WO1988003949A1

WO1988003949A1 - The cs protein of the corpuscles of stannius

Info

Publication number: WO1988003949A1
Application number: PCT/AU1987/000389
Authority: WO
Inventors: Aldonna Butkus; John Paul Coghlan; Peter John Roche
Original assignee: Howard Florey Institute Of Experimental Physiology
Priority date: 1986-11-21
Filing date: 1987-11-19
Publication date: 1988-06-02

Abstract

Essentially pure CS protein, a protein isolated from the Corpuscles of Stannius having ion-transfer properties, precursors thereof and sub-units or fragments thereof. A gene encoding the CS protein and precursors thereof.

Description

THE CS PROTEIN OF THE CORPUSCLES OF STANNIUS

This invention relates to the isolation and characterisation of the CS protein from the Corpuscles of Stannius. The invention is also concerned with the molecular cloning and characterisation of the gene sequence encoding the CS protein.

Note: References referred. to hereafter are collected

» at the end of the description.

In 1839 Stannius described yellow gland-like structures scattered through the kidney of the sturgeon and noted that these structures also occurred in Teleost fishes as two distinct small bodies and suggested that these organs might represent the adrenal glands of fishes. This homology with the adrenal gland of mammals was accepted until more extensive embryological studies were undertaken by Huot (1898) who described the differing origins of the Corpuscles of Stannius and the adrenal cortex. However, confusion still persisted. Giacomini in 1908 (a and b) described a mass of tissue in the anterior region of the kidney which on the basis of morphological characteristics resembled the interrenal system which he designated as the anterior interrenal as distinct from the

SUBSTITUTE SHEET Stannius corpuscles which he designated as ^"the posterior interrenal. Giacomini also made the observation that the corpuscles were present only in those animals where the parathyroids were absent. Vincent (1898) at about the same time had shown that the removal of the Corpuscles of Stannius was not fatal and therefore assumed they were not essential for life. However Petit (1896) had demonstrated that the removal of one corpuscle resulted in compensatory hypertrophy of the remaining one, thus suggesting that the organ might be an endocrine gland. Following these early observations, the function of the Corpuscles of Stannius has fascinated biologists continuously to 1986 and numerous studies have broached this issue.

The corpuscles from various fishes have been extensively examined morphologically both at the light and electron microscopy level (Vincent, 1898; Olivereau and Fontaine, 1965; Krishnamurthy and Bern, 1969; Ristow and Piepho, 1963; Wendelaar Bonga and Greven, 1975; Wendelaar Bonga et al. 1980 and Bhattacharyya et al. 1982). There is a concensus view that they contain at least one type of secretory granule. Variations with life cycle, with level of maturation, with age and sex as well as species differences have created a confused picture.

Overall, ablation, experiments have led to the view that the Corpuscles of Stannius may have an osmoregulatory role, however their close association with the kidney leave open the possibility of surgical damage to the delicate renal area, which may have compounded the issue of function. There is some evidence that they may have a parathyroid hormone like role^' in calcium metabolism (Millet et al. 1980 and 1981; Lopez et al. 1984), a prolactin-like action in the gill (Ogasaware and Hirano, 1984) and possibly be the source of a renin-like enzyme capable of angiotensin II production (Chester Jones 1966;

SUBSTITUTE SHEET Ogawa and Sokabe, 1982).

Infusion of extracts of Corpuscles of Stannius have been reported variously as having actions on water, sodium, calcium, phosphate and magnesium metabolism. ^• The secretory product of the corpuscles of Stannius has until now remained unresolved.

SUMMARY OF THE INVENTION The CS protein of the Corpuscles of Stannius has been purified to homogeneity. The partial protein sequence of the purified CS protein was obtained and used to construct synthetic oligonucleotides which were in turn used to isolate DNA encoding the CS protein of the Corpuscles of Stannius. Characterization of this DNA shows that it encodes the CS protein and a precursor thereof comprising an N-terminal prohormone segment and a signal peptide sequence which are attached to the CS protein sequence.

The precursor of the CS protein having _^an N-terminal prohormone segment and signal peptide is hereinafter referred to as the prepro CS protein. The precursor of the CS protein having an N-terminal prohormone segment is hereinafter referred to as the pro CS protein.

Isolation of the CS protein from the Corpuscles of Stannius will enable further investigations to be carried out on the biological activity of this protein. According to one aspect of the present invention, there is provided essentially pure CS protein.

According to a further aspect of the present invention, there is provided the prepro CS protein, the pro CS protein or the signal or prohormone peptides of the prepro CS protein. The invention is also directed to sub-units or fragments of the prepro CS protein.

According to a still further aspect of the present invention there is provided a gene encoding the prepro CS protein or sub-units or fragments thereof. Particularly,

SUBSTITUTE SHEET there is provided DNA sequences which encode the pro CS protein, the CS protein or the signal or prohormone peptides of the prepro CS protein.

The gene encoding the prepro CS protein or sub-units or fragments thereof may be inserted into appropriate transfer vectors. Suitable vectors include bacterial plasmids, yeast plasmids, phage and other viral vectors. The gene encoding the prepro CS protein or sub-units or fragments thereof may be inserted into appropriate expression vectors for subsequent expression of the prepro CS protein or sub-units or fragments thereof in eurkaryotic or prokaryotic host cells.

Suitable prokaryotic host cells include various E. coli species, Pseudomonas and Serratia marcenes. Suitable eurkaryotic host cells include filamentous fungi, various strains of yeast and mammalian cells_^ (both primary and cell lines) .

The isolation of DNA encoding the prepro CS protein and sub-units or fragments thereof will enabPe large amounts of the corresponding protein products to be produced by recombinant DNA methodology.

According to- a yet further aspect of the present invention, there is provided a method for the production of the CS protein, said method comprising the steps of: (a) inserting the gene encoding -CS protein into an expression vector;

(b) transfecting a cell with the expression vector of step (a) ; and

(c) culturing the cell containing the expression vector to produce the CS protein and subsequently recovering said protein. As the protein sequence of the prepro CS protein has been elucidated by recombinant DNA techniques, the prepro CS protein and fragments or sub-units thereof may be

SUBSTITUTE SHEET j produced using conventional protein synthesis techniques (Barany and Merrifield, 1980).

We have synthesized a peptide fragment of the CS protein precursor corresponding to amino acids 16 to 35 of Figure 3, to investigate the physiological role of this protein. Studies utilising this peptide indicate that the CS protein affects ionic activity in vivo, particularly, in increasing sodium excretion and decreasing plasma potassium concentration. This activity is characteristic of the CS protein, and has been exemplified by, but is in no way restricted to a sheep model. Studies we have carried out in fish indicate this protein is also active in decreasing calcium uptake.

The CS protein and peptide fragments thereof have potential as therapeutic agents in the treatment of cardio-vascular disease, renal disease and electrolyte disorders, particularly,^' oedema, heart failure and high blood pressure. In addition to the above, the N-terminal peptide fragment of the CS protein precursor corresponding to amino acids 16 to 35 of Figure 3, and other fragments of the CS protein, may be useful tools in determining physiological control mechanisms in the kidney, and the control of blood pressure.

According to another aspect of the present invention, there is provided a therapeutic composition comprising the CS protein or peptide fragments thereof in association with a pharmaceutically acceptable carrier or excipient. Such compositions may be useful in the treatment of cardio-vascular disease, renal disease and electrolyte disorders.

According to a further aspect of the present invention, there is provided a method for the treatment of cardio-vascular disease, renal disease and electrolyte disorders, comprising the administration of a

SUBSTITUTE SHEET therapeutically effective amount of the CS protein or peptide fragments thereof, either alone or in association with a pharmaceutically acceptable carrier or excipient.

The isolation and characterization of DNA encoding the prepro CS protein enables labelled DNA or RNA probes to be prepared. These probes, which may contain all or part of the DNA encoding the prepro CS protein, can be used to identify homologous or similar sequences in higher organisms, including man. DNA probes may be labelled isotopically using for example P or I. DNA probes may also be labelled with biotin or avidin, fluorescent or chemiluminescent reagents, or other appropriate labelling molecules.

RNA probes may be produced from the DNA encoding the CS protein using, for example, the SP6 vector system (Pharmacia Corporation, Piscataway, N.J.).

It is well established that the genetic code contains redundancies, that is, certain amino acids are coded for by more than one codon. Accordingly, the invention includes DNA encoding the prepro CS protein or sub-units or fragments thereof where the natural codons are replaced by other codons which code for the same amino acid.

According to a further aspect of the present invention, there is provided a method for the isolation of the CS protein comprising:

(a) detergent extraction of the Corpuscles of Stannius;

(b) electrophoretic separation of the detergent extract in a gel matrix; and

(c) electroelution of the separated CS protein from step (b) and recovery of the purified CS protein.

According to a still further aspect of the present invention, there is provided a method for the isolation of a gene encoding the prepro CS protein comprising the steps of:

SUBST.TUTi SHEET (i) preparing a hybridization probe based on the protein sequence of the prepro CS protein; (ii) screening a cDNA library prepared from RNA isolated from the Corpuscles of Stannius with the probe; and

(iii) identifying and isolating those DNA sequences from the cDNA library of step (ii) which hybridize to the probe.

DESCRIPTION OF THE DRAWINGS FIGURE 1 shows a polyacrylamide gel electrophoresis profile of the isoelectric focusing gel section (pi 5.7 - 6.5) of the CS protein after staining with Coomassie Blue;

FIGURE 2 is the sequencing strategy for the CS protein mRNA: A. Schematic diagram of the CS protein mRNA. The 5' and 3* untranslated regions are indicated by the solid line. The hatched box represents the signal peptide sequence, the solid box represents the prosegment, and the open box represents the mature protein. B. The arrangement of the four positive CS protein cDNA clones. λCS.l was isolated from the RNAse H cDNA Library and λCS.2, λCS.4 were isolated from the Sl-nuclease library.

FIGURE 3 shows the nucleotide sequence and deduced amino acid sequence of the eel CS protein precursor; FIGURE 4 shows a hybridization histochemical analysis of the eel Corpus Stannius-kidney region. 6μm sections of eel CS-kidney region after probing with 32P labelled 75 mer oligonucleotide. The CS region is shown in the lower left region of the autoradiograph.

FIGURE 5 shows the effect of renal arterial infusion into 7 conscious sheep of the N-terrainal 25 amino acid fragment of the CS proteins (peptide 'U'). Peptide ^"U* was infused at a rate of 50 μg/h. Graph (A) is a plot

SUBSTITUTE SHEET of urinary sodium excretion (μmol/min) against time (hours) . Graph (B) is a plot of plasma potassium against time (hours) .

Definitions:

The term "CS protein" used herein refers to a polypeptide isolated from the Corpuscles of Stannius, having a molecular weight approximately between 28,000 and 42,000 Daltons as determined by SDS-PAGE and an amino acid sequence substantially corresponding to amino acids 16-246 of Figure 3.

^* The amino acid sequence of the CS protein depicted in Figure 3 may be varied by the substitution, addition or deletion of one or more amino acids. Variants which possess ion-transfer activity, characteristic of the CS protein, are included within the term "CS protein". Such variants may be produced by solid phase peptide synthesis techniques (Barany and Merrifield, 1980). Alternatively, genetic engineering techniques^" such as site directed mutogenesis (Botstein and Shortle, 1985), restriction endonuclease digestion, and the ligation of DNA fragments, (Maniatis et al., 1982) may be employed to construct DNA expression vectors which express variants of the CS protein. We have isolated the CS protein from the eel; Anguilla australis. It is to be understood that the CS protein may be readily isolated from other animal species following the teaching of the present application. Any such protein having a molecular weight approximately between 28,000 and 42,00 Daltons and an amino acid sequence exhibiting substantial homology with amino acids 16-246 of Figure 3 is to be included within the definition "CS protein".

Substantial homology when used in the above, context refers to at least 75% homology with amino acids 16-246 of

SUBSTITUTESHEET Figure 3.

The term "pro CS protein" used herein, refers to a protein having a sequence corresponding to amino acids 1-246 of Figure 3.

The term "prepro CS protein" used herein, refers to a protein having a sequence corresponding to amino acids -17-246 of Figure 3.

"Essentially pure" when used to define the CS protein produced by the present invention refers to the CS protein substantially free of protein or other materials ordinarily associated with the Corpuscles of Stannius, ordinarily greater than or equal to 95% of the total protein being CS protein by weight.

The term "sub-unit" or "fragment" when used in relation to the CS protein refers to a peptide having an amino acid sequence which is included within the amino acid sequence of the CS ^"protein. Peptides having more than six amino acids are likely to be unique to the CS protein. In order to test whether a peptide is unique to the CS protein, its amino acid sequence may be compared with amino acid sequences on record in amino acid sequence data banks such as the EMBL Data Base (compiled by the European Molecular Biology Laboratory), the Dayhoff Data Base or the Gene Bank Data Base (compiled by the National Institutes of Health, U.S.A.). Sub-units or fragments of the CS protein may or may not possess biological activity. The term "natural codons" refers to those codons which naturally encode amino acids of the prepro CS protein. The term "sub-unit" or "fragment", when used in relation to genes encoding the prepro CS protein, refers to a DNA or RNA sequence included in the gene encoding the prepro CS protein. A fragment may comprise single or double stranded DNA or RNA, and is generally in excess of 10 nucleotides. The sequence of any such fragment may be

SUBSTITUTE SHEET compared with sequences recorded in any of the data banks mentioned above to establish whether it is unique to the CS protein..

The following terms have the meanings set forth below:

DNA - deoxyribonucleic acid

RNA - ribonucleic acid cDNA - complementary DNA (enzymically synthesized from a mRNA sequence mRNA - messenger RNA

A - Adenine

T - Thymine

G - Guanine

C - Cytosine

U - Uracil

SDS-PAGE - Sodium dodecyl sulphate poly- acrylamide gel electrophoresis

EXAMPLES:

EXAMPLE A - Tissue Extraction and Isolation of the CS Protein

(i) Tissue Extracts

Corpuscles of Stannius and samples of several other tissues (kidney, atria, ventricle, muscle and liver) were obtained from decapitated eels. These tissues were placed immediately on dry ice and stored under liquid nitrogen until used. A modified Baier technique (1984) was used for homogenation of the tissues. The zwiterionic detergent CHAPS was substituted for Nonidet P40. The tissue homogenates were centrifuged in an Eppendorf (Trade Mark) centrifuge for 3 minutes and the supernatant used immediately for electrophoresis analysis. (ii) Two dimensional σel electrophoresis techniques

This was performed essentially as originally described by O'Farrell (1975) except that CHAPS was substituted for

SUBST_ITUTESHEET Nonidet P40 as above, and for the electrofocussing gels, the urea solution was deionized just prior to use. After electrofocussing, the gels were equilibrated in O'Farrell's buffer "0" for 10 minutes and then run in the second dimension. These procedures revealed major proteins of the corpuscles of Stannius which are not present in other eel tissues. The portions of the CS electrofocussing gels ( pi 5.7 - 6.5) were cut out, equilibrated and run in the second dimension as before. The line of spots containing the CS protein was visualized with 4 M sodium acetate (Higgins and Dehmus, 1979) or Coomassie Blue (CBB) stained (Figure 1) .

Once identified the spots were punched out, the gel disc was washed in water and stored at -20°C until electroelution. Electroelution was performed at 5°C according to the technique of Hunkapillar et al (1983) in an electroelution dialysis cell (Caltech) . (iv) Characteristics of the CS Protein

The eluted protein was harvested and precipitated overnight with 9 volumes of cold methanol. Following centrifugation at 5°C, the pellet was washed with a small amount of cold methanol, air dried, dissolved in 0.05% SDS and reprecipitated as above. The air dried pellet was then stored in a dessicator at 5°C until analysed. Electrophoretic analysis has established that the CS protein has a native molecular weight of 42,000, and under reducing conditions a major component of 32,000.

The following results were obtained on analysis of the isolated CS protein: 1. Treatment with N-Glycanase demonstrated that the

32,000 monomer is N-linked glycosylated either at a -Asn-X-Thr or -Asn-X-Ser (where X is any amino acid except Pro) position and that the molecular weight was reduced to 28,000 following removal of the

j SUBSTITUTE SHEET carbohydrate. 2. Amino acid analysis was performed according to standard procedures on a Beckman System 6300 Amino

Acid Analyser. An approximation of the ratio of the amino acid residues in the isolated CS protein (based on an assumed total number of amino acids of 210) gave the following results:

Asx (18), Thr (10), Ser (17), Glx (25), Pro (10), Gly (21), Ala (17), Cys (present), Val (15), Met (5), He

(14), Leu (21), Tyr (10), Phe (13), His (3), Lys (8),

Arg (8), indicating a predominance of Glx (Glu or

Gin), Gly, Leu, Asx (Asp or Asn), Ser, Ala and the presence of cysteine residues. 3. Amino acid sequencing

The following partial N-terminal sequence was obtained using an Applied Biosystems gas phase amino acid sequencer: N-Phe-Ser-Ala-Ser-Ser-Pro-Ser-Asp-Val-Ala-Arg-x-Leu-Asπ- Gly-Ala-Leu-Gln-Val-Gly-x-Ser-Ala-Phe-Ala-Leu—, where

-x- respresents cysteine, threonine or possibly tryptophan.

EXAMPLE B - Isolation and Characterisation of a DNA Encoding the prepro CS Protein

(i) Synthetic oliσonucleotide probes

An oligodeoxyribonucleotide probe was synthesized (using an Applied Biosystems DNA Synthesizer) corresponding to the predicted cDNA sequence of the amino terminal 25 amino acid sequence with the assumption that residues 12 and 21 were Thr and Trp respectively and using preferred codon choices for fish.

SUBSTITUTE SHEET The 75-mer probe sequence was as follows: 5'-GGCGAAGGCGGACCATCCCACCTGCAGGGCTCCGTTCAGTGTTCTGGC

CACGTCGGATGGACAGGAGGCGGAGAA-3 ' The 75-mer probe was 5' end labelled with 32P and used to screen the cDNA clone bank constructed from mRNA of the Corpuscles of Stannius. This probe was also used for hybridization histochemistry studies.

(ii) Cloning and Sequence Analysis of cDNA for the Isolated CS Protein

Two different cDNA Libraries were constructed from eel CS poly(A)⁺ RNA. The first library was derived from cDNA which was synthesized using standard procedures; oligo(dT)-ρrimed AMV reverse transcriptase reaction followed by DNA polymerase and S,-nuclease reactions (Maniatis et al, 1982). The second library was derived from cDNA synthesized using the RNase H procedure of Gubler and Hoffman (1983),. which is known to produce full-length cDNA transcripts. Both cDNA libraries were cloned using the bacteriphage vector λgt 10 (Huynh et al, 1985).

Approximately 5,000 clones from each cDNA library were screened with the synthetic 75-mer oligonucleotide which was 5'-end labelled with [γ- 32P]ATP. Hybridization was carried out in 20% formamide, 50 mM NaP0₄ pH 6.8, lmM Na pyrophosphate, 5 x SSC, 5 x Denhardts, 50 μg/ml salmon sperm. DNA at 42°C. Filters were washed (4 times) in 0.2 x SSC at 40°C. Three hybridization-positive clones were isolated from the S,-nuclease cDNA library and .one hybridization-positive clone was isolated from the RNase H library. Analysis of these clones by restriction endonuclease digests and Southern blotting revealed cDNA insert lengths of 0.32 (λCS.2), 0.8 (λCS.4) and 1.1 kb (λCS.3) for the

SUBSTITUTE SHEET three S,-nuclease clones and 2.2 kb for the one RNase H clone (λCS.l), as shown in Figure 2. Part A of Figure 2 represents a schematic diagram of the CS protein mRNA. The 5' and 3^* untranslated regions are indicated by the solid line. The hatched box represents the signal peptide sequence, the solid box represents the prosegment, and the open box represents the mature protein. The nucleotide sequencing strategy and the linear arrangement of these clones is shown in Figure 2. DNA fragments suitable for dideoxy chain termination sequencing (Sanger et al., 1977) were generated by sonication of the appropriate cDNA inserts (Deininger, 1983). These randomly generated fragments were subcloned into M13mpl8. The nucleotide sequence of the CS protein cDNA is shown in Figure 3. The sequence of nucleotide residues 269-343 corresponds to the amino-terminal amino acid sequence region determined for the CS protein. It'is interesting to note that the original 75-mer probe, designed using preferred codon choices, has 75% identity with the authentic sequence.

On the basis of methods for predicting secretory signal sequences (von Heijne, 1986), it is assumed that the site of cleavage between the signal peptide and the excreted protein is between the alanine and tyrosine residues, indicated by an arrow in Figure 3. This leaves a 15 amino acid pro-sequence extension at the amino terminus of the isolated CS Protein. The pro-sequence precursor is presumably processed by specific cleavage between the arginine and phenylalanine residues (at positions 15 and 16) to give -the mature 231 amino acid CS protein shown in square brackets in Figure 3.

The deduced amino acid sequence of the eel CS protein has an Asn-Ser-Thr sequence at amino acids 44-46. The asparagine at position 44 is presumably the

SUBSTITUTE SHEET carbohydrate attachment site. It is interesting to note that there are a total of 15 cysteine residues in the deduced sequence. If all the Cys residues are involved in cysteine bonds, this leaves an extra cysteine which could be free or linked to another peptide chain through a disulfide bond.

EXAMPLE C - Histochemical Localisation of mRNA Encoding the CS Protein

(i) Hypridization Histochemistry This was performed on the Corpuscles of Stannius and other eel tissue, using the labelled 75-mer oligonucleotide probe as described by Coghlan et al.

1985. These studies revealed that the mRNA coding for this protein was located specifically in the Corpuscles of Stannius and not in any other eel tissues examined.

Figure 4 shows specific labelling of the CS and not the kidney with the 75 mer probe.

EXAMPLE D - Synthesis and Biological Activity of a Fragment of the Pro CS Protein

(i) Synthetic Peptide Synthesis

The following peptides, corresponding to amino acids shown in Figure 3, were synthesized by solid-phase synthesis procedures (Barany and Merrifield, 1980) on an Applied Biosystems Model 430A Automated Peptide Synthesizer:

A -1-15 B 1-15 C 16-35 D 96-109 E 118-151

F 217-246 Crude peptides were purified by gel filtration and preparative high performance liquid chromatography (HPLC)

SUBSTITUTE SHEET under reducing conditions to homogeneity, as assessed by analytical HPLC and amino acid analysis. The cysteines were left unblocked and were tested for biological activity in the reduced monomeric form. (ϋ) Biological Activity

Sheep were prepared surgically by removing the ovaries and the right kidney, the carotid artery was exteriorized and cannulas placed into the left renal artery and vein. Synthetic peptide was infused directly into the renal arterial cannula at 50 μg/hr (12 ml/hr) and urine was collected via a Foley bladder catheter. Blood samples were taken from the carotid artery. Urinary sodium and plasma potassium levels were determined by standard procedures. Only peptide C exhibited biological activity in this bioassay, and the results for this peptide a e presented in Figure 5. All statistical analyses were by two-way analyses of variance. Each line in the Figure represents the data for an individual sheep (seven different sheep) , and the shaded area shows the grouped data of one standard error of the mean on either side of the group mean. The results demonstrate that there is a small, but significant, increase in sodium excretion, and also a decrease in plasma potassium concentration in the sheep following infusion of a synthetic peptide fragment of the pro CS protein.

The above results indicate that the CS protein and fragments thereof, particuarly corresponding to peptide C and peptides synthesized from around this region, have potential in the treatment of cardio-vascular disease, renal disease and electrolyte disorders; particularly oedema, high blood pressure and heart failure. Although these experiments have been carried out in sheep, it is to be understood that the therapeutic affect of these agents is not restricted to sheep.

SUBSTITUTE SHEET (iii) An Alternative Assay for Biological Activity

The effect of peptide C, corresponding to amino acids 16-35 of Figure 3, on calcium uptake was tested in the assay described by Wagner et al., 1986. This assay measures inhibitory effects upon branchial calcium uptake and involves monitoring the rate of 45Ca uptake by juvenile rainbow trout.

Results of this assay are shown in Table 1.

TABLE 1 Dose (n) Uptake P % Inhibition

Control (Saline) 7 2 . 62±0 .44 lOμg peptide C 77 00..6677±±00..1144 0.005 (-74%) (4.37nM) lμg peptide C (0.44nM) 88 11..2255**00..1199 P 0.01 (-53%)

These results indicate that the CS protein and a selected peptide fragment thereof have an inhibitory effect on calcium uptake.

(iv) Expression of the CS protein in Escherichia Coli The CS protein cDNA has been cloned into an E. Coli secretion vector, in order to produce large quantities of the CS protein for structural and biological characterization. The CS protein cDNA clone λCS3 was inserted into the EcoRl site of the vector ompA-2, a system developed by Ghrayeb et al. (1984). This secretion vector contains the coding sequence for the ompA signal peptide. This signal peptide guides the cloned gene product across the cytoplasmic membrane into the periplasmic space of the E.Coli cell. The signal peptide is then cleaved from the cloned gene

SUBSTITUTESHEET product by a specific signal peptidase. Thus, the gene product produced has the authentic amino terminus of the original protein.

The coding sequence for the ompA signal peptide was joined onto the coding sequence for the amino terminus of the mature CS protein, i.e. residue 16 in Figure 3, using the site-specific mutagenesis method described by Morinaga et al. (1984). This secretion vector containing the coding sequence for the -ompA signal peptice/CS protein was designated pCS-2. Gel electrophoresis of proteins from E. Coli cells containing pCS-2, and Western blot aalysis using a CS protein antisera, indicate that the cells are producing a gene product which binds the CS protein antibody.

SUBSTITUTESHEET REFERENCES

Baier, L.J., Hanash, S.M. and Erickson, R.P. (1984) Mice homozygous for choromosomal deletions at the albino locus region lack specific polypeptides in two-dimensional gels. Proc. Natl. Acad. Sci. USA ILL: 2132-2136

Barany, G. and Merrifield, R.B., (1980) Solid-Phase Peptide Synthesis, In "The Peptides", Vol. 2 (E. Gross & J. Meienhofer eds.) Academic Press, New York: 1-284

Bhattacharyya, T.K., Buttler, D.G., Youson, J.H. (1982) Ultrastructure of the corpuscles of Stannius in the garpike (Lepisosteus platyrhynchus) . Gen. Comp. Endocrinol. 4___: 29-41

Botstein D. and Shortle D., (1985) Strategies and Applications of In Vitro Mutagenesis, Science, 229: 1193-1201

Chester Jones, I., Henderson, I.W., Chan, D.K.O., Rankin, J.C., Mosley, W. , Brown, J.J., Lever, A.F., Robertson, J.I.S. and Tree, M. (1966)

Pressor activity in extracts of the corpuscles of Stannius from the European eel (Anguilla anguilla) . J. Endocrin. 34,: 393-408 • Coghlan, J.P., Aldred, P., Haralambidis, J., Niall, H.D., Penschow, J.D. and Tregear, G.W. (1985) Review of hybridization histochemistry. Analytical Biomchemistry 149: 1-28 Deininger, P.L. (1983)

Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal. Bioche . , 129: 216-223 Ghrayeb, J., Kimura, H., Takahara, M. , Hsiung, H., Masui, Y. and Inouye, M. (1984) Secretion cloning vectoris in Eschrichia coli, The Embo Journal, 3_: 2437-2442 Giacomini, E. (1908a)

Sulla disposizione del sistema interrenale e del sistema feocromo nelle cieche, e nei leptocephali Rendic. Accad. sc. 1st. Bologna, n.s., 12.: 172 Giacomini, E. (1908b)

SUBSTITUTE SHEET If sistema interrenale e il sistoma cromaffine (sistema feocromo) nelle Anguille adulte, nell eieche, e nei leptocefali.

Mem. Accad. Sc. 1st. Bologna, ser 6, T.5, 407 Abstract in Monit. Zool. Ital. ___<__: 92

Gubler, U. and Hoffman, B.J. (1983)

A simple and very efficient method for generating cDNA libraries. Gene 25: 263-269

Higgins, R.C. and Dahmus, M.E. (1979) Rapid visualization of protein bands in preparative SDS-polyacrylamide gels. Anal. Biochem. £___: 257-260

Huot, E. (1898)

Preliminaire sur l'origine des capsules surrenales des poissons lophobranches_ Compt. Rend. Acad. d. sc. , T.126,49.

Hunkapillar, M.W. , Lujan, E., Ostrander, F. and Hood, L.E. (1983)

Isolation of microgram quantities of proteins from polyacrylamide. gels for amino acid sequence analysis. Methods in Enzymology £1: 227-236

Huynh, T.V., Young, R.A. and Davis R.W. (1985) Constructing and screening cDNA libraries in λgtlO and λgt 11.

In: DNA Cloning Volume I : a practical approach (David Glover, ed.) IRL Press, Oxford, p 49-78

Krishna urthy V.G. and Bern, H.A. (1969) Condative Histologic Study of the Corpuscles of

Stannius and the Juxtaglomerular Cells of Teleost

Fishes.

Gen. Comp. Endocrinol. 1___.: 313-335 Lopez, E. , Tisserand-Jochem, E-M. , Eyqiem, A., Milet, C, Hillyard, C, Lallier, F., Vidal, B. and Maclntyre, I. (1984)

Immunocytochemical detection in eel corpuscles of stannius of a mammalian parathyroid-like hormone. Gen. & Comp. Endocrinol. 53.: 28-36

Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning : A laboratory manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, New York

SUBSTITUTE SHEET Milet, C, Hillyard, C.J., Martelly, E., Girgis, S., Maclntyre, I. and Lopez, E. (1980) Similitudes structurales entre 1'hormone hypocalcemiante des corpuscules de stannis (PCS) par 1'Anguille (anguilla anguilla L.) et 1'hormone parathroidienne mammalienne.

Comptes Rendus Hebd. Seances (Acad. sci. Ser. D. Sci. Nat.) 29J_: (12) 977-980 Morinaga, &. , Franceschini, T., Inouye, S. and Inouye, M. (1984), Improvement of oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA. Biotechnology, 2 : 636-639 O'Farrel, P.H. (1975)

High resolution of two-dimensional electrophoresis of proteins.

J. Biol. Chem. 2_50: (10) 4007-4021 Ogasawara, T. and Hirano, T. (1984)

Effects of prolactin and environmental calcium on osmotic water premeability of the gills of the eel Anguilla japonica. Gen. & Com. Endocrinol. 53.: 315-324

Ogawa, J. (1968)

Osmotic and ionic regulation in goldfish following removal of the corpuscles of Stannius or the pituitary gland. Can. J. Zool. 4.6: (4) 669-676

Ogawa, M. and Sokabe, H. (1982)

Hypocalcemic effect of homologous angiotensin-like substances produced by the renin-like enzyme in the corpuscles of Stannius in the Japanese eel Anguilla japonica. Gen. & Comp. Endocrinol. 4_7: 36-41

Olievereau, M. and Fontaine, M. (1965) Effect de 1'hypophysectomic sur les corpuscles de Stannius de 1'Anguille Comp. Rend. Acad. Sci. 261: 2003-2008

Petit, A. (1896) Recherches sur les capsules surnenales. J. de l'anat. et physiol. T. 32, 391

Ristow, H. and Piepho, H. (1963) Uber die Bildung der Sekretgranula in den Stanniusschen Korperchen des FluBaales.

Naturwissenshaften 5_.: 382-383

Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 7_4: 5463-5467

Stannius, H. (1839)

Die Nebennierenbeiknochenfischen.

Arch. Anat. Physiol. 8.: 233-271

Von Heijne, G. (1986)

A new method for predicting signal sequence cleavage sites.

Nucleic Acids Research, 14.: 4683-4690

Vincent, S. (1898)

The effects of extirpation of the suprarenal bodies of the eel (Anguilla anguilla) .

Proc. Roy. Soc. London, .52.: 354-356

Wagner, G.F., Hampong, M. , Park, CM. and Copp, D.H. (1986) Purification, Characterization, and Bioassay of Teleocalcin, a Glycoprotein from Salmon Corpuscles of Stannius. General and Comparative Endocrinology 63.: 481-491

Wendelaar-Bonga, S.E. and Greven, J.A.A. A Second Cell Type in Stannius Bodies of Two Euryhaline Teleost Species. Cell. Tiss. Res., 159: 287-290

Wendelaar-Bonga, S.E., van der Meij, J.C.A. and Pang, P.K_T. (1980)

Evidence for two secretory cell types in the Stannius bodies of the Teleosts Fundulus Heteroclitus and Carassius Auvatus-.- Cell & Tissue Research 212: 295-306

Claims

CLAIMS :

1. Essentially pure CS protein.

2. Essentially pure pro CS protein.

3. Essentially pure prepro CS protein.

4. The signal or prohormone peptide of the prepro CS protein.

5. A peptide fragment of the pre pro CS protein.

6. A fragment as claimed in claim 5, which possess ion transfer activity characteristic of the CS protein.

7. A fragment as claimed in claim 6 having a sequence corresponding to amino acids 16 to 35 of Figure 3.

8. A therapeutic composition comprising the CS protein or peptide fragments thereof in association with a pharmaceutically acceptable carrier or excipient.

9. A method for the treatment of cardio-vascular disease, renal disease or electrolyte disorders, comprising _he administration of a therapeutically effective amount of the CS protein or peptide fragments thereof either alone or in association with a pharmaceutically acceptable carrier or excipient.

10. A method for the isolation of the CS protein comprising:

(a) detergent extraction of the Corpuscles of Stannius;

SUBSTITUTE SHEET (b) electrophoretic separation of the detergent extract; and

(c) electroelution and recovery of the separated CS protein from step (b) .

11. A gene encoding the prepro CS protein.

12. A sub-unit of the gene claimed in claim 11, which encodes the pro CS protein, the CS Protein or the signal or prohormone peptides of the prepro CS protein.

13. A fragment of the gene claimed in claim 11, excluding those sub-units claimed in claim 12, which is unique to the gene encoding the prepro CS protein.

14. A transfer vector containing a gene, sub-unit or fragment as claimed in any one of claims 11 to 13.

15. A method for the isolation of a gene encoding the prepro CS protein comprising the steps of:

(i) preparing a hybridization probe based on the protein sequence of the prepro CS protein;

(ii) screening a cDNA Library prepared from mRNA extracted from the Corpuscles of Stannius with the probe; and

16. A method for the production of the CS protein, said method comprising the steps of:

(a) inserting the gene encoding CS protein into an expression vector;

(b) transfecting a cell with the expression vector of

SUBSTITUTE SHEET step (a); and (c) culturing the cell containing the expression vector to produce the CS protein and subsequently recovering said protein.

17. A method as claimed in claim 16, wherein the expression vector is a viral vector.

18. A method as claimed in claim 16, wherein the vector is ompA-2 and the transfected cell is E. Coli.

19. The CS protein when produced by the methods of any one of claims 16 to 18.

20. The CS protein, methods for its preparation and pharmaceutical compositions containing it substantially as hereinbefore described.

21. A gene encoding the CS protein and methods for its production substantially as hereinbefore described.