The NeuAc(alpha-2,6)-Gal/GalNAc-Binding Lectin from Elderberry (Sambucus Nigra) Bark, a type-2...

Eur. .I. Biochern. 23.5, 128-137 (1996) 0 FEBS 1996

The NeuAc(a-2,6)-Gal/GalNAc-binding lectin from elderberry (Sumbucus nigra) bark, a type-2 ribosome-inactivating protein with an unusual specificity and structure El\ J M VAN DAMME', Annick BARRE', Pierre ROUGEL, Frcd VAN LEUVEN' and Willy J PEUMANS'

' Laboratory for Phytopathology and Plant Protection, Katholickc Universiteit Leuven, Leuven, Belgium ' Laboratoire de Pharmacologie ct Toxicologie Fondamentales, UniversitC Paul Sabatier, UPR CNRS 8221, Toulouse, France ' Center for Human Genetics, Katholieke Universiteit Lcuvcn, Leuven, Belgium

(Received 18 A~igust/h October 1995) ~ EJB 95 1375/2

The cDNA encoding the NeuAc(a-2,6)Gal/GalNAc binding lectin from elderberry (Samhucus nigrcz) bark (SNAI) was isolated from a cDNA library constructed with mRNA from the bark. Sequence analysis of this lectin cDNA revealed a striking similarity to the previously sequenced type-2 ribosome-inactiva- ting proteins from Ricinus communis and Ahrus precatorius. Molecular modelling of SNAI further indi- cated that its structure closely resembles that of ricin. Since SNAI strongly inhibits cell-free protein synthesis in a rabbit reticiilocyte lysate it presumably is a type-2 ribosome-inactivating protein. However, SNAI differs from all previously described type-2 ribosome-inactivating proteins by its specificity towards NeuAc(rr-2,6)Gal/GalNAc and its unusual molecular structure.

Keywords: Samhucus nigra; elderberry; lectin; ribosome-inactivating protein ; cDNA cloning.

Lectins and ribosome-inactivating proteins (RIP) are two dif- ferent types of defense-related proteins which are widespread in the plant kingdom. Usually, lectins are defined as proteins that bind, specifically and reversibly, carbohydrates without altering their structure (Goldstein and Poretz, 1986). Detailed analyses of several hundreds of lectins have demonstrated that they are a heterogeneous group of proteins which strongly differ from each other with respect to their sugar specificity, molecular structure and biological activities. In general, only related plant species contain similar lectins, such as e.g. legume lectins (Sharon and Lis, 1990). However, there are a few exceptions of related lec- tins which are found in representatives of different plant families (e.g. the mannose-binding lectins from Amaryllidaceae, Allia- ceae, Orchidaceae, Liliaceae and Araceae species ; Van Damme et al., 1995). In contrast to lectins, plant RIP are a homogeneous group of proteins (Barbieri et al., 1993). They are toxins which catalytically inactivate eukaryotic ribosomes by their highly spe- cific rRNA N-glycosidase activity. Basically two types of plant RIP can be distiguished. Type-1 RIP con t of a single catalyti- cally active subunit of about 30 kDa. They are widespread in the plant kingdom and represent a large family of evolutionary-re- latcd proteins. Type-2 RIP are composed of two different sub- units which are linked together by disiilfide bridges. One of these subunits, namely the A chain, has rRNA N-glycosidase activity and shows sequence similarity to the type-1 RIP. The B ~- chain is devoid of N-glycosidase activity but exhibits carbohy-

Co,uc!.sponderzc,e r o E. J. M. Van Darnine, Catholic University of Lcuvcn, Laboratory for Phytopathology and Plant Protection, Willerii dc Croylann 42, B-3001 Lcuven, Bclgium

Fax: +32 16 322976. Ahhrevintions. HCA, hydrophobic cluster analysis; LECSNAI,

cDNA encoding lectin I froin Sarnhuc.~~s nigm ; RIP, ribosome-inactiva- ting protein.

Nole. The novel nucleotide scqucncc data reported here have been submitted to the Genbank' W M B L Data library and are available under >accession number U27122.

drate-binding activity. Only a limited number of type-2 RIP have been described. Besides the well-known toxins ricin (from Ki- cinus communis seeds), abrin (from Ahrus precatorius seeds), viscumin (from Viscum album leaves) and modeccin and volken- sin (from Adenia digitatLz and Adenia volkensii roots, respective- ly) (Barbieri et al., 1993) similar proteins have only been iso- lated from winter aconite (Eranthis hyemalis; Kumar et al., 1993) bulbs, bark of elderberry (Sumhucus nigra; Girbes et a]., 1993a) and leaves of dwarf elder (Samhucus ehulus; Girbes et a]., 1993b).

The discovery of a type-2 RIP in elderberry bark is an inter- esting finding since the same tissue contains large amounts of two different lectins. Earlier work has demonstrated that the bark of this tree is a rich source of an NeuAc(a2-6)Gal/GalNac spe- cific agglutinin (called S. nigra agglutinin I or SNAI; Broekaert et al., 1984; Shibuya et al., 1987) and a GaVGalNAc-binding lectin (called S. nigra agglutinin TI or SNAII; Kaku et al., 1990). In this report we present evidence that SNAI behaves as a type- 2 RIP, which exhibits striking sequence similarity to previously described toxins but differs by its specificity and unusual molec- ular structure.

EXPERIMENTAL PROCEDURES

Materials. Branches of S. nigm were collected locally from a single tree. The inner bark was collected using a knife, taking care to remove the outer corky layer. This tissue was then stored at -70°C or -20°C prior to use. It should bc mentioned that the bark tissue used for the extraction of RNA was collectcd at the cnd of September (since at that time the synthesis of bark proteins is maximal ; Nsimba-Lubaki and Peumans, 19x6) whereas bark material destincd for the isolation of the lectin was sampled immediately after shedding of the leaves (around the end of October).

Van Damme et al. ( E m J. Biochem. 235) 129

Reticulocyte lysate (nucleasc treated) was obtained from Proniega. Oligo(deoxythymidine) cellulose was purchased from Sigma Chemical Co. Radioisotopes were obtained from ICN. A cDNA synthesis kit, the multifunctional phagemid pT,T, I 8 U, rcstriction enzymes and DNA-modifying enzymes were ob- tained from Pharmacia LKB Biotechnology Inc. Escherichia c d i XL1 Blue competent cells were purchased from Stratagene.

Isolation of SNAI. SNAI was isolated from lyophilized bark by affinitiy chromatography on fetuin Sepharose 4B as described previously (Broekaert et al., 1984) except that the lectin was desorbed with 20 mM acetic acid. To obtain an essentially pure preparation, the affinity-chromatography step was repeated.

Gel filtration. Gel filtration of SNAI was performed on a Pharmacia Superose 12 column. Running buffers were NaCIIP, ( 1 .S niM KH,PO,, 10 mM Na,HPO,, 3 mM KCI, 140 mM NaC1, pH 7.4) containing 0.2 M galactose (to avoid binding of the lec- tin to the column) and NaCl/P, containing 6 M urea. About 0.5 mg of protein (in 0.2 ml of buffer) was loaded onto the col- umn. The flow rate was 20 ml/h. Further experimental details are given in the legend to Fig. 1.

Analytical methods. Total neutral sugar was determined by the phcnol/H,SO, method (Dubois et al., 19S6), with glucose as standard.

Lcctin preparations were analyzed by SDS/PAGE using 12.5 %I to 25 % (mass/vol.) acrylamide gradient gels as described by Laeinmli (1970). In some instances lectin samples were re- duced and alkylated a s described previously (Van Damme et al., 1987).

Agglutination assays. Agglutination assays were conducted using human (type A) erythrocytes (Van Damme et al., 1987).

In vitro protein synthesis. A rabbit-reticulocyte-lysate kit was used to determine the inhibitory action of SNAI on in vitvo protein synthesis. The reaction mixture consisted of 35 p1 nuclease-treated lysate, 1 1.11 1 mM amino acid mixture (minus nicthionine), RNA substrate (tobacco mosaic virus RNA at a f i n d concentration of 20 pg/ml), 2 pl Translabel (400 pCi/ml) x id water added to a final volume or SO pl. After incubation of the lysate for 90 min at 30°C activity was measured by deter- mining the incorporation of labeled amino acids into hot tri- chloroacetic-acid-insoluble material using the filter-disk method.

RNA isolation. Total cellular RNA to be used for cDNA synthesis was prepared from plant material stored at -70°C essentially a s described by Van Damme and Peumans (1 993). Poly(A)-rich RNA was enriched by chromatography on oligo- (deoxythymidine) cellulose.

Amino acid sequence analysis. Protein sequencing was conducted on an Applied Biosystems model 477A protein se- quencer intcrfaccd with a n Applied Biosystems model 120A on- line analyzer.

Construction and screening of the cDNA library. cDNA libraries were constructed from poly(A)-rich mRNA isolated from the bark of S. nigrci using the cDNA synthesis kit from Pharmacia. cDNA fragments were inserted into the EcoRI site o f the multifunctional phagemid pT7T3 18 U. The library was propagated in E. coli XL1 Blue.

Recombinant lcctin clones for S. nigru were screened by col- ony hybridization using "P-labeled synthetic oligonucleotides derived from the N-terminal amino acid sequence of the lectin polypeptides. Hybridization was carried out overnight at 40- 50°C as described previously (Van Damme et al., 1991). After washing, filters were blotted dry, wrapped in Saran Wrap and cxposed to Fu.ji film overnight at -70°C. In a later stage the laiidotn-primcr-labeled cDNA clone encoding the S. nigru lectin was used as a probe to screen for more lectin cDNA clones. Hybridization was carried out at 65 "C as described previously (Van Damme et al., 1991). Colonies that produced positive sig-

nals were selected and rescreened at low density using the same conditions. Plasmids were isolated from purified single colonies on a miniprep scale using the alkaline lysis method as described by Mierendorf and Pfeffer (1 987) and sequenced by the dideoxy- nucleotide chain-termination method (Sanger et al., 1977). DNA sequences were analyzed using programs from PC Gene and Cenepro.

Northern Blot. RNA electrophoresis was performed accord- ing to Maniatis et al. (1982). Approximately 3 pg poly(A)-rich RNA was denatured in glyoxal and dimethylsulfoxide and sepa- rated in a l .2% (mass/vol.) agarose gel. Following electrophore- sis, the RNA was transferred to Immobilon N membranes (Milli- pore) and the blot hybridized using a random-primer-labeled lec- t in cDNA insert. Blots were prehybridized for 4 h at 42OC in 50% formamide, 5XNaCI/P,/EDTA (NaClIPJEDTA ; 0.1 8 M sodium chloride, 10 mM sodium phosphate, pH 7.4, 1 mM EDTA), SXDenhardt's solution, 10% dextran sulphate and SO pg/ml denatured salmon sperm DNA and hybridized in the same solution containing 2X 10" cpm/ml labeled DNA insert. Hybrid- ization was performed overnight at 42°C. Afterwards blots were washed consecutively in SXNaCI/P,/EDTA (IS min at 42"C), 1 XNaCI/P,/EDTA containing 0.1 % SDS (30 min at 42°C) and 0.1 XNaCI/P,/EDTA containing 0.1 % SDS (IS min at room tem- perature), respectively. An RNA ladder (0.16-1.77 kb) was used as a marker. Alternatively, blots were hybridized using "P- labeled synthetic oligonucleotide probes derived from the cDNA sequences as described previously (Van Damme et al., 1991).

Molecular modelling of SNAI. The hydrophobic-cluster analysis (HCA; Gaboriaud et al., 1987; Lemesle-Varloot et al., 1990) was performed to delineate the structurally conserved re- gions along the amino acid sequences of the A and B chains of SNAI and the model lectin ricin. HCA plots were generated on a Macintosh LC using the program HCA-Plot2 (Doriane).

Molecular modelling of the SNAI A and B chains was car- ried out on a Silicon Graphics Iris 4D2SG workstation using the programs InsightII, Homology and Discover (Biosym Technol- ogies). The atomic coordinates of rich (code 2aai) were taken from the Brookhaven Protein Data Bank (Rutenber et a]., 1991) and used to build the SNAI three-dimensional model. Energy minimization was carried out by several cycles of steepest de- scent and conjugate gradient using the cvff forcefield of Dis- cover. The program TurboFrodo (Bio-Graphics) was run on a Silicon Graphics Indigo R3000 workstation to perform the superimposition of the models.

The program Cameleon (Oxford Molecular) run on the In- digo R3000 workstation was used to predict the exposure of putative N-glycosylation sites occurring along the amino acid sequences of SNAI, using various algorithms (Janin, 1979; Hopp and Woods, 1981 ; Kyte and Doolittle, 1982; Karplus and Schultz, ,1985; Parker et al., 1986; Thornton et al., 1986).

The amino acid sequence alignments were performed on a MicroVAX 3100 (Digital, Evry, France) using the ialign pro- gram of the Protein Identification Resource data bank of the National Biomedical Research Foundation.

RESULTS

Isolation and characterization of SNAI. SNAI has originally been described as a tetrameric protein (140 kDa) consisting of two subunits [of 3.5 kDa and 30 kDa; Broekaert et al., 19841, which recognizes NeuSAc(rx2-6)GallGalNAc sequences (Shi- buya et al., 1987). Since some plant lectins are retarded on gel- filtration matrices when run in the absence of the complemen- tary sugar, we repeated the determination of the molecular mass of the native lectin by gel filtration in the presence of 0.2 M

Van Damme et al. (Eul: J. Biochem. 235) 130

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Fig. 1. Gel filtration of SNAI an a Superose 12 column in NaClm, (A) and NaCIP, containing 6 M urea (B). Molecular-mass reference markers (for analysis of native SNAI) were catalase (a, 240 kDa), R. communis hemagglutinin (b, 120 kDa), R. communis toxin (c, 60 kDa) and chymotrypsinogen (d, 25 kDa). For the analysis of the denatured proteins (in 6 M urea) myosin (a, 200 kDa), p-galactosidase (b, 130 kDa), R. communis toxin (c, 60 kDa) and chymotrypsinogen (d, 25 kDa) were used as markers.

1 R 2 Fig.2. SDSRAGE of SNAI. Reduced and alkylated, and unreduced SNAI (25 pg each) were run in lanes 1 and 2, respectively. Molecular mass reference proteins (lane R) were lysozyme (14 kDa), soybean tryp- sin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and phosphorylase b (94 kDa).

GCTTCCCAAAACAAGTATAGCAAGATGAGACTGGTAGCAAAATTATTATACCTAGCTGTT a s q n k y s k M R L V A K L L Y L A V

60 20

CTCGCAATTTGCGGGCTTGGAATCCATGGTGCCCTCACACACCCCCGTGTCACCCCACCC 120 L A I C G L G I H G A L T H P R V T P P 40

GTCTATCCCTCGGTTTCCTTTAATTTGACAGGTGCCGACACATACGAACCCTTCCTACGG 180 V Y P S V S F N L T G A D T Y E P F L R 60

GCACTGCAAGAAAAAGTGATACTGGGAAACCATACAGCATTTGATCTACCAGTACTGAAC 240 A L Q E K V I L G N H T A F D L P V L N 80

CCCGAATCTCAAGTCTCGGATTCGAATCGCTTCGTTCTGGTTCCTCTCACCAATCCCAGC 300 P E S Q V S D S N R F V L V P L T N P S 100

GGGGACACCGTCACCTTGGCAATAGATGTCGTTAACCTTTATGTGGTGGCTTTTAGTTCA 360 G D T V T L A I D V V N L Y V V A F S S 120

AATGGCAAGTCCTACTTTTTCAGCGGCTCTACGGCAGTTCAAAGGGACAATTTGTTCGTG N G K S Y F F S G S T A V Q R D N L F V

420 140

GACACCACTCAGGAGGAATTAAACTTCACAGGCAACTATATACTAGCCTCGACGCCAGGTA 480 D T T Q E E L N F T G N Y T S L E R Q V 160

GGTTTTGGAAGGGTGTATATCCCTCTAGGACCCAATCCCTAGATCAGGCCATCTCAAGT 540 G F G R V Y I P L G P K S L D Q A I S S 180

TTGCGGACATACACATTGACTGCTGGAGATACCAAGCCCCTAGCCAGAGGTCTCCTTGTG L R T Y T L T A G D T K P L A R G L L V

600 200

GTGATTCAAATGGTCTCGGAAGCGGCAAGGTTCAGATACATTGAGTTACGAATCCGGACA 660 V I Q M V S E A A R F R Y I E L R I R T 220

AGCATAACTGATGCTAGCGAGTTTACACCAGACCTTTTGATGCTGAGCATGGAG~TAAC 720 S I T D A S E F T P D L L M L S M E N N 240

TGGTCGTCTATGTCCTCGGAGATCCAGCAGGCTCAACCAGGAGGAATTTTCGCTGGAGTG 780 W S S M S S E I Q Q A Q P G G I F A G V 260

GTTCAGCTTCGAGATGAAAGGAACAACTCCATTGAGGTAACCAACTTTAGAAGACTCTTT V Q L R D E R N N S I E V T N F R R L F

840 280

GAGCTGACCTATATTGCGGTTCTTCTCTACGGATGCGCCCCGGTTACTAGTAGTAGTTAT 900 E L T Y I A V L L Y G C A P V T S S S Y 300

AGCAATAATGCTATAGACGCTCAGATAATTAAAATGCCCGTTTTTCGTGGGGGCGAGTAC 960 S N N A I D A Q I I K M P V F R G G E Y 320

GAkARAGTATGTTCGGTGGTAGAGGTAACAAGGCGCATCAGTGGTTGGGATGGATTGTGT E K V C S V V E V T R R I S G W D G L C

1020 340

GTGGACGTGAGGTATGGGCACTACATCGATGGGAATCCCGTCCAGCTGCGGCCGTGTGGA 1080 V D V R Y G H Y I D G N P V Q L R P C G 360

AATGAATGTAACCAACTATGGACGTTCCGCACTGATGGAACAATCCGGTGGTTGGGT~ 1140 N E C N Q L W T F R T D G T I R W L G K 380

TGCCTGACTGCCTCAAGCTCTGTCATGATATACGATTGTATACTGTTCCTCCAGAGGCC C L T A S S S V M I Y D C N T V P P E A

1200 400

ACTAAGTGGGTAGTATCTATTGACGGCACCATCACCAATCCTCACTCAGGACTCGTCCTT 1260 T K W V V S I D G T I T N P H S G L V L 420

ACAGCTCCTCAAGCTGCAGAGGGAACCGCCCTGTCTCTGGAGAACAATATCCATGCCGCT 1320 T A P Q A A E G T A L S L E N N I H A A 440

AGGCAAGGTTGGACTGTAGGAGATGTAGAGCCCCTCGTTACTTTTATTGTGGGATAT~A 1380 R Q G W T V G D V E P L V T F I V G Y K 460

CAAATGTGCTTGAGGGAAACGGTGAAAACATTTTGTATGGTTGGAGGACTGCGTTCTC 1440 Q M C L R E N G E N N F V W L E D C V L 480

AACAGGGTGCAGCAAGAGTGGGCACTCTATGGCGACGGCACCATTCGAGTAAACAGTMT 1500 N R V Q Q E W A L Y G D G T I R V N S N 500

CGTAGCCTATGTGTGACCTCCGAAGACCACGAGCCCAGTGATCTTATCGTCATTCTCAAG 1560 R S L C V T S E D H E P S D L I V I L K 520

TGCGAAGGGTCGGGCAACCAGCGCTGGGTATTCAACACCAACGGTACCATCTCAAACCCA 1620 C E G S G N Q R W V F N T N G T I S N P 540

AACGCTAAACTACTTATGGACGTTGCACAACGCGATGTCTCTCTTCGAAAAATCATTCTC 1680 N A K L L M D V A Q R D V S L R K I I L 560

TATCGGCCCACTGGGAATCCTAACCAGCAATGGATAACTACCACCCATCCAGCTTAGAGA 1740 Y R P T G N P N Q Q W I T T T H P A 578

AGTCGGTAGTCACGTCCTACCTTCTACCCACATTAGAGTATAGCATGATTAATCGGTTAC 1800

AATTAACATGCATATACATGAACACTAAATAAATGTGGATGTTTGAATATTAAGGCCCTG 1860

TTTGATTGCGCAAAAAA 1877

Fig. 3. Nucleotide sequence and deduced amino acid sequence of the cDNA clone LECSNAI encoding SNAI. Since the first ATG codon is probably used as the translation-initiation site, the deduced amino acids preceding this methionine are shown in lower case characters. Putative glycosylation sites are shown in bold. Determined N-terminal amino acid sequences of the A and B chains of SNAI are underlined.

galactose. Under these conditions, SNAI eluted at the same posi- tion as catalase (240 kDa; Fig. I) , which suggests that we under- estimated the molecular mass of native SNAI in our original report. SDS/PAGE of reduced and iodoacetylated SNAI (Fig. 2)

Van Damme et al. (Eus .I. Biochem. 235) 131

-28 1 LECSNAI : MRLVAKLLYLAVLAICGLGIHGALTHPR~TPPVYPSVSF 11 Niqrin: ID------ AGGL-RICCO: MYAVATWLCFGSTSGWSFTLEDNN IF-KQ--1IN- RICI-RICCO: MKPGGNTIVIWMYAVATWLCFGGSTSGWSFTLEDNN IF-KQ--1IN- ABRC-ABRPR: MDKTLKLLILCLAWTCSFSALRCaARTYPPVATN ..QDQV.IK.-

LECSNAI: NLTGA..DTYEPFLRALQEKVILGNHTAFDLPVLNPESQVSDSNRFVLVP 59 Nigrin: --D--VSA--RD--SN AGGL-RICCO: TTAD-TVES-TN-I--VRSHLTT-ADVRHEI---PNRVGLPI-Q--I--E RICI-RICCO: TTA--TVQS-TN-I--VRGRLTT-ADVRHEI---PNRVGLPINQ--IL-E ABRC-ABRPR: TTE--TSQS-KQ-IE--RQRLTG-..LIH-I---PDPTT-EER--YIT-E

LECSNAI: LTNPSGDTVTLAIDWNLYVVAFSSNGKSYFF...SGSTAVQRDNLFVDT 106 AGGL-RICCO: -S-HAELS----L--T-A---GCRAGNSA----HPDNQED-EAITH--T-V RICI-RICCO: -S-HAELS----L--T-A---GYRAGNSA---HPDNQED-EAI~H--T-V ABRC-ABRPR: -S-SERESIEVGI--T-A---AYRAGSa----LR...DAP-SASTY--PG-

LECSNAI: TQE.ELNFTGNYTSLERQVGFGRVYIPLGPKSLDQA1SSLRTYTLTAGDT 155 AGGL-RICCO: QNSFTFA-G---DR--QLG-.L-EN-E--TGP-ED---A-YY-STCGTQI RICI-RICCO: QNRYTFA-G---DR--QLA-NL-EN-E--NGP-EE---A-YY-STGGTQL ABRC-ABRPR: Q.RYS-R-D-S-GO---WAMQT-EE-S--LQA-TH--- ... FLRSG-SND

LECSNAI: KPLARGLLVVIQMVSEAARFRYIELRIRTSITDASEFTPDLLMLSMENNW 205 AGGL-RICCO: PT---SFM-C---I------Q---GEM--R-RYNRRSA--PSVITL--S- RICI-RICCO: PT---SFIIC---I------Q----GEM--RIRYNRRSA--PSVITL--S- ABRC-ADRPR: EEK--T-I-I---A-----Y---SN-VGV--RTGTA-Q--PA---L----

LECSNAI: SSMSSEIQQAQPGGIFAGWQI,RDERNNSIEVTNFRRLFELTYIAVLLYG 255 AGGL-RICCO: GRL-TA--ESNQ-A.--SPI--QRRNGSKFN-YDVSI.L.IPI--LMV-R RICI-RICCO: GRL-TA--ESNQ-A.--SPI--QRRNGSKFS-YDVSI.L.IPI--LMV-R ABRC-ABRPR: DNL-GGV--SVQDT.-PNN-I-SSINRQPW-DSLSHPT.VAVL-LM-FV

LECSNAI: CAPVTSSSYSNNAIDAQIIKMPVFRGGEYE.KVCSW.EVTRRISGWDGL 303 Niqrin: D--TX.TLXTS...F--N-V-R--- AGGL-RICCO: --- PP--QFS.... ... LLIR--VP..NFNAD--MDP.-PIV--V-RN-- RICI-RICCO: --- PP--QFS ....... LLIR--VP..NFNAD--MDP.-PIV--VGRN-- ABRC-ABRPR: -N-PNANQSP.......LLIRSIVE..E..8-I--SRY-P-V--G-R--M

LECSNAI: CVDVRYGHYIDGNPVQLRPCGN..ECNQLWTFRTDGTIRWLGKCLTA ... 3 4 8 Niqrin: X-- AGGL-RICCO: ----TGEEFF----I--W--KSNTDW-----L-K-S---SN-----ISKS RICI-RICCO: -----D-RFHN--AI--W--KSNTDA-----LKR-N---SN-----TYGY ABRC-ABRPR: ----YDDG-HN--RIIAWK-KDRL-E-----LKS-K---SN-----TEGY

LECSNAI: ..SSSVMIYDCNTVPPEATKWWSIDGTITNPHSGLVLTAPQAAEGTALS 396 AGGL-RICCO: SPRQQ-V--N-S-ATVG--R-QIWDNR--I--R-----A-TSGNS--K-T RICI-RICCO: SPGVY--------AATD--R-QIWDN---I--R-S---A-TSGNSG-T-T ABRC-ABRPR: APGNY------TSAVA---Y-EIWDN---I--K-A---S-ESSSM-GT-T

LECSNAI: LENNIHaARQGWTVGD.VEPLVTFIVGYKQMCLRENGENNFVWLEDCVLN 4 4 5 AGGL-RICCO: VQT--YAVS---LPTNNTQ-F--T---LYG---QA-SGK..------ TSE RICI-RICCO: VQT--YAVS---LPTNNTQ-F--T---LYGL--QANSGQ..--I---SSE ABRC-ABRPR: VQT-EYLMR---RT-NNTS-F--S-S--SDL-MQAQ-S-..---A--DN-

LECSNAI: RVQQEWALYGDGTIRVNSNRSLCVTSEDHEPSDLIV1LKCE.GSGNQR~ 4 9 4 AGGL-RICCO: KAE-Q----A--S--PQQ--DN-L-TDANIKGTWK--S-GPA-SG---M RICI - RICCO: KAE-Q----A--S--PQQ--ON-L--DSNIRETVVK--S-GPA-SG---M ADRC-ABRPR: KKE-Q----T--S--SVQ-TNN-L--K--KQGSP--LMA-SN-WAS---L

LECSNAI: FNTNGTISNPNAKLLMDVAQRDVSLRKIILYRPTGNPNQQWITTTHPA 5 4 2 AGGL-RICCO: -KND---L-LYNG-VL--RRS-P--KQ--VHPFH---I-LPLF RICI-RICCO: -KND---L-LYSG-VL--PAS-P--KQ----PLH-D---I-LPLF ABRC-ADRPR: -KNO-S-Y-LHDDMV---KRS-P--KE---HPYH-K---I-L-LF

Fig. 4. Comparison of the deduced amino acid sequences of the cDNA clones encoding SNAI (LECSNAI), the lectin from Kicinus communis (AGGL-RICCO) and the RIP from R. communis (RICI-RICCO) and A. precatorius (ABRC-ABRPR). The arrowhead indicates the processing site for the cleavage of the signal peptide. (-), sequence similarity to LECSNAI. (.) gaps introduced to give maximal similarity Lo IJXSNAT. The first amino acids of the A and B chain of SNAI, the lectin from Ricinus communis and the RIP from R. communis and A. precatorius are shown in bold. Determined N-terminal amino acid sequences of the A and B chains of SNAI are underlined. Reported N-terminal amino acid sequcnccs of the A and B chains of nigrin (Girbes et al., 1993a) are also shown. X indicates that no amino acid could be identified at that position of the sequence.

dcmonstrated that it is composcd of two different polypeptides, namely an A chain of 33 kDa and a B chain of 35 kDa. N- terminal sequencing of the blotted polypeptides yielded a dif- ferent sequence for the A and B chains (Fig. 3), which con-

firmed earlier findings that SNAI is composed of two different types of subunits (Shibuya et al., 1989). Unreduced SNAI yielded a very typical pattern upon SDS/PAGE. Most of the pro- tein migrates with a high apparent molecular mass. However,

1 3 2 Van Dammc et al. ( E m J. Biochern. 235)

saccharide/A chain and two heptasaccharides/B chain (Kimura et al., 1988).

Isolation and characterization of the cDNA clones encoding SNAI. Previous studies of the seasonal changes of the lectin content in the elderberry bark have shown that SNAI accumu- lates during late fall (Nsimba-Lubaki and Peumans, 1986). Therefore, a cDNA library was constructed from poly(A) rich RNA isolated from bark tissue, which actively synthesized the lectin (i.e. bark collected at the end of September).

Northern blot hybridization of the Samhucu.~ bark RNA using oligonucleotide probes complementary to the N-terminal sequence of the A and B chains of SNAI yielded a signal of approximately 1.8 kb. To increase the ratio of full-length clones, the total cDNA was size-fractionated Tor fragments of 1.5- 2.0 kb before ligation in the vector. Screening of the fractionated cDNA library using the synthetic oligonucleotides resulted in the isolation of several cDNA clones encoding the complete coding sequence of SNAI. Approximately 1 o/n of the bacterial colonies in the total (unfractionated) cDNA library contained SNAI sequences, which confirms that the bark accumulates this lectin at the time of sampling of the plant material.

The nucleotide sequence and the deduced amino acid se- quence of the cDNA clone encoding the SNAI (LECSNAI) arc depicted in Fig. 3. The lectin cDNA clone contains a 1734-bp open reading frame encoding a polypeptide of 578 amino acids with one possible initiation codon at position 9 of the deduced amino acid sequence. Translation starting with this codon results in a polypeptide of 570 amino acids (63.1 kDa), which contains the N-terminal amino acid sequences or both the A chain and the B chain of SNAI (Fig. 3). Using the rules of protein process- ing of von Heijne (1 986) a possible cleavage site for the process- ing of the signal peptide was identified between residues 28 and 29 of the lectin precursor, which is in good agreement with the N-terminal amino acid sequence of the A chain. Cleavage of the signal peptide at this site will result in a lectin polypeptide of 60 11 8 Da. Further processing of this polypeptide to yield the A and B chains of the mature mature SNAI implies one or more proteolytic cleavages. Since the B chains start with the sequence GGEYEKVCSV, a cleavage must take place betwcen amino acids 270 and 271 of the lectin precursor ProSNAI after cleav- age of the signal peptide (Fig. 4), resulting in an A chain of 30981 Da and a B chain of 29 154 Da.

The deduced amino acid sequence of LECSNAI contains eight putative N-glycosylation sites at positions 12, 34, 1 12, 1 16,

-1 77 -1:52 -1.28 -0.78 -0.53

Fig. 5. Northern blot of poly(A) rich RNA isolated from the bark of S. nigru. The blot was hybridized using the random-primer-labeled cDNA LECSNAI.

since 65-kDa but little 33-kDa and 35-kDa polypeptides are visi- ble, it is suggested that the A and B chains are linked by disul- phide bonds. Although the combined results of the gel-filtration experiments and SDS/PAGE suggest that native SNAI consists of [our pairs of disulfide bound A-B chains (i.e. [A-s-s-B],), the unusual behavior of the unreduced protein upon SDS/PAGE could not be explained. Further analysis of SNAI by gel filtra- tion in 6 M urea indicated that the denatured (but unreduced) lectin eluted with an apparent molecular mass of about 120 kDa (Fig. 1). Since ricin (which consists of a disulfide-linked A and B chain) eluted under the same conditions with a molecular mass of about 60 kDa (Fig. 1) the basic building block of SNAI appar- ently contains four disulfide-linked 33 and 35 kDa subunits. It is evident, however, that the exact subunit composition of the presumed 120-kDa building block of SNAI cannot be inferred from these gel-filtration experiments. To further elaborate the structure of SNAI we cloned the cDNA encoding SNAI and used the deduced amino acid sequence for molecular modelling of the lectin.

Determination of the total carbohydrate content of SNAT yielded a value of 4.9% (by mass). Assuming a molecular mass of 180 Ddmonosaccharide, the number of sugar residues amounts to about 17/A-B pair. These values are almost identical to those described for ricin, which contains a single penta/hexa-

la 17 N la' 15 D

2a 148 Y

2a' 138 K 18 60 N

18' 56 L 2p 187 R

28' 179 R

l y 103 S

l y ' 94 s 2y 229 S

2 y ' 220 A

- V R D G R F H N G N A - - V R Y G H Y I D G N P - - A - N - - - S - - G Q -

N G - E - - - N - - N F -

- T Y - G - Y S P G V Y -

- _ _ _ _ _ - s - - s s - - S - D S N - I R - E T V

- S E D - - - H E P S D L

- A T - S G N S - G T T -

- A P - Q A A E - G T A -

- V R A S D A S - L K Q -

- V A Q R D V S - L R K -

Q T N I Y A - V

E N N I H A - A

@P L H - G - D

@R P T - G - N

S

G

- - P

P

5 9

55

183

175

100

91

226

217

Fig. 6. Alignment of the amino acid sequence stretches of the homologous subdomains of the ricin B chain (la, 2a, lp, 2p, l y , 27) with the corresponding stretches of the SNAI R chain (la', 2a', 1/y, 2jY, ly', 2y'). Identical and homologous residues are boxed and residues forming the carbohydrate-binding site of riciii and SNAI are circled. (-), gaps introduced to give maximal similarity.

Van Damme et al. (Eur: J . Biochem. 235) 133

A I0 20 Y) (0 SO M 10 110 W IW 110 110 IM 140 IS0 IM 110 110 153 Ha 210 110 w) WI ZIO 2M DO UO

216

SNAI A

B B B B a p a a a a a a

B

I0 20 Y) I $0 W 10 10 W IW I10 IS I10 140 1% 160 170 160 IW 1W 210 220 P4 MI MI u6

SNAI B

Fig. 7. Comparison of the HCA plats of ricin and SNAI A chains (A), and of ricin and SNAI B chains (B). Helices (a) and strands of D sheet ([I) delineated o n the HCA plot or the ricin A chain were reported on the HCA plot of the SNAI A chain. Similarly, the subdomains l a , ID, l y , 2(x, 2p, 2y delineated on the HCA plot of the ricin B chain were reportcd on the HCA plot of the SNAI B chain ( I d , I f l , ly', 2 d . Zg, 27'). These delincalions were used to recognize the structurally conserved regions between the ricin and SNAI chains.

204, 232, 464 and 49X of the lectin precursor ProSNAI. Taking into account the carbohydrate content determined for the puri- fied SNAI, these results suggest that three putative N-glycosyla- tion sitcs are occupied.

Sequence similarity between SNAI and type-2 RIP. A com- parison of the deduced amino acid sequence of LECSNAI with that of other known plant proteins revealed a striking similarity to previously described type-2 RIP (Fig. 4). The overall se- quence similarity between SNAI and the toxin and agglutinin of castor bran i s as high a s 45% and 44%, respectively. Similarly, SNAI shares 43% sequence similarity with abrin. It should be mentioned that the sequence believed to be essential for RIP activity SEAAR, is well conserved.

As in ricin and abrin, the B chain of SNAI is built up of two homologous domains. Moreover, each domain is composed of thrce tandemly arranged subdomains. The position of the cyste- ine residues, which as can be concluded from X-ray crystallo- graphic studies of ricin are essential for the folding of the B chain, are highly conserved between SNAI and the RIP from

castor bean and Abrus precatorius. It should be emphasized, however, that the B chain of SNAI contains an extra cysteine residue at position 327 (Fig. 4).

Northern blot analysis. To establish the total length of the RNA encoding the S. nigm lectin I a blot containing poly(A) rich RNA isolated from the bark was hybridized with the random- primer-labeled lectin cDNA clone LECSNAI. Hybridization of the blot revealed one band of lectin mRNA. Using RNA markers the length of the RNA encoding the lectin was estimated to be approximately 1800 nucleotides (Fig. 5) which is in good agreement with the length of the cDNA clones isolated.

SNAI inhibits proteins synthesis in a reticulocyte lysate but not in a wheat germ cell-free system. Since the structure of SNAI is reminiscent of that of type-:! RIP and, in addition, shows a striking sequence similarity to previously sequenced or cloned type-:! RIP, we tested its possible inhibitory effect on the protein synthesis in a reticulocyte lysate. Native (i.e. unreduced) SNAI strongly reduced the incorporation of labeled amino acids

134 Van Darnnie et al. (Eur: J. Biochem. 235)

when added to a reticulocyte system, the concentration required for 5 0 % inhibition (IC,,,) being about 600 pM (data not shown). Previous experiments with cell-free extracts from wheat em- bryos demonstrated that native and reduced SNAI had no inhibi- tory effect on this plant derived cell-free system (Broekaert et al., 1984). These results suggest that SNAI, like nigrin b (Girbes et al., 1993a), strongly inhibits mammalian but not plant ribo- somes.

A

Modelling of SNAI. Since the overall structure of SNAI and its sequence similarity to r i c h and abrin indicate that this elder- berry lectin is structurally closely related to type-2 RIP, the lec- tin molecule was modelled using the coordinates of ricin, the three-dimensional structure of which has been resolved by X- ray crystallography (Rutenber and Robertus, 1991 ; Rutenber et al., 1991). Although it must be emphasized that the results of these modelling studies have to be interpreted with care, they can give interesting information about structural homologies be- tween related proteins.

In the molecular modelling experiments the numbering of the amino acids of the SNAI B chain slightly differs from the real sequence. Since the coordinates of ricin were used to model SNAI, the sequences of the SNAI and ricin B chains had to be aligned. Consequently the first six amino acid residues of the SNAI B chain had to be omitted.

The calculated percentages of identity and similarity be- tween the amino acid sequences of the carbohydrate-binding B chains of ricin and SNAI are 48% and 69%, respectively. Ac- cording to Rutenber and Robertus (1991) the B chain of ricin is built up of two domains. Each domain is made of four subdo- mains designated 12, In , 18 and l y for domain 1 , and 211, 2n, 2/3 and 2 y for domain 2. Among these subdomains the a, and y subdomains are homologous. They correspond respectively to the amino acid sequence stretches 17-59 ( la) , 60-100 (18) and 103-135 ( l y ) for the first domain, and 148-183 (2n), 187-226 (28) and 229-262 (2y) for the second domain. These subdo- mains were recognized on the basis of both sequence alignments and structural features. However, no regular secondary structures such as n helices and [j sheets were found in these domains, which apparently contain only SIZ loops. Four disulfide bridges (linking cysteine residues 20-39, 63-80, 151-164 and 190- 207) are involved in the folding of the r ich B chain. Similar amino acid stretches with cysteine residues occurring at con- served positions were easily recognized when the amino acid sequence of the B chain of SNAI was aligned with that of ricin:

217 ( 2 p ) and 220-253 (2y’) (Fig. 6). This structural similarity is illustrated by a comparison of the HCA plots of ricin and SNAI B chains (Fig. 7). All these data suggest that the overall folding of the SNAI B chain must be very similar to that of the r i c h B chain. Accordingly modelling of the B chain of SNAI was performed from the atomic coordinates of the ricin B chain.

The calculated percentages of identity and similarity be- tween the A chains of ricin and SNAT are 36% and 60%, respec- tively. Crystallographic studies have demonstrated that the toxic A chain of ricin is composed of three distinct domains. More- over, it contains regular secondary structures, namely eight a helices (A-H) and six strands of 8 sheet (designated a-f) ex- hibiting a lefthanded twist of about 110” when observed along the hydrogen bonds (Katzin et al., 1991). A comparison of the HCA plots of the A chains of ricin and SNAI shows that the a helices and strands of p sheet are readily conserved in both pro- teins. There are, however, some discrepancies in the A chain of SNAI as gaps or insertions of a few amino acid residues occur between the secondary-structure features (Fig. 7). As a result,

15-55 ( I d ) , 56-91 ( I F ) , 94-125 (‘l?’), 138-175 (Zn’), 179-

B

C

Fig. 8. Stereoviews of the three-dimensional models of the A chain (A), B chain (B) and heterodimer (C) of SNAI. Helices (thick lines) and left-handed twisted six-stranded sheet (heavy lines) of A chain are indicated. It is assumed that, like ricin, SNAI results from the association of the A (thin line) and B (thick line) chains by a disulfide bridge be- tween the cysteine residues C256 and C288 of ProSNAT.

the coordinates of the A chain of ricin were used to build a three-dimensional model of the A chain of SNAI.

Despite a few discrepancies, the three-dimensional models of the A and B chains of SNAI exhibit an overall folding very similar to those of ricin A and B chains (Fig. 8). The A chain of SNAI contains eight a helices and a six-stranded [j sheet with a lefthanded twist similar to that found in the A chain of ricin. No secondary structures occur in the B chain of SNAI. This chain can be considered as an assembly of two domains. Its overall shape mimics that of a typical lectin dinier built up of two flattened bell-shaped domes joined by their bottom edges (RougC et al., 1991). As for ricin, it is assumed that the two chains are linked via a disulfide bridge occurring between the two cysteine residues C256-C288 of SNAT which are conserved in the amino acid sequences of both chains.

Determination of the carbohydrate content of SNAI indi- cated that it contains three oligosaccharide chains. The amino acid sequences of the A and B chains of SNAI contain six (N12, N34, N132, N116, N204 and N232) and two “178 (N464 in ProSNAT) and N212 (N498 in ProSNAT)] putative N-glycosyla- tion sites, respectively (Fig. 9). Most of the glycosylation sites

Van Damme et al. ( E m J . Biochem. 235) 13s

A

I

n / B

Fig. 9. Stereoviews showing the location of the putative N-glycosylation sites N-X-T or N-X-S (thick lines) on the three-dimensional models of the A (A) and R (B) chains of SNAI.

of the A chain are located in helices or strands of /I’ sheet but two of them (N34 and N232) occur in well exposed and flexible loops and hence are accessible for glycosylation. The two N- glycosylation sites of the B chain of SNAI are located at the same position as these of the B chain of ricin (Kimura et al., 1988).

X-ray crystallography of ricin resolved the three-dimen- sional structure of the two carbohydrate-binding sites of the B chain. Both sites comprise five amino acid residues. Amino acid residues D22, Q35, W37, N46 and Q47 constitute the first car- bohydrate-binding site. The second sugar binding site i s formed by residues D234, 1246, Y248, N255 and Q256. Although both sites bind galactose through a network of either four (domain 1) or three (domain 2) hydrogen bonds, the site of domain 1 is a low-affinity site whereas the site of domain 2 behaves as a high- affinity site for lactose binding (Yamasaki et al., 1985; Hatake- yama et al., 1986). In addition, hydrophobic interactions occur between the pyranose ring of the sugars and aromatic (W37, Y248) or hydrophobic (1246) residues of the sites. A careful examination of the carbohydrate-binding site 2 of the modelled B chain of SNAI indicates that its composing amino acid resi- dues are readily superimposable to those forming the saccharide- binding site 2 of the B chain of ricin. Although these data have to be interpreted with caution (since they are based on computer modelling) they suggest that both sites are equivalent and could accommodate the same sugar residues. The carbohydrate-bind- ing site 1 of SNAI differs from that of ricin by the replacement of a tryptophan residue (W37) by an arginine residue (R35), that introduces a more hydrophilic character together with a local positive charge. In addition, a deletion of two residues corre- sponding to D44 and A45 of the r ich B chain, occurs in the vicinity of this site in the B chain of SNAI. Accordingly, no steric hindrance could be detected between GalNAc and other residues located around the site when site 1 of the SNAI B chain was superimposed to site 1 of the ricin B chain, after replace- ment of the galactose residue of lactose by a GalNAc residue.

This could explain why results from equilibrium dialysis experi- ments using NeuSAc(a2-6)lactitol as ligand of SNAI suggest that this lectin possesses two equivalent, non-interacting carbo- hydrate-binding sites (Shibuya et al., 1987).

Molecular structure of SNAI. The results of the sequence analysis of LECSNAI and the molecular modelling can be used to explain the structure of the native lectin. Like other type-2 RIP, SNAI is synthesized as a large precursor, which after post- translational processing yields a disulfide linked A and B chain (Fig. 10). Since the molecular modelling of SNAI indicates that its overall structure is very similar to that of ricin, one can rea- sonably expect that C256 (which is the only cysteine residue of the A chain) and C288 (corresponding to C8 of the B chain) are involved in the inter-chain disulfide bridge between the A and B chain (Fig. 10). Similarly, the conserved positions of C304, C323, C345, C357, C427, C442, C468 and C485 (compared with those of the cysteine residues in the B chains of r i c h and abrin) and the similar folding of the B chains of SNAI and r i c h indicate that four intra-chain disulfide bonds are formed between C304 and C323, C345 and C357, C427 and C442, and C468 and C485 of SNAI. In addititon to these 10 cysteine residues, which are presumably involved in inter-chain and intra-chain disulfide bonds, SNAI contains an extra cysteine residu (C327) in its B chain (Fig. 10). This cysteine is not present in ricin or abrin and is located in a loop well exposed on the surface of the B chain. Probably, intermolecular disulfide bridges can be formed between the C327 residues of two adjacent [A-s-s-B] molecules whereby an [A-s-s-B-s-s-B-s-s-A] molecule is formed (which is in agreement with the observation that unreduced SNAI elutes with an apparent molecular mass of 120 kDa upon gel filtration in 6 M urea; Fig. 10). Non-covalent association of two such building blocks yields a molecule with a molecular mass of about 240 kDa ([A-s-s-B-s-s-B-s-s-Aj,), which corres- ponds to the molecular mass of native SNAI (Fig. 10).

136 Van Damme ct al. (ELK J. Biochem. 235)

B chain

Excision of linker sequcnce I

Non rovdciit associdtion of two [A s-s B-s s B-s s-A1 moleculcs

J, into mature SNAI

Fig. 10. Schematic rcpresentation of the molecular structure of the SNAI precursor and the mature lectin. ProSNAl ( i s . the primary translation product of the SNAI mRNA after removal of the signal pep- title) is processed by excision of a linker (LS) between the A and B chains. In the next step, two [A-s-s-B] molecules are covalently linked by a clisulfide bridgc between two adjacent B chains whereby an [A-s- s-B-s-s-H-s-s-A] molecule is Ibrmed. Finally, two such [A-s-s-B-s-s-B- s-s-Al molecules associate (by noti-covalent interactions) into mature SNAI (1 A-s-a-B-s-s-B-s-s-AI,). The numbering of the cysteine residues (scc upper line) corresponds lo their position in the deduced sequence of ProSNAI (Fig. 4). Although the model presented herc explains the expcrimcntal data, the intermediates are hypothetical.

DISCUSSION SNAI is one of the few plant lectins that exhibit specificity

towards sialic acid. Although its biochemical and carbohydrate- binding properties have been studied in detail, no cvidence has been reported that it is structurally or functionally rclated to type-2 KIP. The results of our protein synthesis inhibition experi- ments and the molccular cloning of the cDNA cncoding SNAI cicmonstrate that this lectin presumably is a type-2 RIP, which shows a striking similarity to the previously sequenced toxins from castor bean and A. precatorius. Moreover, molecular mod- elling of the elderberry lectin also suggests that its overall struc- ture strongly resembles that of ricin. However, in spite of thesc obvious similarities to other type-2 RIP, SNAI has some unique propcrties. First, its specificity towards NeuSAc(rx2-6)GallCal- NAc definitely differs from that of the galactose or GalNAc- binding type-2 RIP from castor bean, A. precatorius, misletoe, Arleriirr sp. and Ercrathis hyemci1i.v. Sccond, native SNAI is a tetramcr of four (A-s-s-B] pairs whereas other type-2 RIP are

either monomers or dimers. In addition, the [A-s-s-B] molecules of SNAI are probably pairwise linked through a disulfide bridge between their respective B chains.

The finding that SNAL behaves as a type-2 RIP implies that clderberry bark contains at least two different RIP. Recent work has demonstrated that this plant material contains also a galac- tose-binding type-2 RIP called nigrin (Girbes et al., 199321). Ni- grin contains disulfide-bridge-linked A (26 kDa) and B (32 kDa) chains. It inhibits mammalian protein synthesis but does not af- fect plant ribosomes (Girbes et al., 1993a). Although the N- terminal sequences of both chains of nigrin show similarity to the respective A and B chains of other RIP, the similarity with the N-termini of the SNAl subunits is rathcr low (Fig. 4). It should also be mentioned that SNAI is more abundant in elder- berry bark than nigrin. According to the study of Girbes et al. (1993a) the yield of nigrin was 87 pg/g wet bark whereas that of SNAI varies around 1 mg/g wet bark (Broekaert et al., 1984).

The simultancous occurrence in elderberry bark of two RIP with a different carbohydrate-binding specificity is an interesting finding in view of the possible physiological rolc of these pro- teins. Feeding trials with SNAI demonstrated its (moderate) tox- icity for rats (Pusztai et al., 1991). The (oral) toxicity of nigrin for insects or higher animals has not been documented. How- ever, its strong inhibitory effect on mammalian ribosomes cer- tainly makes it a potential toxin (Girbes ct al., 1993a). The s i n - ultaneous accumulation in the same tissue of two RIP with B chains that have a different carbohydrate-binding specificity prohably offers an evolutionary advantage to the plant. Since both RIP recognize structurally unrelated sugars (namely galac- tose and sialic acid) the mixture of SNAI and nigrin recognizes more glycoconjugates in the digestive tract of predating insects or mammals. Moreover, it is also possible that both RIP act synergistically to penetrate the cells or inactivate the ribosomes.

A final point to discuss concerns the possible hazard of SNAI for human health. Although predominantly located in the bark, SNAI occurs also in the ripe berries (at a concentration of about 20 mg/kg fresh berries; W. Peumans, unpublishcd results), which are in many countries used for the production of foods and drinks. Since SNAI is reasonably heat stable (Broekaert et al., 1984) it is quite possible that some intact lectin is present in the final food products. At present, the oral toxicity of SNAI has only been assessed in rats. In spite of its extensive binding to the epithelial cells in the gut and its almost complete survival in the digestive tract, SNAI was apparently only moderately toxic to this rodent (Pusztai ct al., 1991). Howcver, since the results obtained with rats cannot be extrapolated to human be- ings, the potential hazardous effect of the type-2 RIP SNAI should be recognized.

This work was supported in part by grants from thc Catholic Univer- sity of Leuvcn (OT/94/17) and the National Fund for Scientific Research ( B c I g i u m , Foiirls voor Genmskundig Weten.schn/,jielijk On& rzoek grant 2.0046.93). W. I? is Research Director aiid E. V. D. Postdocloral Fellow of this fund. Furthermore we want to acknowledge grant 7.0047.90 from the Ncilionual Forztls voor Wc~fensckqilielijk 0~irlerzoek-Leven.slijii fund.

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