Structure of equine corticotropin releasing factor

Peptides,Vol. 12, pp. 1437-1440.©PergamonPress plc, 1991.Printedin the U.S.A.

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Structure of Equine Corticotropin Releasing Factor JOHN H. LIVESEY,* ALAN CARNE,'~ CLIFFORD H. G. IRVINE,:~ JANE ELLIS,* M A R G A R E T J. EVANS,* ROGER SMITH* AND RICHARD A. DONALD*

*Department of Endocrinology, The Princess Margaret Hospital, Christchurch, New Zealand tDepartment of Biochemistry, University of Otago, Dunedin, New Zealand 4:Veterinary and Animal Sciences Group, Lincoln University, New Zealand Received 17 August 1990 LIVESEY, J. H., A. CARNE, C. H. G. IRVINE, J. ELLIS, M. J. EVANS, R. SMITH AND R. A. DONALD. Structure of equine corticotropinreleasingfactor. PEPTIDES 12(6) 1437-1440, 1991.--A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determinedby gas phase sequence analysis. Probable carboxyl terminal amidationwas demonstratedby similar retention times for equine and human corticotropinreleasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-ProIle-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala -Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser -AsnArg-Lys-Leu-Met-Glu-Ile-Ile-NH2.The purified peptide is equipotentwith human corticotropinreleasing factor in an in vitro bioassay and in a human plasma binding protein assay. Corticotropin releasingfactor

Neuropeptides

Isolation

IN order to study the hypothalamic control of pituitary adrenocorticotropin (ACTH) secretion in vivo, we have sampled the pituitary venous effluent of the horse (5) and measured plasma arginine vasopressin and ACTH concentrations (10). Wishing to extend our studies to the possible role of corticotropin releasing factor (CRF) in the control of ACTH secretion, we wanted to measure CRF concentrations also. To do this quantitatively by radioimmunoassay requires a knowledge of the chemical structure of equine CRF. This report describes the extraction, the purification and the determination of the probable structure of equine CRF.

Sequence

Horse

Hypothalamus

Brownlee RP-300 C8 reverse phase column (Brownlee Labs, USA). The packing material was 7 ~m in diameter, and the column dimensions were 22 x 0.46 cm with a matching 3 cm guard colunm. Elution was at 1 ml/min and 0.5 ml fractions were collected. Gas phase sequence analysis was performed in an Applied Biosystems 470A sequencer (ABI, USA), with on-line 120A HPLC for detection of the PTH-amino acid derivatives.

Extraction of Hypothalami Hypothalami, including some surrounding tissue, were removed from approximately 70 normal standard-bred horses (Equus caballus) of either sex and various ages within l0 minutes of slaughter and stored frozen until extracted. About 2 kg of hypothalamic tissue was collected in total. The hypothalami were sliced and homogenized while still frozen in 60 g lots in methanol containing 0.1% mercaptoethanol (2.5 ml methanol/g tissue). The homogenate was centrifuged at 2500 × g for 20 rain for 4°C, the supernatant lyophilized almost to dryness and redissolved in 0.05 M phosphate buffer, pH 7.4, containing 0.1% Triton X-100, 0.05% alkali-treated casein (7) and 0.05% NaEEDTA (1 ml buffer/g original tissue). This solution was centrifuged at 5000 x g for 20 min at 4°C, filtered through Whatman GF/A glass fiber paper and stored at -20°C.

METHOD

General Ovine and human corticotropin releasing factor (oCRF and hCRF) were purchased from Bachem Inc. (USA) and Sepharose CL-4B was purchased from Pharmacia (Sweden). Antiserum "Y2Bo '' was a gift from Prof. P. J. Lowry, University of Reading, UK, and hCRF(3-21) was a gift from Dr. D. Harding, Massey University, New Zealand. Tissue homogenization was performed with a Virtus "23" homogenizer (Virtus Co., USA). Protein was measured by the Bradford method (4). Antisera to peptides were raised in rabbits using the peptide conjugated to bovine thyroglobulin with carbodiimide. High pressure liquid chromatography (HPLC) was performed using a 300/~ pore size

Immunoadsorption Rabbit antisera were raised against oCRF and the one se-

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FIG. 1. Reverse phase high pressure liquid chromatography of immunoaffinity-purified equine CRF. Chromatographic details are given in the text. The fraction in the principal ir-CRF peak with the highest concentrationof ir-CRF was used for amino acid sequence determination.

lected for immunoadsorption bound 50% of 125I-hCRF(1-41) at a final antiserum dilution of l:140,000 but only 30% of 1251hCRF(3-21) at a 1:30 dilution. The IgG fraction from 36 ml of this antiserum was purified by 2 × precipitation with 16% NazSO4 and coupled to 75 ml of periodate-activated Sepharose CL-4B by gentle mixing for 2 days at 4°C at pH 9.5 (3). The capacity of the immunoadsorbant was 2.6 Ixg hCRF/ml of matrix. Immunoadsorption was performed batchwise at 4°C as follows. The anti-oCRF-Sepharose (75 ml) was mixed with 700 ml of the hypothalamic extract in a rotating bottle overnight and then poured into a 2.5 cm diameter column and washed with 100 ml of the 0.05 M phosphate/Triton/casein/EDTA buffer and 100 ml water. Immunoreactive CRF (ir-CRF) was eluted by applying to the column, in succession, 30 ml each of acetate buffer pH 5, acetate buffer pH 4 and formate buffer pH 3, which each contained 20% (v/v) CH3CN. Fractions of 3 ml were collected. Prior to each use (including the first), the immunoadsorbent was washed in like manner with the eluent solutions. Fractions containing irCRF were pooled (-----60ml), 20 txl mercaptoethanol was added, and the fractions were stored frozen. Elution of freshly prepared immunoadsorbent with the pH 5, pH 4 and pH 3 buffers eluted undetectable amounts of ir-CRF (<2.3 ng/ml of gel).

Chromatographic Purification of ir-CRF Part (45 ml) of the pool from the immunoadsorption step above was loaded onto the reverse phase HPLC column by repeated injection and eluted with a 0.1% trifluoroacetic acid (TFA)/acetonitrile mobile phase. The acetonitrile gradient was linear from 24% to 60% over 72 min (Fig. 1).

[raised against hCRF(3-21)] was used for measuring ir-CRF in the comparative HPLC of eCRF and hCRF.

Comparative Chromatography of eCRF and hCRF Equine CRF and hCRF, both separately and together (5 pmol of each), were eluted from the reverse phase HPLC column using a 0.1 M NH4HCO3/acetonitrile (pH 8) gradient comprising linear segments of 0-30% acetonitrile over 5 min, 30--40% over 40 min and 40--60% over 5 min. Fractions were assayed by radioimmunoassay.

In Vitro Bioassay Equine anterior pituitary cells were prepared, perifused on a 15-column system and the ACTH concentration in the effluent measured by modifications of the methods described for ovine cells (2). Pulses of extracted peptide or hCRF (3.3, 10, 33 or 100 pM) of 10-min duration were applied to the columns.

Displacement of 12SI-hCRF From Human Plasma CRF Binding Protein Equine, human or ovine CRF (10-8000 pM) was mixed with 125I-hCRF (20,000 cpm) (6) and a 1:20 dilution of pooled human plasma in the phosphate buffer described above (but without the Triton X-100) and stored at -80°C. One by one the mixtures were thawed, incubated at 37°C for 1 h and chromatographed on a 1 × 30 cm Superdex-75 gel filtration column (Pharmacia, Sweden) using the same buffer as eluent at a flow rate of 10 ml/h. Fractions of 0.25 ml were quantitated for radioactivity and the percentage binding calculated.

Radioimmunoassay Immunoreactive CRF was measured during the purification by a radioimmunoassay for hCRF (1) using antiserum "Y2Bo." As this antiserum was in short supply, antiserum "All bleed 10"

RESULTS The hypothalamic extract after lyophilization and reconstitution in the phosphate buffer contained 75 Ixg of ir-CRF and 290

EQUINE CORTICOTROPIN RELEASING FACTOR

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FIG. 2. Gas phase sequencer analysis of 280 pmol of equine ir-CRF. The single letter code for the amino acid found at each cycle is given at the top of the figure.

mg of protein. Following immunoadsorption of this extract, 39 Ixg of ir-CRF was recovered from the immunoadsorbent. Almost all of the ir-CRF was eluted by the pH 3 buffer, along with 6.8 mg of protein. One-quarter of this eluate, when further purified by a single HPLC step, yielded a major and a minor peak of ir-CRF (1.8 ixg and 0.7 ixg, respectively) as shown in Fig. 1. The most concentrated fraction (1.3 Ixg ir-CRF) of the main HPLC ir-CRF peak was subjected to Edman degradation, and a single sequence of 41 amino acids was obtained (Fig. 2). The yield at cycle 2 was 100 pmol (36%). Following comparative reverse phase chromatography at pH 8, the peak width at half height of eCRF alone was 1.5 min, for hCRF alone was 1.8 min and for eCRF plus hCRF was 1.5 rain. We conclude that there is no significant difference in the elution positions of eCRF and hCRF in this system. In the in vitro bioassay, the ir-eCRF/ir-hCRF potency ratio was 0.87, with 95% confidence limits of 0.50 to 1.56. For the displacement of 125I-hCRF from human plasma CRF binding protein, the ir-eCRF/ir-hCRF potency ratio was 1.01 (0.80-1.30). The ir-oCRF/ir-hCRF potency ratio was approximately 0.006. DISCUSSION

After three separation steps [an extraction, one step of immunoafflnity chromatography and one HPLC run (Fig. 1)], a sample of equine CRF, as detected by immunoassay, was subjected to Edman degradation and a single sequence was obtained (Fig. 2). The relatively simple purification is facilitated by an initial methanol extraction, taking advantage of the hydrophobic nature of CRF to achieve an efficient and selective extraction from tissue. We have found methanol to be 7-8 times more efficient than 0.05 M HC1 as an extraction medium for CRF (unpublished data). The amino acid sequence of equine CRF, determined by gas phase Edman degradation, indicates identity to human (6) and rat (11) CRF, but posttranslational modifications cannot be ruled out.

As we had insufficient eCRF to determine directly whether or not the C-terminus is amidated, we compared the retention times of eCRF and hCRF on a reverse phase column at pH 8. At this pH, the difference in retention times of -COOH and -CONH 2 groups is likely to be greater than at an acidic pH, due to ionization of the -COOH terminus. As no detectable difference in the retention times of eCRF and hCRF was found when thus cochromatographed, and since the resolution of the gradient is high (oCRF is separated from hCRF by about 4 min), we conclude that eCRF is probably very similar or identical to hCRF and is probably C-terminal amidated. Our finding that the potency of eCRF is not significantly different from that of hCRF both in the in vitro bioassay and in displacing ~25I-hCRF from the plasma binding protein is also consistent with eCRF having a very similar or identical structure to hCRF. The human plasma CRF binding protein requires a major portion of the hCRF molecule for binding (9), has little affinity for oCRF (9), and thus appears to be a sensitive probe of CRF structure. Hence a peptide of the structure we have elucidated is likely to be an equine corticotropin releasing factor. We cannot, however, rule out the possibility that other corticotropin releasing or inhibiting factors exist in the horse. The finding that equine and human CRF appear to be very similar or identical in structure is significant because it suggests that equine CRF can be measured by the human CRF radioimmunoassay (1). Hence measurement of CRF in the pituitary venous effluent of the fully conscious horse (5) provides valid information regarding the regulation of ACTH secretion. Interestingly, equine ACTH and human ACTH also probably have a common structure (8) and thus the horse may be a better model for the human hypothalamic-pituitary system than the rat or the sheep. ACKNOWLEDGEMENTS We wish to thank Luisa Mattioli for technical assistance. This work was supported by the New ZealandMedical Research Council, the Canterbury Area Health Board and by NIDDK grant 38322.

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