Proprotein convertases in amphioxus: Predictedstructure ... · oligonucleotide primers SQ-3/QS-1 (lane 1), SQ-4/QS-1 (lane 2), SQ-3/QS-2 (lane 3), or SQ-4/QS-2 (lane 4). Primer sequences

Proc. Natl. Acad. Sci. USAVol. 92, pp. 3591-3595, April 1995Evolution

Proprotein convertases in amphioxus: Predicted structure andexpression of proteases SPC2 and SPC3

(precursor processing/subtilisin-related endoproteases/prohormone biosynthesis/protochordates)

ANTHONY A. OLIVA, JR.*, DONALD F. STEINERt, AND SHU JIN CHANHoward Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The University of Chicago, 5841 South Maryland Avenue, Chicago,IL 60637

Contributed by Donald F. Steiner, December 21, 1994

ABSTRACT SPC2 and SPC3 are two members of a familyof subtilisin-related proteases which play essential roles in theprocessing of prohormones into their mature forms in thepancreatic B cell and many other neuroendocrine cells. Toinvestigate the phylogenetic origins and evolutionary func-tions of SPC2 and SPC3 we have identified and cloned cDNAsencoding these enzymes from amphioxus (Branchiostoma cali-forniensis), a primitive chordate. The amino acid sequence ofpreproSPC2 contains 689 aa and is 71% identical to humanSPC2. In contrast, amphioxus preproSPC3 consists of 774 aaand exhibits 55% identity to human SPC3. These resultssuggest that the primary structure of SPC2 has been morehighly conserved during evolution than that of SPC3. Tofurther investigate the function(s) of SPC2 and SPC3 inamphioxus, we have determined the regional expression ofthese genes by using a reverse transcriptase-linked poly-merase chain reaction (RT-PCR) assay. Whole amphioxus wasdissected longitudinally into four equal-length segments andRNA was extracted. Using RT-PCR to simultaneously amplifySPC2 and SPC3 DNA fragments, we found that the cranialregion (section 1) expressed equal amounts of SPC2 and SPC3mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 theSPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determinedthat amphioxus ILP, a homologue of mammalian insu-lin/insulin-like growth factor, was expressed predominatelyin section 3. These results suggest that the relative levels ofSPC2 and SPC3 mRNAs are specifically regulated in variousamphioxus tissues. Furthermore, the ubiquitous expression ofthese mRNAs in the organism indicates that they are involved inthe processing ofother precursor proteins in addition to proILP.

In eukaryotic cells, proteins destined for export or the plasmamembrane surface are often synthesized as larger precursorforms which are proteolytically processed during subsequenttransport and maturation. The initial endoproteolytic cleavageis performed by proprotein convertases which recently havebeen identified as members of the subtilisin family of serineendoproteases. These enzymes recognize a motif of paired ormultiple basic residues with a strong preference for arginine atthe P1 site (1-3). In yeast a single subtilisin-related proproteinconvertase, kexin, has been found (4). However, mammalsexpress at least six members of this family, including SPC1(furin) (5), SPC2 (PC2) (6, 7), SPC3 (PC3/PC1) (8, 9), SPC4(PACE4) (10), SPC5 (PC4) (11), and SPC6 (PC5/PC6) (12,13). These enzymes are related by a similar catalytic core domainbut differ markedly in their carboxyl-terminal sequences.We have investigated the structures of SPC2 and SPC3 and

the functional roles of these enzymes in the processing ofproinsulin to insulin. Studies have shown that both SPC2 andSPC3 are expressed in islet B cells (as well as in other

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

neuroendocrine cells) (6-9), and in vitro transfection assays(12) have shown that these enzymes can cleave proinsulinappropriately. Here we report the cDNA sequencest andexpression of SPC2 and SPC3 from the protochordate am-phioxus. We have previously shown that amphioxus expressesan insulin-like peptide (ILP) mRNA (13). The putative pre-cursor, proILP, is like proinsulin in that it contains a C-peptidewhich is flanked by paired basic residues, but it is also similarto the pro-insulin-like growth factors in that proILP alsocontains a carboxyl-terminal E domain which is probablyremoved during maturation. Our goals are to characterize theproprotein convertases which are expressed in protochordatesand more specifically identify the convertases involved inprocessing proILP to its active form.

MATERIALS AND METHODSMaterials. Oligonucleotides were synthesized on an Applied

Biosystems model 380B DNA synthesizer. Radioisotopes werepurchased from Amersham. Plasmid pGEM4Z was obtainedfrom Promega.RNA and Reverse Transcriptase-Linked Polymerase Chain

Reaction (RT-PCR) Assays. Live amphioxus (Branchiostomacaliforniensis) were purchased from Pacific Bio-Marine(Venice, CA). Total RNA was isolated by tissue homogenizationin guanidine thiocyanate and centrifugation through a 5.7M CsClcushion as described (13). RNAwas quantitated by absorbance at260 nm (A2w = 1 at 40 ,g/ml) and its integrity was verified byUV fluorescence on denaturing (0.66 M formaldehyde) 1%agarose gels stained with ethidium bromide at 1 p.g/ml.cDNA was synthesized with a Moloney murine leukemia

virus (M-MLV) reverse transcriptase kit purchased fromGIBCO/BRL. After 1-hr incubation at 37°C, the reversetranscriptase was inactivated by heating to 100°C for 3 min andan aliquot was removed for PCR. For the subtilisin-relatedproprotein convertases, cDNA fragments which contained thecatalytic domain were amplified by using the following degen-erate oligonucleotide primers: SQ-3 (sense), 5'-CAYGGN-ACNCGNTGYGC; SQ-4 (sense), 5'-CAYGGNACNAGRT-GYGC; QS-1 (antisense), 5'-YTGCATRTCYCTCCANGT;and QS-2 (antisense), 5'-YTGCATRTCNCGCCANGT, inwhich R = G or A, Y = C or T, and N = G, A, T, or C. PCRwas performed for 40 cycles each at 94°C for 1 min, 50°C for1 min, and 72°C for 1 min.For RT-PCR to simultaneously amplify amphioxus SPC2

(480-bp) and SPC3 (334-bp) cDNA fragments, the followingprimers were used: SPC2-1 (sense), GGATGTTGGACCAAC-

Abbreviations: ILP, insulin-like peptide; RT-PCR, reverse tran-scriptase-linked polymerase chain reaction; RACE, rapid amplifica-tion of cDNA ends.*Present address: Division of Neuroscience, Baylor College of Med-icine, One Baylor Plaza, Houston, TX 75226.tTo whom reprint requests should be addressed.tThe sequences reported in this paper have been deposited in theGenBank data base (accession nos. U22051 and U22052).

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Proc. Natl. Acad. Sci. USA 92 (1995)

1 2 3 4

-1353

-1078

-872

l- 603

Table 1. Amino acid sequence identity between amphioxus andhuman (prepro)SPC2 and -SPC3

% sequence identity

Amphioxus SPC2 Amphioxus SPC3Domain vs. human SPC2 vs. human SPC3

Pre 24 16Pro 37 52Catalytic 84 74P 75 59C terminus 61 21Whole protein 71 55

-310

FIG. 1. Amplification of subtilisin-related proprotein convertasecatalytic domains by PCR. Amphioxus cDNA reverse transcribed from0.1 ,ug of poly(A)+ RNA was subjected to 30 cycles of PCR usingoligonucleotide primers SQ-3/QS-1 (lane 1), SQ-4/QS-1 (lane 2),SQ-3/QS-2 (lane 3), or SQ-4/QS-2 (lane 4). Primer sequences arelisted in Materials and Methods. The reaction products were analyzedby electrophoresis in a 5% polyacrylamide gel and stained withethidium bromide. Molecular weight standards (labeled in terms ofbp) were 4X174 DNA digested with Hae III.

CGTTC; SPC2-2 (antisense), CAGTGCCAGCGCGAAAACC;SPC3-1 (sense), TCCATCTTTGCCTGGGCGTC; and SPC3-2(antisense), CGATCAGGTGCTGTACGTCTC. The templatecontained 100 ng of amphioxus RNA reverse transcribed intocDNA. PCR was performed for 30 cycles each at 94°C for 1 min,56°C for 1 min, and 72°C for 1 min in 100 ,ul.

Isolation of poly(A)+ RNA and Northern blot analysis wereperformed essentially as described (14).cDNA Cloning. Cloned amphioxus SPC2 and SPC3 cDNAs

were obtained using the RACE technique as described byFrohman et al. (15) and by screening a phage cDNA libraryprepared from whole amphioxus RNA (13). Other standardmolecular biological techniques were as described by Sam-brook et al. (16).

RESULTS AND DISCUSSIONTo identify subtilisin-related proprotein convertase genes ex-pressed in amphioxus we amplified whole amphioxus cDNA byusing degenerate oligonucleotide primer sets which corre-sponded to highly conserved amino acid sequences in thecatalytic domain of these proteases. When primer set SQ-3/QS-1 was used in PCR a 600-bp fragment was obtained, but nobands of the expected size were observed with the other primercombinations of SQ-3/QS-2, SQ-4/QS-1, or SQ-4/QS-2 (Fig.

D174 H215w w

Pre Pro

* pAPC-2-550

10Cbp

1). However, restriction enzyme analysis showed that the 600-bpfragment was heterogeneous. For example, when the fragmentwas digested with Bgl I multiple bands in the range of 120-480 bpwere observed (data not shown).The heterogeneous nature of the 600-bp fragment was

confirmed by plasmid cloning into pGEM4Z. From individualclones we have obtained four distinct cDNA sequences, ofwhich the deduced amino acid sequence from one cloneshowed the highest identity (80%) to human SPC2, whileanother clone exhibited the greatest identity (78%) to humanSPC3. These sequences were expanded by using the rapidamplification of cDNA ends (RACE) technique (15) and byscreening cDNA libraries (Fig. 2).The complete coding sequences for amphioxus SPC2 and

SPC3 contain 689 and 774 aa, respectively, whereas there are638 and 753 aa in the human (prepro)SPC2 and -SPC3sequences (Figs. 3 and 4). As summarized in Table 1, the aminoacid sequence similarities were highest in the catalytic do-mains. In addition, amphioxus SPC2 contained an Asp-for-Asnsubstitution at position 317 (corresponding to residue 310 inhuman SPC2), as is the case in all other known SPC2 homo-logues (see below). In subtilisin the side chain of this Asn residuehas been demonstrated to stabilize the transition state duringcatalysis (17). To form a similar hydrogen bond with the carbonyloxygen next to the scissle bond, however, the Asp side chain inSPC2 would have to be protonated, and this is consistent with theprobability that SPC2 in all species is active only in an acidiccompartment such as the dense-core secretory granule.A high percentage of identity for SPC2 and SPC3 is also

found in the P domain, which consists of 190-192 aa locatedon the carboxyl side of the catalytic domain. Recently, trans-fection experiments have shown that when successively trun-cated forms of SPC1 (furin) were expressed, deletions withinthe P domain were correlated with loss of catalytic activity(18). Downstream from the P domain amphioxus SPC2 and

Amphioxus SPC2S391lw

Catalytic P-Domain

pAPC-2-650

pAPC-2 -1200 pAPC-2-850

Amphioxus SPC3D174 H215_w _ S389

pAPC3-1800

pAPC3-6000I pAPC3-l1100

FIG. 2. Molecular cloning ofamphioxus SPC2 and SPC3 cDNAs.The organization of amphioxusSPC2 and SPC3 cDNAs is shown

----I schematically. Arrows indicateclones obtained by the RACE tech-nique (15); brackets indicate clonesisolated from a cDNA library.

FAi

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Evolution: Oliva et at Proc. Natl. Acad. Sci. USA 92 (1995) 3593

CAGCACTAATCCTCT(3LCCTCTTCCZ,"AGAAAGAAAAAAAcTcAACCT=I GAAG,---m, CCACAAACCACCAAAGCv 1000

M VK P VG W FLLR 11

ITAAGAAAAAAGCTGACTGTGGTCAACCCGAGC%T-GCTGAATT~TC r-.AAAACGAGrAGAGAGGATAT-GGTGAAGCC~-GGTAGGATGGTTCTCCTCC 200

V Y L L V L A T I L V W Y G A C N S*A P P Y T N D F A V 0 1 R 44

GGTCGACCTGTTGCCTAL ACRTTKGYGACGGGYLrGTACTC RGCEQCRDLCFRGCHCCCTGARTAGG

CGGACCAATCGPGVCG ACTGGGAAAGSTCTCA,TG,CGTCAAAQQAGA QQC FRACTCGRACG

GGGCGCjCCCATGTCCGCGGCAGGGCCAG'rGCGGCGCAGCAGAGCArGGCTGGAGAACGACATGCGGGTACGAGC.-GCAG-TACAACAACAGGGTT-TCCGCC

T WR DM0H L T VL T SKR NQL YD PV H EWR RN G VGL E

TGACTIGGCGGGACATGCAGCACCTGACGGTCCIGACGTCCAAGCGGAACCAGCTGTACGACCCGGTGCACGAGTGGCGGCGGAACGGAGTGGGGCTGGA

F NH L FGFG V LD AG SM V K MA EDWNTmlV PK R FHCT G

G-I-ICAACCATCTGTTCGGGTTCGGCGTGCT.GGACGCGGGCTCCATGGTGAAGATGGCCGAGGACGGAACACTGT"ACCCAAACGGTTCcACT&GCACAGGG

T SM SDA K PI P VEG KV VV KL TT D ACE GQ EN FV RYLL

ACATCCATGTCTGACGCCAAGCCGATCCCAGTGGAGGGGAAAGTAGTGGTCAAGCTGACCACTGACGCGT.LGCGAGGGCCAGGAAAACTTTGTGCGCTATC

CACACrCCCCAATGACCGATCACTCCAGAGGAAGGGCr'TCGACCGCTGGCCCTTCATGACCACTCACACCTGGGGGGAAGACcCTAGAGGAGACTGGGTG

L E V G F Q G D E P Q E G D L L E W T L R L H G T4Q S P P Y I D V

VTGAGTTGGTC TCGG LAGMGCA KGAGGC QCLTCGLGTGSACGCGCTCAGGKCEEEGCECCCACT644AA

300

77

400

ill

144

600

177

700

211

800

244

900

277

1000

311

1100

344

1200

377

1300

411

1400

444

1500

477

1600

511

1700

544

1800

577

1900

611

2000

Q EK E KT EA EE KE TTN EEEE EE EN G P KKG KE GGSS 677- - - - - -- - - - - 113-13 AA~"

CA,TAAAAGAGGAGGCTGATCAGCTGTATACATCTCTGGGTATCTAAAAATACCCTGGTTGCATTGAGrCTcACTCTAACTTATcATGTTATAATATTccGCCAGAGTATTGTATAAAAAAGATATATAATTGTACTTATTGTATCTTGGGTATGTGA,AAGGAAGTTAGTTCATCTGTGGAGTTCTGTGACCGGTTAGCATCTATATTGCGTTGTGGCAATGATTTGTGGAGrAGTCAAATTGTATTAGATCAAAAAAAAAAAAACCCCcAAAAATAGAATGCACACGTGrATTCACAATCAGAGCGAAAAGrGA,TTTTACACACGCACACACACGCACATATAGACACACACAAACTCAACGTGA,TAATTGGATAAGATAAAGAAAAATGATATTTTTGTCGTGAGCTAAATACTGTAcTAAAGATGAAGTACTTTGACAccAGGAACcCTATAcTGTTTAGATGcGGCCGGCGAATTCAGCTTGGA

689

2300

2400

2500

2600

2700

2800

2809

FIG. 3. Nucleotide and deduced amino acid sequence of amphioxus SPC2 cDNA. Putative cleavage site for the signal peptide is assigned to residue

30 (arrow). The subtilisin-related catalytic domain is boxed and dots indicate residues involved in the catalytic mechanism. Arrowheads identify two

potential glycosylation sites, and two potential signals (RDG) for binding to extracellular matrix proteins are overlined. An arrow also identifies the end

of the P domain at residue 602. A carboxyl-terminal glutamic acid-rich sequence, also found in human and rodent SPC2, is underlined.

SPC3 contained carboxyl-terminal sequences which are not

highly conserved, although the carboxyl termini of both human

and amphioxus SPC2 contained a region enriched in Glu

residues which is predicted to form an amphipathic helix (1).

Overall, amphioxus SPC2 exhibited 71% identity to human SPC2,

whereas amphioxus SPC3 is 55% identical to human SPC3.

Very recently, SPC2 and SPC3 sequences have been re-

ported from a number of phylogenetically diverse species,

including frogs (19), fish (20), molluscs (21-23), and hydra

(24). Fig. 5 shows the amino acid identities in the catalytic and

P domains of SPC2 and SPC3 from these species compared to

the human homologues and plotted against the estimated time

of evolutionary divergence. The results clearly show that SPC2

has evolved more slowly than SPC3. It is not known whether

this relatively slow substitution rate observed for SPC2 has a

functional basis, but one possibility is that SPC2 may have a

more ordered structure and/or restricted catalytic specificity.

Northern blot analysis revealed a single 2.7-kb band for

amphioxus SPC3 mRNA, while two bands, a major band at 4.0

kb and a minor band at 7.0 kb, were observed for SPC2 mRNA

(Fig. 6). Preliminary 3'RACE analysis indicate that the 7.0-kb

SPC2 mRNA is due to extended transcription of the 3'

untranslated region (data not shown).

Although the SPC2 and SPC3 mRNAs could be easily

detected on Northern blots, our attempts to perform in situ

hybridization on thin (10- to 20-1km) saggital sections of whole

mounted amphioxus were unsuccessful due to a high back-

ground. As an alternative, we have determined if there were

.K RIG YN EV ND N YRQ I D I ND PL FP KQ0WY L L NTGGTAGGAACGCGGGTATAACGAAGGAATGACrAATCCGTCAGATcGACATCAACGACCCGCTCTTCCCCAACAGrGGTAcCCT,GCTGAATACTGGCCA

0

0

CGGTT.CAACAGCCATGGTACCCGCGTGCAGGAGAGG-TGGTGGGTAAGATCAACAACGGTCT,GTGC,GGAGTGGGCGTCAGG-TATG--GCGCACGAGTGGCAGG

~C'GACGGACGACGGCCGTACTGTGGACGGGCCGCGGGAGCTCACCGTGCAGGCAATGGCGGACGGrGTCAATAAGGGGCGT,GGAGGAAAAGGCAGCATC.¶V W A S G D G G S Q D D C N C D G Y A S S M W T I S I N S A i N D

ACSGTrGGGCATCCGGAGACGGCGGGAGTCAGGACGACTGTAACTGTGACGGATACGCCTCCTCGATGTGGACCATCTCCATCAACTCCGCCATCAATGA

CGGCCGCACCGCCCTTACGACGAGTCCTGC-CCTCCACTCTCGCCTCCACCTTCAGTAACG CGCGACAGcCA-CCGAGCCGcGFCGGCCACCACG'V S l

DL Y GN C TLKH SG T SA AA P EA AG V A LA LQ AN PN L

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GAATTCGCGGCCG;CCCCCCGl-.GGCATCCATCCTATCCAGGGTTGCTCTCCTGTGrGGITr-AGTCACGTCC-TCCGTGTTGGACTCT-TCGGCAGCTCCAAAGTCATC TCTTAGCTCTGTTTATATC ATCACAAGTGTATCACTTCTCGGACTGTGAGACCCACAACCT, GAAC=CGGTCGGGAACAACACGCTGAAACCTCGGTTGGAGACCCACATCTGGC',GCGGCGCGAATCGTGTTGAACAGTlTACGAG^AACGGGCGC I TSCATCTG;;

M G R F Y A W L V L A I V A F S S H G*R A E D G E 0 0 P G H FCGCGGAACATGGCGTTAT TGCCTGCGTCC-TGCGATTGTGw LCTCCTCCACGC-GCGTGCC-GAGGACGGAGACGkC AACCCCGCCACTT

T N T W A V E I Y G G P D R A D L L A L D H G Y E N L G Q I G N LCACCAACCTGGGCGGTGGAATCTACGGAGGGCCCGACAGAGCTGACCTGTTAGCCCTAGACCATG&CTIACGAGAACTTGGGCAGATAGGGAACCTG

E D H Y L F R H K D V P H R S R R G A H Q H T K R L G D D E R I Q WGAGGATCACTACCTCTTCAGACACAAGATGTGCCTCACCGGTCCCGCAGAGGGGCGCATCAGCACACCAGwGGCTGGGGGACATGAAAGGATACAAT

TGC'. GGCGCTGGCTCTGGAGGAACC-CGAAC-:;~GC'-GGACGAGCTACAGCACCT,GATCvTGTGACkCMCGAGTACGAC -.'-1CTCC>GCACCC

G W F Q N G A G L W V N S R F G Y G L L N A E A M V D M A L T W KCGGCTGGCCAGAATGGCGCCGCCTGTGGGTCAACTCCCGGTTCGGCTACGGCCTGCTGAACGCCGAGGtGATGGTGGACATGGCGCTGACGTGGAAG

T V P E K T K C E V R I E N F Q P R D L G N G E E I I I E L E T D GACTGTACCGAGAAGACCAAGTGTGAAGTTAGGATTGAGAACTTCCAGCCGAGGGACCTGGGACGGAGAAGAGATCATCATAGAGCTGGAGACTGACG

C R G Q N H V E A L E H V Q V K T T I D Y S R R G D L R I V L T SGTTGCCGTGGCCAACCACGTGGAAGCTCTGGAGCACGTGCAGGTCAAAACGACGATCGACTACTCCCGCCGGGGGGACCTGAGGATAGWGCTTACGTC

100200300

31400

64500

98600

131700

164800

198900

2311000

2641100

2981200

3311300

3641400

3981500

4311600

4641700

4981800

5311900

P S G T S T T L L D T R R Q D K S Q M G F Q D W P F M S T H N W G 564ACCGTCAGGAACCAGCACCACCCTCCTGGACACCCGCCGTCAGGACAAGTC-CAGATGGGATTCCAGGACT;GGCCCTTCATGTCCACTCACAACTGGGGA 2000

E K P Q G K W T L T I E D K S D H A E N N G V V K D V V L I L H G T* 598GAGAAGCCACAGGCAGrGGAkCACTCACCATAGAGGACAAGTC-TGACCACGCGGAAAACAACGGTGTTG4-GAAGGATGTCGTGCTGATTCTGCACGGAA 2100

P E Q P A Y Q S G G R V Y T D Y N R V Q G D R N V E I S A A K A A 631CCCCGGAACAGCCGGCCTACCAGTCCGGAGGG;&GGGTGTACACTrGACTACAACCGCGrGCAGGGCGJvCAGAAATGTGGAAATATCGGCCGCTAAAGCCGC 2200

P A P A A D A A P A R G S E E R IP G S G P F G S A A S V V I E ECCCAGCCCCCGCTGCGGATGCCGCCCCCGCCCGGGGGTCAGAGGAACGGATCCCCGGCTCTGGTCCCTTCGGCTCTGCTGCGAGCGTGGTTATCGAAGAG

6642300

I P E K E T E F L V N W Q D G M N R Y E T D F N P V N S G P F E P S 698ATTCCTGAAAAAGAAACAGAGTTCTTGGTGAACTGGCAAGACGGAATGAATCGCTACGAGACTGAC= CAACCCCGTCAACTCAGGCCCGTTCGAGCCCT 2400

A D A G S D V Y M D A E D L R P W E A F K Y L 0 R Q M K S P S G Q 731CAGCTGATGCCGGATCAGACGTATACATGGATGCCGAAGACCTTCGTCCTTGGGaGGCTTTCAAGTACCTGCAACGGCAAATGAAATCACCGAGCGGCCA 2500

R H P A H A Y N K P S S Q Y I R D P W V S Q N A L D K E A N L V K 764ACGTCATCCAGCGCATGCGTACAACAAGCCATCGTCACAGTACATCCGGGACCCGTGGGTATCCCAGAATGCCCTGGACAAAGAGGCGAACCTGGTGAAA 2600

Y Y L Q L L G Y E S XTACTACCTACAGCTGCTGGGCTACGAATCATGAAAAAAAAACGTGGATGAAGAAAATAAAACGGAACACCAGAAACTGGAACGAAAAATAAACTGGTCTTAGAT-A

77427002725

FIG. 4. Nucleotide and deduced amino acid sequence of amphioxus SPC3 cDNA. Putative cleavage site for the signal peptide is assigned toresidue 19 (arrow). The subtilisin-related catalytic domain is boxed and dots indicate residues involved in the catalytic mechanism. Arrowheadsidentify two potential glycosylation sites and a potential signal (RDG) for binding to extracellular matrix proteins is overlined. An arrow also identifiesthe end of the P domain at residue 598.

any regional differences in the expression of SPC2 and SPC3.Whole amphioxus were sliced into equal-length sections andtotal RNA was extracted from each section. To obtain the ratioof SPC2 to SPC3 expression we performed multiplexed RT-PCR in which SPC2 and SPC3 DNA fragments were simul-taneously amplified from the same RNA template. In calibra-tion assays it was established that under the RT-PCR condi-tions, the amount of each DNA fragment obtained (as visualizedby ethidium bromide staining) was roughly proportional to theamount of input RNA within the range of 20 to 2000 ng.

Fig. 7 shows the results of a representative experiment inwhich 100-ng amounts of amphioxus RNA were subjected toRT-PCR. With whole amphioxus RNA as template, equallyintense ethidium bromide-stained SPC2 and SPC3 DNA frag-

ments were obtained. This result indicated that whole am-phioxus expressed approximately equal amounts of SPC2 andSPC3 mRNA. However, there were clear regional differencesin the ratio of SPC2 to SPC3 expression. In the anterior(cranial) segment the SPC2-to-SPC3 ratio was 1:1, whereas inthe posterior (caudal) segment the ratio was approximately 5:1in favor of SPC2 (Fig. 7, lanes labeled 1 and 4). In contrast, themid-body segments exhibited a reversed SPC2-to-SPC3 ratioof about 1:5. We also analyzed the expression of ILP mRNAin these segments by Northern blot and RT-PCR, and bothanalyses showed that ILP mRNA was predominately localizedto segment 3 (data not shown).The strong conservation of primary sequence between

mammalian and amphioxus SPC2 and SPC3 strongly suggests

V A O O V G R A R S K RIG P M G OQ R R Q S D D T R P M T F R D PGGGTGGCTCAGCAGGTTGGGCGAGCCCGGAGrAAAGAGGACCCATGGGCCAGCAGCGCCGCCAATCAGACGACACCAGGCCCATGACCTTCCGTGACCC

Y W E K Q W Y L H D T R T S T N L P K L D L H V L P V W R K G r TTT-ACTGAAACAGTGGTATC'GGCaCGACACAAGAACTTCGCAAACCS"GCCCAAGCTAGAC'-CGTACTTCCGGTCTGGAGAAGGCTCACG

G K G I V V A V L D D G I E K D H P D L V D N Y D P D A S Y D F N DGGAAAAGGATCGTCGTGGCCG . GTTGGACGACGGTCGiAAAAGGACCATCCAGACCTCGTGGAACTATGACCCCGiATGCAAGCTATGACTTCAACG

N D D D P Q P R Y E E T N E N K H G T R C A G E I A H A A N N S EACAACGATGACGvACCCGCAACCGGATATGAGGAAACGAACG-AAACAAGCAGGACCTGiTGCGGGAGATCGCCAGCAGCCAACAACTCGG;

C G V G I A F N A R I G G V R M L D G V V ml D A V E A S S I G F NGT-GCGiGCGTGGGATAGCTT.AATGCAAGATAGaGGTGTGOCGiCATGCT.GGACGGiAGiTGGTGACTGACeCCGTGGAGGCAG;:.-CCATCGGTCAA

I Q H V D I Y S A S w G P N D D G K T V E G P E K L A R A A F E K GATCCAGCACGTGGACATCTACAGCGCCTCGTGGGACCCAACGACGACGGCAGACCGTGGAGG;GCC'-CCrGAAGCTLCGCTCGGGCGcCTTCAGAAGG

V R E G R G G K G S I - A W A S G N G G S N G D N C D C D G Y T SG_GTTAGGAGGTGGAGAAGGCA,.TGCCTGGGCGTCCGGCAAC-GGGGCAGTAACG GGCAACTGC rGAC-.GG.GACCGC,C.ACACCAG

S I Y T V S I S S A S Q Q G G S P W Y G E K C A S T L A T A Y S SCTCCATCTACACCGTGTCCATCAGCAGTGCG-,C'-CAGCAGCGGCGC"TUCGC-.GGTACGGCGAGAAGTGCG mCCACGC-TGGCCGUACACG

G E Y K D Q K I S S T D 7r H H E C T D S H T G T S A A A ° L A A G VGGG7GACAAGCAAATAAGiCAGCkCGGCCTGCATCAC-AG. G-ACGGACACUCACGAATCAGCCGCGG CGCAC CGGGG

L.

lw--L--A--L -A-L -E--A-- N--P--N L I-T- W--R--D--V -Q--H- -L -I--V W--T S--E- Y- -D--P L -S--S--N- -P-

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Hy

0 200 400 600 800 1 000Time, Mya

FIG. 5. Evolutionary divergence of SPC2 and SPC3 sequences.Identity of amino acid sequences in the catalytic and P domains ofSPC2 (0) and SPC3 (x) from various species compared with humanSPC2 and SPC3 is plotted against the estimated elapsed time (Mya,millions of years) since the species diverged (25, 26). Species are asfollows: Mo, mouse (7, 8); Ra, rat (25); Xe, Xenopus (19); Af,anglerfish SPC3 (20); Am, amphioxus; Ly, Lymnaea (21); Ap, Aplysia(22, 23); and Hy, hydra SPC3-like (24).

that these enzymes are capable of cleaving the C-peptidedomain from proILP as they do in processing proinsulin.However, it should be noted that we have not yet demonstratedthat these enzymes and ILP are coexpressed in the same cell.Indeed, the broad regional distribution and variable ratio ofSPC2 to SPC3 expression suggest that these enzymes probablyact on additional substrates in amphioxus. Possible substratesinclude other pancreatic and/or neuroendocrine hormoneprecursors such as proglucagon, prosomatostatin, and pro-opiomelanocortin, although none of these hormones has yetbeen identified in amphioxus.Whether SPC2 and SPC3 are necessary or sufficient to

process proILP into a mature insulin-like peptide remains tobe determined. An approach to the solution is to characterizethe processing of proILP expressed in mammalian cells whichcontain SPC2 and SPC3. In particular, it is possible that thecomplete processing of proILP, particularly in the carboxyl-terminal extended sequence, will require an as-yet-unidentified proprotein convertase capable of cleaving atsingle basic residues. In our analyses of SPC DNA fragmentsamplified by PCR, we have observed that amphioxus expresses

A B

9.49 -7.46 -

FIG. 6. Northern blot analysis

4.40of amphioxus SPC2 and SPC3

4.40 - __ mRNAs. A 5-,ug sample of am-phioxus poly(A)+ RNA was elec-

W trophoresed in a 1% agarose/0.66M formaldehyde gel, blotted onto

2.57 - nitrocellulose, and hybridized withradiolabeled SPC2 cDNA (lane A)or SPC3 cDNA (lane B). RNA

1.35 - molecular weight markers (kb)were from BRL.

ci)0

0

I 1 1 2 1 3 1 4 1

_ SPC2

_ SPC3

FIG. 7. Regional expression of SPC2 and SPC3 in amphioxusanalyzed by RT-PCR. Whole amphioxus were sliced into four equalsegments, as shown above (approximately life size); total RNA wasisolated from each segment and subjected to RT-PCR amplification ofSPC2 (480 bp) and SPC3 (334 bp) cDNA fragments. The reactionproducts were electrophoresed in a 1.5% agarose gel and stained withethidium bromide.

at least two other subtilisin-related proprotein convertases inaddition to SPC2 and SPC3.

We thank Sean Martin for assistance in preparing the figures andFlorence Rozenfeld for assistance in preparation of the manuscript.This work was supported by National Institutes of Health Grants DK13914 and DK20595 and by the Howard Hughes Medical Institute.

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