Peptides,Vol. 12, pp. 855-859. ©PergamonPress plc, 1991. Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Isolation and Characterization of Galanin From Sheep Brain R A N N A R SILLARD, 1 I]LO LANGEL~: AND HANS J O R N V A L L * t

Departments of Biochemistry H and *Chemistry L Karolinska Institutet, S-104 01, Stockholm, Sweden "~Centerfor Biotechnology, Huddinge University Hospital, S-141 86, Huddinge, Sweden ~:Department of Biochemistry, University of Stockholm, S-106 91, Stockholm, Sweden Received 26 December 1990 SILLARD, R., t2. LANGEL AND H. JORNVALL. Isolation and characterizationof galaninfrom sheep brain. PEPTIDES 12(4) 855-859, 1991.--Galanin is a well-known,naturally occurringpeptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinallyderived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarityto pig galanin, differing by one amino acid substitution,that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26. Neuropeptide

Galanin

Sheep galanin

Peptideamide

Sheep brain

METHOD

GALANIN, a neuropeptide with 29 amino acid residues and an amidated C-terminus, was first isolated from porcine intestine in 1983 (19). Galanin-like immunoreactivity is widely distributed in the central and peripheral nervous systems of numerous species (2, 3, 7, 14, 15). Autoradiographic and ligand binding studies have shown galanin receptors to exist in different areas of the brain (9,18). The amino acid sequence of galanin is known for three species: pig (19), cow (17) and rat (11). The sequences of cow and rat galanin are deduced from cDNA sequences. The sequence of cow galanin was conf'Lrmed by analysis at the peptide level after isolation from gut. Species variations have been found in the C-terminal half of galanin, at positions 16, 18, 23, 26 and 29 (11, 17, 19). As the structure of galanin bears little similarity to those of other known peptides, galanin is considered as belonging to a separate peptide group. Until this report, descriptions of purification of galanin have regarded only that from intestinal tissue. Isolation of galanin from brain extracts, thereby confirming the existence of this peptide in the central nervous system, and comparison of galanin sequences from different species were expected to render further valuable information about the structure-activity relationships and aid in the design of analogs of pharmacological interest. Knowledge of the amino acid sequence of the species variants is also important in immunological studies where it can prove crucial in antibody recognition. Here we report the purification and amino acid sequence of galanin from sheep brain, using a receptor assay to follow the purification. The amino acid sequence of the isolated sheep galanin is not identical to that of any of the previously known galanins, but is similar to pig galanin.

Materials Sheep brains were obtained from the slaughterhouse at V6ru (Estonia). Sephadex G-25 (fine) was from Pharmacia (Uppsala, Sweden), CM-52 cellulose from Whatman (England), 4-dimethylaminoazobenzene-4'-sulphonyl(DABSYL) chloride and phenylisothiocyanate (PITC) from Pierce (Rockford, IL), carboxypeptidase Y (CPY) from Boehringer-Mannheim (FRG), TPCKtreated trypsin from Worthington (Freehold, NJ), Na125I (2500 Ci/mmol) from NEN (Dreieich, FRG) and porcine galanin from Bachem (Bubendorf, Switzerland). Amino acids were from Sigma (St. Louis, MO) and amino acid amides from Cyclo Chemical (Los Angeles, CA).

Starting Material Sheep brains (570 kg) were prepared and extracted as described for pig brains (16). The concentrate of peptides (500 g, wet weight) was fractionated using isopropanol, as reported for a concentrate of pig intestinal peptides (6), until the removal of fraction F1 by filtration. The filtrate was diluted with 3 volumes of water, after which the peptides were adsorbed onto alginic acid (470 g, wet weight) and eluted with ice-cold 0.2 M HC1. The pH was adjusted to 3.5-+0.1 with solid sodium acetate, and the solution saturated with NaC1. The precipitated fraction, denoted as F2 + F3, was collected by suction filtration (26 g, wet weight) and was the starting material for galanin purification.

High Performance Liquid Chromatography (HPLC) HPLC was carried out with a Waters preparative system on a

1Requests for reprints should be addressed to Rannar Sillard, Departmentof BiochemistryII, KarolinskaInstitutet, S-104 01, Stockholm, Sweden. 855

856

SILLARD, LANGEL AND JORNVALL

100

!,o

0.75

m

5o~. I

0.5 0

aa

-log [PEPTIDE] 0.25

FIG. 1. Displacement of porcine ]25I-galaninfrom rat hypothalamus galanin receptor by porcine galanin (O) and sheep brain crude peptide fraction 5 from gel filtration on Sephadex G-25 (fine) (0). Experimental conditionswere as described under the Method section. Vydac C18 column (22.5 × 250 mm) and with a Waters analytical instrument on an ion exchange Ultropac TSK 535 CM (7.5x 150 ram) and a Vydac C18 (4.6x250 mm). The elution system consisting of A = 0.1% trifluoroacetic acid in water and B = 0.1% trifluoroacetic acid in acetonitrile was used for reversephase HPLC, and that consisting of A = 0 . 2 M acetic acid and B = 1 M ammonium acetate in sovent A was used for ion exchange HPLC. The absorbance of the eluate was monitored at 214 nm and 280 nm for reverse-phase HPLC and at 280 nm for ion exchange HPLC.

Receptor Assay Porcine 125I-galanin (specific activity 1800-2000 Ci/mmol) was labeled by the chloramine-T method (8). Hypothalamic membranes were obtained from male Sprague-Dawley adult rats (8). Receptor assays were performed using a filtration assay (13). Displacement experiments were carded out in a final volume of 400 Ixl of 5 mM Hepes-buffered Krebs-Ringer solution (pH 7.4) containing 0.05% (w/v) bovine serum albumin in the presence of 70-100 p~g of membrane protein, 0.1 nM porcine 125I-labeled galanin and increasing concentrations of the peptide fractions to be studied. Sample incubation, for 30 rain at 37°C, was terminated by the addition of 10 ml ice-cold Hepes-buffered Krebs-Ringer solution followed by filtration through Whatman GF/C filters. The filters were washed with I0 ml of the same solution. In a typical screening experiment, three dilutions (1:1, 1:100, 1:1000 or 1:1, 1:100, 1:10,000) of the same peptide material were used. The specific binding (usually 60-90% of the total binding) was plotted as a function of peptide concentration. The molecular mass (Mr = 3200), similar to that of galanin, was used for peptide concentration calculations. Apparent ICso values were determined from the binding data and the fractions with the highest receptor binding affinities were selected for further purification. In the last two purification steps an aliquot of each HPLC fraction was taken and lyophilized. The lyophilized sampies were used in displacement experiments as described above, with the exception that the specific binding was plotted as a function of peptide dilution factor and the relative receptor binding af-finities were determined for each HPLC fraction.

o

1~ ELUTION

VOLUME, ml

FIG. 2. Ion exchange HPLC of the galanin-containingfraction from preparative reverse-phase HPLC. Load: 2.5 rag. Column: LKB Ultropac TSK 535 CM (7.5 x 150 ram). Solvent system: A, 0.2 M acetic acid; B, 1 M ammonium acetate in solvent A. Flow rate: 1 mt/min. Gradient: 15-55% B in 40 rain. The fraction indicated by the bar was used for further purification. peptide solution, 2 p.1 of 2% NH4HCO3 and 1 p,1 of trypsin solution (0.2 mg/ml) were added. The digestion was carried out at 37°C for 4 hours and the sample thereafter lyophilized. After lyophilization the sample was redissolved in 20 Ixl of water containing 0.1% trifluoroacetic acid and the tryptic fragments were separated by HPLC on a Vydac C18 column (4.6x 250 mm) using an acetonitrile gradient of 5-12% over 20 min and one of 12-58% over 38 min.

Amino Acid Analysis Peptide hydrolysis was carried out at 110°C for 20 h in evacuated tubes with 6 M HC1 containing 0.5% phenol. Amino acid compositions of the tryptic fragments of sheep galanin were determined using PITC precolunm derivatization and subsequent reverse-phase HPLC (4).

Digestion With CPY The lyophilized sample of sheep galanin (100 pmol) was dissolved in 9 p,1 of 0.1 M pyfidine/acetic acid buffer (pH 6.0) in a siliconized Eppendorf tube and 1.5 p,l of CPY solution (0.1 mg/ml in water) was added. The incubation was carried out at 37°C for 1 h and the sample was lyophilized. The amino acids and amino acid arnide released were derivatized with DABSYL chloride as described (5) and separated on a Spherisorb $ 5 0 D S 2 HPLC column (4.6x 150 mm) using an elution system as described (5), with a gradient of 25--65% acetonitrile over 40 min. The peaks were identified using DABSYL-dedvatized amino acids and amino acid amides as standards. RESULTS

Fragmentation With Trypsin

Purification

The lyophilized sample of sheep galanin (ca. 100 pmol) was dissolved in 2 ILl of water in a siliconized glass tube. To this

Peptide fraction F2+F3 from the sheep brain extract was subjected to chromatography on a Sephadex G-25 (fine) column

SHEEP GALANIN

857

TABLE 1

TABLE 2

SEQUENCE ANALYSISOF SHEEPGALANIN

AMINO ACID COMPOSITIONOF THE SHEEP GALANINTRYPTIC FRAGMENTS

Amino Acid

Yield (pmol) Fragments

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Glycine Tryptophan Threonine Leucine Asparagine Serine Alanine Glycine Tryosine Leucine Leucine Glycine Proline Histidine Alanine Isoleucine Aspartic acid Asparagine Histidine Arginine Serine Phenylalanine Histidine Aspartic acid Lysine Histidine Glycine Leucine Alanine*

Amino Acid

130 30 100 110 80 50 80 70 60 60 70 70 70 60 50 40 30 40 20 20 20 25 10 10 5 3 5 3

T1

T2

Ala

1.8 (2)

--

Arg

].0

--

(])

3.0 (3) 2.9 (3) 1.5 (2) 1.1 (1) 3.1 (3) --1.3 (1) 1.1 (1) 1,2 (1) ND* (1) 1.0 (1)

Asx

Gly His lie Leu Lys Phe Pro Ser Thr Trp Tyr

1.3 -0.9 --0.8 0.9

(1)

(1) (1)

1.1 (1) -ND --

-0.9 (1) 0.5 (1) -1.3 (1) --- -

--ND --

in 0.2 M acetic acid. The eluate was divided into 6 fractions and lyophilized. The crude fraction 5 fully displaced porcine 12~I-galanin from rat hypothalamus galanin receptor (Fig. 1), indicating the presence of peptides capable of interaction with the receptor. Fraction 5 (2.2 g) was suspended in 110 ml methanol containing 0.1% 2-mercaptoethanol and filtered. The methanolinsoluble fraction (939 mg) was subjected to ion exchange chromatography on a CM-cellulose (2.5 x 25 cm) column, which was gradually eluted with increasing concentrations (0.01, 0.02, 0.04, 0.08, 0.2 M) of NH4NCO3 (pH 8). The eluates were thereafter lyophilized. The fraction eluted with 0.02 M NH4HCO 3 (168 mg) was further purified by preparative reverse-phase HPLC us-

::i

,An

A*n ~Jn

~,n

E GI!

*An

::1 * *n

::1 iAr ::l

1 GO6

I

--

(1)

- -

::1 * *n

I 20

1.3 ( - )

Values are molar ratios from acid hydrolysis. Values from sequence analysis are within parentheses. *The content of tryptophan in the fragments was not determined (ND).

Values represent the yields of PTH-amino acids in Edman degradation. *Alanine was detected in trace amounts.

I

T3

I 40 TIME,

I

eO

0 8O

mln

FIG. 3. Final purification of sheep galanin by reverse-phase HPLC. Column: Vydac C18 (4.6 x 250 mm). Elution system: A, 0.1% tdfluoroa~etic acid in water; B, 0.1% trifluoroacetic acid in acetonitrile. Flow rate: 1 ml/min. Gradient: 23-27% B in 80 min. %R: the relative binding affinities of the fractions for rat hypothalamus galanin receptor are shown by the patterned columns. The fraction with the highest affinity (R= 100%) was subjected to the structural analysis.

858

SILLARD, LANGEL AND JORNVALL

TABLE 3 COMPARISONOF THE GALANINSTRUCTURES Species Sheep Pig Rat Cow

Sequence GWTLNSAGYLLGPHAIDNHRSFHDKHGLA-NH GWTLNSAGYLLGPHAIDNHRSFHDKYGLA-NH GWTLNSAGYLLGPHAIDNHRSFS DKHGLT-NH GWTLNSAGYLLGPHALDSHRSFQDKHGLA-NH

Reference 2 2

2 2

This work 19 11 17

Letters in italic indicate the variations found.

ing a 15-55% acetonitrile gradient over 40 minutes. The most active fraction (8.76 mg) was subjected to ion exchange HPLC (Fig. 2). The fraction indicated by the bar in Fig. 2 was further purified by analytical reverse-phase HPLC using a shallow acetonltrile gradient, 0.05% per minute (Fig. 3). The material from the peak with the highest receptor binding affinity was subjected to structural analysis. This highly purified fraction contained approximately 0.7 nmol of sheep galanin. Structural Studies

The galanin (300 pmol) was subjected to amino acid sequence analysis on an Applied Biosystems 477 A liquid phase sequencer (Table 1). Residue 29 could not be reliably detected because the C-terminal amino acid in galanin is amidated. In order to confmn the data and to determine the carhoxyl-terminal residue, tryptic fragments were prepared. The fragments T1 (residues 1-20), T2 (residues 21-25) and T3 (residues 26-29) were separated on reverse-phase HPLC and they eluted at 37.0%, 9.5% and 8.5% acetonitrile, respectively. No absorbance at 280 nm was detected on elution of the fragments T2 and T3, suggesting the absence of tyrosine or tryptophan residues. The amino acid compositions of the tryptic fragments, which also indicated the presence of alanine in the fragment T3, were in accordance with the sequence data (Table 2). A batch of sheep galanin was digested with CPY in order to ascertain whether the amino acid alanine in sheep galanin is amidated. The amino acid amide liberated with CPY was identified as alanine amide, which is in agreement with the amino acid composition of the C-terminal tryptic fragment. DISCUSSION We have purified galanin from sheep brain tissue and determined the amino acid sequence, thus demonstrating the exis-

tence of galanin in brain by direct isolation. The purification scheme employed in this study showed no distinct peaks of binding activity other than for galanin. The receptor assay data suggest that sheep galanin interacts with the galanin receptor from rat hypothalamus. Thus the species variants of galanin now known can displace each other in receptor binding assays. This is in accordance with the observations that porcine and rat galanin have the same binding affinity for the receptor in rat hypothalamus (9). The exact values of the parameters for receptor binding of sheep galanin remain to be established after the peptide is synthesized. Comparing the sheep galanin sequence to those from other species (Table 3) shows that sheep galanin has the greatest similaxity to pig galanin, differing only by having a histidine residue instead of tyrosine at position 26. Comparison also shows that the N-terminal end of galanin (residues 1 to 15) is conserved, in contrast to the C-terminal end of the peptide. Significantly, it is this conserved N-terminal end that is responsible for the receptor interaction. The galanin(1-16) fragment has full agonistic properties in inhibiting muscarinlc agonist-mediated stimulation of phosphatidylinositol turnover in slices of the rat ventral hippocampus (8) and the fragment (1-15) can alter forskolin-stimulated cAMP production and inhibit insulin release from RIN m 5F cells (1). Specific binding of ~25I-galanin is fully displaced by galanin(1-16) (8). In contrast, the C-terminal end of galanin, the fragment (10-29), has neither physiological effects (1,10) nor galanin receptor binding properties (12). ACKNOWLEDGEMENTS The authors wish to express their sincere gratitude to Professor Viktor Mutt for his generous help and valuable advice throughout this study and to Carina Palmherg and Anne Peters for helpful assistance. This study was supported by grants from the Swedish Medical Research Council (projects 1010 and 3532), Skandigen AB, KabiGen AB, Wenner-Gren Center Foundation (support to R.S.) and the Swedish Cancer Society (project 1806).

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SHEEP GALANIN

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