Molecular and Cellular Endocrinology, 0 1991 Elsevier Scientific Publishers

MOLCEL

171

78 (1991) 171-178 Ireland, Ltd. 0303-7207/91/$03.50

02524

Primary structure and tissue distribution of anglerfish carboxypeptidase

I-I

William W. Roth I, Robert B. Mackin ‘, Joachim Spiess 2, Richard H. Goodman 3 and Bryan D. Noe ’ ’ Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA, U.S.A., ’ Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Giittingen, F.R. G., and ’ Voiium Institute for Advanced Biomedical Research, Oregon Health Sciences Univer.sity, Portland, OR, U.S.A. (Received

Key words: Prohormone

processing;

23 January

Carboxypeptidase

1991; accepted

H; Carboxypeptidase;

7 March

Enzyme

1991)

structure

comparison

Summary

Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides. This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase, These processing events have been observed in a variety of species, from yeast to mammals. As part of an effort to characterize prohormone processing enzymes in the anglerfish, ~0~~~~ ~~~~cu~us, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH). Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage hgtll. The library was screened with a probe derived from the cDNA for rat CPH. A 2400 base pair AF cDNA clone was isolated. This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH. The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide. The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes. SignificantIy, a11of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme. Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones.

Introduction Most polypeptide hormones are synthesized as inactive prohormone precursors which must then

Address for correspondence: Dr. Bryan D. Noe, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. Tel. 404-727.6251; Fax 404-727.6256.

undergo several modifications in order to yield biologically active peptides (Docherty and Steiner, 1982; Noe, 1985; Lynch and Snyder, 1986). Prohormones are synthesized on membrane-bound ribosomes and directed into the secretory pathway, with cotranslational signal peptide cleavage. It now appears that most posttranslational processing steps occur within the trans-Golgi network or inside newly formed and maturing secre-

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tory granules (Gainer et al., 1985). The initial granule-associated step, for most precursors, is cleavage by one or more endopeptidases which typically cleave at sites marked by basic amino acid residues. The basic residues are then removed by amino- and/or carboxypeptidases. Finally, peptides may be further modified by Nacetylation, C-terminal amidation, or other processing events (Lazure et al., 1983) before they are secreted in active form. Although the cleavage events which occur during processing of many prohormones have been elucidated, the enzymes involved are for the most part poorly understood. Therefore, a primary focus for research in hormone biosynthesis in recent years has been characterization of the enzymes’ responsible for prohormone processing. Although it has been difficult to obtain these proteins in pure form due to their relatively low abundance, the application of molecular biological methods has resulted in several significant contributions to this field. The structures of several prohormone processing enzymes have been elucidated by cDNA cloning. These include the KEXl and KEX2 proteins involved in the synthesis of mating factor and killer toxins in yeast (Fuller et al., 1988, 1989; Mizuno et al., 1988; Cooper and Bussey, 1989). Recently, human (Smeekens and Steiner, 1990) and mouse (Seidah et al., 1990; Smeekens et al., 1991) cDNA clones that have some homology with KEX2 were also described. In addition, bovine (Eipper et al., 1987) and rat (Staffers et al., 1989) cDNAs for the C-terminal peptidylglycine a-amidating monooxygenase have been cloned and sequenced. Several cDNA clones for the prohormone processing carboxypeptidase H (CPH, also known as CPE; EC 3.4.17.10) have been isolated and characterized (Fricker, 1988). One clone was isolated from bovine pituitary (Fricker et al., 1986), another from a rat insulinoma cell line (Fricker et al., 1989; Rodriguez et al., 19891, and a third from a rat pheochromocytoma (Rodriguez et al., 1989). A cDNA for human CPE has also been described (Manser et al., 1990). We report here the isolation and characterization of a cDNA from which the primary structure of the anglerfish (AF) CPH can be deduced.

Materials

and methods

Construction of AF islet cDNA library AF pancreatic islets (630 mg) were homogenized and RNA was extracted using the guanidine thiocyanate procedure of Chirgwin et al. (1979). Poly(A)+ mRNA was selected by two rounds of chromatography on oligo dT cellulose (Aviv and Leder, 1972). The yield was 68 Fg of mRNA after the first selection and 6 pg after the second. One-half of this material was used for cDNA synthesis by the method of Gubler and Hoffman (19831, using the cDNA Synthesis System from Bethesda Research Labs. After the second strand synthesis, the yield of ds cDNA was 0.96 pg. The ds cDNA was blunt-ended with T4 DNA polymerase and synthetic oligonucleotide linkers were added using T4 DNA ligase. These linkers, generously provided by Dr. John Adelman (Vollum Institute) consisted of a 16 mer and a complementary 20 mer which, after ligation, provided the ds cDNA molecules with EcoRI ends and Sal1 restriction sites. The sequence of the linkers is shown below: 5'-AATTCGTTGTCGACTGTCAG GCAACAGCTGACAGTC-5'

After phosphorylation of the 5’ ends with T4 polynucleotide kinase, the cDNA inserts were ligated into hgtll arms. Packaging of recombinant phage into Escherichia coli Y1090 was then accomplished using Gigapack Gold packaging extracts from Stratagene. Packaging efficiencies were 85-95% and averaged 5.3 X 10” recombinants per reaction. Six packaging reactions were completed, yielding a total library size of 3.2 X 10h recombinants. The average insert size was 2100 bp. After a single round of amplification, the library titer was 4 x 10”’ recombinants per ml. Isolation and sequencing of AF cDNA clones The AF cDNA library was screened with portions of a rat CPH cDNA clone obtained from Drs. C. Rodriguez and J. Dixon, Purdue University (Rodriguez et al., 1989). A 1300 bp HincII restriction fragment containing most of the protein coding sequence was used for the primary

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screen. Positively hybridizing clones were replated and tested for ability to hybridize to a 200 bp AvaI fragment derived from the 5’ end of the rat CPH cDNA. Probes were 32P-labeled by nick translation (Sambrook et al., 1989) and hybridized to duplicate plaque lifts at 60°C in a buffer containing 6 X SET (1 X SET = 150 mM NaCl, 30 mM Tris/HCl, 1 mM EDTA, pH 8.0), 500 pg/ml heparin, 0.1% (w/v> sodium dodecyl sulfate, 0.1% (w/v> sodium pyrophosphate (Singh and Jones, 1984). Inserts from clones identified by these methods were excised with Sal1 and subcloned into plasmid pUC8 and phage Ml3 mp18 for the purpose of sequence determination by the Sanger chain termination method (Sanger et al., 1977). Ml3 clones expressing the CPH insert in both orientations were sequenced using modified T7 DNA polymerase (Sequenase, U.S. Biochemicals) and the Ml3 universal primer. Additional sequence data were generated by use of synthetic oligonucleotide primers complementary to insert DNA. Primer extension sequencing of AF cDNA

Primer extension reactions were performed using a method similar to that described by Wise et al. (1984). A 26-base oligodeoxynucleotide complementary to nucleotides 63-88 of the AF CPH cDNA (see Fig. 1) was synthesized. This nucleotide primer was labeled with 32P using T4 polynucleotide kinase and hybridized to 5 pg AF pituitary poly(A)+ RNA in a volume of 16 ~1. The primer/template mixture was heated to 90°C for 20 min and allowed to cool slowly to 45°C. 4 ~1 aliquots were distributed into reaction tubes containing mixtures (5 ~1) of the four nucleoside triphosphates (NTPs) and one dideoxy NTP in 1.6 X reverse transcriptase buffer. Final concentrations of the NTPs for primer extension were 800 PM, except for the nucleotide to be terminated, which was 300 PM, in a reaction volume of 10 ~1. Dideoxy NTP analogues were also added at a concentration of 300 FM. Primer extension reactions were started by the addition of 1 unit of AMV reverse transcriptase (U.S. Biochemicals). The reactions were incubated 90 min at 45”C, and stopped by addition of 1 ~1 0.5 M EDTA. The reaction contents were diluted with 20 ~1 0.5 M sodium acetate containing 10 pg yeast tRNA and

extracted with phenol-chloroform prior to ethanol precipitation of nucleic acids. Primer extension products were resuspended in 4 ~1 of formamide dye mix and resolved by electrophoresis on 5% acrylamide-urea sequencing gels. Northern hybridizations

RNA prepared from anglerfish tissues was electrophoresed on 1% denaturing agarose gels containing 2.2 M formaldehyde (Sambrook et al., 19891, transferred to nylon membranes (Magnagraph, MSI) in 10 X SET, and crosslinked to the membranes by baking for 2 h in a 75°C vacuum oven. The blots were probed with a 1395 bp HincII fragment of the cloned AF CPH cDNA, which was labeled with 32P to a specific activity of 8 X lo8 cpm/pg using T7 DNA polymerase (Hodgson and Fisk, 1987) and random synthetic 9 mers (Prime-It kit, Stratagene). These hybridizations were carried out in buffer containing 6 x SET, 5 X Denhardt’s solution, 200 pg/ml sheared salmon sperm DNA, 0.1% (w/v> sodium dodecyl sulfate, 0.1% (w/v) sodium pyrophosphate, and 50% (v/v> formamide. The blots were incubated 16 h at 47°C with lo6 cpm/ml of the “‘P-labeled AF CPH probe DNA. After hybridization, the blots were washed in 1 X SET, 0.05% SDS for 30 min at room temperature, followed by a 30 min wash at 60°C in the same buffer. Results

The initial screen of 250,000 pfu of the AF cDNA library with the rat HincII probe yielded 11 positively hybridizing phage clones. To select those clones which might be full length, the positive phage clones were rescreened with a smaller probe, an AuaI fragment of the rat cDNA containing the sequence for the amino terminal portion of the rat CPH. As a result, two clones were selected. These clones, designated hCP3 and hCP6, contained inserts of 2407 bp and 2050 bp, respectively. The AF cDNA inserts were excised by digestion with Sal1 and subcloned into appropriate plasmid and phage vectors for further analysis. The complete nucleotide sequence of the larger clone, CP3, was determined (Fig. 1, nt 49-2453). This clone contains a 1380 nt open reading frame which can be translated to yield a

peptide with remarkable homology to mammalian CPH (Fig. 2). Partial sequencing of the clone CP6 showed that it is identical, except that it lacks 373 nt at the 5’ end, Clone CP3, though obviously a cDNA for the AF CPH, is not a full length cDNA clone. Computer-simulated translation of the insert DNA resulted in a peptide large enough to be the mature form of the enzyme; but there was no translation initiation site, nor was there a nucleotide sequence near the 5’ end that might specify a signal peptide, a necessary component of a secretory granule protein. Therefore, another screen of the AF library was performed utilizing an AF CPH specific oligonucleotide derived from the 5’ end of the cDNA sequence. After a screen of 300,000 pfu resulted in no positive clones, it was decided to determine the additional sequence at the 5’ end of the AF CPH by primer extension of mRNA. This approach has been used for the determination of short stretches of 5’ sequence not found in cDNA clones (Wise et al., 1984; Clauser et al., 1988). A 26 base oligonucleotide complementary to a region near the 5’ end of the AF cDNA clone CP3 was hybridized to AF pituitary poly(A)+ RNA. Extension of this primer by reverse transcriptase gave a product approximately 125 nt in length, which is about 95 nt longer at the 5’ end than the cloned AF cDNA. If one adds these 95 nt to the 2407 nt derived from the CP3 sequence (Fig. 11, this accounts for the size of the CPH mRNA that was seen in Northern hybridizations (see below). When a set of primer extension reactions was performed in the presence of dideoxynucleotide analogues, most of the sequence of the template RNA could be determined directly. The sequence at the 5’ end of the AF CPH RNA, appended to the cloned DNA, is shown in Fig. 1 (nt l-48, underlined). This does not include approximately 40 nt at the 5’ end of

Fig. 1. Sequence of AF CPH cDNA. The nucleotide sequence of the cloned AF CPH is shown. The portion determined by primer extension of AF mRNA is underlined. Computer simulated translation of the cDNA shows a long open reading frame followed by the 3’ nontranslated region, which contains several AATAAA cleavage/polyadenylation signals (bold).

175

the full length primer extension product which could not be determined clearly in the sequencing experiments. The extended coding strand sequence in Fig. 1 contains an ATG codon in the appropriate reading frame to be the initiation codon for the CPH precursor. The nucleotide sequence preceding this ATG compares favorably with the proposed consensus sequence for initiation of protein synFISH HUMAN BOVINE RAT

thesis (Kozak, 1984). Translation of the mRNA beginning at this point would result in a 454 amino acid precursor peptide with a hydrophobic signal sequence at the N-terminus. The AF CPH precursor protein sequence was aligned with that of the bovine enzyme using a computer protein sequence alignment program (Genepro version 4.20, Riverside Scientific Enterprises, Seattle, WA, U.S.A.). When the amino

MKQICSIVLL---GA-AWS--LVSA-A-------------MAGRGGS'ALLALCGALAACGWLLGAEAQEPGAPAAGMRRRRR MARRGGCALLVLCGSLAACAWLLGAEARGPGGPVAGARRRRR MAGRGGRVLLALCAALVAGGWLLAAEAQEPGAPAAGMRRRRR

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t A ENTKLAPETKAVIHWIMEIPFVLSANLHGGDWANYPYDETSDPQ S..A....S....A..Q...R.Y.SF..A....N Q . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . L .,........ .L..........S..A....SC...D..Q...R.Y.SF..P....D ~:,::::::::::::::~::::::::::::.L..........S.TA....SC...A..Q.. .R.Y.SF.......N A 0 A RPPCRKHDDDSSFKDGITNGGAWYSVPGGMQDFNYLSSNCFEITLELSCDKFPNEDTLKTYWEQNRNSLVNYIEQV . . . . . .N . . . . . .V..T...........................V....E...P.E.......D.K...IS.L..I . . . . . .N . . . . . .VE.T...A.......................V....E...P.E...N...D.K...IS..Q.I . . . . . .N . . . . . .V..T...........................V....E...P.E...S...D.K...I..L..I

25

101

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LESLMERKEEEREELMDWWKMMSETLNF . FS......K.. ..E........... . . .FS......K....E........... .FS......K....E...........

405

433 434 433 434

Fig. 2. Alignment of translated sequence of AF CPH cDNA with sequences of mammalian CPEs. This figure shows the complete primary amino acid sequence of AF CPH, compared with that of the human, bovine and rat hormone processing CPHs. Putative signal peptides and ‘pro-peptides’ are shown in italics. Dashes indicate residues that are found in the mammalian sequences, but are missing from the fish CPH. Dots indicate residues in the mammalian sequences that are identical with those in the fish CPH. Additional symbols indicate residues involved in Zn binding ft), substrate binding (0). or catalysis f A ) (Fricker et al., 1986; Fricker, 1988). Glycosylation consensus sites are also marked f * ).

176

gion of the AF CPH clone with very little nontranslated sequence. This fragment hybridized to a 2500 nt RNA in preparations from AF pancreatic islets, brains and pituitaries. In the lanes containing RNA from pituitary and islets, a second band of smaller size was also visible. No autoradiographic bands were seen in lanes containing RNA from AF liver, spleen, gill, skeletal muscle or gut. 4.4 -

Discussion 2.4 -

1.4 -

Fig. 3. Expression of CPH in AF tissues. Total RNA from AF tissues was electrophoresed in a denaturing agarose gel, blotted to a nylon membrane and probed with a “P-labeled fragment of the AF CPH cDNA. RNA was prepared from AF liver (lane 1f. spleen (lane 2). brain (lane 3). pituita~ (lane 4), gill (lane 51, pancreatic islets (lane 6). skeletal muscle (lane ?), and gut (lane 8). Each lane was loaded with 15 pg RNA except for lane 4 (pituitary) where only 5 pg were loaded. Migration positions of the RNA markers (BRL RNA ladder) are indicated in kb at the left.

acid sequence of the mature form of bovine CPE (Fricker et al., 1986) was used for the alignment, the homology between the two sequences (Fig. 2) was 81%. If conservative substitutions are included, this figure increases to 88%. Amino acid residues which are thought to be important for activity, by analogy to carboxypeptidases A and B (Fricker et al., 1986; Fricker, 1988), are marked in the figure. Two potential N-linked glycosylation sites are also indicated. All of these residues are conserved in the fish CPH. A portion of the AF CPH cDNA clone CP3 was used to probe Northern blots of RNA prepared from various AF tissues (Fig. 3). A plasmid subclone of CP3 was digested with HincII, which cuts within the linkers, and also within the AF cDNA insert at a site 3’ to the translation termination point. The 1395 bp fragment prepared from this digest contains the protein coding re-

The pancreatic islets of anglerfish are an excellent model system for the study of prohormone synthesis and processing. Hormone precursors made in AF islet cells include proinsulin, proglucagons I and II, prosomatostatins I and II, and anglerfish propeptide Y (aPYI (Goodman et al., 1980, 1982; Hobart et al., 198Oa, b; Lund et al., 1983; Noe et al., 1986). In addition, the AF pancreatic islet is distinct from the exocrine pancreas, allowing recovery of large amounts of essentially pure endocrine tissue from these animals by dissection. A number of important observations regarding hormone biosynthesis have been made using AF islets. Previously, we characterized several AF islet enzymes involved in prohormone processing. These include two endopeptidases involved in the processing of AF somatostatin precursors (Mackin and Noe, 1987a) and an amidating enzyme (Mackin et al., 1987: Noe et processing caral., 1991). Also, a hormone boxypeptidase was identified and characterized from AF islet secretory granules (Mackin and Noe, 1987b). In the present work, we have determined the primary structure of the AF CPH. The similarity between the deduced amino acid sequences of AF CPH and either rat or bovine CPH (81% identity; 88% homology, considering conservative substitutions) indicates a high degree of structural, and presumably functional, conservation of this enzyme during evolution. The sequence homologies extend only to those areas included in the mature, active CPH enzymes (Fig. 2). Interestingly, the ‘pre-pro’ region of the fish CPH does not exhibit the extended propeptide containing five consecutive arginine residues that is found in mammalian CPH, but does have a hydrophobic

177

sequence at the N-terminus. Although signal sequences of AF proteins have not been extensively examined, the structures of the signal peptides for the AF pancreatic prohormones are known. They are very much like other eukaryotic signal peptides, as is the proposed signal sequence for the CPH (Fig. 2, italicized residues). There is a positively charged N-terminus, followed by a stretch of hydrophobic amino acid residues, and several small nonpolar amino acids which fit proposed criteria (van Heijne, 1983) for signal cleavage sites. Application of the matrix described by von Heijne (1986) suggests that cleavage by the signal peptidase could occur in the vicinity of the sequence Ala-Ala-Gly. The mature AF CPH produced by this cleavage would be 433 amino acids long, the same size as bovine CPH. The significance of the fact that the fish CPH lacks the highly basic ‘pro-peptide’ (residues - 1 to -6 in the mammalian CPH species; Fig. 2) is not known. However, one can infer that no posttranslational processing event is necessary to remove this peptide during the maturation of the fish CPH. Like its mammalian homologues, the AF CPH exists in soluble and membrane bound forms (Mackin and Noe, 1987b). Recently, a mechanism for membrane association involving a-helix formation at the carboxyl-terminal end of bovine CPE has been proposed by Fricker et al. (1990). Because of the distribution of amino acids in this C-terminal region, the hydrophobic amino acids are exposed on one side of the helix, thus providing the putative membrane association site. This mechanism may provide the explanation for the membrane association of the AF CPH as well, since the fish and mammalian enzymes have nearly identical amino acid sequences at their carboxyl termini. As shown in the amino acid alignment (Fig. 21, 21 of the 23 C-terminal residues are identical; the other two are conservative changes. Moreover, if one applies the algorithms of Chou and Fasman (using the Genepro program) to predict the probability of a-helix formation to the C-terminal 28 amino acids of both the mammalian and fish CPH sequences, the fish peptide has a higher propensity to form a helix. The substitution in the fish CPH of leucine and methionine for phenylalanine and serine at positions 409 and 410 in bovine CPH, respectively

(Fig, 2), results in the prediction of initiation of a-helix formation at serine 408 in the fish CPH. HeIix formation in bovine CPH is predicted to begin at arginine 412 using the same window parameters in the Genepro program. Helix maintenance in both peptides is predicted through glutamic acid 429. Therefore, it is probable that the same mechanism for membrane association proposed by Fricker et al. (1990) for mammalian CPH applies for the fish CPH as well. We also examined the tissue specific accumulation of the AF CPH mRNA. The major form of AF CPH mRNA is 2.5 kb in length, compared with 3.3 kb for bovine (Fricker et al., 1986) and 2.2 kb for the rat CPH (Rodriguez et al., 1989). As expected, the enzyme was expressed only in endocrine tissues (Fig. 3). The amount of CPH mRNA per pg of total RNA recovered was highest in the AF pituitary, where this enzyme is thought to be necessary for processing of several hormones and neuropeptides. The faster migrating, less intensely labeled bands observed in the pituitary and islet preparations (lanes 4 and 6 in Fig. 3) may result from the presence of smaller AF CPH mRNAs generated by cleavage and polyadenylation at either of the two AATAAA sites upstream from the polyadenylation site at bases 2422 to 2427 (see Fig. I>. Although no comprehensive study to identify enteroendocrine cells in the anglerfish intestine has been performed, it has been demonstrated that mRNAs coding for both preprosomatostatin (G~~odman et al., 1981) and preproglucagon (Lund et al., 1981) are expressed in AF intestine. Therefore, it is somewhat surprising that no CPH-specific mRNA was detected in our extracts of intestine. A possible explanation for this result is that the enteroendocrine cells in the AF gut are widely dispersed and not very numerous, making the levels of CPH mRNA in this tissue extremely low. In support of this suggestion is the finding of Lund et al. (1981) that, when expressed in terms of total mRNA recovered, there is greater than 100-fold more glucagon-specific mRNA in AF islets than in intestine. The AF CPH mRNA was not expressed in any of the non-endocrine tissues examined, suggesting that the enzyme we have characterized is the form of carboxypeptidase used selectively for prohormone processing in AF

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tissues. Overall, it can be seen that the AF CPH is very similar to the CPH enzymes which are believed to be responsible for processing mammalian prohormones. Accordingly, our rest&s strongly support the use of AF islets as a model system in which to investigate prohormone processing enzymes. It will be of great interest to determine the primary structures of other AF enzymes involved in posttranslational processing of prohormones. Acknowledgements

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