Molecular cloning and nucleotide sequence of a human renin cDNA fragment

Volume 11 Number 20 1983 Nucleic Acids Research

Molecular cloning and nadeotide sequence of a human renin cDNA fragment

Florent Soubrier* + , Jean-Jacques Panthier+, Pierre Corvol* and Francois Rougeon

+ Unite de Genetique et Biochimie du Developpement, Institut Pasteur, ERA CNRS 851, 25 rue duDocteur Roux, 75724 Paris Cedex 15, and * Institut National de la Sante et de la RechercheMedicale, Unite 36, 17 rue du Fer a Moulin, 75005 Paris, France

Received 1 August 1983; Revised and Accepted 22 September 1983

ABSTRACTWe have studied human renin messenger RNA by hybridiza-

tion with the mouse submaxillary gland (SMG) renin cDNA probe.The human kidney messenger RNA is about 1.6 kilobase (kb) long,similarly to the mouse SMG renin mRNA. A kidney renin cDNA cloneof 1.1 kb length was obtained. A comparison of nucleotide se-quences of mouse and human cDNA clones reveals conservation ofresidues involved in catalytic mechanisms and a potential glyco-sylation site. The human renin molecular probe allowed us tostudy renin expression in human chorionic tissue. The chorionicand kidney renin messenger RNAs are similar in length. TheSouthern blot analysis reveals the presence of a single reningene in human DNA.

INTRODUCTION

Interest in the renin angiotensin system is related to

the facts that renin plays a major role in the regulation of

blood pressure and water and salt metabolism, and that pharmaco-

logical blockade of this system is extremely effective in the

treatment of hypertension. The main source of human renin is the

juxtaglomerular cells of the afferent arterioles in the kidney.

Other sources of renin or renin-like enzymes are the chorion (1)

and the central nervous system (2). In the kidney, renin is

found mainly as an active enzyme, whereas chorionic cells con-

tain an inactive form of the enzyme which is trypsin activable.

Biosynthetic experiments with chorionic cell cultures suggest

that inactive renin is the proform of the active enzyme (3).

Similarly in renin-secreting tumors, an inactive 54 KD renin

precursor is converted into an active 44 KD enzyme (4). Although

human renal renin has been purified to homogeneity (5), there

are no data concerning its primary structure, due to its low

concentration in the kidney. To date none of the extra renal

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Nucleic Acids Research

renin-like enzymes in man have been extensively purified, and

their relation to the kidney enzyme remains speculative,

although iramunohistochemical studies have shown imraunological

similarities. The purpose of this communication is to report

the partial amino-acid sequence of human renal renin deduced

from the nucleotide sequence, which shows a striking homology

with both mouse submaxillary gland (SMG) renin and other aspar-

tyl proteases. In addition, we provide evidence that human

renal and chorionic renin mRNAs are the same length and that in

these different organs, they are transcribed from a single gene.

MATERIALS AND METHODS

Mouse SMG renin probe

Clone pRnl-4, 1427 nucleotide length, contains an essen-

tially complete renin mRNA transcript and is inserted in the

PstI site of pBR322 (6) . Double-stranded DNA was labeled by

nick-translation (7) . Purified insert was isolated from a 3,5 %

acrylamide gel for Southern blot hybridization and screening of

the cDNA library.

DNA preparation

Human female and mouse BALB/c male DNAs were extracted

respectively from frozen placenta and liver. High molecular

weight DNA was prepared by a modification of the methods of

Blin and Stafford (8). Restriction digest, gel electrophoresis

and Southern blot hybridization were performed as described by

Heldmann and Rougeon (9). Mouse SMG renin cDNA insert and a

EcoRI-SacI restriction fragment isolated from human cDNA insert,

ligated and nick-translated, were used as probes. Filters were

washed using high stringency conditions, as defined in this

paper.

mRNA Isolation and Northern blot technique

SMG, kidney, chorion and leukemic lymphoid total RNA was

prepared by the lithium chloride-urea method as described (10).

Fractionation of poly(A)-containing RNA on oligo(dT)cellulose

was performed as described (10) . Glyoxal-dimethylsulfoxide

treated RNA was electrophoresed, transferred to nitrocellulose

filter and hybridized with the mouse SMG renin probe according

to Thomas (11). Washing conditions were 1 x SSC (50°C) for inter-

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species hybridization and 0.1 x SSC (50°C) for homologous hybri-

dization (Fig. 1) .

Construction of a cDNA library from kidney mRNA

Poly(A) mRNAs were fractionated on a 5-20 % sucrose

gradient. Fractions were checked for renin mRNA by hybridization

of an aliquot with mouse SMG cDNA probe by the Northern blot

technique (11) . Fractions enriched for renin sequence were used

as a template for reverse transcription (12). The cDNA obtained

was converted into double-stranded SI nuclease resistant DNA (121

Double-stranded cDNA was fractionated on a 5-20 % sucrose gra-

dient and larger cDNA was inserted into the PstI site of pBR322

by the oligo-d(CG) tailing method (12). Host bacteria were

transformed and colonies were screened by hybridization with

the nick-translated cDNA insert of pRnl-4 as a probe. =< 10,000

clones were obtained and the recombinant clone pHRn22, contai-

ning the larger insert, 1,100 base pairs, was kept for further

analysis among five clones exhibiting common restriction sites.

DNA sequence analysis (Fig. 2)

DNA fragments were labeled at their 5' ends by the poly-

nucleotide kinase exchange reaction or at their 3' ends, either

by the terminal deoxynucleotidyl-transferase or the Klenow frag-

ment of DNA polymerase I. The chemical degradation reaction was

performed using the method of Maxam and Gilbert (13).

1743 •» ~ m1431 <—

1 2 3 4

Figure 1 : Characterization of human renin mRNA by Northern blothybridization with the mouse SMG renin cDNA probe.Lane 1, pBR322 digested by Avail. Lane 2, human kidneymRNA (15 yg). Lane 3, kidney mRNA from SWISS female(10 ug). Lane 4, SWISS SMG mRNA (0.1 yg).In another experiment, human mRNA from leukemic cellswas used as a negative control and no hybridizationwas detectable (data not shown).

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RESULTS AND DISCUSSION

Molecular cloning of SWISS mouse submaxillary gland renin

has allowed the determination of the complete amino acid sequen-

ce of the precursor (44 KD). This precursor is converted into

active renin, which consists of two chains of 35 and 3 KD (6).

The general strategy was to use the mouse SMG cDNA as a molecu-

lar probe to study human renin mRNA in the kidney and the cho-

rion, as well as the human renin structural gene. Since infarc-

ted kidney synthesizes increased amounts of renin, messenger RNA

was prepared from a surgically removed infarcted kidney due to

arterial thrombosis and causing severe arterial hypertension.

Characterization of the renin mRNA was performed by the

Northern blot technique. The autoradiogram presented in Figure 1

shows that among kidney poly(A) containing RNA, a mRNA popula-

tion hybridizes specifically with the mouse SMG probe. The 1.6

kb length of this mRNA is similar to that of the mouse kidney

and SMG renin mRNA.

Because of its low abundance, kidney mRNA was enriched

for renin message by fractionation on a sucrose gradient.

Aliquots of each fraction were tested by Northern blot hydridi-

zation with the mouse SMG renin probe. Fractions containing

renin mRNA were pooled and used to construct a cDNA library of

approximately 10,000 clones. Colonies were screened by hybridi-

zation with the mouse SMG renin P cDNA as a probe. One clone,

pHRn22, containing a 1,100 base pairs insert, was chosen among

five hybridizing colonies for further experiments.

That pHRn22 is derived from the 1.6 kb renin mRNA was

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demonstrated by nucleotide sequence analysis of the clone

(Fig. 3). Alignment of the nucleotide sequence with the corres-

ponding sequence of mouse SMG renin cDNA reveals considerable

homology. The overall homology between human renal and mouse SMG

renin is 77 % at the nucleotide level in the coding region, as

expected from mouse-human divergence. The rate of silent subs-

titutions is 6.3 %. A deletion of 9 nucleotides occurred in SMG

renin sequence after residue 228 and a deletion of 3 nucleotides

in human renin is present at residue 310. At the amino acid

level, the homology in the sequenced region is 69.6 %. In the

protein sequence encoded by the clone, amino acid residues

known to be involved in the catalytic mechanisms are conserved.

Aspartate 286, corresponding to aspartate 215 of pepsin, is

present as well as the two following residues which are also

present in mouse SMG renin and other aspartyl proteases. Other

araino acids involved in the catalytic mechanisms, such as tryp-

tophan 108 and tyrosine 146, corresponding to tryptophan 39 and

tyrosine 75, respectively, of pepsin are present. Cysteines in-

volved in disulphide bonds 114-121 and 277-281 are conserved at

the same position.A potential N-glycosylation site is shown by

the presence of the sequence Asn-Gly-Thr located at residues

138-139-140. This results was expected since human renal reninis

stained by Schiff reaction (5) and binds to Con-A-sepharose (14).

Comparison with the mouse SMG renin sequence shows that the di-

basic Arg352 - Arg353 pair is replaced by a Ser - Ser dipeptide.

Figure 3 : Sequence of human renin cDNA and comparison with mouseSMG renin cDNA.The two upper lines show the nucleotide sequence(below) and predicted amino acid sequence (above) ofhuman renin. The two lower lines indicate nucleotidesequence of the homologous region (above) and thepredicted amino acid sequence (below) of mouse SMGrenin. Only nucleotides which differ from the humanrenin chain are shown. Homologous amino acids areunderlined. Deletions are indicated by parentheses. Themouse SMG renin nucleotide and amino acid sequencespreviously published (6) have been revised from residue195 to residue 198 in view of the amino acid sequencepublished by Misono et al. (17) . Since the pHRn22clone does not include the 5' region of the codingsequence, nucleotides and amino acids numberscorrespond to the mouse SMG renin sequence, beginningwith the first residue of renin precursor.

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17431431

1 2 3Figure 4 : Extra renal expression of renin.

The human renin cDNA clone pHRn22 was used as a probe.Lane 1, pBRn22 digested by Avail. Lane 2, kidney mRNA(10 pg). Lane 3, chorion mRNA (40 pg).

The cleavage site between chain A and B of submaxillary renin

occurs after this Arg - Arg pair and might be located after the

dibasic pair Lys35it- Lys355 in the case of human renin. However,

there is no evidence, at the present time, for such a processing

of the human enzyme. It can be noticed that there is a 87 % homo-

logy between the B chain of mouse SMG renin and its correspon-

ding region in the human enzyme (Leu35g Argmji) •

The human renin clone was also used to investigate the

extra renal expression of renin. Chorion was isolated from a

healthy woman undergoing full term cesarean section. Poly(A)

containing RNA was prepared and analyzed by the Northern blot

technique. As shown in Figure 4, a unique mRNA, that is the same

size as renal renin mRNA, hybridizes with human renin cDNA probe.

To determine if renin mRNA from both kidney and chorion

is transcribed from different genes, we made a human DNA blot

hybridization with mouse SMG cDNA as a probe, since this cDNA

contains an almost complete mRNA transcript. Human DNA was pre-

pared from a placenta of a female fetus after cesarean section

and digested with EcoRI restriction enzyme, which cuts in human

renin cDNA at one site. As shown in Figure 5, two fragments of

6.0 and 3.5 kb are identified by the mouse cDNA probe.

Furthermore, with the human cDNA EcoRI-Sacl restriction fragment,

a unique 6.0 kb fragment was detected showing that there is only

one renin gene per haploid genome in humans. This single gene

appears to be transcribed into mRNAs of similar size in at least

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Figure 5 : Genomic pattern of human renin gene.Human DNA is digested by EcoRi. Lane 1, humanDNA hybridized with the mouse SMG renin cDNAprobe. Lane 2, human DNA hybridized with theEcoRi-SacI fragment of the human renin cDNAprobe.

two organs. However, until chorionic renin mRNA structure is

known, we cannot exclude the possibility of differential spli-

cing leading to slightly different mRNAs, but similar in sizes.

Because human renin mRNA from kidney and chorion, and

mouse SHG mRNA are approximately the same size, the molecular

weight of the human renin precursor should be similar to that of

the mouse SMG renin precursor. Based on acellular translation

and calculation from the primary amico acid sequence deduced

from the nucleotide sequence, we have previously found that the

molecular weight of mouse SMG is 44 KD (6, 15). The discrepancy

between the expected 44 KD prorenin and the tentative 54 KD human

renin precursor could well be explained by the presence of glyco-

sylated chains interfering with the molecular weight determina-

tion by SDS gel electrophoresis. Furthermore, these results

suggest that higher molecular weight renins (16) are not present

in kidney as precursors of the active renin. Renal and chorionic

renins are transcribed from a single gene into mRNAs of similar

sizes. Differences in the level of activation of renin in these

two tissues could be related either to a differental splicing of

the renin mRNAs or to different post-translational processing of

the precursor.

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ACKNOWLEGMENTS

This work was supported by grants from C.N.R.S. (A.T.P.

n° Al-5054 and 960008), INSERM (PRC-Genie Genetique), and the

Fondation pour la Recherche M6dicale Francaise.

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3 - Acker G.M., Galen F.X., Devaux C , Foote S., Papernick E.,Pesty A., Mfinard J. and Corvol P. (1982) J. Clin. Endocrinol.Metab. 55, 902-909.

4a- Leckie B.J., McGhee N.K., Lever A.F. and Robertson J.I.S.(1981) in : Sambhi M.P. (ed.) Heterogeneity of renln andrenin substrate. Elsevier North-Holland Inc. New York159-174.

4b- Corvol M.T. et al., submitted.5 - Galen F.X., Devaux C , Guyenne T., Mfinard J. and Corvol P.

(1979) J. Biol. Chem. 254, 4848-4854.6 - Panthier J.J., Foote S,, Chambraud B., Strosberg A.D.,

Corvol P. and Rougeon F. (1982) Nature 298, 90-92.7 - Rigby P.W.J., Dieckmann M., Rhodes C. and Berg P. (1977)

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10 - Auffray C. and Rougeon F. (1980) Eur. J. Biochem. 107,303-314.

11 - Thomas P.S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205.12 - Auffray C , Nageotte R., Chambraud B. and Rougeon F. (1980)

Nucl. Acids Res. 8, 1231-1241.13 - Maxam A.M. and Gilbert W. (1980) Methods Enzymol. 65, 499-

560.14 - Yokosawa A.H., Holladay L.A., Inagami T., Haas E. and

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16 - Barett J.D., Eggena P. and Sambhi M.P. (1977) Circ. Res.suppl. 2, 41, 7.

17 - Misono K.S., Chang J.J., Inagami T. (1982) Proc. Natl. Acad.Sci. USA 79, 4858-4862.

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Molecular cloning and nucleotide sequence of a human renin cDNA fragment

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