C T G C A C C A C C A A C T G C T T A G C A C C C T T G G C A

V I H D N F G I V E G L M T T V H A I

G T C A T C C A T G A C A A C T T T G G T A T C G T G G A A G G A C T C A T G A C C A C T G T C C A T G C C A T C

A T Q K T V D G P S A K L W R D G A G

G C C A C C C A G A A G A C G G T G G A T G G C C C C T C C G C G A A G C T G T G G A G A G A T G G C G C T G G G A

Y T A C

A G C T

P C C C

F T T C

$ T C A

K A A G

T A C A

A G C T

A Q N I I P A S T G A A K A V G K V I P

G C C C A G A A C A T C A T C C C T G C T T C C A C T G G G G C T G C C A A G G C T G T G GC4] A A G G T C A T C C C T

E L N G K L T G M A F R V P T A N V S V

G A G C T G A A T G G G A A G C T C A C T G G A A T G G C C T T C C G T G T C C C C A C C G C C A A C G T G T C T G T G

V D L T C R L E K P A K Y D D I K R V V

G T T G A C C T G A C C T G C C G T C T G G A G A A A C C A G C C A A G T A T G A T G A T A T C A A G A G G G T G G T A

K Q A C D G P L K G M L G Y T E H Q V V

A A G C A G G C G T G C G A T G G C C C C C T C A A G G G C A T G C T A G G A T A C A C T G A G C A C C A G G T T G T C

S S D F N G D S H S S T F D A G A G I A

T C C T C T G A C T T C A A T G G T G A C A G C C A C T C C T C C A C C T T T G A C G C G G G G G C T G G C A T T G C C

L N D H F V K L V S W Y D N E F G Y S N

C T G A A C G A C C A T T T T G T C A A G C T T G T T T C C T G G T A T G A C A A C G A G T T T G G C T A C A G C A A C

R V V D L M V H M A S K E

C G T G T G G T G G A C C T C A T G G T C C A C A T G G C C T C C A A G G A G T G A g a c c c c t g g a c c a g c a a g a g c a c a a g a g g a c c t c a c t g c t g g g g a g t c c c t g c c a c a c t c a g t c c c c c a c c t c c c c t c c t c a c a g t t c c a a g c a a g g a t a t g

t a g a c c c c g a g g g g c c t a g g g a g c c g c a t a c c a t c a a t a a a g t a c c c t g t g c t c a a c c g a a a

20 40 48 60 108 80 168 i00 228 120 288 140 348 160 408 180 468 2O0 528 220 588 240 648 260 708 280 768 300 828 320 888 333 953 1032 1094

B

Muscle GAPDH Liver GAPDH

i0 20 30

M V K V ~ G R L V T R A A E N S G K V D I V A I N D P F V K V ~ G R I G R L V T R A A F I q S G K V D I V A I N D P F

Fig. 1. (A) Full-length aa sequence ofJ. orientalis skeletal muscle G A P D H subunit and nt sequence of the partial GapC cDNA clone (EMBL/X87226).

The complete aa sequence was achieved by a combination of the N-terminal region of the purified protein (residues 1-54, sp/P80534) obtained by the Edman method and the predicted aa sequence of the partial GapC cDNA clone extending from residue 25 to the C terminus. The same aa sequence (residues 25 54, underlined) was obtained in the overlapping N-terminal region both from the nt sequence and by protein sequencing.

The stop codon is indicated by an asterisk. The nt sequence is numbered starting at the first nt of the clone. The untranslated 3' region of the cDNA is shown in lower-case letters and the polyadenylation signal is underlined. (B) Alignment of the N-terminal aa sequences of the main G A P D H isoforms purified from skeletal muscle and liver of euthermic jerboa (sp/P80447).

(4)

142 A. Soukri et al./Gene 181 (1996) 139 145

mated sequencer) and the 309 aa long sequence, starting at residue 25, deduced from the partial O R F of the c D N A clone. The aa of the overlapping region (from residue 25 to 54, underlined in Fig. 1A) were identical in both sequences. The nt sequence of the O R F should correspond to a functional GapC gene and not to a pseudogene since no in-frame stop codons are found and all essential residues involved in catalysis, strictly conserved among all eukaryotic and eubacterial G A P D H s (Tso et al., 1985), are present in the predicted aa sequence. Moreover, since no difference was observed in the aa sequences of the N-terminal regions of the main G A P D H isoforms purified from jerboa muscle and liver (Fig. 1B) identical proteins, probably encoded by the same gene, should be expressed in both tissues.

Nevertheless, Southern blot analysis of jerboa genomic D N A showed at least three major bands with each digest (Fig. 2), indicating that at least three GapC copies are present in the jerboa genome. This is the case for other mammals, since although several GapC copies have been found in human and rat genomes, a single GapC copy seems to be functional, being the same G A P D H - e n c o d - ing mRNA present both in human lung and liver (Benham et al., 1984; Tokunaga et al., 1987; Tso et al., 1985).

The 927-bp coding sequence of the partial GapC c D N A clone of jerboa was compared with other GapC sequences in GenBank and E M B L databases using the G C G software program FASTA analysis (Program Manual, 1994), showing the highest degree of homology with other mammalian genes, namely 90% identity with man (M33197) and 86% identity with rat (M17701). A high sequence homology is also found at the 3'-noncoding region up to the poly(A) track with other mammalian GapC cDNAs, namely 86% identity with man and 75% identity with rat, although in the case of jerboa this sequence is clearly shorter (161 bp versus 216 bp for man and 207 bp for rat) (Tso et al., 1985).

The complete aa sequence of jerboa G A D P H , which

K DNA (kb)

5.1 4 . 2 3.5 2 . 0 ~ 1.9 - - 1 . 6 ~ 1 . 3 8 0 . 9 4 0.83

EcoRI EcoRV BamHI PstI

Fig. 2. Southern blot analysis of restriction endonuclease-digested jerboa genomic DNA. Jerboa muscle genomic DNAs (10 pg each) were digested to completion with the indicated restriction enzymes, fraction- ated on a 0.7% agarose gel, blotted to a nylon filter, hybridized with the jerboa cDNA clone labeled with 32p by a random-primer kit (Boehringer Mannheim, Germany), and finally washed at high stringency.

comprises 333 residues (corresponding to the 36-kDa protein) is the first protein sequence of this rodent reported so far and shows a high degree of identity with other eukaryotic G A P D H s (Hensel et al., 1989). The high degree of homology of the J. orientalis G A P D H aa sequence to other G A P D H s (Fothergill-Gilmore and Michels, 1993) was evident on first sight and permitted an almost unambiguous alignment. Alignment with 20 other G A P D H sequences of the SwissProt database using the computer program cited above revealed that the highest homology was with other mammalian G A D P H s , namely 96% identity with man (P00354) and 89% identity with rat (P04797).

2.2. lmmunoblotting and Northern analysis of tissues from euthermic and hibernating J.orientalis

We have previously reported that the G A P D H specific activity values of soluble protein fractions from both skeletal muscle and liver were in hibernating jerboa two- to threefold lower than in euthermic animals (Soukri et al., 1995, 1996). However, immunoblotting analysis of these protein fractions using an a n t i - G A P D H antibody showed that only in hibernating muscle a parallel decrease (about threefold) in the amount of G A P D H protein, measured by the intensity of the immunostained 36-kDa band, takes place (Fig. 3A). In contrast, no significant changes in G A P D H protein amount were found in liver (Fig. 3A). In fact, a posttranslational covalent modification of G A P D H (probably an ADP- ribosylation), which renders a more acidic protein and decreases enzyme activity, occurs in the hibernating liver tissue (Soukri et al., 1996). Therefore, both the comparative analysis of the G A D P H amount and the enzymatic activity in soluble protein fractions of muscle and liver tissues from euthermic and hibernating animals sug- gested differential regulation of the G A P D H during hibernation.

Mammalian hibernation provides an ideal system to study the role of differential gene expression in adaptive evolution. In order to determine if hibernation affected GapC transcription, the cDNA clone described above was used as a probe in Northern blot analysis of poly(A)+mRNA samples isolated from both liver or skeletal muscle of euthermic and hibernating animals.

In all tissues a single G A P D H transcript of 1.2 kb was detected in Northern blot experiments (Fig. 3B).

Noteworthy, Northern blot quantitations revealed a decreased level (two- to threefold lower) of the GapC mRNA expression in hibernating skeletal muscle of jerboa compared to that of the euthermic tissue (Fig. 3B).

In contrast, GapC mRNA levels remained in liver tissues virtually constant in the two physiological states (Fig. 3B). A single GapC gene should be expressed, as discussed above, in all these tissues since the same transcript was found in both physiological states and no

(5)

®

^muscle ^liver

• h • h

I - li - i

( ~ muscle

• h

liver

• h

+ i

~I---GAPDH

p-actin

Fig. 3. Comparative analysis of G A P D H protein and GapC transcript levels in skeletal muscle and liver from euthermic and hibernating J. orientalis. The same starting material of each tissue type (samples from four to five individuals) was used for both determinations. (A) Western blots, using a monospecific a n t i - G A P D H antibody, of proteins of crude extracts from skeletal muscle and liver of euthermic (e) and hibernating (h) animals. Soluble proteins of cell-free extracts from euthermic and hibernating tissues (about 60 gg of protein) were subjected to SDS-PAGE on 12% polyacrylamide gels and transferred to

nitrocellulose filters. The filters were probed with an antiserum (1/500 dilution) raised against the muscle GAPDH of euthermic jerboa prior to incubation with a goat anti-rabbit IgG antibody-horseradish peroxi- dase conjugate (1/1000 dilution, Sigma Chemical Co., St. Louis, MO).

The only protein band detected corresponds to the 36-kDa GAPDH subunit. Immunostained protein bands were quantified in at least three different tissue preparations with an analytical imaging instrument (Bio Image, Millipore Corporation, Ann Harbor, MI, USA). (B) Northern blots of skeletal muscle and liver mRNAs using as probes the GapC eDNA clone here described (top) or a human 13-actin cDNA (American Type Culture Collection) (loading control, bottom). All lanes contained the same amount of poly(A)+mRNA samples, about 1.5 ~g, isolated from both tissues of euthermic (e) and hibernating (h) animals.

Poly(A) + mRNA was isolated from whole tissues by guanidinium thio- cyanate treatment (total RNA fraction) followed by oligo(dT)-cellulose chromatography, quantified using a UV-visible densitometer, then denatured, electrophoresed in 1% agarose-formaldehyde gels and finally blotted on nylon filters according to standard procedures (Sambrook et al., 1989). The mRNAs were detected by sequential hybridization of the filters using random-priming 32p-labeled eDNA probes and washing at high stringency. The only GapC mRNA band detected corresponds to a single 1.2-kb transcript. Relative GapC transcript levels were estimated with respect to the corresponding [~-actin controls in autoradiographs from three different preparations using the analysis imaging system described above.

significant differences were detected between the N-terminal aa sequences of G A P D H from tissues of euthermic and hibernating animals (Fig. 1B; Soukri et al., 1996). This agrees with what was found in other m a m - mals, since it is well established that a single gene encoding G A P D H is functional in different tissues in man, mouse, rat and chicken ( B e n h a m et al., 1984;

Piechaczyk et al., 1984; T o k u n a g a et al., 1987; Tso et al., 1985). The same 1.2-kb transcript and similar quantita- tive results were found when the hybridization was done with total R N A (data not shown).

The decrease of the GapC m R N A observed in hibernating skeletal muscle correlates well with the decrease in b o t h G A P D H protein and enzyme activity found in this tissue (Fig. 3; Soukri et al., 1995). This result is consistent with a specifically regulated decrease in the rate of GapC m R N A synthesis or a degradation of R N A during p r e p a r a t i o n for the maintenance of the hibernating state. The existence of a nonspecific m R N A degradation in hibernating muscle could be ruled out since no significant changes have been found in the level of

~-actin m R N A , used as control in N o r t h e r n blots, between euthermic and hibernating tissue (Fig. 3B).

Moreover, animals in hibernation have been reported to exhibit surprisingly little nucleic acid or protein degradation ( L y a m a n et al., 1982).

O u r results indicate that in skeletal muscle of j e r b o a G A P D H regulation upon hibernation should be at the m R N A level. The finding that GapC m R N A decreases specifically during hibernation is consistent with the hypothesis that the adaptation to hibernation involves, at a molecular level, changes in the gene expression pattern (Stere et al., 1992; T a k a m a t s u et al., 1993).

The fact that the GapC m R N A level in liver was not altered in either physiological state is in sharp contrast with the clear decrease in enzyme activity found in the hibernating tissue (Soukri et al., 1996). Actually, in contrast to what happens in skeletal muscle, liver G A P D H activity should be regulated by a differential and specific mechanism as the animal enters into hibernation, maintaining the levels of b o t h GapC m R N A and a m o u n t of protein constant. The mechanism implied here should be a posttranslational covalent modification, p r o b a b l y a m o n o - A D P - r i b o s y l a t i o n (Soukri et al., 1996), by the reversion of which the central carbon metabolism of liver is ready to function immediately upon animal arousal. Several studies also suggest that covalent modification of enzymes m a y be a key molecular mechanism involved in liver metabolic regulation during hibernation (Storey, 1987; Storey and Storey, 1990). In fact, the changes in physico-chemical properties observed between jerboa G A P D H s from liver of hibernating and euthermic animals (Soukri et al., 1996) are similar to those described for covalently modified enzymes of different living systems under harsh environmental conditions (Brttne et al., 1994; Plaxton and Storey, 1984;

Storey, 1984, 1987; Storey and Storey, 1990).

Hibernating m a m m a l s show a n u m b e r of metabolic responses to this resting state including the repression of the glycolytic flow and initiation of glyconeogenesis from aa ( L y a m a n et al., 1982). This is in agreement with the fact that the main function of glycolysis is to provide ATP and the metabolic pool for respiration. G A P D H is

(6)

144 A. Soukri et al./Gene 181 (1996) 139-145 a glycolytic allosteric e n z y m e c a t a l y z i n g a reversible

o x i d a t i o n a n d p h o s p h o r y l a t i o n that is a c t u a l l y subjected to m e t a b o l i c r e g u l a t i o n ( F o t h e r g i l l - G i l m o r e a n d Michels, 1993). I n this respect, different m e c h a n i s m s have b e e n p r o p o s e d to be i n v o l v e d in h i b e r n a t i o n - i n d u c e d m e t a b o l i c depression. At the p r o t e i n level b o t h p o s t t r a n s l a t i o n a l c o v a l e n t modifications, n a m e l y phos- p h o r y l a t i o n (Storey, 1987) or A D P - r i b o s y l a t i o n ( S o u k r i et al., 1996), a n d e n z y m e b i n d i n g to the s u b c e l l u l a r p a r t i c u l a t e fraction (Storey a n d Storey, 1990) have b e e n p r o p o s e d to decrease m e t a b o l i c flux in different p a t h - ways by r e d u c i n g the activity of r e g u l a t o r y key enzymes.

O n the other h a n d , tissue specific r e g u l a t i o n of genes e n c o d i n g key r e g u l a t o r y e n z y m e s c o u l d also play a r e l e v a n t role i n h i b e r n a t i o n - a s s o c i a t e d m e t a b o l i c depression, as o u r results with J. orientalis G a p C s t r o n g l y suggest.

3. Conclusions

We have d e m o n s t r a t e d t h a t G A P D H of J. orientalis is i n f l u e n c e d b y the physiological state ( e u t h e r m i c / h i b e r n a t i n g ) in b o t h skeletal muscle a n d liver tissues. It seems clear t h a t there are two types of c o n t r o l of the G A P D H t h a t are associated with the h i b e r n a t i n g state:

o n e at the m R N A level which occurs in muscle, a n d a second o n e at the p r o t e i n level (a p o s t t r a n s l a t i o n a l p r o t e i n m o d i f i c a t i o n ) that takes place in the liver.

Acknowledgement

This w o r k is p a r t of a c o l l a b o r a t i v e Research Project b e t w e e n C N R ( M o r o c c o ) a n d C S I C (Spain), a n d has b e e n p a r t i a l l y s u p p o r t e d b y D G I C Y T (Spain) ( g r a n t s P B 91-85 a n d PB 94-033) a n d J u n t a de A n d a l u c i a ( P A I 3182, a n d C o l l a b o r a t i v e G r a n t J u n t a de A n d a l u c i a - M i n i s t 6 r e d ' E d u c a t i o n et de la Recherche Scientifique of M o r o c c o ) . T h e a u t h o r s t h a n k Prof. M. L o s a d a for his interest a n d help, Dr. N. B o u r h i m for p r o v i d i n g i n d u c e d h i b e r n a t i n g a n i m a l s a n d Mr. M. B e n j e n a t t for his help in sequencing.

References

Alexander, M.C., Lomanto, M., Nasrin, N. and Ramaika, C. (1988) The effect of insulin on GAPDH mRNA in adipocyte cells. Proc.

Natl. Acad. Sci. USA 85, 5092-5096.

Benham, FJ., Hodgkinson, S. and Davies, K.E. (1984) A glyceraldehyde-3-pbosphate dehydrogenase pseudogene on the short arm of the human X chromosome defines a multigene family. EMBO J. 3, 2635-2640.

Bourhim, N., E1 Kebbaj, M.S., Ouafik, L.H., Chantard, T.H., Giraud, P. and Olivier, C. (1993) Opioid binding sites in jerboa (JacuIus orientalis) brain: a biochemical comparative study in the awake-

active and induced hibernating states. Comp. Biochem. Physiol.

104B, 607 615.

Brtine, B., Dimmeler, S., Vedia, L.M. and Lopelina. E.G. (1994) Nitric oxyde: a signal for ADP-ribosylation of proteins. Life Sci. 54, 61-70.

Castex, C. and Hooc-Paris, R. (1987) Insulin and glucagon in hibernating mammals. Diabetes Metab. 13, 176-181.

El Hilali, M. and Veillat, J.P. (1972) Jaculus orientalis: a true hibernator.

J. Mammal. 53, 574-593.

Fothergill-Gilmore, L.A. and Michels, P.A.M. (1993) Evolution of glycolysis. Prog. Biophys. Biol. 59, 105 235.

Hensel, R., Zwickl, P., Fabry, S., Lang, J. and Palm, P. (1989) Sequence comparison of glyceraldehyde-3-phosphate dehydrogenase from the three urkingdoms: evolutionary implication. Can. J. Microbiol. 35, 81-85.

Lyaman, C.P., Willis, J.S., Malan, A. and Wang, L.C.H. (1982) Hiberna- tion and Torpor in Mammals and Birds. Academic Press, New York.

Piechaczyk, M., Blanchard, J.M., Riaad-E1-Sabouty, S., Dani, C., Marty, L. and Jeanteur, P. (1984) Unusual abundance of vertebrate glyceraldehyde-3-phosphate dehydrogenase pseudogenes. Nature 312, 469 471.

Plaxton, W.C. and Storey, K.B. (1984) Phosphorylation in vivo of red muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anionic stress. Eur. J. Biochem. 143, 267-272.

Quail, E.A. and Yeoh, G.C.T. (1995) The effect of iron status on glyceraldehyde-3-phosphate dehydrogenase expression in rat liver. FEBS Lett. 359, 126 128.

Russel, D.A. and Sachs, M.M. (1989) Differential expression and sequence analysis of the maize glyceraldehyde-3-phosphate dehydrogenase gene family. Plant Cell 1,793 803.

Russel, D.A., Wong, D.M.L. and Sachs, M.M. (1990) The anaerobic response of soybean. Plant Physiol. 92, 401 407.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning.

A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467.

Shih, M.C. and Goodman, H.M. (1988) Differential light regulated expression of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenase in Nicotiana tabacum.

EMBO J. 7, 893-898.

Soukri, A., Valverde, F., Hafid, N., El Kebbaj, M.S. and Serrano, A.

(1995) Characterization of muscle glyceraldehyde-3-phosphate dehydrogenase isoforms from euthermic and induced hibernating Jaculus orientalis. Biochim. Biophys. Acta 1243, 161-168.

Soukri, A., Hafid, N., Valverde, F., El Kebbaj, M.S. and Serrano, A.

(1996) Evidence for a posttranslational covalent modification of liver glyceraldehyde-3-phosphate dehydrogenase in hibernating jerboa (Jaculus orientalis). Biochim. Biophys. Acta 1292, 17%187.

Srere, H.K., Wang, L.C.H. and Martin, S.L. (1992) Central role for differential gene expression in mammalian hibernation. Proc. Natl.

Acad. Sci. USA 89, 7119 7123.

Storey, K.B. (1984) Phosphofructokinase from foot muscle of the whelk, Busycotypus canaliculatum: evidence for covalent modification of the enzyme during anaerobiosis. Arch. Biochem. Biophys. 235, 665-672.

Storey, K.B. (1987) Regulation of liver metabolism by enzyme phosphorylation during mammalian hibernation. J. Biol. Chem. 262, 1670 1673.

Storey, K.B. and Storey, J.M. (1990) Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation.

Q. Rev. Biol. 65, 145 174.

Takamatsu, N., Ohba, K.I., Kondo, J, Kondo, N. and Shiba, T. (1993) Hibernation-associated gene regulation of plasma proteins with a collagen-like domain in mammalian hibernators. Mol. Cell. Biol. 13, 1516 1521.

(7)

Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori, K., Ohkubo, M., Sawada, K. and Sakiyama, S. (1987) Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancer.

Cancer Res. 47, 5616-5619.

Tso, J.Y., Sun, X.-H., Kao, T., Reece, S.R. and Wu, R. (1985) Isolation and characterization of rat and human glyceraldehyde-3-phosphate

dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Mol. Gen. Genet. 13, 2485-2502.

Velasco, R., Salamini, F. and Bartels, D. (1994) Dehydration and ABA increase in RNA levels and enzyme activity of cytosolic GAPDH in the resurrection plant Craterostigma plantagineum. Plant Mol. Biol.

26, 541 546.