Animal and Plant Members of a Gene Family with Similarity to Alkaloid-Synthesizing Enzymes

Animal and Plant Members of a Gene Family withS

M*U

R

wopmibdgspiIpa

ns

at(1itceacfiwp

(

oCE

Biochemical and Biophysical Research Communications 271, 191–196 (2000)

doi:10.1006/bbrc.2000.2598, available online at http://www.idealibrary.com on

imilarity to Alkaloid-Synthesizing Enzymes

arco Fabbri,* Gabriele Delp,† Otto Schmidt,* and Ulrich Theopold*,1

Department of Applied and Molecular Ecology and †Department of Soil and Water,niversity of Adelaide, Glen Osmond, South Australia 5064, Australia

eceived March 29, 2000

from Drosophila melanogaster (5). Hemomucin is com-ptei(wao

M

f

aipfubi

ppmTsim

oqdMwc(

m

R

h

Here we describe novel members of a gene familyhich have similarity to strictosidine synthase (SS),ne of the key enzymes in the production of monoter-ene indole alkaloids. In addition to the first animalember of the family described previously (Drosoph-

la hemomucin), a second Drosophila member haseen identified, which appears to differ in subcellularistribution from hemomucin. In Arabidopsis, SS-likeenes form a multigene family, compatible with a pos-ible function as antifeedants and antibacterial com-ounds. In Caenorhabditis, two members have been

dentified and one member each in mouse and human.nterestingly, the human SS-like gene is strongly ex-ressed in the brain, the very organ many of the indolelkaloids act upon. © 2000 Academic Press

Key Words: alkaloids; dopamine; insect immunity;eurotransmission; biogenic amines; strictosidineynthase.

Amongst plant alkaloids, some monoterpenoid indolelkaloids have attracted particular interest due toheir ability to act as toxins (strichnin), antimalarialquinine), antineoplastic (vincristine and vinblastine,) or antipsychotic drugs (reserpine, 2). This led to thedentification of a number of enzymes that are part ofheir biosynthetic pathway including tryptophane de-arboxylase and strictosidine synthase (SS), two keynzymes in the production of monoterpenoid indolelkaloids (3). SS has been isolated from two plant spe-ies which are known for their pharmaceutical bene-ts, namely Catharanthus roseus (madagascar peri-inkle, 4) and Rauvolfia serpentina (sarpagandhalant, 3).We have previously described a cell-surface molecule

hemomucin) isolated from a hemocyte-like cell line

1 To whom correspondence should be addressed at Departmentf Applied and Molecular Ecology, University of Adelaide, Waiteampus, Glen Osmond, SA 5064, Australia. Fax: 161-8-8379 4095.-mail: [email protected].

191

osed of two domains, one with mucin-type repeats andhe other with sequence similarity to SS. We also foundvidence for the existence of hemomucin in two othernsect species, the hymenopteran Venturia canescens6) and the lepidopteran Galleria mellonella (7). Heree report on a novel gene family with similarity to SSnd hemomucin using expression data and informationbtained from genomic sequencing and EST projects.

ATERIALS AND METHODS

Flies. D. melanogaster w118 flies were kept on cornmeal/yeastood at 25°C with a 10/14 h light/dark cycle.

Preparation of antisera. For the production of an antiserumgainst recombinant hemomucin, a PCR amplified fragment cover-ng amino acids 178–299 (5), position 216–341 in Fig. 1, was ex-ressed in the expression vector pQE32 (Quiagen). The resultingusion protein was purified according to the instructions of the man-facturer and excised from a preparative polyacrylamide gel. Rab-its were immunized according to Harlow and Lane (8) with approx-mately 10 mg protein/immunization.

Electrophoretic techniques. SDS–polyacrylamide gel electro-horesis on a Mini-Protean II electrophoresis unit (Bio-Rad) waserformed according to Laemmli (9). Molecular weights were deter-ined using prestained SeeBlue molecular weight markers (Novex).he proteins were blotted onto a nitrocellulose membrane (Amer-ham) as described before (7). The amount of protein loaded was asndicated in the figure legends. The blotting efficiency was deter-

ined by staining the blot with Ponceau S.Sequence similarity searches were performed on the NCBI server

r the BDGP server using the blastp or tblastn program (10). Se-uences with a significant similarity to the original sequence wereownloaded into the Lasergene program package (DNASTAR Inc.,adison, WI) and further aligned using Megalign. Genomic regionsere analysed using Genfinder. Chromosomal localisations for P1

lones and BACs were available from the BDGP/HHMI EST Project11, 12).

Northern blots. RNA blots were performed as described (7). Theultiple tissue Northern blot was purchased from Invitrogen.

ESULTS

In an attempt to identify genes that are related toemomucin, we used the hemomucin amino acid se-

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

u

Vol. 271, No. 1, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Sequence alignment of 3 members of the SSl family. Drosophila (Dm) and human (H.s.), members of the SS family were alignedsing the Megalign program.

192

qfitMtwwecsqUfatBscmTmteWap(fd(wpDp

the mucin domain and differs in subcellular localisa-tDal

osittltmdlaeqismuwfhllmsAshp

tsbJlEtcfit(ebhg

D

gta

Ttwad


uence to run comparisons with sequences obtainedrom sequencing projects of the major model organ-sms, including Drosophila melanogaster, Arabidopsishaliana, Caenorhabditis elegans, Homo sapiens and

us musculus. In order to reduce background due tohe mucin domain, which shows sequence similarityith a large number of glycoproteins, only the domainith similarity to SS was used for comparison. Inter-stingly, one additional ORF with similarity to SSould be identified in Drosophila (Fig. 1). Further in-pection of the genomic region comprising this se-uence revealed no ORFs with mucin-type character.sing Genefinder, a complete ORF could be assembled

rom the genomic sequence, which also did not containny mucin-like sequences. The novel gene localises tohe same chromosomal position as hemomucin (98F).ecause of the sequence similarity to SS and the ab-ence of a mucin domain, we decided to call the geneoding for the second member of this family Drosophilaelanogaster strictosidine synthase-like 2 (DmSSl2).he ORF predicts a product of approximately 46 kDaolecular mass. Since the sequence conservation be-

ween hemomucin and DmSSl2 is strong enough toxpect antibody cross-reactions, we performedestern-blot analyses using an antiserum produced

gainst recombinant hemomucin. Moreover structuralrediction programs predicted DmSSl2 to be secreted13, 14). Therefore, we analysed hemolymph samplesor hemomucin-like proteins. We were indeed able toetect a labeled band of the predicted molecular massand different from the 100 kDa of hemomucin), whichas completely absent in a blot developed with there-immune serum (Fig. 2). We therefore conclude thatmSSl2 is antigenically related to hemomucin andresent in Drosophila hemolymph. It does not contain

FIG. 2. (A) A candidate for DmSSl2 in Drosophila hemolymph.wo different concentrations of Drosophila hemolymph (equivalento the hemolymph of 40 and 20 third instar larvae) were analysedith an antiserum specific for hemomucin and the preimmune serums a control. A band of the size expected for DmSSl2 was onlyetected with the immune serum.

193

ion from hemomucin, which is a cell surface protein.mSSl2 also lacks two of the three N-linked glycosyl-tion sites present in hemomucin and is expected to beess glycosylated or not glycosylated at all.

In order to identify members of the SS family fromther organisms, we performed sequence searches oneveral organisms with genomic and/or EST sequenc-ng projects. In A. thaliana, 13 members could be iden-ified, two with the highest similarity to SS from Ca-haranthus roseus (AtSSl11 and AtSSl12) and otheress related members (Figs. 3A and 3B). Amongsthese, four sequences seem more closely related to he-omucin than to SS (AtSSl4-7). Three sequences we

erived from soybean (Glycine max) ESTs that wereong enough to allow an alignment were included in thenalysis. Additional members were identified from C.legans and both mouse and human. The human se-uence looks complete whereas the mouse ORF wasncomplete at the time of preparation of this manu-cript. None of the newly identified members contain aucin domain as with hemomucin, which seems to be

nique to insects. Since SS has been grouped togetherith paraoxonases and gluconolactonases into a super-

amily based on sequence similarity, we also includeduman paraoxonase in our comparison (15). In a phy-

ogenetic analysis, all SS-like sequences are more re-ated to each other than to paraoxonase (Fig. 3B). The

ajority of the plant sequences group together and areeparate from the animal sequences. SequencestSSl4-7 from A. thaliana, and one of the soybeanequences (GmSSl3) are an exception as they showigher similarity to the animal members than to otherlant members of the family even from A. thaliana.Since the EST sequencing projects are performed on

issue-specific libraries, a preliminary indication of thepecificity of expression can be obtained from the num-er of ESTs obtained from one particular tissue.udged from this, high expression for the human SS-ike EST is expected in the brain (a majority of theSTs in the clot with hemomucin similarity are from

he brain, see: http://www.ncbi.nlm.nih.gov/UniGene/lust.cgi?ORG 5 Hs&CID 5 22391). In order to con-rm high expression in the brain, a human multipleissue Northern blot was probed with one of the ESTsclone image 258726), confirming that the strongestxpression amongst the tissues chosen is in fact in therain (Fig. 4). The chromosomal localisation of theuman SSl-sequence is to chromosome 20 between re-ions 11:21 and 11:23.

ISCUSSION

Here we present evidence for the existence of a novelene family with similarity to the plant enzyme stric-osidine synthase, one of the key enzymes in plantlkaloid biosynthesis. Since secondary metabolites like

ilosftop

mmaifo1o

rdpi


ndole alkaloids are widely distributed in plants butess often found in animals, the existence of membersf this family in a number of animal species came as aurprise. We speculate that the animal members of theamily have a different function than SS. The fact thathe Arabidopsis members fall in different groups, withne group being more similar to hemomucin than otherlant genes, indicates that also in plants, SSl proteins

FIG. 3. Relationship between members of the SSl family: The saoseus (C.r.) strictosidine synthase as well as Arabidopsis sequencesisplay, where branch distances correspond to sequence divergence) uarts (positions 155–179 in Fig. 1) is shown in A. Some of the sequenn B is based on the sequence between positions 116 and 281 in Fig

194

ay perform more than one function and be involved inore than one biochemical pathway. The existence of

t least 13 members of the SSl family in Arabidopsis isn agreement with the identification of multiple iso-orms of the enzyme in C. roseus (16) and with thebservation that this plant is known to contain over00 different monoterpenoid indole alkaloids (1). Eachf these SSl family members may differ in their enzy-

sequences as in Fig. 1 and sequences from C. elegans (C.s.) and C.re analysed for their possible phylogenetic relationship (unbalancedg the CLUSTAL program as part of Megalign. One of the conservedare identical in this part but differ otherwise. The phylogenetic tree

mewesin

ces. 1.

mdtsifhAdpil

tdgTmp(wcidtatfbcdiopsin(rSaq

ase and the product of the l(2)amd gene, which arekta(l(mcpl

msltatlasntst

A

cgAt

R

Rsca


atic activities and/or substrate specificities for theifferent products of secondary metabolism. In addi-ion, they might differ in their expression, which washown to be induced by fungal elicitors and jasmonaten the case of SS from C. roseus. The selective pressureor the production of a variety of phytoalexins mayave lead to the duplication of an ancestral SSl gene inrabidopsis and other plants (like soybean). In Arabi-opsis, multiple gene duplication events have hap-ened leading to the 13 observed members of the fam-ly (for example, AtSSl8 and 9 and AtSSl4-7 are closelyinked and have arisen by gene duplication).

The gene coding for the next enzyme in the biosyn-hetic pathway leading to indole alkaloids, strictosi-ine glucosidase (17) also seems to be part of a multi-ene family in Arabidopsis (unpublished observations).he most intensely studied members of this family areyrosinases, which are believed to be part of the

lant’s defense against insects and possibly pathogens18). In contrast to that and similar to the situationith SS, we could only identify one gene with signifi-

ant similarity to strictosidine glucosidase in Drosoph-la (unpublished results). Strictosidine itself and theeglucosylation product of strictosidine could be showno have antibacterial activity but seem to lack thentifeedant activity of further downstream products inhe biosynthetic pathway for indole alkaloids (19). Asor insects, hemomucin, the previously identified mem-er from Drosophila seems to be exceptional in that itontains a mucin-like domain attached to the SS-likeomain. Therefore in Drosophila and possibly in othernsects, there seem to exist two members of the family,ne on the cell-surface and a second one which isresent in hemolymph. At this stage, it can only bepeculated what the substrates for SSl members innsects might be, but it is interesting to note that aumber of components of the phenoloxidase cascadelike dopamine) show structural similarity with indoleing-containing substances. The enzyme upstream ofS in the biosynthetic pathway for monoterpene indolelkaloids (tryptophane decarboxylase) shows high se-uence similarity with the enzymes dopa decarboxyl-

FIG. 4. Expression pattern of the human SSl member. TotalNA from brain (br), kidney (ki), liver (li), lung (lu), pancreas (pa),pleen (sp), and skeletal muscle (sm) was analysed using the ESToding for the human SSl protein as a probe. The 28S RNA is showns an internal control for the amount loaded.

195

nown to be involved in both the generation of neuro-ransmitter substances (like dopamine and serotonin)nd central components of the phenoloxidase cascade20, 21). The amd product is also expressed in theymph glands suggesting a possible role in immunity21). In agreement with a possible function of hemo-

ucin in defense reactions which may include antimi-robial activity and/or protein crosslinking, we havereviously gained evidence that it is involved in hemo-ymph coagulation (7).

An interesting finding is the fact that the humanember of the SSl family shows the strongest expres-

ion in the brain, the very organ some indole alkaloidsike reserpine act upon by interfering with the synapticransmission mediated by dopamine (2). Learningbout the function of the human SSl protein(s) mightherefore help to understand the effect of these alka-oids in the brain. It will also be interesting to analyse

possible function of the insect genes in the nervousystem and to look into a possible correlation betweeneurological defects in the genomic and mutations inhe corresponding gene. With the availability of theequence information presented here it is now possibleo address these questions.

CKNOWLEDGMENTS

We thank Peter Langridge, Ute Baumann, and Mark Dowton forritically reading the manuscript. This work was supported by arant from the Australian Research Council. U.T. is supported by anRC Research Fellowship, M.F. by a postgraduate scholarship from

he University of Adelaide.

EFERENCES

1. Kutchan, T. M. (1995) Alkaloid biosynthesis—The basis for met-abolic engineering of medicinal plants. Plant Cell 7, 1059–1070.

2. Bennett, M. R. (1998) Monoamineric synapses and schizo-phrenia—45 years of neuroleptics. J. Psychopharmacol. 12,289–304.

3. Kutchan, T. M., Hampp, N., Lottspeich, F., Beyreuther, K., andZenk, M. H. (1988) The cDNA clone for strictosidine synthasefrom Rauwolfia serpentina: DNA sequence determination andexpression in Escherichia coli. FEBS Lett. 237.

4. McKnight, T. D., Roessner, C. A., Davagupta, R., Scott, A. I., andNessler, C. L. (1990) Nucleotide sequence of cDNA encoding thevacuolar protein strictosidine synthase from Cantharanthus ro-seus. Nucleic Acids Res. 18, 4939.

5. Theopold, U., Samakovlis, C., Erdjument-Bromage, H., Dillon,N., Axelsson, B., Schmidt, O., Tempst, P., and Hultmark, D.(1996) Helix pomatia lectin, an inducer of Drosophila immuneresponse binds to hemomucin, a novel surface mucin. J. Biol.Chem. 271, 12708–12715.

6. Kinuthia, W., Li, D., Schmidt, O., and Theopold, U. (1999) Is thesurface of endoparasitic wasp eggs and larvae covered by alimited coagulation reaction? J. Insect Physiol. 45, 501–506.

7. Theopold, U., and Schmidt, O. (1997) Helix pomatia lectin andannexin V, two molecular probes for insect microparticles: Pos-sible involvement in hemolymph coagulation. J. Insect Physiol.

8. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratoy Man-

1

1

1

1

1

1

ysis, novel RNA editing, and evolutionary implications. Proc.

1

1

1

1

2

2


ual, Cold Spring Harbor: Cold Spring Harbor Laboratory.9. Laemmli, U. K. (1970) Cleavage of structural proteins during the

assembly of the head of the bacteriophage T4. Nature 227, 680–685.

0. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman,D. J. (1990) Basic local alignment tools. J. Mol. Biol. 215, 403–410.

1. Hartl, D. L., Nurminski, D. I., Jones, R. W., and Lozovskaya,E. R. (1994) Genome structure and evolution in Drosophila:applications of the framework P1 map. Proc. Natl. Acad. Sci.USA 91, 6824–6829.

2. Kimmerly, W., Stultz, K., Lewis, S., Lewis, K., Lustre, V., Ro-mero, R., Benke, J., Sun, D., Shirley, G., Martin, C., and Palaz-zolo, M. (1996) A P-1 based physical map of the Drosophilaeuchromatic genome. Genome Res. 6, 414–430.

3. Nielsen, H., Brunak, S., and von Heijne, G. (1999) Machinelearning approaches for the prediction of signal peptides andother protein sorting signals. Protein Eng. 12, 3–9.

4. Nakai, K., and Horton, P. (1999) PSORT: A program for detect-ing sorting signals in proteins and predicting their subcellularlocalisation. Trends Biochem. Sci. 24, 34–35.

5. Kobayashi, M., Shinohara, M., Sakoh, C., Kataoka, M., andShimizu, S. (1998) Lactone-ring-cleaving enzyme: Genetic anal-

196

Natl. Acad. Sci. USA 95, 12787–12792.6. Dewaal, A., Meijer, A. H., and Verpoorte, R. (1995) Strictosidine

synthase from Catharanthus roseus—Purification and charac-terisation of multiple isoforms. Biochem. J. 306, 571–580.

7. Geerlings, A., Ibanez, M. M. L., Memelink, J., van der Heijden,R., and Verpoorte, R. (2000) Molecular cloning and analysis ofstrictosidine b-D-glucosidase, an enzyme in terpenoid indole al-kaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275,3051–3056.

8. Rask, L., Andreasson, E., Ekbom, B., Eriksson, S., Pontoppidan,B., and Meijer, J. (2000) Myrosinase: Gene family evolution andherbivore defense in Brassicaceae. Plant Mol. Biol. 42, 93–113.

9. Luijendijk, T. J. C., Vandermeijden, E., and Verpoorte, R. (1996)Involvement of strictosidine as a defensive chemical in Catha-ranthus roseus. J. Chem. Ecol. 22, 1355–1366.

0. Wright, T. R. F. (1996) The Wilhelmine E. Key 1992 invitationallecture: Phenotypic analysis of the dopa decarboxylase gene clus-ter mutants in Drosophila melanogaster. J. Heredity 87, 175–190.

1. Wang, D., and Marsh, J. L. (1995) Developmental regulation ofthe a-methyldopa-hypersensitive gene of Drosophila melano-gaster. Dev. Biol. 168, 598–612.

Documents

Animal and Plant Members of a Gene Family with Similarity to Alkaloid-Synthesizing Enzymes