Occurrence of a furin-like prohormone processing enzyme in Aplysia neuroendocrine bag cells

Comp. Biochem. PhysioLVol. 105B,No. 2, pp. 345-348, 1993 Printed in Great Britain

0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd

OCCURRENCE OF A FURIN-LIKE PROHORMONE PROCESSING ENZYME IN A P L Y S I A NEUROENDOCRINE BAG CELLS* GREGG T. NAGLE,t~ WALTER R. A. VAN HEUMEN,§ SUSAN L. KNOCK,~¶ ANNA T. GARCIA,§ DAVID A. McCULLOUGHII and ALEXANDERKUROSKY§ tMarine Biomedical Institute; Departments of :~Anatomy and Neurosciences; §Human Biological Chemistry and Genetics; and IIRadiation Therapy, the University of Texas Medical Branch, Galveston, TX 77555, U.S.A. (Tel. 409-772-2771; Fax 409-772-4865)

(Received 26 October 1992; accepted 27 November 1992) Abstract--1. Strong evidence is accumulating that the endoproteases which process prohormones at dibasic residue cleavage sites are members of a subtilisin-related class of proteases. 2. Using the polymerase chain reaction (PCR), we have isolated and characterized an Aplysia californica neuroendocrine bag cell cDNA product (270 base pairs) that encodes a sequence which is highly homologous to the subtilisin-related class of processing proteases that includes yeast Kex2, human/mouse/Drosophila furin, human PC2, and mouse PC1/PC3 and PC2. 3. The characterized cDNA PCR product showed the highest degree of residue identity with the furin-related group of proteins (human/mouse furin 71%; Drosophila furin 63%). 4. These results establish that Aplysia contain a subtilisin-like gene and suggest that the expression of this gene may play a role in processing Aplysia precursor proteins in the bag cells and likely also in the exocrine atrial gland. 5. Furthermore, the Aplysia nucleotide sequence results, together with available sequence information from human, mouse, and Drosophila furins, provide reasonable evidence that the furin-like enzymes may represent a separate subclass of the subtilisin-like processing enzymes.

discharge, a prolonged and synchronous burst of bag cell electrical activity that lasts 30 min or longer (Kupfermann and Kandel, 1970). ELH acts not only on the ovotestis to trigger release of mature oocytes (Rothman et al., 1983), but it also acts as a nonsynaptic neurotransmitter within the abdominal ganglion causing excitation of LB and LC cluster neurons and burst augmentation of neuron RI5. Aplysia calfornica also express three ELH-related genes in the atrial gland, an exocrine organ in the reproductive tract (Nagle et al., 1986). The function of the atrial gland in reproductive activity is unresolved but appears to be related to pheromonal activity (Painter et aL, 1991). The ELH-related genes encode the ELH-related peptides A-ELH, [Ala27]A-ELH, and [Gln 23, Ala27]A-ELH. These peptides are also able to elicit egg laying upon injection into Aplysia. Because the atrial gland produces large amounts of peptides ( ~ 1 mg) and is a relatively simple organ with few other major precursor proteins it provides an excellent model to study precursor processing. Moreover, contrasting the processing of genetically related precursors in the bag cells, a relatively complex neuroendocrine system, to procussing in the atrial gland, a simple exocrine system, should provide more than additive information concerning precursor processing mechanisms in Aplysia.

INTRODUCTION

A remarkable feature of many precursor proteins generating regulatory peptides, such as peptide hormones, is the fact that proteolytic processing frequently occurs at a unique dibasic residue cleavage site (combinations of Lys and Arg). Recently, there has been considerable evidence to indicate that the protease responsible for cleavage at the dibasic site is a subtilisin-like enzyme. A family of these enzymes is indicated and includes, for example, Kex2, furin, and prohormone convertases (PC) (Mizuno et al., 1988; Hatsuzawa et al., 1990; Smeekens and Steiner, 1990; Seidah et al., 1990; van den Ouweland et al., 1990). The neurosecretory bag cells of the mollusc Aplysia calfornica synthesize a 37-kDa egg-laying hormone (ELH) precursor containing a number of dibasic cleavage sites (Scheller et al., 1983). Proteolytic procussing of this precursor gives rise to several peptides including ELH and ct-, fl- and g-bag cell peptides. ELH is a 36-residue peptide that is released from bag cell processes into the hemolymph during an after*Supported by National Institutes of Health Grant NS 29261 (to A.K.) and by Grant H-1190 from the Robert A. Welch Foundation (to A.K. & G.T.N.). ¶S.L.K. is a Jeane B. Kempner Fellow. CBPB 105/2--J

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346 MATERIALS A N D M E T H O D S

Animals Specimens of Aplysia californica were purchased from Alacrity Marine Biological Services (Redondo Beach, California) and were used as a source of bag cells and atrial glands. The animals were housed in individual cages in aquaria containing recirculating artificial seawater (Instant Oceans, Mentor, Ohio) at 14 _ 2°C. All animals used in this study were sexually mature as determined by injection of an extract of the atrial gland (Painter et al., 1991).

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R N A isolation and polymerase chain amplification. The consensus nucleotide sequences of the primers used in the polymerase chain reaction were derived from the amino acid sequences surrounding the asparagine and serine active site residues of the yeast K E X 2 gene (Mizuno et al., 1988), the human fur gene (van den Ouweland et al., 1990), the human insulinoma hPC2 cDNA (Smeekens and Steiner, 1990), and the mouse pituitary mPC1 and mPC2 cDNAs (Seidah et al., 1990). The sense asparagine site primer was: 5'-TCT(A/T)TGT(G/C)TGGGCCTCIGGG (G/A)A(T/C)GGIGG-3'. The antisense serine site primer was: 5'-C(G/A)GCAGCC(T/A)IGGGAGCA G A I G C A G A I G T I C C - 3 ' . The cDNA templates for PCR were prepared from Aplysia californica bag cell and atrial gland total RNA (5 pg) using 10 units of avian myoblastosis virus reverse transcriptase (Promega) essentially as described in the Invitrogen cDNA Cycle Kit protocol. Total R N A was isolated using the RNAzol B procedure (Chomcyznski and Sacchi, 1987). One-quarter of the cDNA products of reverse transcription were used for each subsequent PCR reaction. Reaction mixtures for PCR contained cDNA template, 50pmol of each primer, and 2.5 units of Taq D N A polymerase (Perkin-Elmer Cetus) in 10mM Tris-HCl (pH8.8), 50mM KC1, 1.5mM MgC12, 0.5 mM dNTPs, 0.1% (w/v) Triton X-100 in a volume of 50 #1. Reactions were performed in a Perkin Elmer Cetus D N A thermal cycler for 35 cycles of denaturation (94°C, 1.5 min), annealing (55°C, 2.5 min), and extension (72°C, 2.5 min). An aliquot of each PCR reaction was analyzed by fractionation on a 1.5% (w/v) agarose gel.

DNA sequence analysis The remaining PCR products were blunt-ended with T4 D N A polymerase, fractionated on a 3% NuSieve/1% SeaPlaque agarose gel (FMC BioProducts), and the 270-base pair (bp) product was ligated into the Sma I site of M 13mp 19 (GIBCO BRL). D N A sequencing of single-stranded D N A was performed by the Sanger dideoxy chain termination method using the Sequenase Version 2.0 kit (United States Biochemical Corp.).

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Fig. I. Agarose gel analysis of PCR products from Aplysia bag cell and atrial gland total RNA. Electrophoresis of the products obtained following polymerase chain amplification of bag cell and atrial gland cDNA using the Asn and Ser primers described in Materials and Methods. First strand cDNA was synthesized from total RNA using I00 ng (bag cell, lane 1) and 10 ng (atrial gland, lane 2) Ser primer. The position of the 270-bp band is indicated by the arrow. Size markers are HaeIII-digested ~X174 (GIBCO BRL). R E S U L T S

Two oligonucleotide primers were synthesized based upon consensus nucleotide sequences of the subtilisin domain of Kex2, mPC1, mPC2, hPC2, and hfurin surrounding the serine catalytic site residue and the asparagine residue that stabilizes the enzymesubstrate intermediate (Seidah et al., 1990). These olgonucleotides were used to prime PCR amplification of cDNA synthesized from Aplysia californica bag cell and atrial gland total RNA. Agarose gel electrophoresis of the PCR products revealed a single major 270-bp band in each case (Fig. 1). Since the length of this product was consistent with the distance between the Asn and Ser residues encoded by the subtilisin-related genes, the bag cell PCR product was subcloned for further nucleotide sequence analysis. The sequence of the cloned PCR product (Fig. 2) indicated that one of the two potential open reading frames exhibited significant amino acid sequence identity to the corresponding regions of Kex2, mPCI, mPC2, hPC2, Drosophila furinl, and hfurin (Fig. 3). The comparison of the 270-bp Aplysia PCR product to the furin-related sequences was especially striking. There was 71% residue identity with human/ mouse furin and 63% identity with the Drosophila furin 1 (Table 1). Comparison with human and mouse Xo

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Fig. 3. Alignment of the predicted amino acid sequence deduced from the Aplysia bag cell 270-bp PCR product (Afnrin) with the homologous regions of human furin (van den Ouweland et aL, 1990), Drosophila furin 1 (Roebroek et al., 1991; Hayflick et al., 1992), human insulinoma PC2 (Smeekens and Steiner, 1990), mouse pituitary PCI and PC2 (Seidah et al., 1990), and yeast Kex2 (Mizuno et al., 1988). Gaps ( - ) were introduced to maximize alignment. The asterisk (*) indicates the location of the active site Ser residue. The mouse and human furin sequencesare identical in the region shown. Similarly, mPC1 (Seidah et aL, 1990) and mPC3) Smeekens et al., 1991) are identical. PC sequences showed fewer identities (49-56%). Yeast Kex2 was 37% identical. DISCUSSION These results provide strong evidence that Aplysia californica express a subtilisin-like gene in the bag cell neurons and likely as well in the atrial gland. The predicted amino acid sequence of the PCR-amplified eDNA synthesized from Aplysia bag cell total RNA was remarkably homologous to the furin-like enzymes characterized from human, mouse and Drosophila. This sequence region represents about 30% of the subtilisin-like proteolytic domain. The Table 1. Aminoacid comparisonof Aplysia Afurin to other subtilisin-likeenzymes Enzyme Identities* % hfurio 48/68 71 DfurinI 43/68 63 hPC2 34/70 49 mPC1 38/68 56 mPC2 34/70 49 yKex2 26/70 37 *See Fig. 3 for reports of sequences.

347

addition of the Aplysia subtilisin-like sequence to the furin class of processing enzymes gives considerably more credence to the possibility that the furin-like enzymes may represent a separate subclass of processing enzymes. The PC-related enzymes of human and mouse PC1/PC3 and PC2 appear to be evolutionarily more distant. Furthermore, the overall relatively high degree of homology evident between the various furin-related enzymes of human, mouse, Drosophila, and now Aplysia indicates that the subtilisin-like proteolytic domain of these enzymes is well conserved. The residue identity between human and mouse furin was 99%. Further evidence that adds support to the occurrence of furins as a separate subclass of subtilisin-related enzymes is indicated in their structure. The furin enzymes possess a potential transmembrane consensus motif and with the exception of Dfurin have a cysteine-rich sequence segment in the carboxy-terminal region. The PC-related enzymes have neither of these sequence motifs. Clearly, more structural information of subtilisin-like gene products will be required to establish these relationships more definitively. Smeekens et al. (1991) have suggested that the furin-like putative processing enzymes may function mainly in the trans Golgi on precursor proteins secreted via unregulated or constitutive pathways whereas the role of PC2 and PC1/PC3 may relate more to processing of endocrine hormones and neuropeptides. This conclusion was drawn from the observation that furin is expressed in a number of different tissues, including heart, muscle, lung, and testis, in several animals studied (Schalken et al., 1987). This is an interesting possibility that still remains an open question; however, there is evidence that the early cleavage event in the processing of proELH may be a constitutive event occurring in the trans Golgi (Sossin et al., 1989). In this regard, it will be important to inquire whether or not Afurin processes the ELH-related precursors in the bag cell neurons and in the atrial gland. Since the ELH-related peptides occur in high concentration in the atrial gland ( ~ 1 mg) and represent the majority of the peptides produced, the atrial gland should provide an excellent model to evaluate the mode of action of this important candidate dibasic amino acid processing enzyme. Acknowledgements--We thank Drs A. Neil Howell and David W. Niesel for helpful discussions and Angelina Mouton for preparation of the manuscript. REFERENCES

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