THE JOURNALOF BIOLOGICAL CHEMISTRY Vol. 258, No. 15, Issue of August 10,pp.

9544-9549,1983
Printed in U.S.A.

The Biosynthesis and Primary Structure of Pea Seed Lectin*

(Received for publication, October 29, 1982)

Thomas J. V. Higgins, Peter M. Chandler, Gerard Zurawski, Susan C. Button, and Donald Spencer
From the Commonwealth Scientific and IndustrialResearch Organization, Division of Plant Industry, Canberra, Australian Capital Territory 2601, Australia

Plant Material-Peas (Pisum satiuum L. cv. P1/G 086, a selection from cv. Greenfeast from Rumsey and Son, Sydney, Australia) were grown in artificially lit cabinets with a 16-h photoperiod asdescribed previously (16) but a t 20 C rather than25 C. Zsolation of Pea Seed Lectin-Lectin was isolated from developing or maturepea cotyledons by the saccharide affinity method described for the isolation of concanavalin A (17). Briefly, this involved extracting cotyledons in 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH 7.5, 0.5 M NaCI, and 1 mM phenylmethylsulfonyl fluoride followed by centrifugation a t 10,000 X g for 30 min to remove insoluble material. The supernatant (total extract) was treated with saturated ammonium sulfate and the fraction precipitating between 30% and 80% saturation was retained. This fraction was dissolved in Hz0 and dialyzed first against H,O and then against1 M NaCl before beingadsorbed to Sephadex G-50 previously equilibrated with 1 M NaC1. The Sephadex columnwas washed with 1 M NaCl for 48 h and the lectin was then displaced with 0.2 M D-glucose in 1 M NaCI. The fractions containing protein were pooled and dialyzed first against 1 M NaCl and finally against 30 mM Na phosphate, pH 7.4, containing 0.45 M NaC1. Serology and Preparation of Affinity Gels-IgG against pea seed Lectins, although widely distributed in nature, are found lectin was isolated from the serum of sheep wbicb had been injected most abundantly in the seeds of legumes. Pea seed lectin with9mg of lectin in Freunds completeadjuvant followed by a belongs to agroupwhichincludes concanavalin A(from booster injection of 2 mg in Freunds incomplete adjuvant 1 month Canaualia ensiformis), lentil lectin (from Lens culinaris), and later. The animal was bled after 1 further week. Antilectin IgG was favin (from Vicia faba). All four lectins are mitogenic and isolated from crude serum using a column consisting of pea lectin bind specifically to D-mannose and D-glucose (1).Native pea covalently linked to Sepharose4B. T o prepare this column, lectin (3 lectin has anM , of approximately 49,000 with subunitsof M , mg/ml of swollen gel) was coupled to CNBr-activated Sepharose 4B as recommended by Pharmacia (Uppsala, Sweden) except that the = 17,000 ( p ) and 6,000 ( a ) (2). Favin and lentil lectin are also pH used was 7.4. Coupling, blocking unreacted sites with 1 M glycine, two-chain lectins and are similar in size to pea lectin (3, 4). and washing with 5 cycles alternatively at pH 4.0 and pH 8.0 was Concanavalin A, however, is a tetramer composed of a single followed by one wash each in 3 M KCNS in 0.1 M Tris-HC1, pH 8, 8 M urea in 0.1 M Tris-HC1, pH 8, and 0.5 M NaCl in 0.1 M Tris-HC1, subunit of M , = 25,500 (5). In mature pea seeds, the lectin is localized within protein pH 8, then two washes in 30 mM Na phosphate, pH 7.4, with 0.45 M bodies (6) along with legumin and vicilin, the major storage NaCl and 1% Tween 20. The gel was then treated for 15 min with bovine serum albumin (5 mg/ml) in PNT,washed in PNT, and stored proteins (7-9).Legumin and vicilin both contain subunits with 0.02% Na azide. This antigen gel was incubated with total lectin which arise by post-translational processing of high molecular antiserum by rotating gently for 30 min a t room temperature and weight precursors (10-13). This endoproteolyticprocessing then by standing on ice for 30 min. Unbound proteins were removed has beenshown to occur intheprotein bodies (14). We by washing with ice-cold 0.01 M Na phosphate, pH 7.4, 0.15 M NaCl therefore investigated whether lectin is also synthesized as a and the specifically bound antilectin IgG was eluted with 0.1 M citric adjusted to pH 3 with 0.1 M Na2HP04. Fractionswere collected single chain which is later processed to form the two compo- acid into 1 M K phosphate, pH 7.4, inorder to raise the pH rapidly. nent subunits. Using pulse-chase labeling, in vitro protein Fractions containing IgG were pooled, adjusted to pH 7.4 with 5 N synthesis andsequencing of cDNA plasmids, we have shown KOH, concentrated to about 10mg/ml in a pressure filtration appathat pea seed lectin is initially synthesized as a high molecular ratus (Amicon, Sydney,Australia) witha P M 30 membraneand weight precursor containing a leader sequence with thea and dialyzed against PNT.IgG was coupled to CNBr-activated Sepharose 4 8 as described above for coupling of antigen, again at pH 7.4. Immunoaffinity Chromatography-The Sepharose-IgG gel was

Isolation of pea lectin from immature seeds, together with in vivo pulse-chase labeling and in vitro translation of RNA from such seeds, all gave results which indicated strongly that pea lectin is initially synthesized as a large precursor (pre-pro-form)(Mr= 25,000) which is cleaved both co- and post-translationally to yield the CY and /3 subunits of mature lectin. Two overlapping cDNA plasmids complementary to lectin mRNA were sequenced. They coded for both /3 and a subunits of lectin, in the orientation /3 + CY from the NH2 terminus, together with a hydrophobic NH2-terminal leader sequence and included a 3 untranslated region of 124 nucleotides. In the appropriate reading frame, no stop codon was found between the coding sequences for the /3 and CY subunits, confirming the synthesis of pea lectin via a pre-pro-form. The entire pea lectin sequence showed a high degree of homology with the published amino acid sequences of lectins from lentil, broad bean, and, to a lesser extent, with concanavalin A from jack bean. Sequence variation occurred mainly in those regions thought not to be involved in metal- and sugar-binding or in the formation of 8pleated sheets.

P subunits covalently linked in theorientation NH,-p-aCOOH. Comparison of the deduced amino acid sequence with the published amino acid sequence of the a chain (15) also indicates that there may be further post-translational modification of the carboxyl terminus of the a chain.
EXPERIMENTAL PROCEDURES

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: IgG, immunoglobulin G; PNT, 30 mM Na phosphate, pH 7.4, with 0.45 M NaCl and 1% Tween 20; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

9544

Synthesis and Structure of Pea Seed Lectin

used to purifylectin from complex mixtures obtained by cell-free translation of pea cotyledonRNA or from extracts of pulse- and chase-labeled pea cotyledons. Such mixtures were dialyzed against P N T and the Sepharose-IgG was equilibrated with the same buffer. Batchwise immunoadsorbtion of the lectin was carried out using 50 to 200 pl of Sepharose-IgG and up to 1 ml of protein solution. After binding for 30 min a t room temperature, the unbound proteins were removed by seven 1-ml washes withPNT and the bound protein was eluted withfour 0.5-ml washes with 5% SDS, each for 30min a t 37 "C. The pooled bound fractions were precipitated with 4 volumes of cold acetoneon ice for 15min. The precipitatedprotein was collected by centrifugation a t 10,000 X g for 10 min and fractionated by SDS-PAGE (18) followed by fluorography (19). Construction of Lectin cDNA Clones-cDNA clones were constructed as described in detail elsewhere (20). Briefly, poly(A)-containing RNA was isolated from cotyledons 22 days after flowering and used for cDNA synthesis with avian myeloblastosis virus reverse transcriptase (Dr. d. Beard, Life Sciences Inc.). After second strand synthesis by the same enzyme the DNA solution was extracted with phenol and precipitated with ethanol. The DNA wasdissolved in water and digested with S1 endonuclease in the presence of 0.3 M NaCI, 0.03 M Na acetate, 3 mM ZnS04, pH4.5. After extraction with phenol and precipitation with ehtanol, the DNA was fractionated on a Bio-Rad A150m column (280 X 7 mm) equilibrated with 0.01 M Tris-HCI, pH 7.5, 1 mM EDTA. The leading half of the DNA peak was precipitated with ethanol, dissolved in water, anda homopolymer tract of dC was added (21). An estimated 15 dC residues were added per 3"OH terminus. PstI-digested pBR322 was similarly modified with a dG tract. The homopolymer-tailed cDNA and pBR322 were phenol-extracted, ethanol-precipitated, and mixed in approximately equimolar proportions in50 pl of 0.2 M NaC1. The mixture was heated a t 60 "C, then cooled to room temperature over a 2-h period before addition of 200 pl of competent Escherichia coli K-12 RR1 cells (22). Following 30-min incubation on ice and then 5 min a t 42 "C, 1 ml of L broth (10 g of Difco Bacto-Tryptone, 5 g of Difco Yeast Extract, 10 g of NaCl/liter) was added, the cells were incubated 30 min at 37 "C, and the transformants selected on L agar-containing tetracycline (20pglml). Colonies containingplasmids with inserts were detected by subsequent failure togrow on L agar-containing ampicillin (100 pglml). Colonies were screened by hybridization (23) using '"P-labeled cDNA prepared from poly(A)-containing RNA to reveal those inserts representing abundant mRNA sequences. Plasmids were isolated from colonies giving the strongest hybridization signal and tested for cross-hybridization of inserts. One of the hybridization families detected had properties expected of a cloned lectin mRNA sequence as judged by hybrid release translation. Full details of this procedure are described elsewhere (24). Briefly, an insert from pPS1550 (a lectin cDNA plasmid) was subcloned into the PstI site of phage fd103 replicative form DNA. The phage derivative containing the strand complementary to lectin mRNA was identified (fd103-50A) and 1 pg of this single-stranded DNAwas mixed with 0.5 pg of poly(A)-containing RNA from cotyledons 22 days after flowering in 25 pl of 0.2 M NaCI, 0.02 M Tris-HCI, pH 7.5, 2 mM EDTA, 0.1% SDS. Following hybridization at 65 "C for 20 min, the mixture was fractionatedon a Bio-Rad A150m column. Nucleic acids in the excluded peak (fd103-50A plus mRNA hybridized to the insert)were precipitated with ethanol and added to an in vitro translation system before andaftertreatment with7 mM methylmercuric hydroxide (Alfa Chemicals, Ventron Corporation, Danvers, MA). Fractionation of the radioactive polypeptides by SDS-PAGE followed by fluorography revealed polypeptides whose mRNAs were hybridized to the insert in the phage DNA and released after melting of the hybrids. Twocross-hybridizing cDNA clones wereselected for sequencing which was done by the method of Maxam and Gilbert (25). Determination of NucleotideSequence at the 5' End of Lectin mHNA-The inserts in the lectin cDNA plasmids do not represent the extreme 5' end of lectin mRNA. T o obtain the sequence for this region a 49-base pair primer, consisting of the fragment between the MboII and SauIIIa sites at aminoacid positions -5 and +8, respectively (see Fig. 31, was purified from pPS15-104 and 5' end-labeled with polynucleotide kinase and [y-:'2P]ATP (25). RNA was prepared from pea cotyledons (17 to 20 days after flowering) and 6 mg were passaged once over oligo(dT)-cellulose in 0.1 M NaCI, 0.01 M TrisHCI, pH 7.5, 1 mM EDTA, 0.1% SDS. Bound RNA was eluted with water, sodium acetate (pH 6.0) added to 0.3 M, and the RNA precipitated with 2 volumes of ethanol. Primer (approximately 50 ng) was mixed with the poly(A)-containingRNA in 150 pl of 0.16 M KCI, 0.1 M Tris-HCI,pH 8.3, andthesolution boiled for2min andthen

9545

allowed to cool in a water bath from 70-40 "C overa2-h period. Magnesium chloride and dithiothreitol were each added to 10 mM and dATP, dGTP, dCTP, TTP to 400 p~ (finalconcentrations). Reverse transcriptase (75 units) wasadded; the mixture was incubated a t 41 "C for 1 h and then extracted with buffer-saturated phenol and the nucleic acids were precipitated with ethanol. RNA was hydrolyzed a t 65 "C for 1 h in 250 pl of 0.1 M NaOH and, after neutralization of the solution with Tris.HCI, DNA was precipitated with ethanol in the presence of 30 pg of calf thymus carrier DNA and sequenced as described above. Other Methods-The isolation of RNA, cell-free translation in the wheat germ system and the preparation of microsomal membranes
a

..'.
I .

17-

" e

- 0 - 1 7

6-

-fl

-6

31

FIG. 1. Pea lectin extracted from mature andimmature seeds and fractionated by SDS-polyacrylamide gel electrophoresis. a, lectin isolated by saccharide affinity chromatography; lune I , from mature seeds; lane 2, from immature seeds, 25 days after flowering; lane 3, from mature seeds, heavy gel loading. All lanes stained with Coomassie blue. b, pea lectin synthesis in vivo under pulse-chase labeling conditions. Intact cotyledons from immature pea seeds (15 days after flowering) were labeled for 1.5 h with a mixture of 14 "C-aminoacids and then transferred to a nonradioactive medium for an additional 22 h. Cotyledon extracts were prepared a t intervals, lectin was isolated, fractionated on anSDS-polyacrylamide gel, and detected by fluorography. Lane 1, total unfractionated cotyledon extract after 1.5-h labeling; lane 2, lectin isolated by saccharide affinity chromatography after1.5-h labeling; lane 3, lectin isolated by immunoaffinity chromatography after 1.5-h labeling; lanes 4 to 7, lectinisolated by immunoaffinitychromatography from cotyledon extracts after a chase period of, respectively, 2, 4, 11, and 22 h. The of the lectinpolypeptides. numbersindicatethe M,X

25 -23

FIG. 2. Pea seed lectin synthesis in uitro. Total polysomal RNA from immature cotyledons (15 days after flowering) was translated in a wheat germ cell-free system eitherwith or without addition of microsomal membranes from dog pancreas. Lectin-related products were isolated by immunoaffinity chromatography.Lune I , unfractionated in vitro products, no added pancreas membranes; lane 2, lectinrelated translation products, no addedmembranes;lane 3, lectinrelated translation products synthesized in the presence of membranes; lane 4 , lectin synthesized in vivo after 1.5-h labeling. The numbers indicate theM,X lo-" of lectin polypeptides.

9546

Synthesis and Structure

of Pea Seed Lectin

lectin from immature seeds contained a third subunit of M, = 23,000 (Fig. la, lane 2). With heavier loadings a small amount of the M, = 23,000 component was also detectable in lectin from mature seeds (Fig. la, lane 3). The increased proportion of the n/i, = 23,000 component in immature seeds suggested the possibility that lectin is first synthesized as a polypeptide of this size and subsequently cleaved to generate the two subunits of M, = 17,000 and 6,000 which are characteristic of mature pea seeds (2). Synthesis of Pea Seed Lectin in Vivo-Detached, but otherwise intact, cotyledons from developing (immature) pea seeds take up radioactive amino acids and incorporate them into a range of proteins (13, 18). Lectin was isolated from extracts of such immature cotyledons which had been pulselabeled with a mixture of Y-aminoacids for 1.5 h. Using either saccharide affinity chromatography (Fig. lb, lane 2) or immunoaffinity chromatography (Fig. lb, lane 3) to isolate lectin, the only radioactive polypeptide detected following SDS-PAGE was of M, = 23,000. Neither the M, = 17,000 nor the M, = 6,000 subunit was present. When similarly labeled cotyledons were transferred to a nonradioactive medium and incubated for chase periods of up to 22 h, isolation of lectin by immunoaffinity chromatography revealed a progressive decrease in the proportion of the polypeptide of M, = 23,000 and a corresponding increase in the M, = 17,000 and 6,000 -30 -20

from dog pancreas were described earlier (12), as was also the pulsechase labeling of detached cotyledons with C-labeled aminoacids (18, 26).

RESULTS Subunit Composition of Lectin from Mature and Immature Seeds-Pea lectin, isolated from mature seeds by saccharide affinity chromatography and fractionated by SDS-PAGE, showed the expected subunit composition, namely, two subunits of M, = 17,000 and 6,000 (Fig. la, lane I). However,

4 0 . s= -

. Q N b --

. 0 . D

FIG. 3. Diagram showing the sequencing strategy used in determining the nucleotide sequence of the inserts in pPS1550 and pPS15-104. The overlapping regions of these two inserts (approximately 690 nucleotides) had identical sequences. Solid synbols represent 3-labeled ends (DNA polymerase I) and open symbols 5-labeled ends (polynucleotide kinase).

NNNNNNW

RNAANNARNR

ATG GCT TCT CTT CAA ACC CAA ATG ATC TCG TTC Met Ala Ser Leu Glu Thr Glu Met Ile Ser phe

TAC GCG ATA TTT CTA TCC ATT CTC TTA Tyr Ala Ile Phe Leu Ser Ile Leu Leu

1 10 -10 ACA ACA ATC CTT TTC TTC AAG GTG AAC TCA ACT GAA ACC ACT TCC TTC TTG ATC ACC AAG TTC Thr l'hr Ile Leu Phe Phe Lys Val Asn Sex E Glu Thr Thr Ser Phe Leu Ile Thr Lys phe

20

30

40

AGC CCC GAC CAA CAA AAC CTA ATC TTC CAA GGA GAT GGC TAT ACC ACA AAA GAG AAG CTG ACA CTG ACC A.&G GCA GTA AAG AAC ACT GTT Ser Pro Asp Gin Gln Asn Leu Ile Phe Gln Gly Asp Gly Tyr Thr Thr Lys Glu Lys Leu Thr Leu Thr Lys Ala Val Lys Asn Thr Val

GGC AGA GCC CTC TAT Gly Arg Ala Leu Tyr

60 70 50 TCC TCA CCT ATC CAT ATC TGG GAT AGA GAA ACA GGC AAC GTT GCT AAT TTT GTA ACT TCC TTC ACT TTT GTC ATA Ser Ser Pro Ile His Ile Trp Asp Arg Glu Thr Gly Asn Val Ala Am Phe Val Thr Ser Phe Thr Phe Val Ile

100 80 90 AAT GCA CCC AAC AGT TAC AAC GTT GCC GAC GGG TTT ACG TTC TTC ATC GCA CCT GTA GAT ACT AAG CCG CAG ACC GGC GGT GGA TAT CTC Am Ala Pro Am Ser Tyr Asn Val Ala Asp Gly Phe Thr Phe Phe Ile Ala Pro Val Asp Thr Lys Pro Gln Thr Gly Gly Gly Tyr Leu 110 120 130 GGA GTT TTC AAT AGC GCA GAG TAT GAT AAA ACC ACT CAA ACT GTT GCT GTG GAG TTT GAC ACT TTC TAT AAT GCT GCA TGG GAT CCA AGC Gly Val Phe Am Ser Ala Glu Tyr Asp Lys Thr Thr Gln Thr Val Ala Val Glu Phe Asp Thr Phe Tyr Asn Ala Ala Trp Asp Pro Ser AAC AGA GAT AGA CAT ATT Am Arg Asp Arg His Ile

160 150 140 GGA ATC GAT GTG AAC AGT ATC AAA TCC GTA AAC ACT A4G TCG TGG AAG TTG CAG AAT GGT GAA GAG GCT AAT Gly Ile Asp Val Am Ser Ile Lys Ser Val Asn Thr Lys Ser Trp Lys Leu Gln Am Gly Glu Glu Ala Am 170 180 190

GTT GTG ATA GCT TTT AAT GCT GCT ACT AAT GTG TTA ACT GTT AGT TTG ACC TAT CCT AAT TCA CTT GAG GAA GAG AAT GTA ACT AGT TAT Val Val Ile Ala Phe Am Ala Ala Thr Asn Val Leu Thr Val Ser Leu Thr Tyr Pro Asn Ser Leu Glu Glu Glu Am Val Thr Ser Tyr

200

210

220

ACT CTT AGC GAC GTT GTG TCT TTG AAG GAT GTT GTT CCT GAG TGG GTA AGG ATT GGT TTC TCA GCT ACC ACA GGA GCA GAA TAT GCA GCA Thr Leu Ser Asp Val Val Ser Leu Lys Asp Val Val Pro Glu Trp Val Arg Ile Gly Phe Ser Ala Thr Thr Gly Ala Glu Tyr Ala Ala

230

240
TTCATCAT

CAT GAA GTT CTT TCA TGG TCT TTT CAT TCT GAG TTG AGT GGA ACT TCA AGT TCT AAG CAA GCT GCA GAT GCA TAG TTTTTTGCTT His Glu Val Leu Ser Trp Ser Phe His Ser Glu Leu Ser Gly Thr Ser Ser Ser Lys Gln Ala Ala Asp Ala Stop CA TGCATGTCAA GTCATGTGTG ACAGATCCAG TTTCTATAAA TAAACTGCGC ATATGCAGTA CTTTTGTAAT GTTGTTATGT ATGTTACTTG ATGCGTTTAT

TA15C13

FIG. 4. The nucleotide sequence of cloned DNA complementary to acids deduced from this sequence. The published amino acid sequences terminal amino acids of the p subunit (15, 27) showed complete agreement the 5 untranslated region showed some ambiguity; unidentified bases in this by R (see Results). All other assignments were unambiguous. Amino acid respective NH, termini of, pre-pro-lectin, pro-lectin (and the fl subunit), and assignment of NH2 terminus of pre-pro-lectin.)

pea lectin mRNA and the amino for allthe LY subunit and 25 NH2except for residue 23. Sequencing in region are indicated by N and purines residues at -30, 1, and 188 are the the 01 subunit. (See Discussion for

Synthesis and Structure of Pea Seed Lectin

components (Fig. lb, lanes 4 to 7).These results are consistent with the suggestion that lectin initially appears as a M, = 23,000 polypeptide (pro-lectin) which is post-translationally cleaved to form the smaller subunits over a period of many hours. Cell-free Synthesis of Pea Lectin-When total polysomal RNA from immature pea cotyledons wastranslated ina wheat germ system, a single lectin-related polypeptide was selected by immunoaffinity chromatography (Fig. 2, lane 2). On SDSPAGE thispolypeptide was significantly larger (M, = 25,000) than the putative lectin precursor selected pulse-labeling after in vivo (Fig. 2, cf. lanes 2 and 4 ) . The inclusion of dog pancreas microsomal membranesinthe i n uitro translationsystem resulted in two lectin-related products (Fig. 2, lane 3 ) . The major one, M, = 23,000, coincided with the i n uiuo precursor and the minor one coincided with M, = 25,000 product formed in the absence of added membranes. These results provide presumptive evidence for the presence of a leader sequence on the primary lectin translation product. The in vivo and i n uitro studiesthereforeindicatethe involvement of at least twoprocessing steps in pealectin synthesis, namely, the co-translational cleavage of a leader sequence from a pre-pro-formandthepost,-translational cleavage of the resultant pro-lectin to generate the a and /3 subunits. NucleotideSequence of PeaLectin mRNA-In orderto confirm the existence of the pre-pro-form of lectin, a population of cDNA inserts was constructed in the plasmid pBR322 using total poly(A)-containing RNA from immature pea cotyledons (22 days after flowering). Two clones (pPS15104 and pPS15-50) with overlapping inserts were selected which, in hybrid release translation experiments, specifically selected the mRNA for an i n vitro translation product of M, = 25,000 (data not shown). The DNA inserts in both clones were sequenced using the restriction sites and strategy depicted in Fig. 3. The overlapping regions of these two inserts have identical sequences. The complete nucleotide sequence of these inserts and the amino acid sequences derived from the only open reading frame areshown in Fig. 4. Comparison of the derived amino acid sequence with the published amino acidsequence for the entire pea lectin a subunit (M, = 6,000) (15) and for the NH,-terminal 25 residues of the 6 subunit ( M , = 17,000) (27) confirmed that the cloned cDNA was indeed derived from the mRNA for pea lectin (Fig. 4). When considered together thetwo cDNA inserts accounted for 917 nucleotides of lectin mRNA. Thisconsisted of a sequence coding for 19 amino acids at the 5 end, a sequence of 735 nucleotides encompassing both the @ and a subunits, and a noncoding sequence of 124 nucleotides at the 3 end. To study sequences at the extreme 5 end of lectin mRNA which were notrepresentedinthe cDNA inserts,primer extension experimentswere performed. ASauIIIa-MboII fragment from the 5 end of the insert in pPS15-104 was 5-end labeled, hybridized to lectin mRNA in a total poly(A)-containing RNA population, and used to prime a reverse transcriptase reaction. The extended primer was then sequenced. An unambiguous sequence was obtained extending as far as the methioninecodon at aminoacid position -30. We assume that translation starts at this codon (-30) rather than at the methionine codon at position -23 for reasons detailed under Discussion, The leader sequence of pre-pro-lectin is therefore 30 amino acids in length. In the same experiment the sequence determined for the 5 untranslated region of the message cqntained many instances of apparent reaction in all four lanes (denoted as N in Fig. 4), and several instances of pyrimidinereactions which were

9547

intermediate between T+C and C reactions (denoted as the complementary purine nucleoside R in Fig. 4). We interpret this change in the apparent specificity of the cleavage reactions as representing sequence heterogeneity in the 5 untranslated region of two or more lectin mRNAspecies and/ or from degradation of lectin mRNA at the extreme 5 end. Unfortunatelythe sequence of thecomplementarystrand cannot be obtained by primer extension. The major termini in cDNA synthesis occurred 12,15, and16 nucleotides beyond the methionine codon at -30 suggesting that the5 untranslated region of lectin mRNA is very short. The lack of a stop codon in the appropriatereading frame between position 1 and position 245 (Fig. 4) confirmed that pea lectin is initially translated as a pro-protein from which the a and 0 subunits must be derived by post-translational cleavage. Within the pro-lectin molecule, the subunits are arranged in the order p -+ a from the NH, terminus.
DISCUSSION

Our results from i n vivo pulse-chase experiments indicated that pea lectin is synthesized asa single translation product which is cleaved post-translationally to yield the a and 0 subunits typical of pea lectin in mature seed. I n vitro translation studies further suggested that the primary translation product of lectin mRNA contained a leader sequence which is removed co-translationally. These conclusions were confirmed and extended when two cDNA clones complementary to pea lectin mRNA were sequenced. The combined cDNA insert of 917 bases had an openreading frame of 792 nucleotides and the deduced amino acid sequence showed perfect agreement with published sequences for the entire a subunit (15) and, with the exception of 1 residue (Asp forAsn a t residue 23 of Fig. 4), for the NH2-terminal25 residues of the /3 subunit of pea lectin (27). The deduced amino acid sequence for the remainderof the p subunit showed substantial homology with that published for the p subunit of lentil lectin (4) was favin (3). No stopcodon in the appropriate reading frame was present between the sequences codingfor the two subunits which were oriented 0-+ a from the NH, terminus. The presence of a leader sequence was confirmed by the finding of a sequence in thecDNA insert coding for 19 amino acids with a predominantly hydrophobic composition which was 5 relative to the NH, terminus of the p subunit. This sequence did not contain an ATG codon corresponding to the start of translation. When the 5 region of lectin mRNA was sequenced by extension of a primer from the cDNA insert, methionine codons were found at positions -23 and -30. It is uncertain which of these 2 methionine residues is the actual start of translation but two lines of evidence favor the residue at -30. In the first place it is rare for translation not to start at the first methionine codon in a mRNA; of 153 eukaryotic mRNAs surveyed by Kozak (28) only 11 did not begin translation at the first methionine codon. Secondly, in the same survey, it was found that 91% of the initiator methionine AUG codon; this is codons had a purine three bases 5 to the the case for the methionine codon at -30, but is not so for the codon at -23. The most likely start of translation is therefore at position -30, which would generate aleader sequence 30 amino acids in length of which 17 are hydrophobic. This interpretation is strongly supported by the partial amino acid sequencing of the NH, terminus of i n vitro synthesized favin (29). The signal sequence of favin is 29 amino acids in length and, with respect to the 4 amino acids (Met, in vitro, is highly homologous Leu, Ile, and Phe) incorporated with the pea lectin signal sequence. The deduced amino acidsequence of the pea /3 subunit

9548

Synthesis and Structure Seed of Pea

Lectin

differed in only 27 out of 157 residues from that reported by Foriers et al. (27) for the 8 subunit of lentil lectin. The actual length of the pea p subunit is uncertain since the COOHterminal amino acid sequence is unknown. Pea lectinmRNA codes for a further 30 amino acids beyondthe COOH terminus of the lentil subunit (residues 158 to 187, Fig. 5 ) . The favin /3 subunit is 24 residues longer than that from lentil and the deduced amino acid sequence for the pea 8 subunit shows close homology with those 24 residues in favin (Fig. 5 ) . There is conservation of a triplet (Tyr-Pro-Asn) which is at the COOH terminus of favin and isalso common to concanavalin A (residues 179 to 181, Fig. 5). From this it seems likely that the next6 amino acids (Ser-Leu-Glu-Glu-Glu-Asn) in the pea sequenceforma"linker" region in thepro-lectin (Mr = 23,000) precursor and that this sequence is removed during post-translational processing. This sequence is highly hydrophilic and may therefore be exposed to protease attack. It is interesting to note that a very similar sequence (Glu-Glu-AlaAsn, also followed by Val) is present in the pea lectin mRNA at positions 158 to 162. Position 158 corresponds to the COOH terminus of lentil lectin. It ispossible that lentil lectin mRNA codes for the closely related Glu-Glu-Glu-Asn-Val sequence at this position and that that this sequence acts as a signal for endoproteolytic cleavage in lentil lectin to generate two its subunits. Concanavalin A, which is not cleaved endoproteolytically, is completely divergent in this region.

Pea lectin mRNA codes for 4 amino acid residues at the COOH terminus of the a subunit that were not reported in theamino sequence of themature polypeptide (15). It is possible that these residues(Ala-Ala-Asp-Ala) are also removed post-translationally andin this context is it interesting to note that acarboxypeptidase has been localized in the protein bodies of mature dryseeds of the legume Vigna radiata (30). Lectins from lentil, V . faba (favin), pea, and C. ensiformis (Concanavalin A) have similar sugar-bindingspecificities and the extensive homology of their amino acid sequences has been discussed (4, 15). This homology exists in spite of the fact that concanavalin A is a single chain lectin whereas the other three lectins each consist of two subunits. Cunningham et al. (3) achievedmaximumsequence homology between concanavalin A and favin by proposing a circularly permuted model in which the NH2- and COOH-terminal aminoacids of Concanavalin A were located consecutively within the p subunit of favin. Our results confirm the homology of the pea lectin @-subunitwith the other lectins and extend this homology through the entire sequence of the p subunit. In pea and lentil lectin, favin, and concanavalin A there is a striking conservation of amino acid sequences which are believed to be important to the structure and function of these lectins (Fig. 5 , see also Refs. 3 and 4). In particular, there is conservation of the amino acid residues which contribute to

10 20 30 Pea T E T T S F L I T K F S P D Q Q N L I F Q G D G Y T T K E K [ ] L T L T K A V K [ Lentil S G-G[ 1 v s -[ Favin S - P - T RD --IP G [ I t Con A Q-DALK-MFNQ-K- KD" L A T - G T N G N - E R V S S N G S P

1 1
1

Pea Lentil Favin Con A Pea Lentil Favin Con A Pea Lentil Favin Con A Pea Lentil Favin Con A Pea Lentil Favin Con A

50 60 70 40 ] N T V G R A L Y S S P I H I W D R E T G N V A N F V T S F T F V I N A [ ] P N S Y N IE-G T NGSQ-FR DE -S V- G L S D-Q-T-ID[ ]A - G [ I EGSSV- FYA-V- ES[ ]SAT-SA-EAT-A-L"K[ ]S-D-[ ]H

[ [

80 90 100 110 V A D G F T F F I A P V D T K P Q T G G G Y [ ] L G V F N S A [ ] E Y D K T T Q T V A V [ -1 Y N G K S
P-

swYNGKD-" IA- SNI-SSIPSGSTGRL-L-PD-NA[

A
]-[

b [ 11-

120 160 E F D T F Y N A A [ ] W D P S N R D R H I G I D V N S I K S V N T K S W K L Q N G E E A [ I K E N [ [ I G K I S N -L-YP-TDIGy P[ 1 I K S V R S K K - A K - N M - D - K V G 170 180 190 200 N V V I A F N A A T N V L T V S L T Y P N S L E E E N V T S Y T L S D V V S L K D V V 1 NE-P [ ]L-GE-P H-A-SST -T-LT A H - I Y - S V D K R - S A V V S 1 IADATSV-YD-D-N-L 210 240 220 230 P E W V R I G F S A T T G A E Y A A H E V L S W S F H S E L S G T S S S K Q A A D A F-Q-H-N-Q-GH-KT-P-N T T-LVGLsL Y K E T TN -T KI - K S N - T H FIG. 5. A comparison of the published amino acid sequence of lentil lectin, favin, and concanavalin
et al. (4)) with the amino acid sequence of pea lectin derived from the cDNA clones. Residues homologous with pea lectin are indicated with a solid line. Square brackets indicate the absence of amino acids in this position when sequences are aligned for maximum homology. The numbers refer to positions in the pro-lectin sequence. The NH, termini of mature pea and lentil lectins, favin, and concanavalinA occur as follows: lentil and pea LY subunit a t position 188, lentil and pea p subunit a t 1, favin N a t 188, favin 6 a t -1, concavalin A (a single chain) at 108.

A (after Foriers

Synthesis and Structure Seed of Pea

Lectin

9549

REFERENCES the structure of the hydrophobiccavity and the 8-pleated sheets, and those residues which are involved in the metal1. Lis, H., and Sharon, N. (1981) in The Biochemistry of Plants (Marcus, A,, ed) Vol. 6, pp. 372-449, Academic Press, Sydney binding and sugar-binding sites. The new information on the 2. Trowbridge, I. S. (1974) J. Bid. Chem. 2 4 9 , 6004-6012 pea lectin 8 subunit presented hereprovides further evidence 3. Cunningham, B. A,, Hemperly, J. J., Hopp, T. P., and Edelman, of conservation of sequences in relation to these sites. The G. M. (1979) Proc. Natl. Acad. Sci. U. S.A. 7 6 , 3218-3222 region of greatest divergence in sequenceof pea and the other 4. Foriers, A., Lebrun, E., Van Rapenbusch, R., de Neve, R., and lectins (residues 65 to 77 in the 8 subunit, Fig. 5 ) coincides Strosberg, A. D. (1981) J . Bid. Chem. 2 5 6 , 5550-5560 with a region which is thought not to be involved in 8-pleated 5. Wang, J. L., Cunningham, B. A,, and Edelman, G. M. (1971) Proc. Natl. Acad. Sci. U. S. A . 6 8 , 1130-1134 sheet formation (4) or metal- or sugar-binding (31). 6. Van Driessche, E., Smet, G., DeJaegere, R., andKanarek, L. Unlike pea and lentil lectin, favin is aglycoprotein, the (1981) Planta 153,287-296 carbohydrate moiety of which is attached to theAsn residue 7. Craig, S., Millerd, A., and Goodchild, D. J. (1980) A u t . J .Plant at position 167 in Fig. 5 (3). This residue is lacking in lentil 339-351 Physiol. 7, lectin but is present in pea lectin. However, lack of glyco8. Graham. T . A,, and Gunning, B. E. S . (1970) Nature ILond.) sylation in pea lectin is consistent with the known require228,81-82 9. Varner, J. E., and Schidlovsky, G. (1963) Plant Physiol. 3 8 , 1 3 9 ment for Asn-X-Thr (Ser) for N-glycosylation of asparagine 144 (32). Whereasin favin the sequence is Asn-Ala-Thr,the 10. Croy, R.R. D., Gatehouse, J. A., Evans, I. M., and Boulter, D. corresponding sequence in pea lectin is Asn-Ala-Ala. (1980) Plants 1 4 8 , 49-56 The structure of pea lectin mRNA, as revealed by cDNA 11. Gatehouse, J. A., Croy, R. R. D., Morton, H., Tyler, M., and sequencing, includes an untranslated region at the 3' end of Boulter, D. (1981) Eur. J. Biochem. 118,627-633 poly(A) 12. Higgins, T. J. V., and Spencer, D. (1981) Plant Physiol. 6 7 , 205the molecule consisting of 124 nucleotides prior to the 211 tail. Within the 3' untranslated sequence the putative poly(A) recognition sequence (AAUAAA) (33) occurs 59 residues from 13. Spencer, D., and Higgins, T. J. V. (1980) Biochem. Int. 1, 502509 the poly(A)sequence. The more usual position of this se14. Chrispeels, M. J., Higgins, T. J. V., and Spencer, D. (1982) J. quence is 11-30 nucleotides from the poly(A)sequence (34). Cell Biol. 93, 306-313 Taken together, these results indicate that pea lectin under15. Richardson, C., Behnke, W. D., Freisheim, J . H., and Blumenthal, goes a number of processing steps: the co-translational reK. M. (1978) Biochim. Biophys. Acta 537,310-319 moval of a leader sequence from a pre-pro-form of the protein, 16. Millerd, A,, and Spencer, D. (1974) A u t . J. Plant Physiol. 1 , 33 1-34 1 post-translational cleavage of the pro-lectin to yield 01 and 8 subunits and, possibly, removal of 4 amino acid residues at 17. Agrawal, B. B. L., and Goldstein, 1. J. (1967) Biochim. Biophys. Acta 1 4 7 , 262-271 the COOH terminusof the a subunit. Our findings of a pro- 18. Spencer, D., Higgins, T . J. V., Button, S. C., and Davey, R. A. lectin precursor molecule ( M , = 23,000) confirms suggestions (1980) Plant Physiol. 6 6 , 510-515 madeearlier by Trowbridge (2) andaddssupporttothe 19. Gill, D. S., Kumarasamy, R., and Symons, R. H. (1981) Virology prediction that a similar biosynthetic pathway might operate 1 1 3 , 1-8 20. Chandler, P. M., Higgins, T. J. V., Randall, P. J., and Spencer, for the closely related lentil lectin (4). Recent data on the D. (1983) Plant Physiol. 7 1 , 47-54 cell-free synthesis of favin demonstrate that the broad bean R., Jay, E., and Wu, R. (1976) Nucleic Acids Res. lectin is also synthesized via a high molecular weight precursor 21. Roychoudhury, 3,101-116 containing a signal sequencefollowed by the 8 and (Y subunits 22. Bolivar, F., Rodriguez, R. L., Greene,P. J., Betlach, M. C., (29). Co-translational and post-translationalmodification of Heyneker, H. L., Boyer, H. W., Crosa, J. H., and Falkow, S. the lectins of castor bean have also been described(35).These (1977) Gene 2 , 95-113 involve the removal of a putative signal sequence, addition of 23. Grunstein, M., and Hogness, D. S. (1975) Proc. Natl. Acad. Sci. U. S.A. 72, 3961-3965 carbohydrates, and possibly the endoproteolyticcleavage of a 24. Chandler, P. M. (1982) Anal. Biochem. 1 2 7 , 9-16 high molecular weight precursor. Soybean lectinis also likely 25. Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65, to be synthesized with a signal sequence (36) but does not 499-560 appear to be modified further after translation. Peumans et 26. Chrispeels, M. J., Higgins, T. J . V., Craig, S., and Spencer, D. al. (37) described the synthesis of the 8 subunit of pea lectin (1982). J . Cell Biol. 93, 5-14 by an homologous cell-free translation system from pea axes. 27. Foriers, A., Wuilmart, C., Sharon, N., and Strosberg, A. D. (1977) Biochem. Biophys. Res. Commun. 75,980-986 However, the a subunit did not appear tobe synthesized and 28. Kozak, M. (1981) Nucleic Acids Res. 9 , 5233-5252 the putative 8 subunit contained methionine, an amino acid 29. Hemperly, J. J., Mostov, K. E., and Cunningham, B. A. (1982) J. known to be absentfrompea lectin (2). Further work is Biol. Chem. 257,7903-7909 therefore required to explain these results. 30. Harris, N., and Chrispeels, M. J. (1975) Plant Physiol. 5 6 , 292The synthesis of the pea seed lectin described here shares 299 many characteristics with the synthesis of the two major pea 31. Becker, J. W., Reeke, G. N., Cunningham, B. A,, and Edelman, G. M. (1976) Nature (Lond.) 259, 406-409 seed storage proteins, legumin and vicilin. These include an D. K., Lennarz, W. J., and Brew, K. (1978) J . Biol. Chem. initial translation product with a leader sequence, synthesis 32. Struck, 253,5786-5794 via a large precursor which is slowly processed post-transla- 33. Fitzgerald, M., and Shenk, T.(1981) Cell 24, 251-260 tionally to yield subunits, and the subcellular localization of 34. Proudfoot, N. J., and Brownlee, G . G(1976) . Nature (Lond.)263, these translational and post-translational events. These prop- 211-214 erties are further documented and discussed in the accompa- 35. Roberts, L. M., and Lord, J. M. (1981) Eur. J . Biochem. 119, 31-41 nying paper (38). 36. Vodkin, L. (1981) Plant Physiol. 6 8 , 766-771 Note Added in Proof-The earlier sequence (3) for the 6 subunit 37. Peumans, W. J., Delaey, B. M., Manickam, A., and Carlier, A. R. of favin has been revised (Hopp, T. P., Hemperly, J. J., and Cunningham, B. A. (1982) J. Bid. Chem. 257, 4473-4483), butthe revisions do not affect the comparisons made here.
(1980) Plunta 150, 286-290 38. Higgins, T. J. V., Chrispeels, M. J., Chandler, P. M., and Spencer, D. (1983) J. Biol. Chem. 258, 9550-9552