The isolation and characterization of rabbit motilin precursor cDNA

341

Biochimica et Biophysica Acta, 1131 (1992) 341-344 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

BBAEXP 90378

Short Sequence-Paper

The isolation and characterization of rabbit motilin precursor c D N A David K. Banfield a, Ross T.A. MacGillivray a, John C. Brown b and Christopher H.S. McIntosh b Department of Biochemistry and b MRC Regulatory Peptide Group, Department of Physiology, University of British Columbia, Vancouver (Canada) (Received 4 May 1992)

Key words: Motilin associated protein; cDNA; (Rabbit)

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.

Motilin was originally isolated from porcine intestinal extracts, and shown to be a 22 amino acid peptide [1-4]. Canine motilin differs from the porcine peptide at five amino acid positions [5,6]. The cDNAs encoding human and porcine motilin precursors have been isolated from intestinal libraries and the predicted amino acid sequences indicated that the motilin precursor consists of a 25 amino acid N-terminal signal peptide followed by motilin and a C-terminal peptide (motilin associated peptide; MAP) [7-10]. Cloning of the human and rhesus monkey motilin genes demonstrated that the sequence encoding motilin was split by an intron, which is unusual for such a small peptide [11]. The objective of the current study was to isolate the cDNA, and thus determine the sequence of motilin, from the rabbit. This animal is widely used in gastrointestinal motility studies, and the intestine has been shown to possess motilin receptors [12,13] and contract in response to porcine motilin [14]. Rabbit total duodenal cellular RNA, obtained from mucosal scrapings, was used to isolate polyadenylated

Correspondence to: C.H.S. Mclntosh, MRC Regulatory Peptide Group, Department of Physiology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3. The nucleotide sequence data presented in this paper have been submitted to the EMBL/Genbank database under the accession number X63860.

RNA, by chromatography on oligo(dT) cellulose. This RNA was used to synthesize single-stranded cDNA (sscDNA) using MMLV reverse transcriptase and the oligonucleotide T17xs P (Table I) as primer. Oligo(dT) primed cDNA libraries were prepared in Agtl0 and Agtll and screened with a 32p-labeled fragment of human motilin cDNA as hybridization probe. The human motilin cDNA fragment was isolated using the polymerase chain reaction (PCR) to amplify human RNA with Thermus aquaticus (Taq) DNA polymerase and primers MT4 and T17xs P (Table I). Prior to use as probes, amplified DNA was made blunt-ended using the Klenow fragment of Escherichia coli DNA polymerase I, ligated into the HinclI site of pUC19, and the DNA sequence determined using templates for double-stranded DNA sequence analysis prepared as described [15]. Nucleic acid and protein sequence data were analyzed with the Clustal, Pestfind, Palign, and Antigen programs of P C G E N E (Intelligenetics). Using the human motilin cDNA as a hybridization probe, a single positive phage was purified from the rabbit Agtll library. One of the predicted translation products shared amino acid sequence similarity with the motilin associated peptide sequences of human [7,8,11] and porcine motilin [9]. The sequence of the cDNA fragment consisted of nucleotides 249-582 (Fig. 2), excluding the polyadenylate tail. The remaining cDNA sequence was obtained by PCR amplification of

342 TABLE ! Oligodeoxyribonuch'otide primers used to ampli~' rabbit rnotilin precursor eDNA "

Primer

Sequence

MTI MT4 MT7 MT8 gt 10F Tl7xsi,

5'-CCTACGGCGAACTTCAGAGGATG-3' 5'-CTGGCCTCCCAGACGGAAGCCTT-3' 5'-TCCAATTTCCACAGGAGCAG-3' 5'-CCCGTTTTCCTCTTCCAGGG-3' 5'-TGGCGACGACTCCTGGAGCCCG-3' 5'-ACACTGCAGGAGCTCTCTAGATTTTTTTTTTTTTTTTT-3'

" The D N A sequence of primers MTI and MT4 is based on c D N A sequence alignments of human [7] and porcine [9] motilins. The D N A sequence of primers MT7 and MT8 was taken from the partial rabbit motilin precursor c D N A sequence (see text for details).

overlapping c D N A fragments from the rabbit duodenal Agtl0 library (Fig. 1B and Table I). The 3' end of the rabbit motilin c D N A was cloned into pUC 19 following a round of amplification with primers gtl0F and MT 4, and a second using gtl0F and MT 1 (Fig. 1). The 5' end of the rabbit motilin c D N A was cloned using primers gtl0F and MT 7 for the first, and primers gtl0F and MT 8 for the second round of amplification. The D N A sequence of the 5' and 3' PCR products was determined and the c D N A sequence of rabbit motilin is shown in Fig. 2. The length of the rabbit motilin precursor c D N A sequence was 582 nucleotides. The c D N A encodes an open reading frame of 402 base pairs including a 25 amino acid signal peptide, 22 amino acids corresponding to motilin, and an 86 amino acid MAP (Fig. 2). The D N A sequence was determined for five independent PCR products. No sequence discrepancies were observed between the PCR

A signalpeptidase endoprotease ~ Motilin ~ V/I////."I

Signal peptide

Molilinassociatedpeptide

B gtl0F

4

1

8

gtl0F

7

Xgtl0

Xgtl0

Fig. l. (A) Schematic representation of prepromotilin [7-10]. The arrows indicate the positions of the putative signal peptidase and endoproteinase cleavage sites, respectively. (B) Amplification scheme for rabbit motilin c D N A precursor. The arrows indicate the position and direction of amplication for the PCR primers. The thick lines represent a portion of the AgtlO vector sequence arms.

GAGGCTCCTCCAGGCCCACTCGGATCACGCTCACCTCACGCCGCAGACGGGAGCCGCTGGCCGTTGCCTAGCTCCACG A

V

A

A

L

L

L

V

H

V

T

A

M

L

A

GTG

GCT

GCC

CTG

CTG

CTG

GTG

CAC

GTG

ACC

GCC

ATG

CTG

GCC

-6

M

V

S

R

K

79

ATG

GTG

TCC

CGC

AAG

S

Q

T

E

V

P

I

F

T

Y

S

E

L

Q

R

M

Q

E

139

TCC

CAG

ACG

GAA

GCC

TTC

GTC

CCC

ATC

TTC

ACC

TAC

AGC

GAA

CTC

CAG

AGG

ATG

CAG

GAA

R

E

R

N

R

G

H

K

199

AGG

GAG

CGG

AAC

AGA

GGG

CAC

AAG

A

A

P

R

P

A

E

P

T

L

E

E

E

N

G

R

M

Q

L

T

259

GCC

GCT

CCA

AGG

CCT

GCG

GAG

CCC

ACC

CTG

GAA

GAG

GAA

AAC

GGG

AGG

ATG

CAG

CTG

ACT

A GCT

P CCT

V GTG

E GAA

I ATT

G GGA

M ATG

R AGG

M

N

S

R

Q

L

E

K

Y

R

A

A

319

ATG

AAC

TCC

AGG

CAG

CTG

GAA

AAG

TAC

CGG

GCC

GCC

L

E

A

A

E

R

A

V

H

P

D

A

P

S

R

P

C

W

P

A

379

CTG

GAA

GCT

GCA

GAG

CGG

GCT

GTC

CAC

CCA

GAC

GCT

CCG

AGT

CGG

CCC

TGC

TGG

CCT

GCC

G GGG

G GGA

E GAA

S AGT

G GGA

W TGG

S AGC

G GGG

E

P

S

P

T

439

GAG

CCC

TCT

CCC

ACC

504

CCCACCCACACAGCTGGGCTGGGAAAGAAAACCCCTTCTCCTCAGCTACCCCGTCCAGCAAATAAAGCATGAACTTTAA

583

CGGAAAAAAAAAAA

± GCC

A~F

15

I K~ AAG

S

L

S

V

Q

Q

R

S

D

A

A

TCC

CTG

AGC

GTG

CAG

CAG

AGG

TCT

GAC

GCA

GCG

STOP TGA

35

55

75

95

108 GCGAGGCCCCAGAAGTCCGCCGG

(N)

Fig. 2. The c D N A sequence of rabbit motilin precursor c D N A . Nucleotides and amino acids are numbered on the left and right hand sides of the figure, respectively. The putative positions of the signal peptide cleavage site (between Ala - 1 and Phe + 1) and endoproteinase cleavage site (following Lys-24) are indicated by the arrows. Lys-23-Lys-24 are in bold text. The translational stop codon and the polyadenylation consensus sequence A A T A A A are in bold text.

343

A

5

10

15

20

["VPN"TYSELQPdVI~ H

Rabbit

Human

FVP i F T Y G E L Q R M ~ E K E R N K ~ Q FVP I F T Y G E t , Q R H ~ E H E R N ~ 3 Q

mcP~H ' " S m ~ 0 M ~ R N T e

Pig Dog

Consensus F',,'PT7 7 * "

Ei/.~ ~* *~KERN *p*

Exon II/111 Exon Ill/IV

B ~

30

40

50~

QRS DAAA/~ RP AE P T ~ 4 Q L

Rabbit Human Pig Consensus

60

70

T~PVE I GMRMNS RQL EK YRA

GEEGPVDPAEPI~EEENEMIKLI~PLEIGMRMNSRQLEKYPA EELGPLDPSEPT~EEERVVIKLL~PVDIGIP.MDSRQLEKYRA

1

2

3

Exon IV/V / 80

Rabbit Human Pig Consensus

~V 90

1O0

~ERAVHPDAP SRPCWPAGGESGWSGEP SPT@ [LE~3LLSEMLPQHAAK@ T L ~ L L G Q A P Q S TQNQNAAK @ **

Fig. 3. Comparisons of amino acid sequences of motilin and the motilin associated peptide (MAP). (A) Amino acid sequence alignment of rabbit, human [7,8,11], porcine [4,9] and canine [5,6] motilins. Amino acid residues common to all four species are indicated in the consensus sequence. The * symbol represents a conservative amino acid substitution. The amino acids in the box represent a putative antigenic determinant of motilin predicted with the Antigen program of PCGENE (Intelligenetics). The position of the exon I I / I I I boundary in the human motilin precursor gene [11] is indicated with an arrow. (B) Amino acid sequence alignment of rabbit, human [7,8,11], and porcine [9] MAP. The arrow indicates the putative position of endoproteinase cleavage. The symbol (*) indicates an identical amino acid is found at that position in all species compared. The symbol (.) indicates that a similar amino acid residue is found at that position in all species compared. The positions of the exon I I I / I V and I V / V boundaries in the human motilin precursor gene [11] are indicated by arrows. For both (A) and (B) the following groups of residues were defined as similar: A,S,T., D,E., N,Q., R.K., I,L,M,V and F,Y,W.

amplified cDNA fragments and the cDNA fragment isolated by hybridization to the human cDNA. Alignment of the sequences of human, canine and rabbit motilin (Fig. 3A) shows that they share 64% sequence identity. All of the eight amino acid differences represent conservative substitutions. The amino acids in the box represent a putative antigenic determinant of motilin, and this region corresponds to the highest point of hydrophilicity in the motilin precursor. In the human motilin precursor gene, the motilin coding sequence is split by an intron [11]. The position of the intron/exon boundary separates this region of motilin from the amino-terminal 14 amino acids of motilin (Fig. 3A). When the predicted MAP sequences are aligned it is evident that they are variable in both composition and length (Fig. 3B): the rabbit MAP is 86

amino acids long, and human and porcine 68 and 72 amino acids, respectively. The differences in length result from variability in the carboxy-terminal region. The MAPs share 40% amino acid sequence identity and 60% amino acid sequence similarity. The variability in the length of the precursors may be the result of intron sliding events which occurred since the three species last shared a common ancestor [16]. While the function of the MAP is unknown [8], alignment of the known sequences identifies three potential structural/functional regions (Fig. 3B, boxes 1-3). Box 1 outlines a putative endoproteinase dibasic cleavage site Lys-23-Lys-24 and the adjacent carboxyterminal amino acids. This may be a portion of the recognition site required for correct processing of the motilin precursor in vivo [17]. The second region (box 2) consists of a putative PEST site [18,19]. PEST sites are regions rich in proline, glutamic acid, serine and threonine which are subject to rapid intracellular degradation [18,19]. This site may direct the rapid degradation of either the motilin precursor or the MAP following endoproteinase cleavage. The third structural/functional region (box 3) contains 22 amino acids and is the most conserved region of the MAP (73% amino acid sequence identity and 100% similarity. This region may play a role in the posttranslational processing events leading to motilin secretion. Knowledge of the sequences of rabbit motilin and its associated peptide will allow their production by peptide synthesis or expression cloning. Availability of these peptides will facilitate the development of species and regional specific antibodies, and studies on the secretion and mode of action of motilin. Finally, the utilization of cDNA probes in hybridization studies should throw light on a number of controversies in the literature including gastrointestinal ceil localization [20-22], existence of brain motilin [23] and regional homology of porcine motilin and rabbit skeletal tropomyosin [24]. The authors are grateful for the technical assistance of Annabelle Chong, Karen Ogryzlo, and Tina Umelas. This research was supported by grants from the Medical Research Council of Canada to RTAM (MT7716) and the MRC Regulatory Peptide Group. References 1 Brown, J.C., Cook, M.A. and Dryburgh, J.R. (1972) Gastroenterology 62, 401-404. 2 Brown, J.C., Cook, M.A. and Dryburgh, J.R. (1973) Can. J. Biochem. 51,533-537. 3 McIntosb, C.H.S. and Brown, J.C. (1990) In Motilin (Itoh, Z., ed.), pp. 13-30, Academic Press, San Diego. 4 Schubert, H. and Brown, J.C. (1974) Can. J. Biochem. 52, 7-8. 5 Poitras, P., Reeve, Jr. J.R., Hunkapillar, M.W., Hood, L.E. and Walsh, J.H. (1983) Regul. Pept. 5, 197-208. 6 Reeve, J.R. Jr., Ho, F.J., Walsh, J.H., Ben-Avram, C.M. and Shively, J.E. (1985) J. Chromatogr. 321,421-432.

344 7 Seino, Y., Tanaka, K., Takeda, J., Takahashi, H., Mitani, T., Kurono, M., Kayano, T., Koh, G., Fukumoto, H., Yano, H., Fujita, J., Inagaki, N., Yamada, Y. and Imura, H. (1987) FEBS Lett. 223, 74-76. 8 Dea, D., Boileau, G., Poitras, P. and Lahaie, R.G. (1989) Gastroenterology 96, 695-703. ~,~ Bond, C.T., Nilaver, G., Godfrey, B., Zimmerman, E.A. and Adelman, J.P. (1988) Mol. Endocrinol. 2, 175-180. 1/t Nilaver, G., Beinfeld, M.C., Bond, C.T., Daikh, D., Godfrey, B. and Adelman, J.P. (1988) Synapse 2, 266-275. 11 Daikh, D.I., Douglass, J.O. and Adelman, J.P. (1989) DNA 8, 615-621. 12 Bormans, V., Petters, T.L. and Vantrappen, G. (1986) Regul. Pept. 15, 143-153. 13 Depoortere, I., Peeters, T.L. and Vantrappen, G. (1991) Peptides 12, 89-94. 14 Peeters, T.L., Bormans. V., Matthijs, G. and Vantrappen, G. (1986) Regul. Pept. 15, 333-339. 15 Gatermann, K.B., Rosenberg, G.H. and Kaufer, N.F. (1988) Biotechniques 6, 951-952.

16 Craik, C.S., Rutter, W.J. and Fletterick, R. (1983) Science 220, 1125-1119. 17 Barr, B.J. (1991) Cell 66, 1-3. 18 Rogers, S., Wells, R. and Rechsteiner, M. (1986) Science 234, 364-368. 19 Rechsteiner, M., Rogers, S. and Rote, K. (1987) Trends Biochem. Sci. 12, 390-394. 20 Pearse, A.G.E., Polak, J.M., Bloom, S.R., Adams, C., Dryburgh, J.R. and Brown, J.C. (1974) Virch. Arch. B. Cell. Path. 16, 111-120.

21 Helmstaedter, V., Kreppein, W., Domschke, W., Mitznegg, P., Yanaihara, N., Wiinsch, E. and Forssman, W.G. (1979) Gastroenterology 76, 897-902. 22 Usellini, L., Buchan, A.M.J., Polak, J.M., Capella, C. Cornaggia, M. and Solcia, E. (1984) Histochemistry 1,363-368. 23 O'Donahue, T.L., Beinfeld, M.C., Chey, W.Y., Chang, T.M., Nilaver, G., Zimmerman, A., Yajima, H., Adachi, H., Roth, M., McDevitt, R.P. and Jacobowitz, D.M. (1981) Peptides 2, 467-477. 24 Beinfeld, M.C. and Korchak, D.M. (1986) Neuroscience 5, 250225119.