Pepndes, Vol 11. pp 123-128 ©PergamonPress plc, 1990 Prmtedm the U S A
0196-9781/90 $3 00 + 00
Amino Acid Sequence of VIP, PHI and Secretin From the Rabbit Small Intestine D. G O S S E N , L. B U S C A I L , A. C A U V I N , P. G O U R L E T , P. D E N E E F , J. R A T H E , P. R O B B E R E C H T , M . - C . V A N D E R M E E R S - P I R E T , A. V A N D E R M E E R S A N D J. C H R I S T O P H E 1
Department o f Biochemistry and Nutrition, Medical School Universitg Ltbre de Bruxelles, B-IO00 Brussels, Belgium
GOSSEN, D , L BUSCAIL, A CAUVIN, P GOURLET, P DE NEEF, J RATHE, P ROBBERECHT, M -C VANDERMEERSPIRET, A VANDERMEERS AND J. CHRISTOPHE. Amino actd sequence of VIP, PHI and secretmfrom the rabbtt small mtestme PEPTIDES 11(1) 123-128, 1990 --VIP, PHI and secretln were punfied from rabbit small intestine throughout a maximum of 6 chromatographic steps After elutmn on a reverse phase C18 column, the 3 peptldes were separated on a Fractogel column using specific radlo~mmunoassays for detectmn After caUon exchange chromatography on Mono S, the final steps were performed using a reverse phase RP8-e column For these steps, radloreceptor assays were utilized to detect VIP and PHI We confirmed that the VIP sequence of rabbit was ldenttcal to that of porcine VIP The PHI sequence was also found identical to that of porcine PHI By contrast, rabbit secretln was highly original, differing from porcine secretm m hawng Leu, Arg and Leu-NH2 residues instead of Phe, Ser and Val-NH z m, respectively, positron 6, 16 and 27 VIP
Secretln
Ammo acid sequence
PHI
Rabbits
METHOD
WE report here the purificatxon and amino acid sequence of the three parent peptldes VIP, PHI [peptlde h~st~dme ~soleucmamlde (1-27)] and secretin extracted from rabb~t gut Our aim was two-fold 1) The structure of porcine, bovine, dog, guinea p~g, rat, rabbit, and human VIP is identical with the exception of guinea pig VIP [(12) and review in (19)]. For PHI of porcine, bovine, rat, and human origin, amino acid vanatmns are more numerous and occur m position 10, 12, 17 and/or 27 (4, 19, 20). The structure of rabbit PHI has not yet been reported. In general, VIP and PHI are cosynthesized and coreleased from the same nerve endings (1) so that both neuropeptldes have a good chance to act on the same receptors (23). Indeed, in almost all target tissues so far studied, these receptors brad VIP as well as PHI but are VIP preferring. However, unexpected variations in the PHI structure, in a mammalian species not yet documented, could conceivably lead to a situatmn where PHI might be more potent than VIP. 2) We were tempted to complete our study on gut peptides in the rabbit by estabhshmg the amino acxd sequence of the parent hormone secretin for 3 reasons. 1) secretin can interact with VIP receptors in pancreas and gut in rat (18,22); 2) adenylate cyclase activity m the rabbit pancreas is poorly stimulated by porcine secretin (9), and 3) pepsinogen secretxon from molated rabbit gastric glands is not stimulated by porcine secretin (16), suggesting a umque structure for secretm itself and/or secretm receptors in this mammahan species.
Ttssue Extract Preparation Six male adult New Zealand rabbits were killed by a blow on the neck and exsangumated. The entire small intestine was removed, rinsed with 0.15 M NaCI at room temperature, frozen in llqmd nitrogen, and stored at - 7 0 ° C . This material (250 g) was then boiled in 2 1 water for 10 min, homogenized with an Ultra-Turrax, cooled at 4°C, and acidified with acetic acid to a final 1 M concentration. The 15,000× g supematant served as starting material for the purification procedure.
Chromatographwal Procedure Four types of columns were used as follows
Reverse phase C18 columns. 15 × 1.5 cm and 6 × 1.5 cm (Bulk C18 from Waters, Milford, MA) eqmhbrated in 10 mM HC1, 10% CH3CN and eluted with 50% CH3CN in 10 mM HCI at a flow rate of 3 ml/min. Sizefracnonanon Fractogel column. 90 × 1.6 cm made of the superposition of Fractogel TSK HW40S (50 cm) and TSK HW50S (40 cm) (Merck, Darmstadt, F.R G.) equdibrated and eluted with a 60 mM phosphate buffer (pH 7.2) containing 0.3% bovine seurm albumin (wt..vol.), 0.02% Tween 20 (vol..vol.) and 0.1% sodium azlde (wt :vol.) at a flow rate of 8.8 ml/hr.
1Requests for repnnts should be addressed to J Chnstophe, Department of Blochenustry and Numtlon, Medical School, Unlverslt6 Llbre de Bruxelles, Boulevard of Waterloo 115, B-1000 Brussels, Belgium
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124
GOSSEN ET AL
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FIG 1 Displacement of ~25I-Data-secretm bound to rabbit antiserum SNI 5 by increasing concentrauons of porcine secrenn (El), secretm(2-27) ([~), secretln(7-27) (+), secretm(l-21) (x), secretm(14-27) (V), and related peptldes (PHI, VIP, GRF and glucagon O) (final aanserum dilution 1 500,000) Binding was expressed as percent lnltml bound to free ratio, in absence of any added pepnde Citron exchange HPLC on HR 5/5 Mono S column. (Pharmacm, Uppsala, Sweden) equilibrated in solvent A. 10 mM HCI, 20% CH3CN and eluted with a hnear gradient of solvent B: 10 mM HC1, 20% CH3CN, 0.8 M NaCI, 0-60% B in 60 mln at a flow rate of 1 ml/mm Reverse phase HPLC on Ltchrospher 100 RP8-e column 25 × 0.4 cm (Merck, Darmstadt, F.R.G.) equilibrated with 0.1% trifluoroacetic acid, 3% CH3CN and eluted with a linear gradient of CH3CN (the slope of the gradient used is described in legends for figures). PHI and VIP Detectton Radtotmmunoassays used for VIP and PHI detection during the first steps of purification were based on, respectively, antiserum R501 (kindly offered by Dr N Yanalhara, Dept. of Organic Chemistry, Shizuoka School of Pharmacy, Shizuoka, Japan) and antiserum AC24, as detailed elsewhere (4,27) Radtoreceptor assay using [125I]VIP as tracer and rat hepatic membranes as source of VIP/PHI receptors was performed as previously described (23,24). This method allowed a rapid detection but was not used for quantitative evaluation of the neuropept~d~c material. Tracers. Porcine [125I]VIP and porcine [~2~I]PHI were iodanated as descnbed in (4) Secretin Detection A radiolmmunoassay was used for secretm detection during the entire purification procedure. The specificity of rabbit antiserum SNI5 raised against synthetic porcine secretin coupled to bovine serum albumin by carbodllmlde is illustrated in Fxg. 1. The tracer used was Data-secretm iodlnated as previously described (14). Sequence Analysts The amino acid sequence was estabhshed by Edman degradation of 100 to 200 pmol of peptide in an Applied B~osystems 477 A pulsed liquid phase sequencer coupled to a 120 A PTH (phenylthiohydantoln) amino acid analyzer (A.B.I., Foster City, CA). Carboxypepttdase Y Digestion and Ammo Actd Analyst~ Two hundred pmol of peptlde were Incubated with 0.06 i,zg
Synthetic porcine VIP and PHI were kindly given by Dr. D. H. Coy (Tulane University, New Orleans, LA) and synthetic porcine secretln by Dr W. Konlg (Hoechst, Aktiengesellschaft, Frankfurt/ Main, F R G ) Data-secretin was obtained from Fluka (Buchs, Switzerland) Normal rabbit serum and sheep antlrabbit immunoglobulins were purchased from the Laboratory of Hormonology (Marloie, Belgium) and [~2sI]iodine carrier-free (IMS 300) from the Amersham Radlochemlcal Center (Bucks, U.K ) RESULTS
Peptides present in the acidic extract of 250 g rabbit small intestine were adsorbed on a reverse phase Bulk C18 column (15 × 1.5 cm) and eluted with 50% CH3CN in 10 mM HC1 Two consecutive runs on this preparative column allowed concentrating the extract to 60 ml that were divided into 10 aliquots. After CH3CN evaporation, the eluate was lyophdlzed, then each aliquot was successively applied on a Fractogel column as described in the Method section PHI, VIP and secretln were identified by radlolmmunoassay PHI-IR eluted in two peaks of different molecular weight while VIP-IR and secretln-IR eluted as single peaks clearly distinct from the two PHI-IR peaks (Fig. 2). Fractions 55 to 58, 59 to 63, and 76 to 81 were pooled for further purification of, respectively, secretm, VIP and PHI. The three pools were separately adsorbed on a 6 × 1 5 cm reverse phase C18 column, then eluted in concentrated form with 50% CH3CN m 10 mM HC1. After CH3CN evaporation and ionic strength adjustment, each eluate was chromatographed in a single run on a cation exchange Mono S column, as described in the Method section and illustrated in Figs 3, 4 and 5 (upper panel) The PHI-IR material eluted as a major peak with 0.25 M NaC1, the VIP material (detected by radioreceptor assay) eluted with 0.35 M NaC1, and secretin-IR material with 0.40 M NaC1. Each peak was pooled and submitted to reverse phase HPLC on RP8-e. Two or three runs were performed with increasingly flatter linear gradients of CH3CN (Figs 3, 4 and 5, middle and lower panels). These steps were performed in one day, PHI and VIP materials being detected by radioreceptor assay. The total amount of VIP, PHI and secretln purified from 250 g rabbit small intestine was 5 txg, 3 5 Ixg and 1 i~g, respectively, based on absorption at 280 nm (for VIP and PHI) and 214 nm (for secretln) One hundred to two hundred pmol of each purified peptlde were applied to a polybrene filter and sequenced. The sequencer identified unambiguously the 28 amino acids of VIP (Asn-NH2 as Asn), 26 amino acids of PHI, and 25 amino acids of secretin The identification of PTH-derivatized amino acids and their recovery in the case of secretin is illustrated in Fig 6 A single time digestion by carboxypept~dase Y (see the Method section) confirmed the C-terminal structure of each peptlde. The sequence of VIP previously documented (19) was confirmed as being: Hls-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-LeuArg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-LeuAsn-NH 2 We established the PHI sequence as being HIs-AlaAsp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-GlnLeu - Ser- Ala - Lys - Lys - Tyr- Leu - Glu - Ser- Leu - Ile - NH2. Rabbit VIP and rabbit PHI were, therefore, identical to the corresponding porcine peptides Hydrolysis of purified rabbit secretin (200 pmol)
VIP, PHI AND SECRETIN IN RABBIT
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