Proc. NatI. Acad. Sci. USAVol. 89, pp. 6678-6682, August 1992Biochemistry

Conservation of the prohormone convertase gene family in m'tazoa:Analysis of cDNAs encoding a PC3-like protein from hydra

(subtilisin-related proprotein convertases/furin/PACE/kex2/precursor processing)

SHU JIN CHAN*, ANTHONY A. OLIVA, JR.*, JOSEPH LAMENDOLA*, ANN GRENSt, HANS BODEt,AND DONALD F. STEINER**Howard Hughes Medical Institute, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637; and tDepartment of Developmental and CellBiology, University of California, Irvine, CA 92717

Contributed by Donald F. Steiner, March 24, 1992

ABSTRACT A subclass of proteolytic enzymes that cor-rectly cleave precursor proteins at paired basic residues and arestructurally related to the bacterial subtilisins has recently beenidentified. In yeast, a single membrane-bound proteolyticprocessing enzyme encoded by the kex2 gene has been found,whereas in higher vertebrates cDNAs encoding four distinctenzymes (PC2, PC3, furin, and PACE 4) have been identified.Like kex2, furin (also known as PACE) contains a hydrophobictransmuembrane domain, but PC2, PC3, and PACE 4 lack thisfeature. All five enzymes exhibit striking similarities in theircatalytic domains, and this suggests that they have arisen froma common ancestral subtilisin-like gene. We report here theidentification of cDNAs encoding a protein that is similar instructure to PC3 from a simple metazoan, Hydra vulgaris(formerly Hydra attenuata). cDNAs encoding two isoforms ofthis PC3-like enzyme were obtained that differ only in theircarboxyl-terminal sequences, probably due to alternative splic-ing of a common pre-mRNA. Neither form contains a trans-membrane domain. Predicted amino acid sequence compari-sons revealed that the hydra PC3-like enzyme is 55.4% and56.7% identical in the catalytic domain to mouse PC3 andhuman furin, respectively. RNA blot analyses revealed that thePC3-like RNA is expressed predominantly in the hydra bodycolumn and not in the head region, although the hydra headcontains a hipg density ofnerve cells, which synthesize a varietyof neuropeptides. For this reason, we suspect that anotherproprotein cleavage enzyme isoform may be expressed in headnerve cells. The isolation of a PC3-like cDNA from hydra isconsistent with the presence of neuroendocrine cells and indi-cates that the PC/furin gene family has been well conserved inall metazoa. A simplife nomenclature for the group ofmammalian processing proteases is proposed.

In eukaryotes many extracellular or plasma membrane pro-teins, including peptide hormones and their receptors, aresynthesized as preproproteins, which must be proteolyticallyprocessed to attain their mature forms. An initial cleavageoccurs in the endoplasmic reticulum to remove the amino-terminal signal peptide, and the proprotein is further pro-cessed in the Golgi or secretion granule (1). Structural studieshave shown that although the proproteins have differentprimary sequences, they share a common motif for process-ing; i.e., endoproteolytic cleavage usually occurs on thecarboxyl side of paired basic residues (Lys-Arg or Arg-Arg)and is followed by the action of a carboxypeptidase B-likeenzyme, which removes the basic amino acids from the newcarboxyl terminus (1, 2). In vertebrates, the carboxypeptid-ase activity has been identified as carboxypeptidase E (or H),

and its cDNA and gene have been cloned in several species(3, 4).More recently, cDNAs and genes encoding the endopro-

tease activity have been identified in two disparate classes oforganisms, fungi and mammals. In yeast, the kex2 geneencodes a membrane-bound enzyme that was shown to be aserine proteinase structurally related to the bacterial subtili-sins (5, 6). In mammals, cDNAs encoding four subtilisin-likeprocessing enzymes, PC2 (7-9), PC3/PC1 (10, 11), furin (12,13), and PACE 4 (14),t have been cloned. Moreover, cDNAsencoding furin-like enzymes have also been recently identi-fied in Drosophila melanogaster (15) and Caenorhabditiselegans (16). The catalytic domains of the yeast kex2 andmammalian SPC1, SPC2, SPC3, and SPC4 enzymes, as wellas the insect and nematode furin-like enzymes, all share aclose similarity in amino acid sequence, which suggests thatthey are both functionally and evolutionarily related. Tobetter define evolutionary relationships within this superfam-ily, it is important to investigate the phylogenetic distributionof these genes in more primitive metazoans. We report herethe isolation ofacDNA that encodes a PC3-like enzyme froma representative cnidarian (coelenterate), Hydra vulgaris.§Since the cnidarians are considered the most simple orga-nisms that possess specialized tissues, including nerve cells,this finding indicates that the prohormone convertase genefamily has been conserved throughout metazoan evolution.

MATERIALS AND METHODSMaterials. Oligonucleotides were synthesized on an Ap-

plied Biosystems model 380B DNA synthesizer. PlasmidpEFla contains a 1.2-kilobase (kb) cDNA insert encodinghydra elongation factor 1 (A.G., unpublished results). DNAfragments were labeled with [a-32P]dCTP (3000 Ci/mmol; 1Ci = 37 GBq) by using a nick-translation kit from Amersham.RNA and PCR Analysis. H. vulgaris were grown and

harvested under standard culture conditions (17). RNA wasprepared by using the guanidinium thiocyanate method (18);poly(A)+ RNA was isolated by passage twice through anoligo(dT)-cellulose column. RNA blots were prepared from a

Abbreviation: SPC, subtilisin-related proprotein convertase.tTo provide a simple uniform terminology for these mammalianserine proteases, we propose that they be designated subtilisin-relatedproprotein convertases (i.e., SPCs) and specifically that theybe numbered in the order of their discovery as follows: furin/PACE= SPC1, PC2 = SPC2, PC3/PC1 = SPC3, and PACE 4 = SPC4. Itis suggested that this designation be used along with lowercaseabbreviations for various species (i.e., mouse SPC3 or mSPC3).(Note that SPC can also designate secretory pathway convertase.)This nomenclature will be used throughout the remainder ofthis textwith the alternative names given in parentheses as necessary, toavoid confusion.§The sequences reported here have been deposited in the GenBankdata base (accession nos. M95931 and M95932).

6678

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Proc. Natl. Acad. Sci. USA 89 (1992) 6679

5'UT 1 PRO CATALYTIC DOMAIN V Na>a_ I 3'U T

-i XHy-2

I-INTR'N I

5'UT| S I PRO I CATALYTIC DOMAIN tSb 3'UT

X Hy-1

XHy-14

FIG. 1. Cloning of hydra SPC3-like cDNA. The organization of cDNAs encoding hydra SPC3-like type a and b isoforms is drawnschematically, and the regions covered by cloned cDNAs isolated from a Agt11 hydra cDNA library are shown. AHy-3 contains an unsplicedintron located at the 5' end. The signal peptide (S), propeptide domain (PRO), and catalytic domain are labeled; hatched boxes denote the differenta and b carboxyl-terminal coding sequences. UT, untranslated.

1.0% agarose/0.66 M formaldehyde gel, and hybridizationswere performed as described (19).PCR was performed in a Perkin-Elmer/Cetus DNA cycler.

Degenerate oligonucleotide primers corresponding to con-

served amino acid sequences found in PC2, PC3, furin, andkex2 were used: SQ-3, 5'-CAYGGNACNCGNTGYGC-3';SQ-4, 5'-CAYGGNACNAGRTGYGC-3'; QS-1, 5'-YTGCA-TRTCYCTCCANGT-3'; QS-2, 5'-YTGCATRTCNCGC-CANGT-3'; QS-3, 5'-YTGNACRTCYCTCCANGT-3';QS-4, 5'-YTGNACRTCNCGCCANGT-3', where R = G or

A, Y = C or T, and N = G, A, T, or C. SQ-3 and SQ-4 are

sense primets that encode the amino acid sequence His-Gly-Thr-Arg-Cys-Ala, and QS-1, QS-2, QS-3, and QS-4 are

cDNAs that correspond to the sequence Thr-Trp-Arg-Asp-Met/Val-Glh.The cDNA template for PCR was synthesized from hydra

poly(A)+ RNA or total RNA by using dT12_18 primer andmurine leukemia virus reverse transcriptase (BRL). PCRamplification was programmed for 30 cycles, with each cycleconsisting of 94°C for 1 minm 50°C for 2 min, and 70°C for 2min. PCR was also used to generate specific DNA fragmentsfor hybridization in which case the annealing temperaturewas increased to 56°C.cDNA Cloning. A hydra cDNA library cloned in Agtll,

complexity of 1.5 x 106 plaques, was constructed by DougFisher (University of California, Irvine). The library wasscreened, and plaques were isolated by standard techniques(20). cDNA inserts were excised from phage DNA by diges-tion with EcoRI, subcloned into pGEM4Z (Promega), andsequenced by using a Sequenase kit (United States Biochem-ical).

RESULTS

To determine whether hydra express proteolytic enzyme(s)belonging to the SPC family, we utilized the PCR techniqueto amplify DNA fragments that encode a region within thesubtilisin-like catalytic domain. Degenerate oligonucleotideprimers (see Materials and Methods), were used to amplifytotal hydra cDNA. One primer set, SQ-3 and QS-1, yieldedthe predicted 600-base-pair (bp) fragment, which was sub-cloned. Six independent plasmid clones were analyzed andwere found to contain an identical subtilisin-related se-quence.The 600-bp sequence was then used to screen a hydra

cDNA phage library. From approximately 360,000 recombi-nants, 58 positives were identified; of these, 14 were exam-ined by restriction mapping, and five plaques were purifiedand sequenced. Sequence analysis revealed that the cDNAclones encoded two isoforms of a hydra PC-like enzyme,which differ in their carboxyl-terminal amino acid sequences,although the nucleotide sequence encoding the catalytic

domain was identical in all five clones. AHy-3 and AHy-7contained a 380-bp carboxyl coding sequence (type a) and 3'untranslated region that differed from a 135-bp coding se-quence (type b) and 3' untranslated region found in clonesAHy-1 and AHy-14 (Fig. 1). We also deduced that AHy-3contained an unspliced intron located at the 5' end of thecDNA based on sequence comparison with the other clonesand the finding that the junction site contains a consensusintron acceptor sequence (TCCCTGTGTAATAAi). More-over, an intron is located at the same position in both thehuman SPC1 and SPC2 genes (21, 22).The nucleotide and predicted amino acid sequences for the

two hydra SPC cDNA isoforms are presented in Fig. 2. Thetype a sequence contains a 2379-nucleotide open readingframe encoding 792 amino acids, which we propose is orga-nized as follows: 29-amino-acid signal peptide, 119-amino-acid propeptide domain, and 641 amino acids in the matureenzyme. In the type b isoform, the predicted mature enzymeis smaller and contains 558 residues. Neither isoform con-tains an internal hydrophobic segment, and thus these are notpredicted to be integral membrane proteins.The amino acid sequence of the hydra protein was com-

pared with mammalian SPC1, -2, -3, and -4 and yeast kex2 inFig. 3, and the calculated sequence identities are summarizedin Table 1. Within the catalytic domain, the highest percent-age sequence identity was obtained with mouse SPC3 (55.4%)and human SPC1 (56.7%) followed by human SPC4 (52.3%),human SPC2 (50.2%), and yeast kex2 (46.8%). Significantsimilarity was also observed in the propeptide region and ina 163-residue intermediate domain immediately adjacent tothe catalytic domain. Possibly these regions may be impor-tant for the correct folding of the enzyme and in substratebinding (24, 25). However, based on the sequence similarityin the catalytic domain and the finding that the hydra protein,unlike SPC1 and kex2, does not contain a transmembranesequence, we have designated the hydra isoforms as SPC3-like enzymes.Northern blot analysis confirmed that the two hydra SPC3-

like isoform cDNAs were not cloning artifacts but were most

Table 1. Identity of hydra SPC3-like amino acid sequencecompared with mammalian SPC1, -2, -3, and -4 and yeast kex2

% identity

Propeptide Catalytic Intermediatedomain domain domain

mSPC3 40.0 55.4 37.4hnSPC1 38.8 56.7 35.0hnSPC4 33.8 52.3 41.1hnSPC2 28.8 50.2 40.5ykex2 9.9 46.8 28.8

Percent identity was calculated from sequences shown in Fig. 3. m,mouse; hn, human; y, yeast.

XHy-3

-.4X Hy-7

Biochemistry: Chan et al.

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6680 Biochemistry: Chan et al. Proc. Natl. Acad. Sci. USA 89 (1992)

1 10 20Hot AMn Tyr Arg Gly Ile Tyr Arg Arg Arg Tyr Va1 Ph. Val Lou Lou Lou Lou Val Ala Val Val Asn Ile S.r Tyr Gly

ATTATAATT ATG ARC TAT MGG GGT ATA TAT COC CGA AGA TAT GTA TTT OTT TTA CTG CTT CTG OTT OCT GTA OTT AAT ATA TCA TAT GOTTh+30 40 50

Trp hzVal Lou Lys Ann Lye Asp Tyr Lysf Arg Arq Tyr Lou Mrt Pro SMr Gly Va1 Glu Lys Va1 Arg Lys Rix Lou Mrt Arq Lys TyrTOG ACT GTC TTA AAA MT MAA OAT TAC MAA COT AGA TAC CTT TCG CCG TCG GSA GTA GAA MAA OTT COC MAA CAT TTA ACT AGA MAA TAT

60 70 s0Va1 Ala SMr Arg Asa Mn Thr Gin Thr Ph. Lys Lys Rie Tyr Ph. Mrt Mn Thr Trp Ala Val His Ile Asp Pro Pro Amp mn Asp Va1OTT OCA TOT COC MRT MRT ACA CAA ACG TTT MAA MAA CAT TAT TTC TCT MAT ACT TOG OCO GTG CAT ATT GAC CCC CCA GAC MRT OAT OTA

90 100 110Ala Asp Arg Ile Ala Lys Lys His Gly Ph. Thr Msn Ile Gly Lye Ile Gly Msn Ile Glu Gly His Tyr Rim Ph. Lye His Glu Glu IleOCA OAT AGA AT? OCA MAA MAA CAT GOT TTT ACA MT ATA GMA AAG ATA MGA MRT ATT GRA GOGT CAC TAT CAT TTC MG CAT GMA GMA ATA

120 130 140Gly Glu Arg Glu Lou Glu Lys Ala Arg His Lys Thr Ala Lou Lou Msn Lou Glu Asp Glu Va1 Lye Ph. Ala Glu Gin Gin Lys Ile LouGSA GAG COA MAO tTA GM MAA OCT MA"CAC MAA ACA OCT CT? CTT MRT CTA GM GAT GAG OTA MAA TTT OC-A GAG CAG CAG MRA ATC TTA

150 E 160 ~170-Glu krg Val Lys Arg*Aep Gly Ile Pro AMn Asp Pro Tyr Ph. Lye Asp Hot Trp Tyr Lou Lou Mnn Thr Gly Gin Ala Mrt Gly Pro AlaGM MGA GTT MAA MGA OAT 00? AT? CCA MRT OAT CC? TAC TTC MG OAC ATO TOG TAT TTA CTA MRT ACT GOT CAG OCT MGT WOC CC? OCT

160 190 200Gly Va1 Asp Hot AMn Va1 Va1 Pro Va1 Trp Lys Lys Msn Ile Thr Sly Arg Gly Ile Va1 Ile Mrt Va1 Lou Asp Asp Gly Lou Asp TipGOC OTA OAT ATO MT OTT OTT CCT OTT TOG MG MAA AAC ATT ACT GOC MGA GOA ATC OTT ATA TCA GTM CTG OAT OAT GOC TTA OAT TOG

210 220 230Thr Hie Pro Asp Lou Glu Al. Msn Tyr Asp Gin Thr Ala Mrt Ile Va1 Lou Msn Asp Msn Asp Msn Asp Pro Hot Pro Arg Asp Mrt Asp-ACA CAC CCA OAC CTO GM WCA MC TAT OAT CMA ACA WCA MGT AT? GTC CT? MRT OAT MRT GAC MRT OAT CCA ATG CCA COT OAT AMC OAT

240 250 260Al. Asp AMn Cye Rio Gly Thr Arg Cye Ala Gly Glu Ala Al. Al* 11e Ala Msn AMn Gly Ile Cye Gly Thr Gly Va1 Ala Tyr Msn Al.OCT OAT MRT TCT CAT GOT ACC MGA TGT GCA GWA GAG OCA OCT OCA ATT OCT MRC MRT GOT ATC TOT GOT ACT 000 OTT OCO TAC MT OCT

270 290 290Lye Ile Gly Gly Va1 Arg Hot Lou Asp Gly Gin Al. Thr Asp Al. Lou Glu Al. Mrt Al. Lou Gly Ph. Arg Gly Asp Rie Ile Asp IleMAA ATT GSA GSA OTA COT ATG CTA OAT GOT CM WCA ACT OAT OCA TTA GM OCT MGT OCT TTA GOGG TTT CGA GSA GRC CAT AT? OAT AT?

300 310 320Tyr Ile AMn Cys Tip Gly Pro Lye Asp Asp Gly Lye Thr Ph.s Gly Lye Pro Gly Pro Hot Al. Ala Lye Al. Lou Arg Lou Oly Al. OiuTAC ATA MT TOT TOO GSA CCA MAA OAT OAT GSA MAA ACA TT GSA MAA CCA GSA CCG ATG OCT MCA MAA OCA CTG MGG CTT GOT OCT GMA

330 340 350Gin Gly Arg Mna Arg Lou Gly Mrt Ile Ph. Va1 Tip Ala Thr Gly Msn Gly Gly Lou Thr Asp Asp Asp Cye AMn Cye Asp Gly Tyr ThrCMR GSA MGA MT COT TTA GSA TCA AT? TTT OTA TOO WCA ACT GOT MRT GOT GOT TTA ACA OAT OAT OAC TOT MAC TGT OAT 000 TAT ACA

360 370 300Thr Mrt Ile Ph. Thr II* Mrt II. Gly Cye Ile Gly Asp Hie Gly Lou Mrt Ala Tyr Tyr Thr Giu Lye Cy etMr M Thr Lou Ala ValACA MOT ATA TT ACA ATA TCA ATA 0CC TOC ATA GSA GAC CAT GSA TTA TCA 0CC TAT TAT ACA GAG MAA TOT TCA TCA ACT CTT OCT GTC

390 400 410Thr Ph. Msn Gly Ala Mrt Hie Lye Glu Gly Arq Olu Msn Lye Net Val Thr Thr Asp Lou Tyr Rie Gin Cye Thr Glu Glu Ph. Lye GlyACA TTT MT GOT OCA TCA CAT MAA GMA GSA COA GM MRC MAA ATG OTA ACA ACA OAT TTA TAT CAT CMA TOC ACT GMk OAG if? MAA GSA

420 430 440Thr Mrt Ala Mrt Ala Pro Lou Ala Ala Gly Ile Ile Ale Lou Thr Lou Glu Ala Msn Pro Lou Lou Thr Tip Arg Asp Val Gin Ale LouACO TCA OCT TCA MCA CCA ifA OCA OCT GOT AT? ATT OCT TA ACA TTG GM OCA MhT CCA TTG ifO ACT TOOGAMA OAT OTT CMA OCA CTO

450 460 470Ile Va1 Hie Thr Ala Gin Ii. Thr Mrt Pro Va1 Asp Giu Gly Tip Lye Arg Msn Gly Ala Gly Ph. Hie Ph. Msn lie Lye Ph. Gly Ph.AT? OTA CAT ACT OCA CMA ATA AC? TCT CCA OTT OAT GMA GSA TOG MAA MGA MT GOT OCT GOT TTC CAC TTT MRT CAT MAA TTT GOT iTT

460 490 500Sly Arg Lou Asp Ala Msn Ala Hot Val Msn Ala Ala Gin Mrt Tip Lye Msn Lou Pro Ala Gin Arq Lye Cys Thr Ala Ala Mrt Gly Ph.GSA MGA TA OAT OCA MRC OCA ATG GTG MAT OCA OCT CMA MT TOG MAA MAC TTG CC? 0CC CM MGA MAA TOC ACA OCT OCT ICA GSA IT?

510 520 530Asp lie Gin Asp II* Pro Arq Gly Asp Mrt Lou Ph. Il. Mnn Ile Pro Thr Val Ala Cys Glu Mrt Mrt Mr Ala Gin Ile Ala Lye Va1OAT CAC CAG OAT ATT CCA COA GOOGOC TOT TA TT ATT MRC AT? CC? ACA OTT MCA TOT GMA MC MGC TCA OCA CMA AT? GCA MAA OTT

540 550 560Glu lie Val Val Lou Thr Val Mar Ph. Val lie Arq AMg Arg Gly Asp Val Mrt Ii. Asp Lou Ile Mrt Pro Lye Asp Thr Lye Mrt GlnOAG CAT GTM OTT TTG ACA GTC MGT TTT OTT CAT MGA COT COA GSA OAC GTC MGT ATA OAT TTG AT? TOT CCA MAG OAT ACA MAA MGT CMA

570 560 590Not Lou Mtr Pro Mrg Lye Tyr Asp Asp Mrt Asp Glu Gly Lou Asp Glu Tip Mtr Ph. Met Thr Val Tyr Msn Tip Gly Glu Man Pro LyeATO TTO MOT CCC MGA MAA TAT OAT OAC TCT OAT GM GOS? TTA OAT GMA TOG MGT if? ATG ACT OTT TAT MRC TOG GOT GM MRT CCG MAA

600 610 620Gly Ile Tip Mrg Lou Lye Ii. Thr Asp AMn Pro Msn Gin Asp Asp Va1 Hot Msn Lou Ph. Men Gly Asp Mna Thr Asp Asp Va1 Giu MorGOT AT? TOO COT ifA MRA AT? ACT OAT MT CCA MRT CM OAT GhC OTT ATG MAC CTC ifT MAT GO? OAT MRT ACA OAT OAT OTA OAG TCT

630 640 650Lou Glu Glu Mrg Va1 Ile Asp Thr Gin Thr Lye Gin Msn Lye Ala Glu Tip Glu Lye Hot Mrg Lye Glu Msn Pro Tyr Ph. Asp Val ProCTC GAG GAO COA GTC ATA OAC ACT CMA ACT MAA CAG MC MAA OCT GAG ToG GM MAA ATG COC MAA OAG MC CCA TAC if? OAT OTT CCA

660 670 660Tyr Pro Thr Gly Va1 Mgq Lye Asp Lye Va1 Lou Gly Mtr Thr Glu Ile Mnn Asp Mn Mrt Ph. Asp Thr Pro lie Thr Glu Thr Ph. LyeTAC CCA ACO GSA OTA COC MAA OAT MAA GTG CT? GAn TOG ACA GMA ATA MRT OAT ARC MOT TTT OAT ACA CC? CAT ACA OAG ACC if? AMA

T AC ACG ATT GM GOT MGC ACT CMA GAC CAT GTG MA CC? MRA GM GSA OCT MAA GMA CCC TOGMsn Thr Il. Oiu Gly Mrt Thr Gin Asp lie Val Lye Pro Lye Glu Gly Ala Lye Glu Pro Tip690 700 710

Il. Ile Mrg Msn lie Il. Pro Glu Val Msn Lou Gin AMn Mn Asp Mn Net Msn Thr Lou Msn Ph. Asp Pro Val Thr Sly Mgq Lye LyeATA ATC COT MRT CAC A?? CCA GAG OTT MT TTA CAG MRT MRT OAT MRC ATG MAT ACA ifA MT TTC OAT CCT GTC ACT GSA COG MAA MAAGSA MRT TA? MGA MRT ACT MRC MRT ATC MRT MRC MRT TCC MGT ACA OCT if? MAA COG MAA AMAMA CAG TMA TAMTATArTTATOOAOATAMAGly Msn Tyr Mrg Msn Thr Mnn Asn I1. Mn Mnn Mn Mar Mrt Thr Ale Ph.e Lye Arg Lye Lye Lye Gin CC

720 730 740Mna Mrt 1i. Mn Lye Lye Ile Ile Msn Mrt Arq Lye Mrg Msn Ph. Lou Thr Ph. Mrg Mnn Ph* Lou Lye Lye Mrt Lye Lye Val Gin ValMRT TOG ATC MRT MRA MG AT? ATA AmC TOT MG MAA MGA MC if? ifA ACA TTC MGA MRT ITT CTG MAA MRA MT MAA MG OTA CM GTG

750 760 770Gin Gin GIU Glu Thr Gly Thr Gin Arg Val Gin Va1 Mna Ala Gly Tyr G1u Msn Pro Arg Ile Mrt Cye Glu Mrt GIy Tyr Thr Thr CYsCMA CM GMA GMA ACG GSA ACG CMA COT OTA CMA 0?? MT QCC GOC TAC GMA MT CCA MA ATA TCA TOT GM MCT OGG TAT ACA AkCT TOTCCGCTATAGr~TTAATTCOTT TAhCCAA~~tATTTCAATTCT11jiTAGAC&TGTTTTAO760 790 793'MrV Gly Val Lou 21. Msn Tyr Lye Lou Thr Ph. Tyr Gly Thr Gly Glu OCACATCTTGTTGTOT GOT OTT TTA ATA MRT TAC MAA CTA ACT if? TAT GOT ACT GO? GMA TACCTTOTOCAMTGiTTTATATTOTGOTTTTTTTTTTTTACCTTMTAOTGCOTMTOWTTTAWAMATTTTAA~MA

FIG. 2. Nucleotide and deduced amino acid sequences of cDNAs encoding hydra SPC3-like a and b isoforms. The sequence of the type aisoform is given with sequence differences found in the b isoform written below. The subtilisin-related catalytic domain is boxed; arrows indicateputative cleavage sites that remove the signal peptide and propeptide domain.

likely due to alternative splicing ofa single precursor mRNA.By using the catalytic domain cDNA fragment as the probe,hybridization of hydra poly(A)+ RNA revealed three bands:a faint 9.4-kb band and strong bands at 3.5, 3.0, and 1.4 kb(Fig. 4, lane a). We are uncertain as to the nature ofthe 1.4-kbRNA species since it is clearly too small to contain the entirecoding sequence. In a separate experiment, the type a-spe-cific cDNA fragment hybridized only to the 3.5- and 3.0-kbRNAs (Fig. 4, lane b). Subsequently, when the same blot wasstripped and reprobed with type b cDNA, the 9.4-kb RNAwas visualized (Fig. 4, lane c). Since the autoradiogramrevealed that the 3.5- and 3.0-kb RNAs were the dominantspecies, we infer that the type a sequence is the major hydraSPC3-like isoform. However, sequence differences betweenthe 3.5- and 3.0-kb RNAs have not been determined.

In higher vertebrates, it has been shown that SPC1 (furin)mRNA is expressed in most tissues, whereas the expression

of SPC2 and SPC3 mRNAs is limited to neuronal andneuroendocrine cells (8, 10, 11, 26). Thus, it was of interestto determine whether the expression of hydra SPC3-likemRNA was tissue or cell specific. In the case of hydra, thedensity of nerve cells in the head region of this organism isabout 5 times higher than in the body column (27). Total RNAwas isolated from excised hydra head and body columnsections and used for Northern analysis. Surprisingly, SPC3-like mRNA was detected only in the body column RNA (Fig.5B). As a control, the RNA blot was also probed with a hydraelongation factor (EFla) cDNA. As shown in Fig. SC, EFlamRNA was clearly detected in both head and body columnRNA, albeit the signal was somewhat stronger from the bodycolumn RNA. As an additional check, we amplified a SPC3-like cDNA fragment by PCR from equivalent amounts ofhead or body column RNA that had been reverse transcribedinto cDNA. After 30 repetitive cycles, an ethidium bromide-

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pro-peptide domainKHYFSNTWAVHIDPPDNDVADRIAKKHGFTNISKIGNIEGHYHFKHEEIGERELEKARHKKRQFVNEUAAEI -PGGQEMSAIAEELGYDLLGQIGSLENHYLFK)0(SHPRRSRRSALHIQKVFTNTIUAVRI -PGGPAVANSVARKHGFLNLGQI - -GFDYYHFIHRGVTKRSLSPHRPRRPVYTNHNAVQV-LGGPAEADRVAAAHBYLNLGQIGNLEDYYHFYHSKTFKRSTLSSRGPRPVFTNHFLVELHKG6EDKARQVMEHGFG-VRKLPFAEGLYHFYHNGLAKAKRRRSLHHLEEMHPNtKYEHDVRG- -LPNHYVFSKELLKLGKRSSLEELQGDNNDHILSVHDLFPRNDL

Proc. Natl. Acad. Sci. USA 89 (1992) 6681

a b c128868312585106

catalytic domainhySPC3-l ike TALLNLEDEVKFAEQWKILI -------DGIPNDPYFKOI YLLNTGQASGPAGVD 180SPC3 TKRLSDDDRVTWAEQQYEKE SIZ SVQKDSALDLFNDPMNQQWYLQDTRMTAALPKLD 145

hnSPKI HSRLQREPQVQWLEQQVAKIT -----DVYQEPTDPVKFPQQWYLSVTQ------RD 131hnSPC4 HTFLRMDPQVKWLQQQEVKI VKN- QVRSDPQALYFNDPIWSNIIYLHCGDKNSRCR-SE 183hnSPC2 KQQLERDPRVYKMLQQE6FJ(JCYRDINEIDINJhLFTKQVYLINTGQADGTPGLD 145

kex2 FKRLPVPAPPHDSSLLPVKEAEDK--------LSI-INPLFERQIHLVNPSF----PGSD 153VhySPC3-like NVVPVIW KKNITGRGIVISVLDGLDWTHPDLEAEYDQTASIVNDNDN P.-DSDAD 239

SPC3 LHVIPVVEKGITGKWVVITVLDDGLEWNHTDIYANYDPEASYDFNDHPFPRYDLTNE 205hnSPC1 LNVKAAMQCYTGHGIVWSILDDGIEVNHPDLAGUYDPGASFDVDQDPDPQPyTQMR D 191hnSPC4 W0AA NYTOMVMILD IERNFPDLAUMSYASYDOVYUni YnASNF M

LIWAYEAELGYTSKGVTIi 1DI-,Y"LHPDLASUINAEASYDFSS PyIPRY1DDWFIWLDLWYNNITGAGVVAAIVDOSLDYENEDLKDOFCAEGSWDFNDNTNLPKPR-LSD- -

3.50k3.01"

205210

NCHGTKKRCEAAMIAIUGIC6TVAYNAKIGGVMLDGQA-TDALEASALGFRGDHIDIY 298NKH1TRCMEIKMQANUIKC VCVAYNSKVGGI.LDSIV-TDAIEASSIGFNPGHVDIY 264NRHTRCMEVAVANNGVCSV6AYNARIGBVUTDGYEV-TDAVEARSLGLNPNHIHIY 250NKNHTrCEVMSAMSYC IVGIAYNAKIGIMlDSDV-TDVVEAKSLGI RPNYIDIY 302NSHCThCMEVSAAAIINIC AYNVSKAVAY IWADQPFNTDI IEASSISH1MPQLIDIY 265DYHGTRCMEIAAKKG6NFCGVGVGYNIKISGIRILSCDI-TTEDEAASLIYGLDVNDIY 269

INCWVPD6KTFGKPGPHAAKALRLGAEQGLRNRLRSLIFWAT UfLTWOOCNCG6M 358SASCMP EGPGRLAQKAFEYSVKQGRQGKGSIFVAtSUNGGRQGDNCDOGYTD 324SASWSPEDGKTVDGPARLAEAFFRGVSQGGGLGSIFDWS8W EDHSCNCDGYTN 310SASW6PODLAKTV QPGRLAKF EYCIKKGRQGLSIFV6ASUYSREGDYCSCDYTN 362SASWTDNGKTVDGPRDVTLQNDStVNKGRGCSIYWAS6D-SYDCCNC YAS 324SCS9PADODGRHLQGPSDLVKKALVKSVTEGRDSKGAIYVFASG TRGDNCNYDGYTN 329

SIFTISIGCIGIDHBLSAYYTEKCSSTUVTFNGASHKEGRENKNVTTDLYHQCTEEFKGT 418SIYTISISSASQQ6LSPWYAEKCSSTUTSYSSGDYTDQR--- ITSADLINDCTETHTGT 481SIYTLSISSATQFGNVPVYSEACSSTLATTYSS6NQNEKQ --- IVTTDLRQKCTESHTGT 367SIYTISVSSATENCYKPWYLEECASTLATMSSAFYERK --- IVTTDLRQRCTDGHTGT 419SNWTISINSAINDGRTALYDESCSSTLASTFSNSNPE-AGVATTDLYGNCTLRHST 383SIYSITIGAIDHKDLHPYSEGCSAVMAVTYSSGS----- GEYIHSSDINGRCSNSHGGT 384

intermediate domainSASAPLAAGIIALTLEAIPLLTVQ/UI VIHTAQITS--PVDEGMR FHFNHKFG 478SASALAACIFALALEANPNLTWMQHI WTSEYDP- LASNPGWtUWKGL"SRFG 440

SASAPLAAI IALTLEAIKNLTWDMQHI WQTSKPAH- -LNANDMATNSV6RKVSHSYG 425SVSA-VACI IALALEAISQLT1DVQHL LVKTSRPAH--LKASDSIKVNGAGHKVSHFYG 477SAAAPEAAGVFALALEANLGLTNDMQHL LTSKRNQLHDEVHQVRRNGVGLEFNHLFG 443SAAWLAAGVYTLLLEANPNLTWVQY ILSAVGLE-KNADGIDVRDSAMGKKYSHRYS 443

FBRLDANAMVN- -MQSVKNLPAQRKCTA-ASGFDIQOIPRGDSLFINIPTVACESSSAQ 533FCLLNAKALVDLADPRTVRNVPEKKECVVKDNNFEPRALKANGEVIVEIPTRACE6QENA 500YCLLDVAGANA--LAQNWTTVAPQRKCII-DILTEPKDI--GKRLEVRKTVTACLGEPNH 480FCLVDAEALVV - - EAKIKUITAVPSQHMCVA-ASDKRPRS IPLVQVLRTTALTSACAEHSDQR 535YCVLDAGVK--NAK0WKTVPERFHCVG-GS6QSPEKIPSTGKLVLTLTTDACEKENF 500F6KIDAHKLIE- -MSKTVENVNAQTWFYLPTLYVSQSTNSTEETLESVITISEKSLQDAN 501

IAKVEHVVLTVSFVHRRRNDVSIDLISPKDTKSQNLSPR-KYDDSDEGLDEIVSFNlTVYN 592IKSLEHVQFEATIEYSRRGDLHVTLTSAVGTSTVLLAER-ERDTSPNtUFK WMSVHTh 559ITRLEHAQARLTLSYNRCDLAIHLVS1N6TRSTLLAR- PHDYSADGFIAFTTHSV 539VWYLEHVRTSISHPIRRDLQQIYLVSPSCTKSQLLA- -LLDLSNEFTNEEFNTVT V 594VRYLEHVQAVITVNATRN6DLNINNTSPMGTKSILLSRRPRDDOSKVyUFJgMPFTTHTh 560FKRIEHVTVTVDIDTEIR6TTTVDLISPAGI ISNLGVVRPR-DVSSEGFtDWTFNSVAHy 560

BENPKGIWRLKITD 606BENPVCTVTLKITD 573DEDPSBEVVLEIEN 553GEKAE6QVTLEIQD 608GEDARGTVTLELGF 574SENGVGDWKIKVKT 574

FIG. 3. Alignment of the deduced amino acid sequence of thehydra SPC3-like protein with mammalian SPC1, -2, -3, and -4 andyeast kex2 sequences. The hydra SPC3-like (hySPC3-like) sequence(residues 69-606), including approximately half of the propeptidedomain, the catalytic domain, and a 163-residue intermediate do-main, was compared with corresponding sequences from mouseSPC3 (mSPC3) (10), human SPC1 (hnSPC1) (12), human SPC4(hnSPC4) (14), human SPC2 (hnSPC2) (7), and yeast kex2 (ykex2)(5). The carboxyl-terminal regions of these enzymes, which are veryheterogeneous in length, were not included in the comparison. Thesingle-letter amino acid code is used. Arrowheads indicate positionsof aspartic acid, histidine, and serine that form the catalytic triad andthe asparagine (aspartic acid in SPC2) that occupies the oxyanionhole in catalysis (23). Amino acid residues that are identical in at leastfive sequences are shown in boldface type.

stained DNA fragment of the correct size was visible in anagarose gel only with body column cDNA as the template. ASouthern blot of the gel, however, revealed that a smallamount of SPC3-like cDNA had also been amplified fromhead cDNA (data not shown).

DISCUSSIONThe isolation of a SPC3-like cDNA from hydra providesfurther evidence that the SPC gene family has been highlyconserved throughout evolution of multicellular organisms.In mammals, at least four distinct SPC genes have beenfound, whereas the yeast Saccharomyces cerevisiae contains

FIG. 4. Analysis of hydraSPC3-like RNA. Hydra poly(A)+RNA (3 Zg) was electrophoresedon a 1.0%6 agarose/0.33 M form-

-9.49 aldehyde gel, blotted onto nitro-7.46 cellulose, and hybridized with a

2.0-kb cDNA insert from Hy-2-4.40 (lane a) or a 320-bp fragment con-

taining the type a coding sequenceand 3' untranslated region (laneb). In lane c, the blot from lane b

-2-37 was stripped by boiling in waterfor 5 min and rehybridized with a

-1 35 1.2-kb hydra elongation factorcDNA fragment. Autoradiogramswere exposed overnight with anintensifying screen. Size markers(in kb) were from a RNA ladder

-0.24 (BRL).

a single proprotein convertase gene, kex2. Recently, Siezenet al. (25) have proposed that mammalian SPC1, -2, and -3(and presumably 4) evolved from a common ancestral genethat diverged from yeast kex2 at an earlier date. Our findingssupport this view in that sequence comparisons reveal thathydra SPC is more similar to SPC1, -2, -3, and -4 and lesssimilar to kex2.We have identified the two isoenzymes encoded by hydra

cDNAs as SPC3-like, based on their structural organization,their lack of transmembrane domains, and the significantamino acid sequence identity of their catalytic domain to thatof mouse SPC3 (55.4%). However, the hydra SPC3-likecatalytic domain shows an essentially equal degree of identity(56.7%) to that of human SPC1 (furin), whereas mouse SPC3and human SPC1 are 62.3% identical in this region (10, 11).These results are consistent with the possibility that mam-malian SPC3 and SPC1 are the products ofa gene duplicationevent that occurred subsequent to hydra in evolutionarydevelopment. However, we do not infer that a solubleSPC3-like converting enzyme preceded the evolutionarydevelopment of the membrane-bound SPC1 form since, asshown in Fig. 2, alternative processing can generate at leasttwo hydra SPC3-like isoforms. It is possible that alternativesplicing of the pre-mRNA may generate an additional hydra

A 1 2 3 B 1 2 3

_~~

C

FIG. 5. Analysis of SPC3-like RNA in hydra head and bodycolumn. RNA blot was prepared from a 1.0%o agarose/0.33 Mformaldehyde gel that contained 0.3 ,g of hydra poly(A)+ RNA (lane1), 10 ,g of hydra head RNA (lane 2), or 10 ,ug of hydra body columnRNA (lane 3). (A) Gel stained with ethidium bromide. (B) Autorad-iogram of blot hybridized with hydra SPC3-like cDNA, exposed 48hr. (C) Autoradiogram of same blot as in B stripped by boiling inwater for 5 min and rehybridized with hydra elongation factor lacDNA and exposed 8 hr. Some smearing of the hybridizing bandsoccurred with both probes 2 and 3 (B and C), which indicates that themRNAs from hydra head and body column were partially degraded.

hySPC3-1 i keSPC3

hnSPC1hnSPC4hnSPC2

kex2

hnSPCykex2

hySPC3-1 i keSPC3

hnSPC1hnSPC4hnSPC2ykex2

hySPC3-1 i keSPC3

hnSPCIhnSPC4hnSPC2ykex2

hySPC3-1 i keSPC3

hnSPC1hnSPC4hnSPC2ykex2

hySPC3-I i keSPC3

hnSPC1hnSPC4hnSPC2ykex2

hySPC3-1 i keSPC3

hnSPC1hnSPC4hnSPC2ykex2

hySPC3-I i kemSPC3hnSPCIhnSPC4hnSPC2ykex2

hySPC3-1 i keSPC3

hnSPC1hnSPC4hnSPC2ykex2

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Proc. Natl. Acad. Sci. USA 89 (1992)

isoform with a carboxyl transmembrane domain that wouldbe SPC1-like.Although SPC1, -2, and -3 are clearly homologous en-

zymes, recent studies have shown that they exhibit differ-ences in cell-specific expression, topological localizationwithin the cell, and substrate specificity. In rats and mice,SPC2 and SPC3 mRNAs are expressed only in neuronal andneuroendocrine cells, and immunocytochemical and bio-chemical studies indicate that they are localized in thesecretion granules (10, 11, 28, 29). Recently, transfectionstudies by Thomas et al. (30) and Benjannet et al. (31) havedemonstrated that SPC2 and SPC3 cleave proopiomelano-cortin at distinct sites. In contrast, SPC1 (furin) mRNA isfound in most tissues, and the enzyme seems to be localizedintracellularly to the trans Golgi region (32). Hosaka et al. (33)and Molloy et al. (34) have reported that SPC1 may cleavepreferentially at sequences having the motif Arg-Xaa-Xaa-Arg.Although it seems likely that the hydra SPC3-like en-

zyme(s) will be found in neuroendocrine secretion granules,we have not yet identified a specific expressing cell type orany possible proprotein substrates in hydra. An unexpectedfinding has been that the hydra SPC3-like RNA is expressedpredominantly in the body column. This is in contrast tomorphological studies that have shown that the head regioncontains a much higher density of nerve cells, although it hasbeen shown that neurons in the body column also containsecretory granules or vesicles (27). However, Grimmelik-huijzen et al. (35) and Falkner and Van Noorden (36) haveshown that some of the nerve cells in the head and peduncle(foot) regions in hydra react strongly with antisera to Arg-Phe-NH2, cholecystokinin, substance P, neurokinin, andoxytocin; the precursor forms of all of these neuroendocrinepeptides are known to be processed at paired basic residues(37). These findings suggest that there may be anotherproprotein convertase present in hydra that is expressed inthese hormone-producing cells. We have searched for asecond SPC cDNA by PCR using hydra head cDNA astemplate but without success (S.J.C., unpublished results). Itmay be that the hydra head region contains a proproteincleavage enzyme that is unrelated to subtilisin, such as theaspartyl proteinase recently described in yeast (38) andanglerfish (39). However, further studies will be required toidentify such a proteinase as well as to more fully character-ize the hydra SPC3-like activity, its localization, and itsnatural substrates.

We thank Paul Gardner for synthesis of oligonucleotides, StevenP. Smeekens for helpful comments, and Florence Rozenfeld forpreparing this manuscript. This work was supported by the HowardHughes Medical Institute and by Public Health Service Grant HD24511 to H.B. and PHS Grants DK 13914 and DK 20595 to D.F.S.

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