Peptides,Vol. 12, pp. 289-295. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0196-9781/91 $3.00 + .00
Structural Characterization of Calcitonin Gene-Related Peptide Purified From Rabbit Intestine V I K T O R E. E Y S S E L E I N , *l J O S E P H R. R E E V E , J R . , ? C A T I A S T E R N I N I , $ F A B I O C O M I N E L L I , * W I L L I A M M . D A V I S , * M I C H A E L T. D A V I S , § T E R R Y D. L E E , § F A N - J E N H O , ? D U S T I N R I D O U T * A N D J O H N E. S H I V E L Y §
*Inflammatory Bowel Disease Center, Harbor-UCLA Medical Center Division of Gastroenterology, Torrance, CA 90509 i'Center for Ulcer Research and Education, VA Wadsworth Medical Center, Los Angeles, CA 90073 and Department of Medicine UCLA, Los Angeles, CA 90024 ¢Center for Ulcer Research and Education, VA Wadsworth Medical Center, Los Angeles, CA 90073 and Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA 90024 §Division of Immunology, City of Hope Research Institute, Duarte, CA 91010 R e c e i v e d 20 A u g u s t 1990
EYSSELEIN, V. E., J. R. REEVE, JR., C. STERNINI, F. COMINELLI, W. M. DAVIS, M. T. DAVIS, T. D. LEE, F.-J. HO, D. RIDOUT AND J. E. SHIVELY. Structural characterization of calcitonin gene-relatedpeptide purifiedfrom rabbit intestine. PEPTIDES 12(2) 289-295, 1991.--Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAFamide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP. Calcitonin gene-related peptide (CGRP) High performance liquid chromatography
Gastrointestinal hormones Neuropeptides Microsequence analysis Fast protein liquid chromatography Peptide purification Mass spectral analysis
CALCITONIN gene-related peptide (CGRP), a 37 amino acid peptide, has been predicted from the sequence of mRNA generated from alternative processing of the primary transcript of the rat calcitonin gene present in a medullary thyroid carcinoma (2,14). The calcitonin gene is alternatively expressed in a tissuespecific fashion producing either the calcium regulatory hormone calcitonin in the thyroid or the neuropeptide alpha-CGRP in nervous tissues. Alternative splicing of the primary transcript of the calcitonin gene has also been described in human medullary thyroid carcinoma. Nucleotide sequence analysis revealed a 90% homology to the region in the corresponding rat gene encoding for alpha-CGRP (or CGRP-I) (16). Besides alpha-CGRP, another
form, beta-CGRP (or CGRP-II), is expressed in man (15) and rat (1) which is encoded by a different gene. In the rat, beta-CGRP differs from alpha-CGRP by one single amino acid in position 35 (Lys instead of Glu). In man, Lys is found in position 35 in both CGRP forms. The amino acid sequences of human CGRP-I and II differ in the mid-region of the peptide, in positions 3, 22 and 25. The purification and chemical characterization of CGRP-I from human medullary thyroid carcinoma tissue provided the proof that CGRP is actually expressed (11). The prohormone of both CGRP forms contains, in man and rat at the amino terminus and the carboxyl terminus, pairs of basic amino acids (1, 2, 1416) which are known processing sites for trypsin-like enzymes.
~Requests for reprints should be addressed to Viktor E. Eysselein, M.D., Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90509.
289
290
EYSSELEIN ET AL.
At the carboxyl terminus, the pair of basic amino acids is preceded by Gly. The pair of basic amino acids is removed by a carboxy peptidase B-like enzyme followed by cleavage of glycine to form carboxyl terminal amide and glyoxalate (4). Amidated CGRP is the final processing product (10,11). The two forms, CGRP-I and II, were also chemically characterized from human spinal cord (13). Only one form was isolated from porcine spinal cord which was more similar to human CGRP-II than CGRP-I (10). Porcine CGRP contained Glu in position 35 which is identical in rat alpha-CGRP, but differs from rat beta-CGRP which contains Lys. In the rat, Mulderry et al. (12) showed that an immunoreactive form eluting on ion exchange chromatography in the region of alpha-CGRP is the predominant form in extracts of the dorsal root ganglia. In the intestine, however, about 70% of the total immunoreactivity found eluted in the region of beta-CGRP. Northern blot and in situ hybridization with alpha- and beta-CGRP cRNA probes showed that only beta-CGRP mRNA was expressed in the intestine, where it was localized to enteric intrinsic neurons (12,17). These data support the concept that, at least in the rat, a peptide with immunological and chromatographical similarity to beta-CGRP is expressed in the intestine. CGRP has not been chemically characterized in the intestine, nor has the encoding gene been cloned from enteric neurons. The aim of the present study was to purify CGRP from the intestine and characterize it chemically by microsequence and mass spectral analysis. Rabbit intestines were used since we concomitantly study in this species the role of CGRP in experimentally induced colitis (7). ABBREVIATIONS
CGRP: calcitonin gene-related peptide; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; FPLC: fast protein liquid chromatography; RIA: radioimmunoassay. METHOD
Radioimmunoassay of CGRP The radioimmunoassay was performed as previously described (18,19) with slight modifications. CGRP antiserum used in this study was raised in rabbits against synthetic [Tyr] rat CGRP(2337) (Bachem, Torrance, CA) coupled to keyhole limpet hemocyanin via glutaraldehyde as previously characterized (18,19). The antibody was used in the radioimmunoassay at a final dilution of 1:500,000. [Tyr] rat CGRP(23-37) (Bachem, Torrance, CA) was labeled with 125I by chloramine T and purified by HPLC as previously described (6). Human CGRP-I (Peninsula, Belmont, CA) was used as standard. The RIA was performed at pH 7.5 in 0.05 M phosphate buffer containing 0.17% (w/v) bovine serum albumin. After an incubation period of 16-25 hours at 4°C, free CGRP was separated from antibody-bound CGRP by dextran-coated charcoal. With this assay condition, the detection limit was 10 pM and the IDso 50 pM.
Extraction of CGRP From Rabbit Small Intestine and Dorsal Root Ganglia Analytical HPLC and ion-exchange FPLC of rabbit CGRP. To study the forms of immunoreactive CGRP in fresh (not stored) intestine, one rabbit small intestine was removed immediately after the animal was sacrificed by an overdose of pentobarbital, rinsed with cold water and placed on dry ice. Two grams of the frozen intestine were then extracted by 5 ml boiling acetic acid for 5 min, homogenized, placed on ice and extracted by additional 5 ml of cold 4% TFA. Dorsal root ganglia (L1-S3) were
removed surgically, pooled and extracted in 5 volumes boiling acetic acid and cold TFA as described for extraction of the intestine. The extract was centrifuged at 2000 x g for 10 rain, and the supernatant was applied to a C-18 Sep Pak cartridge (Waters) previously eluted sequentially with 10 ml of acetonitrile and 10 ml aqueous 0.1% trifluoroacetic acid (TFA, buffer A); immunoreactive CGRP was eluted from the cartridges by 2 ml 50% acetonitrile containing 0.1% aqueous TFA. The Sep Pak eluate was either used for 1) analytical HPLC or 2) ion-exchange FPLC. Analytical reverse phase HPLC. The Sep Pak eluate was diluted with 4 volumes buffer A and loaded at 1 ml/min onto an analytical reverse phase HPLC C-18 column (Waters, i.LBondapak, 10 micron, 3.9 mm x 30 cm), which was eluted at 1 ml/min by increasing concentrations of acetonitrile (0.25%/min). Fractions of 1 ml were collected, and aliquots of 5, 10, and 50 ILl were used to measure immunoreactive CGRP. The column was equilibrated on separate runs with human CGRP-I and -II and rat alpha- and beta-CGRP. Analytical ion-exchange FPLC. The Sep Pak eluate was diluted 1:10 with 0.025 M acetate buffer, pH 4.8, containing 10% acetonitrile and 0.1% Tween 80 (buffer A) and loaded at 1 ml/min onto a cation exchange fast protein liquid chromatography (FPLC) column (LKB, TSK SP-5PW, 8 x 75 mm), which was equilibrated with buffer A. Immunoreactive CGRP was eluted at 1 ml/min by increasing concentrations of sodium chloride using a 50-minute gradient to 0.5 M sodium chloride in buffer A. Aliquots of 50 Ixl were used for radioimmunoassay. The column was calibrated in separate runs with human CGRP-I and -II. Tissue procurement. Rabbits were sacrificed for use of colonic muscle strip studies by an overdose of pentobarbital. After removal of the colon, the small intestines were immediately excised, rinsed with cold water, frozen on dry ice, and stored at -70°C until extraction. Purification of CGRP. The frozen intestines (1 kg) were placed into 2.5 liter boiling 3% acetic acid, blended in a Polytron homogenizer for 5 min and boiled for 10 min. The extract was cooled, and 2.5 liter 4% aqueous TFA were added. The extract was centrifuged twice at 2000 x g for one hour. The supernatant was loaded at a flow rate of 40 ml/min onto a preparative reverse phase C-18 column (Dynamax, ixBondapak, 5 micron, 41.4 mm x 25 cm), equilibrated with 0.1% TFA. The column was rinsed with 0.1% aqueous TFA at a flow rate of 10 ml/min and eluted with a linear gradient to 50% acetonitrile in 100 rain. Fractions of 10 ml were collected. Fractions containing CGRP immunoreactivity were diluted with 0.1% aqueous TFA (1:3; v/v) and applied to a second preparative C-8 column (Dynamax, ~xBondapak, 5 micron, 21.4 mm x 25 cm), equilibrated with 0.1% TFA and eluted with the same gradient. In the next step, immunoreactive CGRP was diluted in three volumes of 0.1% TFA and purified with a semipreparative reverse phase C-18 HPLC column (Waters, txBondapak, I0 micron, 7.8 x 300 mm), which was eluted at 2 ml/min by increasing concentrations of acetonitrile: 5 minutes with 0.1% aqueous TFA, a 5-minute gradient to 20% acetonitrile, then a 70-minute gradient to 37.5% acetonitrile. The fractions (4 ml) containing immunoreactive CGRP were pooled and applied to a cation exchange fast protein liquid chromatography (FPLC) column (LKB, TSK SP-5PW, 8 x 75 mm), which was equilibrated with 0.025 M acetate buffer, pH 4.8, containing 10% acetonitrile and 0.1% Tween 80 (buffer A). Immunoreactive CGRP was eluted at 1 ml/min by increasing concentrations of sodium chloride using a 100-minute gradient to 1 M sodium chloride in buffer A. Beta-mercaptoethanol (0.1 ml) and TFA (0.05 ml, 10% v/v) were added to the collected fractions (2 ml). The semipreparative reverse phase C-18 HPLC and the FPLC step were repeated. Immunoreactive CGRP was further purified by a series of reverse phase gradient HPLC steps, including a C-18
RABBIT INTESTINAL CGRP
(Waters, txBondapak, 10 micron, 3.9 mm x 30 cm), a Phenyl (Waters, ixBondapak, 10 micron, 3.9 mm × 30 cm), and a C-4 (Vydac, 10 micron, 4.6 mm × 25 cm) column. The purification was monitored by radioimmunoassay and absorbance at 220 and 280 nm.
Chymotryptic Digestion and Reverse Phase HPLC Approximately 200 pmoles of intact peptide were digested with HPLC-purified chymotrypsin (Sigma, enzyme: substrate = 1:25) in 0.5 ml of 0.1 M NH4HCO 3, pH 8.0, for 18 h at 37°C. The digestion was stopped by addition of acid then fractionated on a Brownlee Labs Aquapore 300 C-18 column (1 mm × 25 cm, 7 micron) using a 35-minute linear gradient from 2 to 72% B (A, 0.1% TFA; B, 0.1% TFA/90% acetonitrile; v/v) at a flow rate of 0.08 ml/min. The purified digestion products were manually collected into 0.5 ml polypropylene microcentrifuge tubes.
Amino Acid Sequence Analysis Samples of purified peptides were subjected to automated Edman degradation on a City of Hope built gas phase sequencer (9) with identification of the PTH derivatives performed as described (8).
Mass Spectral Analysis Samples collected from reverse phase HPLC were concentrated to dryness using a Savant vacuum centrifuge and redissolved in a few microliters of DMSO. Approximately 0.002 ml of the sample solution was added to 0.001 ml of the sample matrix on a 1.5 x 6 mm stainless steel sample stage. A mixture of dithiothreitol:dithioerythritol (5:1) (21) and camphor sulfonic acid (6 mM) (5) was used as sample matrix. Positive ion spectra were obtained using a JEOL HX-100HF high resolution, double focusing, magnetic sector mass spectrometer operating at 5 kV accelerating potential and a nominal resolution of 5000 for the intact peptide while the chymotryptic fragments were analyzed at a nominal resolution of either 3000 (CT2 and CT3) or 500 (CTI). The mass value reported for CT1 corresponds to the average mass of the protonated molecule. Values for the others correspond to the monoisotopic mass of the protonated molecule. Sample ionization was accomplished using a 6 keV Xe atom beam. A JEOL DA5000 data system was used to control instrument parameters and collect spectral data. RESULTS
Analytical HPLC and FPLC of Rabbit CGRP in Extracts of the Intestine and the Dorsal Root Ganglia One major immunoreactive peak was found in the HPLC eluate of the intestinal extract which comprised 86% of the total immunoreactivity found (Fig. la). Two additional, minor and earlier eluting immunoreactive peaks were detected which comprised 2 and 12% of the total immunoreactivity found. Standard human CGRP-I eluted 2 min earlier than rabbit CGRP. Both human CGRP forms, CGRP-I and CGRP-II, were not separated on this column using these elution conditions. In extracts of the dorsal root ganglia, also only one major immunoreactive CGRP peak was detected which eluted in the same region as the intestinal form (data not shown). On analytical ion-exchange FPLC of intestinal extract, only one immunoreactive CGRP was detected at 46 min in the eluate, which eluted near human CGRP-II (at 44 min) but far after human CGRP-I (at 35 min) (Fig. lb). FPLC of extracts of the dorsal root ganglia revealed one immunoreactive CGRP peak which eluted in the region of standard human CGRP-I
291
(Fig. lc). No immunoreactivity in the region of the intestinal form was detected.
Purification of CGRP Recovery of immunoreactive CGRP is shown for each purification step in Table 1. In the third purification step, the semipreparative C-18 HPLC, two immunoreactive peaks were detected (Fig. 2). The predominant, later eluting peak comprised 78% of the total immunoreactivity found. The major loss occurred at the 6th step which was an ion-exchange step of a fairly pure peptide. The higher pH (4.8) compared to the pH during reverse phase HPLC separations (pH 2.1) may allow for more absorption of the peptide on column and plastic tube surfaces. Figure 3 shows the final C-4 HPLC purification (step 9, Table 1) of the immunoreactive CGRP peak. One single absorbance peak was detected, which was associated with CGRP-like immunoreactivity. This material was used for subsequent microsequence and mass spectral analysis yielding intact CGRP (see below). The minor, earlier eluting peak labeled rCGRP ox? in Fig. 2 (step 3, Table 1) was also purified (data not shown). This form may represent oxidized CGRP.
Chemical Characterization of CGRP Mass spectral analysis (Table 3) of the single absorbance peak in Fig. 3 gave a value of 3779.33 for the mass of the protonated molecular ion, which is consistent with the value calculated for rabbit CGRP(1-37) (Fig. 4). Microsequence analysis results for CGRP and its chymotryptic peptides are shown in Table 2. The first thirty-three residues could be unambiguously assigned from microsequence results for intact CGRP and its chymotryptic peptides (Table 2). No microsequence data were obtained for the last four residues. By homology to porcine CGRP and rat alpha-CGRP, the sequence was expected to be Ser-Glu-Ala-Phe-amide. This sequence is consistent with the mass value obtained for the intact CGRP. More importantly, this sequence is consistent with the mass value obtained for the carboxyl terminal peptide which was determined with an accuracy of _+0.3 mass units. Substitutions for Ser, Ala, or Phe would require an additional substitution at one of the other positions in order to maintain the same molecular weight. On the other hand, the mass of Glu is one unit higher than Lys or Gin. Either Lys or Gin could be substituted for Glu provided the carboxyl terminus is a free acid rather than an amide. Only amide form has been found for purified CGRP structures whose carboxyl termini have been unambiguously determined (10,11). For these reasons, the carboxyl terminus of CGRP has been assigned as Ser-Glu-Ala-Phe-amide but confirmation will require further analysis. The earlier eluting form (Fig. 2) was also purified and chemically characterized in part. Amino terminal sequence analysis was carried out through 21 cycles. The sequence was GA~ITAT_VTHRLAGLLSR_GG (data not shown). Cysteine in position 2 and 7, and Ser in position 17 were not detectable. The low abundance of this peptide did not allow accurate mass spectral analysis. However, a weak signal was observed 16-18 mass units above the molecular weight of CGRP, suggesting that this peptide corresponded to rabbit CGRP whose methionine had been oxidized during the extraction or purification steps. DISCUSSION
This is the first report describing the purification and chemical characterization of CGRP from the intestine. The most abundant form of CGRP in rabbit intestine was purified by several
292
E Y S S E L E I N ET AL.
a
100 -
TABLE 1
50
hCGRP-I
RECOVERY OF IMMUNOREACTIVE CGRP DURING PURIFICATION FROM RABBIT INTESTINE (l kg)
I i,i ,d m tr
..°o°°o*°°~' =,J
F-
°°°.°°.°'°'°°"
.d
50
o
25
.oo° °°o°°°
20
40 TIME, min
I-nan
b
-
60
8
hCGRP-II
200"
-60 hCGRP-I
~
/ ¢
/
¢
40 B
0
Extract 1) Preparative C-18 HPLC 2) Preparative C-8 HPLC 3) Semipreparative C-18 HPLC 4) FPLC 5) Semipreparative C-18 HPLC 6) FPLC 7) C-18 HPLC 8) Phenyl HPLC 9) C-4 HPLC
13 10.8
83
9.8
91
10.3 10.8
105 104
7.6 7.2 2.4 2.2 2.5
70 95 33 92 113
¢
E
¢
Q.
/
0,-="
/
/
¢
• 20
/
-
~ o
0 20 TIME,
40 min
0
hCGRP-II hCGRP-I ~/
300"
60 i
,,""
//
=E 200"
.I'"
--I
40
,.J
O
Ix
Step Recovery (%)
v
/
100
Immunoreactive CGRP (nmoles)
O
O.
i
Purification Step
100"
0
m
,"" --" -
0
, 20 TIME,
I
20
o~
steps of reverse phase HPLC and ion-exchange chromatography. Chemical characterization by microsequence and mass spectral analysis o f the intact peptide and its c h y m o t r y p t i c f r a g m e n t s revealed that rabbit intestinal C G R P is identical to h u m a n C G R P - I I (or beta) in 35 o f 37 a m i n o acid residues (Fig. 4). T w o a m i n o acid differences were detected, one in position 1, with Gly in rabbit C G R P instead of Ala in h u m a n C G R P , and the other in position 35, with Glu instead o f L y s , respectively. T h e last four a m i n o acids, w h i c h were not d e t e r m i n e d by seq u e n c e analysis, were a s s i g n e d based on m a s s spectral analysis of C G R P ( 1 - 3 7 ) and its c h y m o t r y p t i c f r a g m e n t CT3 and f r o m hom o l o g y with rat alpha and porcine C G R P . If the carboxyl terminal a m i n o acid w o u l d not be amidated but contain free acid, this a s s i g n m e n t w o u l d be not correct since amidated phenylalanine differs f r o m the free acid by 0.98 m a s s units. H o w e v e r , this is an unlikely possibility since both calcitonin g e n e s encode in h u m a n s
0 40 min
60
- 30
rbCGRP
1200
uJ -J
r-
FIG. 1. (a) Elution profile of CGRP-like immunoreactivity (CGRP-LI) from analytical reverse phase C-I 8 HPLC of rabbit intestinal extract. The intestine was not stored but immediately processed as described in the Method section. The column was eluted by increasing concentrations of acetonitrile as indicated. The elution positions of standard human (hCGRP) and rat CGRP-I and CGRP-II were determined on separate runs. Standard human CGRP-I was not separated from human CGRP-II using this column and these elution conditions. Also, HPLC of extracts of rabbit dorsal root ganglia revealed only one immunoreactive CGRP peak which eluted in the same region as the intestinal form (data not shown). Standard rat CGRP-I and CGRP-II eluted 0.5 and 2 min in front of human CGRP-II, respectively (data not shown). (b) Analytical ion-exchange FPLC profile of CGRP-LI in rabbit intestinal extracts. The column was eluted by increasing concentrations of NaC1 (B, buffer B: 1 M NaC1 in 0.025 M acetate buffer, see the Method section). The elution positions of standard human CGRP-I and -II are shown. (c) Analytical ion-exchange FPLC profile of CGRP-LI in extracts of rabbit dorsal root ganglia. The column was eluted by increasing concentrations of NaCI as in (b). The elution positions of standard human CGRP-I and -II are shown.
20
iI
800
~:
I-
!
,--I
d.
0
iI i
n.-
i rbCGRP
400
I
i
10
ox?
III l
0 0
FLII
.
10 TIME,
20
-, ......... 30
0
40
min
FIG. 2. Elution profile of CGRP-like immunoreactivity (CGRP-LI) from reverse phase semipreparative HPLC (step 3, Table 1). The further purification of the major peak (marked rbCGRP = rabbit CGRP), which comprised 78% of the immunoreactive material found in the column eluate, is shown in Table 1. The earlier eluting immunoreactive peak, marked rbCGRP ox?, was also further purified and presumably represents oxidized rabbit CGRP.
RABBIT INTESTINAL CGRP
100
A
293
-
-40
TABLE 2
I
I I I
v
30
> :3. o3 o
SEQUENCE ANALYSIS OF RABBIT INTESTINAL CGRP AND ITS CHYMOTRYPTIC FRAGMENTCT-3*
LIJ Amino Acid
..I
50
20
Iv-
1 0
~0
~ o
0196-9781/91 $3.00 + .00
Structural Characterization of Calcitonin Gene-Related Peptide Purified From Rabbit Intestine V I K T O R E. E Y S S E L E I N , *l J O S E P H R. R E E V E , J R . , ? C A T I A S T E R N I N I , $ F A B I O C O M I N E L L I , * W I L L I A M M . D A V I S , * M I C H A E L T. D A V I S , § T E R R Y D. L E E , § F A N - J E N H O , ? D U S T I N R I D O U T * A N D J O H N E. S H I V E L Y §
*Inflammatory Bowel Disease Center, Harbor-UCLA Medical Center Division of Gastroenterology, Torrance, CA 90509 i'Center for Ulcer Research and Education, VA Wadsworth Medical Center, Los Angeles, CA 90073 and Department of Medicine UCLA, Los Angeles, CA 90024 ¢Center for Ulcer Research and Education, VA Wadsworth Medical Center, Los Angeles, CA 90073 and Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA 90024 §Division of Immunology, City of Hope Research Institute, Duarte, CA 91010 R e c e i v e d 20 A u g u s t 1990
EYSSELEIN, V. E., J. R. REEVE, JR., C. STERNINI, F. COMINELLI, W. M. DAVIS, M. T. DAVIS, T. D. LEE, F.-J. HO, D. RIDOUT AND J. E. SHIVELY. Structural characterization of calcitonin gene-relatedpeptide purifiedfrom rabbit intestine. PEPTIDES 12(2) 289-295, 1991.--Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAFamide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP. Calcitonin gene-related peptide (CGRP) High performance liquid chromatography
Gastrointestinal hormones Neuropeptides Microsequence analysis Fast protein liquid chromatography Peptide purification Mass spectral analysis
CALCITONIN gene-related peptide (CGRP), a 37 amino acid peptide, has been predicted from the sequence of mRNA generated from alternative processing of the primary transcript of the rat calcitonin gene present in a medullary thyroid carcinoma (2,14). The calcitonin gene is alternatively expressed in a tissuespecific fashion producing either the calcium regulatory hormone calcitonin in the thyroid or the neuropeptide alpha-CGRP in nervous tissues. Alternative splicing of the primary transcript of the calcitonin gene has also been described in human medullary thyroid carcinoma. Nucleotide sequence analysis revealed a 90% homology to the region in the corresponding rat gene encoding for alpha-CGRP (or CGRP-I) (16). Besides alpha-CGRP, another
form, beta-CGRP (or CGRP-II), is expressed in man (15) and rat (1) which is encoded by a different gene. In the rat, beta-CGRP differs from alpha-CGRP by one single amino acid in position 35 (Lys instead of Glu). In man, Lys is found in position 35 in both CGRP forms. The amino acid sequences of human CGRP-I and II differ in the mid-region of the peptide, in positions 3, 22 and 25. The purification and chemical characterization of CGRP-I from human medullary thyroid carcinoma tissue provided the proof that CGRP is actually expressed (11). The prohormone of both CGRP forms contains, in man and rat at the amino terminus and the carboxyl terminus, pairs of basic amino acids (1, 2, 1416) which are known processing sites for trypsin-like enzymes.
~Requests for reprints should be addressed to Viktor E. Eysselein, M.D., Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90509.
289
290
EYSSELEIN ET AL.
At the carboxyl terminus, the pair of basic amino acids is preceded by Gly. The pair of basic amino acids is removed by a carboxy peptidase B-like enzyme followed by cleavage of glycine to form carboxyl terminal amide and glyoxalate (4). Amidated CGRP is the final processing product (10,11). The two forms, CGRP-I and II, were also chemically characterized from human spinal cord (13). Only one form was isolated from porcine spinal cord which was more similar to human CGRP-II than CGRP-I (10). Porcine CGRP contained Glu in position 35 which is identical in rat alpha-CGRP, but differs from rat beta-CGRP which contains Lys. In the rat, Mulderry et al. (12) showed that an immunoreactive form eluting on ion exchange chromatography in the region of alpha-CGRP is the predominant form in extracts of the dorsal root ganglia. In the intestine, however, about 70% of the total immunoreactivity found eluted in the region of beta-CGRP. Northern blot and in situ hybridization with alpha- and beta-CGRP cRNA probes showed that only beta-CGRP mRNA was expressed in the intestine, where it was localized to enteric intrinsic neurons (12,17). These data support the concept that, at least in the rat, a peptide with immunological and chromatographical similarity to beta-CGRP is expressed in the intestine. CGRP has not been chemically characterized in the intestine, nor has the encoding gene been cloned from enteric neurons. The aim of the present study was to purify CGRP from the intestine and characterize it chemically by microsequence and mass spectral analysis. Rabbit intestines were used since we concomitantly study in this species the role of CGRP in experimentally induced colitis (7). ABBREVIATIONS
CGRP: calcitonin gene-related peptide; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; FPLC: fast protein liquid chromatography; RIA: radioimmunoassay. METHOD
Radioimmunoassay of CGRP The radioimmunoassay was performed as previously described (18,19) with slight modifications. CGRP antiserum used in this study was raised in rabbits against synthetic [Tyr] rat CGRP(2337) (Bachem, Torrance, CA) coupled to keyhole limpet hemocyanin via glutaraldehyde as previously characterized (18,19). The antibody was used in the radioimmunoassay at a final dilution of 1:500,000. [Tyr] rat CGRP(23-37) (Bachem, Torrance, CA) was labeled with 125I by chloramine T and purified by HPLC as previously described (6). Human CGRP-I (Peninsula, Belmont, CA) was used as standard. The RIA was performed at pH 7.5 in 0.05 M phosphate buffer containing 0.17% (w/v) bovine serum albumin. After an incubation period of 16-25 hours at 4°C, free CGRP was separated from antibody-bound CGRP by dextran-coated charcoal. With this assay condition, the detection limit was 10 pM and the IDso 50 pM.
Extraction of CGRP From Rabbit Small Intestine and Dorsal Root Ganglia Analytical HPLC and ion-exchange FPLC of rabbit CGRP. To study the forms of immunoreactive CGRP in fresh (not stored) intestine, one rabbit small intestine was removed immediately after the animal was sacrificed by an overdose of pentobarbital, rinsed with cold water and placed on dry ice. Two grams of the frozen intestine were then extracted by 5 ml boiling acetic acid for 5 min, homogenized, placed on ice and extracted by additional 5 ml of cold 4% TFA. Dorsal root ganglia (L1-S3) were
removed surgically, pooled and extracted in 5 volumes boiling acetic acid and cold TFA as described for extraction of the intestine. The extract was centrifuged at 2000 x g for 10 rain, and the supernatant was applied to a C-18 Sep Pak cartridge (Waters) previously eluted sequentially with 10 ml of acetonitrile and 10 ml aqueous 0.1% trifluoroacetic acid (TFA, buffer A); immunoreactive CGRP was eluted from the cartridges by 2 ml 50% acetonitrile containing 0.1% aqueous TFA. The Sep Pak eluate was either used for 1) analytical HPLC or 2) ion-exchange FPLC. Analytical reverse phase HPLC. The Sep Pak eluate was diluted with 4 volumes buffer A and loaded at 1 ml/min onto an analytical reverse phase HPLC C-18 column (Waters, i.LBondapak, 10 micron, 3.9 mm x 30 cm), which was eluted at 1 ml/min by increasing concentrations of acetonitrile (0.25%/min). Fractions of 1 ml were collected, and aliquots of 5, 10, and 50 ILl were used to measure immunoreactive CGRP. The column was equilibrated on separate runs with human CGRP-I and -II and rat alpha- and beta-CGRP. Analytical ion-exchange FPLC. The Sep Pak eluate was diluted 1:10 with 0.025 M acetate buffer, pH 4.8, containing 10% acetonitrile and 0.1% Tween 80 (buffer A) and loaded at 1 ml/min onto a cation exchange fast protein liquid chromatography (FPLC) column (LKB, TSK SP-5PW, 8 x 75 mm), which was equilibrated with buffer A. Immunoreactive CGRP was eluted at 1 ml/min by increasing concentrations of sodium chloride using a 50-minute gradient to 0.5 M sodium chloride in buffer A. Aliquots of 50 Ixl were used for radioimmunoassay. The column was calibrated in separate runs with human CGRP-I and -II. Tissue procurement. Rabbits were sacrificed for use of colonic muscle strip studies by an overdose of pentobarbital. After removal of the colon, the small intestines were immediately excised, rinsed with cold water, frozen on dry ice, and stored at -70°C until extraction. Purification of CGRP. The frozen intestines (1 kg) were placed into 2.5 liter boiling 3% acetic acid, blended in a Polytron homogenizer for 5 min and boiled for 10 min. The extract was cooled, and 2.5 liter 4% aqueous TFA were added. The extract was centrifuged twice at 2000 x g for one hour. The supernatant was loaded at a flow rate of 40 ml/min onto a preparative reverse phase C-18 column (Dynamax, ixBondapak, 5 micron, 41.4 mm x 25 cm), equilibrated with 0.1% TFA. The column was rinsed with 0.1% aqueous TFA at a flow rate of 10 ml/min and eluted with a linear gradient to 50% acetonitrile in 100 rain. Fractions of 10 ml were collected. Fractions containing CGRP immunoreactivity were diluted with 0.1% aqueous TFA (1:3; v/v) and applied to a second preparative C-8 column (Dynamax, ~xBondapak, 5 micron, 21.4 mm x 25 cm), equilibrated with 0.1% TFA and eluted with the same gradient. In the next step, immunoreactive CGRP was diluted in three volumes of 0.1% TFA and purified with a semipreparative reverse phase C-18 HPLC column (Waters, txBondapak, I0 micron, 7.8 x 300 mm), which was eluted at 2 ml/min by increasing concentrations of acetonitrile: 5 minutes with 0.1% aqueous TFA, a 5-minute gradient to 20% acetonitrile, then a 70-minute gradient to 37.5% acetonitrile. The fractions (4 ml) containing immunoreactive CGRP were pooled and applied to a cation exchange fast protein liquid chromatography (FPLC) column (LKB, TSK SP-5PW, 8 x 75 mm), which was equilibrated with 0.025 M acetate buffer, pH 4.8, containing 10% acetonitrile and 0.1% Tween 80 (buffer A). Immunoreactive CGRP was eluted at 1 ml/min by increasing concentrations of sodium chloride using a 100-minute gradient to 1 M sodium chloride in buffer A. Beta-mercaptoethanol (0.1 ml) and TFA (0.05 ml, 10% v/v) were added to the collected fractions (2 ml). The semipreparative reverse phase C-18 HPLC and the FPLC step were repeated. Immunoreactive CGRP was further purified by a series of reverse phase gradient HPLC steps, including a C-18
RABBIT INTESTINAL CGRP
(Waters, txBondapak, 10 micron, 3.9 mm x 30 cm), a Phenyl (Waters, ixBondapak, 10 micron, 3.9 mm × 30 cm), and a C-4 (Vydac, 10 micron, 4.6 mm × 25 cm) column. The purification was monitored by radioimmunoassay and absorbance at 220 and 280 nm.
Chymotryptic Digestion and Reverse Phase HPLC Approximately 200 pmoles of intact peptide were digested with HPLC-purified chymotrypsin (Sigma, enzyme: substrate = 1:25) in 0.5 ml of 0.1 M NH4HCO 3, pH 8.0, for 18 h at 37°C. The digestion was stopped by addition of acid then fractionated on a Brownlee Labs Aquapore 300 C-18 column (1 mm × 25 cm, 7 micron) using a 35-minute linear gradient from 2 to 72% B (A, 0.1% TFA; B, 0.1% TFA/90% acetonitrile; v/v) at a flow rate of 0.08 ml/min. The purified digestion products were manually collected into 0.5 ml polypropylene microcentrifuge tubes.
Amino Acid Sequence Analysis Samples of purified peptides were subjected to automated Edman degradation on a City of Hope built gas phase sequencer (9) with identification of the PTH derivatives performed as described (8).
Mass Spectral Analysis Samples collected from reverse phase HPLC were concentrated to dryness using a Savant vacuum centrifuge and redissolved in a few microliters of DMSO. Approximately 0.002 ml of the sample solution was added to 0.001 ml of the sample matrix on a 1.5 x 6 mm stainless steel sample stage. A mixture of dithiothreitol:dithioerythritol (5:1) (21) and camphor sulfonic acid (6 mM) (5) was used as sample matrix. Positive ion spectra were obtained using a JEOL HX-100HF high resolution, double focusing, magnetic sector mass spectrometer operating at 5 kV accelerating potential and a nominal resolution of 5000 for the intact peptide while the chymotryptic fragments were analyzed at a nominal resolution of either 3000 (CT2 and CT3) or 500 (CTI). The mass value reported for CT1 corresponds to the average mass of the protonated molecule. Values for the others correspond to the monoisotopic mass of the protonated molecule. Sample ionization was accomplished using a 6 keV Xe atom beam. A JEOL DA5000 data system was used to control instrument parameters and collect spectral data. RESULTS
Analytical HPLC and FPLC of Rabbit CGRP in Extracts of the Intestine and the Dorsal Root Ganglia One major immunoreactive peak was found in the HPLC eluate of the intestinal extract which comprised 86% of the total immunoreactivity found (Fig. la). Two additional, minor and earlier eluting immunoreactive peaks were detected which comprised 2 and 12% of the total immunoreactivity found. Standard human CGRP-I eluted 2 min earlier than rabbit CGRP. Both human CGRP forms, CGRP-I and CGRP-II, were not separated on this column using these elution conditions. In extracts of the dorsal root ganglia, also only one major immunoreactive CGRP peak was detected which eluted in the same region as the intestinal form (data not shown). On analytical ion-exchange FPLC of intestinal extract, only one immunoreactive CGRP was detected at 46 min in the eluate, which eluted near human CGRP-II (at 44 min) but far after human CGRP-I (at 35 min) (Fig. lb). FPLC of extracts of the dorsal root ganglia revealed one immunoreactive CGRP peak which eluted in the region of standard human CGRP-I
291
(Fig. lc). No immunoreactivity in the region of the intestinal form was detected.
Purification of CGRP Recovery of immunoreactive CGRP is shown for each purification step in Table 1. In the third purification step, the semipreparative C-18 HPLC, two immunoreactive peaks were detected (Fig. 2). The predominant, later eluting peak comprised 78% of the total immunoreactivity found. The major loss occurred at the 6th step which was an ion-exchange step of a fairly pure peptide. The higher pH (4.8) compared to the pH during reverse phase HPLC separations (pH 2.1) may allow for more absorption of the peptide on column and plastic tube surfaces. Figure 3 shows the final C-4 HPLC purification (step 9, Table 1) of the immunoreactive CGRP peak. One single absorbance peak was detected, which was associated with CGRP-like immunoreactivity. This material was used for subsequent microsequence and mass spectral analysis yielding intact CGRP (see below). The minor, earlier eluting peak labeled rCGRP ox? in Fig. 2 (step 3, Table 1) was also purified (data not shown). This form may represent oxidized CGRP.
Chemical Characterization of CGRP Mass spectral analysis (Table 3) of the single absorbance peak in Fig. 3 gave a value of 3779.33 for the mass of the protonated molecular ion, which is consistent with the value calculated for rabbit CGRP(1-37) (Fig. 4). Microsequence analysis results for CGRP and its chymotryptic peptides are shown in Table 2. The first thirty-three residues could be unambiguously assigned from microsequence results for intact CGRP and its chymotryptic peptides (Table 2). No microsequence data were obtained for the last four residues. By homology to porcine CGRP and rat alpha-CGRP, the sequence was expected to be Ser-Glu-Ala-Phe-amide. This sequence is consistent with the mass value obtained for the intact CGRP. More importantly, this sequence is consistent with the mass value obtained for the carboxyl terminal peptide which was determined with an accuracy of _+0.3 mass units. Substitutions for Ser, Ala, or Phe would require an additional substitution at one of the other positions in order to maintain the same molecular weight. On the other hand, the mass of Glu is one unit higher than Lys or Gin. Either Lys or Gin could be substituted for Glu provided the carboxyl terminus is a free acid rather than an amide. Only amide form has been found for purified CGRP structures whose carboxyl termini have been unambiguously determined (10,11). For these reasons, the carboxyl terminus of CGRP has been assigned as Ser-Glu-Ala-Phe-amide but confirmation will require further analysis. The earlier eluting form (Fig. 2) was also purified and chemically characterized in part. Amino terminal sequence analysis was carried out through 21 cycles. The sequence was GA~ITAT_VTHRLAGLLSR_GG (data not shown). Cysteine in position 2 and 7, and Ser in position 17 were not detectable. The low abundance of this peptide did not allow accurate mass spectral analysis. However, a weak signal was observed 16-18 mass units above the molecular weight of CGRP, suggesting that this peptide corresponded to rabbit CGRP whose methionine had been oxidized during the extraction or purification steps. DISCUSSION
This is the first report describing the purification and chemical characterization of CGRP from the intestine. The most abundant form of CGRP in rabbit intestine was purified by several
292
E Y S S E L E I N ET AL.
a
100 -
TABLE 1
50
hCGRP-I
RECOVERY OF IMMUNOREACTIVE CGRP DURING PURIFICATION FROM RABBIT INTESTINE (l kg)
I i,i ,d m tr
..°o°°o*°°~' =,J
F-
°°°.°°.°'°'°°"
.d
50
o
25
.oo° °°o°°°
20
40 TIME, min
I-nan
b
-
60
8
hCGRP-II
200"
-60 hCGRP-I
~
/ ¢
/
¢
40 B
0
Extract 1) Preparative C-18 HPLC 2) Preparative C-8 HPLC 3) Semipreparative C-18 HPLC 4) FPLC 5) Semipreparative C-18 HPLC 6) FPLC 7) C-18 HPLC 8) Phenyl HPLC 9) C-4 HPLC
13 10.8
83
9.8
91
10.3 10.8
105 104
7.6 7.2 2.4 2.2 2.5
70 95 33 92 113
¢
E
¢
Q.
/
0,-="
/
/
¢
• 20
/
-
~ o
0 20 TIME,
40 min
0
hCGRP-II hCGRP-I ~/
300"
60 i
,,""
//
=E 200"
.I'"
--I
40
,.J
O
Ix
Step Recovery (%)
v
/
100
Immunoreactive CGRP (nmoles)
O
O.
i
Purification Step
100"
0
m
,"" --" -
0
, 20 TIME,
I
20
o~
steps of reverse phase HPLC and ion-exchange chromatography. Chemical characterization by microsequence and mass spectral analysis o f the intact peptide and its c h y m o t r y p t i c f r a g m e n t s revealed that rabbit intestinal C G R P is identical to h u m a n C G R P - I I (or beta) in 35 o f 37 a m i n o acid residues (Fig. 4). T w o a m i n o acid differences were detected, one in position 1, with Gly in rabbit C G R P instead of Ala in h u m a n C G R P , and the other in position 35, with Glu instead o f L y s , respectively. T h e last four a m i n o acids, w h i c h were not d e t e r m i n e d by seq u e n c e analysis, were a s s i g n e d based on m a s s spectral analysis of C G R P ( 1 - 3 7 ) and its c h y m o t r y p t i c f r a g m e n t CT3 and f r o m hom o l o g y with rat alpha and porcine C G R P . If the carboxyl terminal a m i n o acid w o u l d not be amidated but contain free acid, this a s s i g n m e n t w o u l d be not correct since amidated phenylalanine differs f r o m the free acid by 0.98 m a s s units. H o w e v e r , this is an unlikely possibility since both calcitonin g e n e s encode in h u m a n s
0 40 min
60
- 30
rbCGRP
1200
uJ -J
r-
FIG. 1. (a) Elution profile of CGRP-like immunoreactivity (CGRP-LI) from analytical reverse phase C-I 8 HPLC of rabbit intestinal extract. The intestine was not stored but immediately processed as described in the Method section. The column was eluted by increasing concentrations of acetonitrile as indicated. The elution positions of standard human (hCGRP) and rat CGRP-I and CGRP-II were determined on separate runs. Standard human CGRP-I was not separated from human CGRP-II using this column and these elution conditions. Also, HPLC of extracts of rabbit dorsal root ganglia revealed only one immunoreactive CGRP peak which eluted in the same region as the intestinal form (data not shown). Standard rat CGRP-I and CGRP-II eluted 0.5 and 2 min in front of human CGRP-II, respectively (data not shown). (b) Analytical ion-exchange FPLC profile of CGRP-LI in rabbit intestinal extracts. The column was eluted by increasing concentrations of NaC1 (B, buffer B: 1 M NaC1 in 0.025 M acetate buffer, see the Method section). The elution positions of standard human CGRP-I and -II are shown. (c) Analytical ion-exchange FPLC profile of CGRP-LI in extracts of rabbit dorsal root ganglia. The column was eluted by increasing concentrations of NaCI as in (b). The elution positions of standard human CGRP-I and -II are shown.
20
iI
800
~:
I-
!
,--I
d.
0
iI i
n.-
i rbCGRP
400
I
i
10
ox?
III l
0 0
FLII
.
10 TIME,
20
-, ......... 30
0
40
min
FIG. 2. Elution profile of CGRP-like immunoreactivity (CGRP-LI) from reverse phase semipreparative HPLC (step 3, Table 1). The further purification of the major peak (marked rbCGRP = rabbit CGRP), which comprised 78% of the immunoreactive material found in the column eluate, is shown in Table 1. The earlier eluting immunoreactive peak, marked rbCGRP ox?, was also further purified and presumably represents oxidized rabbit CGRP.
RABBIT INTESTINAL CGRP
100
A
293
-
-40
TABLE 2
I
I I I
v
30
> :3. o3 o
SEQUENCE ANALYSIS OF RABBIT INTESTINAL CGRP AND ITS CHYMOTRYPTIC FRAGMENTCT-3*
LIJ Amino Acid
..I
50
20
Iv-
1 0
~0
~ o
