Biosynthesis of peptides in the skin of Xenopus laevis: Isolation of novel peptides predicted from the sequence of cloned cDNAs

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Biosynthesis of Peptides in the Skin of Xenopus laevis : Isolation of Novel Peptides Predicted From the Sequence of Cloned cDNAs K. RICHTER, H. A S C H A U E R * A N D G. K R E I L ' Institute o f Molecular Biology, Autstrian A c a d e m y o f Sciences, A-5020 Salzburg, Austria and * M a x - P h m c k Institute fin" Biochemistry, D-8033 Martinsried, F R G

RICHTER. K.. H. ASCHAUER AND G. KREIL. Biosynthesis ofpepti~h's in the skin ~' Xenopus laevis: lsohltion of n,n'el ;wptides predicted t)'om the sequence of chined cDNAs. PEPTIDES 6: Suppl. 3, 17-21, 1985.--From skin of XctT,p:¢.~ hwvis, a few peptides have been isolated which are identical or homologous to gastrointestinal hormones and/or neurotransmitters of mammalian origin. We have studied the biosynthesis of these peptides using recombinant DNA techniques. From cDNA librariers constructed from skin mRNA, clones with inserts coding for the precursors of caerulein, thyrotropin releasing hormone and a new peptide termed PGL ~have been isolated and sequenced. In the case of caerulein, a small family of precursors containing one, three or four copies of the end product have been detected. The caerulein sequences are separated by homologous sequences which potentially could give rise to additional constituents of skin secretion. Three such peptides have been detected which are presumably liberated from caerulein precursors by cleavage at single arginine residues.

Xcm~pus laevis

Caerulein Recombinant DNA techniques

PGL ~'

Thyrotropin releasing hormone

THE pioneering studies of Erspamer and his Italian colleagues have shown that amphibian skin synthesizes a variety of biologically active peptides [4, 5, 6]. In many instances it was found that these skin peptides were homologous or identical to mammalian peptides which act as hormones or neurotransmitters. This "brain-gut-skin triangle" [7] has intrigued biologists, biochemists and pharmacologists for many years. Yet. while it may seem plausible that different cells of ectodermal origin can produce similar sets of peptides, their function in amphibian skin has not been elucidated. In fact. the amount of a given peptide per gram of skin can vary widely even between closely related species [5]. This variation in the composition of the skin secretion is difficult to reconcile with its potential role as a repellent released upon stress. More recent studies on the biosynthesis of some of these peptides have shown that they are derived from larger precursors and that in the course of their proteolytic cleavage other fragments are generated as well, x~hich can in fact be detected in skin secretion. This then raises the interesting question as to which constituents are of physiological importance in this secretion and which ones are mere by-products of precursor processing. We consider amphibian skin to be a promising model for studies on the biosynthesis of peptides as many of them are

Precursor sequencing

made in much larger quantities than their mammalian counterparts. Moreover, during suitable stimulation, intact secretory granules are released from skin which can be isolated in fair yield. This opens the possibility to study the entire content of such granules, including processing enzymes and matrix proteins, without the problems, caused by contaminating lysosomes etc.. encountered when isolating secretory granules from intact cells. In this brief review we describe some of our studies on the biosynthesis of peptides in the skin of Xem~pus laevis. Using recombinant DNA techniques, the precursors for caerulein and thyrotropin releasing hormone as well as for a newly found peptide could be sequenced. Striking homologies have been detected between some of these precursors as well as between different segments of individual precursor polypeptides.

Caeruh, in Precursors The decapeptide caerulein was first detected in the skin of the Australian frog Litoria (Hyla) caerulea [3], and subsequently in other species as well, including X. laevis [5]. Caerulein has the same carboxy-terminal sequence as the mammalian hormones cholecystokinin and gastrin and it shares many of their biological actions.

~Requests for reprints should be addressed to G. Kreil, Inst. of Molecular Biology. Billrothslrasse 1I. A-5020 Salzburg Austria.

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FIG. 1. Amino acid sequence of preprocaerulein IV. This sequence was derived from the nucleotide sequences of the inserts of clones 11-53 and 11-130. The signal peptide probably ends at Ala-16 or Ser-20. The four copies of caerulein are underlined, the pairs of arginines are underlined by dots.

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FIG. 2. Schematic representation of the structures of preprocaeruleins I, I*, III and IV. As shown in the top line, all start with an amino-terminal signal peptide and (except for 1') end with a caerulein sequence followed by a COOH-terminal pentapeptide. Dark bars represent the sequence Asp-Gly-Caerulein-Gly-Arg-Arg. Open bars are highly homologous sequences, both inter-and intramolecularly. Single slanted lines mark deletions (see [ 171 for details).

From skin ofX. laevis, a c D N A library was constructed and searched for clones containing inserts derived from m R N A s coding for caerulein precursors [9]. A synthetic oligodeoxynucleotide complementary to the codons for the sequence Trp-Met-Asp-Phe of caerulein was used to find such clones. Three clones could be detected with inserts coding for the carboxy-terminal region of caerulein precursors. The insert found in clone pUF37 could be translated into a polypeptide which contained two copies of caerulein and which also indicated a high degree of internal homology [9]. More recently, about two dozen caerulein clones were isolated and characterized on the basis of the fragments generated with the restriction endonuclease HindlII [17]. The inserts of several clones were sequenced by the chemical cleavage method [14]. Three different types of precursors containing one, three and four copies of caerulein, respectively, have been found and these were accordingly termed type I. III and IV. A single clone was found to contain a

variant, shortened form of the type I called t y p e I*. The sequence of a clone with an insert derived from the mRNA for the type IV precursor and the deduced amino acid sequence of the precursor polypeptide are shown in Fig. 1. The different types of precursor polypeptides are schematically shown in Fig. 2. In the regions common to an}, two precursors, the homology of the nucleotide and amino acid sequences is always greater than 90%. The same is true for segments preceding the caerulein sequences in a given precursor (see Fig. 3 for some more details). These precursors must have arisen by a complex series of unequal crossing over and duplications and deletions of genes and gene segments. The liberation of caerulein from these precursors must invoh'e an number of different processing reactions. Cleavage at. and excision of. the pairs of arginine residues (see Fig. 1). a hallmark of prohormone activation, would yield an intermediate form of caerulein with an extension of two or four amino acids at the amino end and an extra glycine at the carboxy terminus. The latter must then be removed by oxidative cleavage to yield the terminal amide [2]. The further processing of the amidated intermediate with the amino-terminal extension Phe-Ala-Asp-Gly or Asp-Gly is probably catalyzed by a dipeptidylaminopeptidase. This type of processing through stepwise cleavage of dipeptides has previously been demonstrated for honeybee promelittin [13] and }'east alpha-mating factor precursor [12]. A dipeptidylaminopeptidase has been isolated from skin secretion of X. laevis which possesses the substrate specificity required for the cleavage of these amino-terminal extensions (Hutticher and Kreil, in preparation). The pyroGlu residues found at the amino end of caerulein is encoded as glutamine in the precursor. Whether its formation occurs spontaneously or is catalyzed by an enzyme is currently not known. In the course of this processing of caerulein precursors. several other fragments must be liberated and these ought to

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FIG. 3. Fragments of caerulein precursors detected in skin secretion of Xenopus laevis. Skin secretion was isolated and fractionated as described for the isolation of PGL" [1]. Their amino acid compositions and amino-terminal sequences were determined (lines 2) and compared with sequences predicted from clones cDNAs (lines I). The first peptide corresponds to residues 113-139 of preprocaerulein !1I, the middle one to residues 34-60 of the type I precursor, and the bottom peptide to residues 34--60 of preprocaeruleins III and IV.

be present in skin secretion as well. During our search for PGL", another peptide predicted on the basis of c D N A sequences (see below), several such fragments were indeed found. In these experiments, skin secretion ofX. laevis was extracted with n-butanol at alkaline pH and the organic extract was then separated by paper electrophoresis and high pressure liquid chromatography. As is shown in Fig. 3, the amino acid composition and amino terminal sequences of some of these peptides could be aligned exactly with segments of caerulein precursors. A detailed analysis of these and other unknown peptides found in skin secretion of X. laevis has been performed by Gibson, Poulter and Williams [8]. Their function and biological activity, if any, is currently not known.

coding for the precursor of PGL ~' is one of the main species of skin mRNA [10]. At present, nothing is known about the physiological properties of PGL". It is noteworthy, however, that in common with many peptides that do interact with membranes, PGL ~,can potentially form an amphipathic helix [10]. This is also true for the homologous fragment from the xenopsin precursor (see Fig. 4, lines b) as well as for the fragments of caerulein precursors as shown in Fig. 3. This indicates that a number of potentially membrane active peptides exist in skin secretion. PGL" and some of its fragments have also been detected by Gibson et al. (see following communication). Prepro- TRH

PGL", a .Vew Peptide Found in Skin Secretion

During our screening of the cDNA library for clones containing inserts derived from mRNAs for caerulein precursors. several positive clones were analyzed and sequenced which could potentially code for the precursor of a peptide which had not been described previously. The sequence of the largest insert is shown in Fig. 4, lines (a) [10], which contains a single open reading frame of 64 consecutive triplets. The polypeptide corresponding to this sequence would be comprised of 64 amino acids, starting with a methionine and a typical signal peptide. This putative precursor also contains a segment of 24 amino acids flanked by Lys-Arg and Gly-Arg-Arg at the amino and carboxy side, respectively. It was thus postulated that a peptide starting with tyrosine and ending with leucine amide, accordingly termed PYL" [19], should be present in skin secretion ofX. laevis. This peptide was subsequently synthesized and used as reference to search for the natural counterpart. [1]. A shorter peptide which lacked the first three amino acids of PYL" could indeed by detected. The 21 amino acid peptide with an amino terminal glycine, termed PGL", could be liberated from the postulated precursor if processing would also occur at single arginine residues. That this type of processing does indeed occur in frog skin is exemplified by the fact that the fragments of caerulein precursors mentioned in the previous section (see Fig. 3) must also be formed by cleavage at single arginine residues. Hybrid selected translation has shown that the mRNA

About ten years ago, it was found that Bombina orientalis and Rana pipiens contain large amounts of thyrotropin releasing hormone (TRH) in their skin [11,20]. Contrary to the peptides mentioned earlier, TRH appears to be secreted not only to the outside but also into the bloodstream. Using a similar approach as described for caerulein, a c D N A library from skin ofX. laevis was screened for clones derived from mRNA of the TRH precursor. Among 1400 clones, a single one could be found which codes for the amino-terminal part of the TRH precursor [16]. The predicted polypeptide starts with a methionine and a typical signal sequence. It contains three complete and one incomplete copy of the sequence Lys-Arg-Gln-His-Pro-Gly-!¢~i~-Arg.Typical prohormone processing at these sites would yield pGlu-His-Pro-NH~, i.e., TRH. We have so far not been able to find a clone containing the coding sequence for the carboxy terminal region of prepro-TRH in its insert. DISCUSSION

Our studies have shown that in the skin of X. laevis a small family of mRNAs coding for different preprocaeruleins is present. These differ from each other mainly through deletions/insertions of different segments. According to our analysis, the mRNA coding for the type IlI precursor is the most abundant one accounting for about 80% of the caerulein clones. Types I and IV each respresent about 10% while type I* was found only once. It is noteworthy that the deletions found in these clones always include one or even two caeru-

20

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Fig. 4. Comparison of eDNA and amino acid sequences of (a) prepl~-PGL" {[ 10], K. Richter. unpublished experiments) and {b) p~pro-xenopsin {18]. Identities in the nucleotide sequences are marked by dots. identical amino acids are underlined. One gap, marked/, was introduced into each of the Y-untranslated regions to maximize homology. Note that the homology between the nucleotide and amino acid sequences of the two precursors ends after Lys-57. PGL" starts at Gly-39, xenopsin corresponds to the last eight amino acids of its p~cursor. Stop codons are marked b~ (***).

lein sequences (see Fig. 2). The precursors are otherwise highly homologous to each other and they only differ by a few point mutations. These small differences, however, make it unlikely that the mRNAs originate through different splicing of a common pre-mRNA. Rather, we assume that several genes for prepro-caeruleins exist in the genome of X.

laevis. In the course of our search for clones coding for caerulein precursors, several false positives were isolated and sequenced. Among these, the precursor of a new peptide, termed PGL% was found and this peptide has since also been isolated from skin secretions. It is noteworthy that the predicted precursor of PGL" would contain only 64 amino acids and thus represent the smallest prepro-peptide hitherto found in nature. Last year, the sequence of the precursor of xenopsin, another peptide present in skin o f X . laevis, was elucidated [18]. In this case as well, a complex cascade of processing reaction must take place to liberate the final product. These are postulated to include cleavage at single arginine residues and action of the dipeptidylaminopeptidase. It is particularly interesting that the precursors of caerulein, PGL" and xenopsin exhibit sequence homology in the 5'-untranslated regions of their m R N A s as well as in the amino terminal region of the predicted polypeptides. This similarity is highest between the PGL" and xenopsin precursors (see Fig. 4). Up to residue 57 of the polypeptide sequence, the homology is 60% for the amino acid and 66% for the nucleotide sequence. After residue 57, this homology ends abruptly and the two sequences diverge completely.

This represents a rather unique example in that homologous precursors give rise to different end products. It will be interesting to check whether these regions of homology are on separate exons in the corresponding genes. It looks like a piece of genetic information coding for a signal peptide and part of a pro-region can serve as an "'export licence'" after transposition to different genetic loci. On the other hand, prepro-TRH shows no resemblance to the three precursors mentioned above, neither in the 5'untranslated region of the mRNA nor in the signal or propeptides. The TRH precursor does, however, also exhibit internal homology. The four TRH copies can be arranged in two pairs separated by homologous hexapeptides [16]. It is obvious that these studies represent only a modest beginning. The biosynthesis of many of the peptides discovered by Erspamer and his colleagues could be investigated by using similar methods. Of particular interest would be the biosynthesis of dermorphin, a heptapeptide with potent opiate-like activity found in the skin of certain South American frogs [15]. This peptide has a most unusual feature in that it contains a D-alanine in its sequence. It appears likely that dermorphin is also derived from a larger precursor made on ribosomes and that. by an unknown posttranslational reaction. D-alanine is generated from L-alanine or some other amino acid. This is in fact the first case that a D-amino acid has been found in a peptide synthesized by animal cells. We also want to explore the possibility that the intact secretory granules released from frog skin could serve as a source for isolating processing enzymes, matrix proteins etc.

P E P T I D E S IN A M P H I B I A N S K I N

21

O n e such e n z y m e , a d i p e p t i d y l a m i n o p e p t i d a s e , h a s a l r e a d y b e e n purified a n d partly c h a r a c t e r i z e d ( H u t t i c h e r a n d Kreil, in p r e p a r a t i o n l . Finally, it is o u r h o p e t h a t the c D N A s for t h e s e precursors o f a m p h i b i a n p e p t i d e s c a n be u s e d to s e a r c h for h o m o l o g o u s p r e c u r s o r s in l o w e r v e r t e b r a t e s a n d i n v e r t e brates. T h e r e h a v e b e e n n u m e r o u s r e p o r t s a b o u t the immunological d e t e c t i o n of p e p t i d e s h o m o l o g o u s to m a m m a lian and a m p h i b i a n p e p t i d e s in i n v e r t e b r a t e tissues a n d e v e n in unicellular o r g a n i s m s . W i t h few e x c e p t i o n s , h o w e v e r , no

data o n the s e q u e n c e o f t h e s e c r o s s - r e a c t i n g m o l e c u l e s h a v e b e e n available, w h i c h would clearly b e n e c e s s a r y to subs t a n t i a t e i m m u n o l o g i c a l a n d o t h e r findings. ACKNOWLEDGEMENTS We would like to thank R. Egger for excellent technical assistance and Prof. G. Braunitzer for help and advice in the analysis of peptides. This work was supported by grants from the -Politzer Stiftung'" of the Austrian Academy of Sciences and the Fonds zur F6rderung der wissenschaftlichen Forschung (grant $29 T41.

REFERENCES

I. Andreu, D.. H. Aschauer. G. Kreil and R. B. Merrifield. Solidphase synthesis of PYL" and isolation of its natural counterpart, PGL" I PYL~'-4-241 from skin secretion o f , \ c n , p t t s laevi~. Ettt'.l Biochem 149: 531-535, 1985. 2. Bradbury, A. F., M. D. A. Finnie and D. G. Smyth. Mechanism of C-terminal amide formation by pituitary enzymes. Nature 298: 686--688. 1982. 3. DeCaro, G., R. Endean, V. Erspamer and M. Roseghini. Occurrence of caerulein in extracts of the skin of Hyht cat, rtdea and other Australian hylids. Br J Pharmacol 33: 48-58, 1968. 4. Erspamer, V. The tachykinin peptide family. Tremls .Vettrosci 4: 267-269, 1981. 5. Erspamer, V.. G. Falconieri Erspamer. G. Mazzanti and R. Endean. Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea. Comp Biochem Physi,I [c] 77: 9%108, 1984. 6. Erspamer, V. and P. Melchiorri. Active polypeptides of the amphibian skin and their synthetic analogues. Pure Appl ('hem 35: 463-494, 1973. 7. Erspamer, V. and P. Melchiorri. Active polypeptides, from amphibian skin to gastrointestinal tract and brain of mammals. Trends Pharmacol Sci !: 391-395. 1980. 8. Gibson, B. W.. L. Poulter and D. H. Williams. A mass spectrometric assay for novel peptides: Application to Xem,p,s laevis skin secretions. Pcptides 6: Suppl 3. 23-27. 1985. 9. Hoffmann, W., T. C. Bach, H. Seliger and G. Kreil. Biosynthesis of caerulein in the skin of Xem~pu,s laevis: partial sequences of precursors as deduced from cDNA clones. EAIBO .I 2: 111-114. 1983. 10. Hoffmann, W., K. Richter and G. Kreil. A novel peptide designated PYL" and its precursor as predicted from cloned mRNA of Xenopus lacvis skin. EAIBO ,I 2:711-714. 1983. 11. Jackson. I. M. D. and S. Reichlin. Thyrotropin releasing hormone: abundance in the skin of the frog R , m t pipicns. Science 198: 414---415. 1977.

12. Julius, D.. L. Blair, A. Brake, G. Sprague and J. Thorner. Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase. Cell 32: 83%852, 1983. 13. Kreil, G., L. Haiml and G. Suchanek. Stepwise cleavage of the pro-part of promelittin by a dipeptidyl aminopeptidase: evidence for a new type of precursor-product conversion. Eur J Biochem 1 It: 4%58, 1980. 14. Maxam. A. M. and W. Gilbert. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65: 49%560, 1980. 15. Montecucchi, P. C., R. De Castiglione, S. Piani, L. Gozzini and V. Erspamer. Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of PhylIomed, sa .wmvagei. Int d Pept Protein Res 17: 275-283. 1981. 16. Richter, K. E. Kawashima, R. Egger and G. Kreil. Biosynthesis of thyrotropin releasing hormone in the skin ofXenopus htevis: partial sequence of the precursor deduced from cloned cDNA. EMBO J 3: 617-621, 1984. 17. Richter, K.. R. Egger and G. Kreil. Sequence of preprocaerulein cDNAs cloned from skin of Xenoptts laevis: a small family of precursors containing one, three, or four copies of the final product. J Biol Chem. in press. 1986. 18. Sures, I. and M. Crippa. Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor. Proc Natl Acad Sci USA 81: 380--384, 1984. 19. Tatemoto. K and V. Mutt. Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 285: 417-418. 1980. 20. Yasuhara, T. and T. Nakajima. Occurrence of thyrotropin releasing hormone in frog skin. Clwm Pharm Bull eTokyoJ 23: 3301-3303. 1975.