The α- and β-subunits of the jacalins are cleavage products from a 17-kDa precursor

219

Biochimica et Biophysica A cta, 1156 (1993) 219-222 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

BBAGEN 23761

The a- and/3-subunits of the jacalins are cleavage products from a 17-kDa precursor Lien Do Ngoc

a,b

Mich~le Brillard

a

and Johan Hoebeke

a

Laboratoire d'Enzyrnologie et de Chimie des Protdines, URAI334 du C.N.R.S., Unicersit~ Franqois Rabelais, Tours (France) and h Laboratory of Biochemistry, Unicersity of Hanoi, Hanoi (Viet Nam) (Received 29 June 1992)

Key words: Jacalin; amino acid sequence; Proteolysis

The jacalins of three Artocarpus species were purified by affinity chromatography on a desialylated mucin-CNBr-Sepharose 4B column. The/3-chains and the 14 kDa a-chains were separated by high pressure liquid chromatography and the 17 kDa chains by preparative electrophoresis. The 17 kDa and 14 kDa chains had a similar highly conserved N-terminal sequence. The /3-chains were different for the three species and Artocarpus champeden contained two different /3-chains. CNBr cleavage of the 17 kDa polypeptide of Artocarpus tonkinensis yielded one peptide more than the 14 kDa. The N-terminal sequence of this fragment was similar to that of the/3-chain proving that this chain results from a proteolytic cleavage at the C-terminus of the 17 kDa peptide. The large heterogeneity of the/3-chains of jacalins from different species could be used as a marker for evolutionary studies on the Artocarpus family.

Introduction Seeds of jack fruits contain a lectin, named jacalin [1], that is, specific for the T-antigen structure Gal/313GalNac [2]. Recently, it was shown that jacalin from A. integrifolia consisted of two polypeptides [3], an a-chain of approx. 12 kDa, which was completely sequenced [4] and a mixture of /3-chains of 20 amino acids long. In view of the heterogeneity of the polypeptide chains of jacalin [5-6], the hypothesis was made that the /3-chain could be cleaved from a 17-kDa precursor protein [3]. The results presented here on the N-terminal sequencing of both polypeptide chains of three different Artocarpus species from Vietnam, explain the previous reported heterogeneity of the /3chains isolated from A. integrifolia and give direct evidence for the hypothesis that they result from a post-traductional cleavage. Materials and Methods Seeds from jack fruits of three different species, A.

integrifolia, A. champeden and A. tonkinensis, were freshly collected, dried and grinded to meal. The meal

Correspondence to: J. Hoebeke, Laboratoire d'Enzymologie et de Chimie des Prot~ines, URA 1334 du C.N.R.S., Facult~ de M~decine, 2 bis, Bd. Tonnell~, F-27032 Tours C~dex, France.

was extracted in phosphate buffered saline (10 mM phosphate, 150 mM NaC1, p H 7.4) and the extracts were adsorbed on a desialylated mucine-CNBr-Sepharose 4B column as described [7].The jacalins were desorbed by a 0.1 M methyl-a-galactoside solution in the same buffer. Purity of the preparations was checked by SDS-polyacrylamide electrophoresis according to Laemmli [8]. The /3-chains and the a-chains were purified by H P L C on a C 4 column using a 30-min acetonitrile gradient in water-trifluoroacetic acid 0.1% of 2 % / m i n (concentration of acetonitrile in the gradient buffer was 85%) after 15 min of washing in the water solution. The a-chain could be purified at 77% of the acetonitrile gradient while the/3-chain was eluted at 57% of the gradient. The peak eluted at 83% of the gradient contained a mixture of the a-chain and the 17 kDa precursor as demonstrated by SDS-polyacrylamide electrophoresis. The 17 kDa polypeptide was thus purified by preparative gel electrophoresis. After detection on a cut-out fragment of the localisation of the protein bands with Coomassie Blue, gel bands corresponding to this localisation were eluted in 10 ml of 2% trifluoroacetic acid in water. The solution was concentrated under vacuum, water was added and the solution was again concentrated under vacuum to eliminate most of the trifluoroacetic acid. CNBr cleavage of the polypeptides was performed in 70% formic acid at room temperature for 24 h at a

220 molar ratio C N B r / p r o t e i n of 10000.The resulting products were separated by HPLC under the same conditions used for the a- and /3-chains but with a gradient of 2.8%/min. Although the cleavage yield was not assessed, determination of the amount of pmoles obtained from the N-terminal sequence of the cleavage product allowed to estimate that the yield was approx.

N%

.OD220

o°o" ° oOOO'°°

1

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23

oo

iO

.i

100%.

Automated gas-phase sequencing was performed on an Applied Biosystems model 477A with on-line identification of the phenylhydantoin derivatives. The initial yield of the sequencer was assessed as 95%. Aminoacid analysis was performed on lectin hydrolysates in a Beckman automatic aminoacid analyzer 119CL [9].

24

I

I

I

27

30

45 Minutes

Results and Discussion

ACN%

The aminoacid composition of the jacalins of A.

-OD220

2

11"

3 o

tonkinensis and A. champeden were compared with that of the A. integrifolia lectin [10] (Table I). No major

.o

.--"°'""

oO

o

differences could be found with the exception of the presence of cysteic acid in the hydrolysate of the A. champeden lectin. The presence of two cysteines in this lectin was confirmed spectrophotometrically with dithionitrobenzoate [11]. The affinity-purified jacalin of the three species showed two heavily stained polypeptide bands, both under reducing and non reducing conditions, in SDS-

5O

24

TABLE I

Aminoacid analysis of the jacalins from A. champeden and A. tonkinensis Aminoacids (17 kDa)

A. champeden

A. tonkinensis

A. integrifolia a

Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Trp b Cysteic acid

15 12 16 14 9 25 6 16 1 10 10 8 11 1 10 2 2 2

15 14 15 11 8 24 6 16 1 11 10 8 12 2 11 4 2 0

16 12 15 12 8 25 4 15 2 12 11 12 11 1 11 2 N.D. N.D.

~' Aminoacid composition of the A. integrifolia jacalin according to Ref. 10. 6 Tryptophan content was measured spectrophotometrically according to 16.

1

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30

33

36

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39 Minutes

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42

45

Fig. 1. Separation of the polypeptides of the affinity-purified jacalins from A. tonkinensis (upper profile) and from A. champeden (lower profile). H P L C profile of the polypeptides on a C 4 reverse phase column in an acetonitrile gradient. Peak 1 (57% acetonitrile) corresponds to the fl-chain, peak 2 to the 14 kDa and peak 3 to a mixture of the 14 kDa a-chain and the 17 kDa precursor. The insert shows the SDS-PAGE profile of the affinity purified jacalin. Arrows correspond to molecular weight standards of respectively 14000 and 20000. The iacalin from A. champeden has a double fl-chain (peaks 1 and 1').

P A G E at 14 and 17 kDa with a small amount of a polypeptide of 18 kDa (Fig. 1). This suggests that the possible disulfide bridge in A. champeden is intracatenar and does not link the postulated a:/32 heterodimers [3] together. No protein staining was observed at the front suggesting that the /3-chains were too small to adsorb Coomassie Blue in sufficient amounts for visualisation. When the same jacalins were subjected to HPLC, a major polypeptide was purified at 57% acetonitrile (recognized as the /3-chain by sequencing) while two overlapping peptide peaks were eluted respectively with 77 and 83% of acetonitrile. Both peaks yielded a same

221 N-terminal sequence corresponding to that of the c~chain of A. integrifolia [4]. In contrast with a previous report of three different /3-chains in A. integrifolia [3], the /3-chains isolated from A. tonkinenis and A. integrifolia were unique. Only the/3-chains of A. champeden showed a doublet (Fig. 1). The sequences of the isolated /3-chains are shown in Table II and compared with the sequence of the three /3-chains previously reported for A. integrifolia. The sequence of the /3chain of A. integrifolia from Vietnam corresponds unequivocally to the jacalin /31 from a commercial preparation. The /3-chains of the jacalins of the two other species were similar neither with each other nor with one of the three /3-chains reported. The /3-chain doublet of the A. champeden jacalin could be ascribed to a change of I to K at position 6. The residues conserved in the /3-chains of the lectin of Pomifera maclura and A. integrifolia were also conserved in the /3-chains of the jacalins of the two other species, corroborating the hypothesis that these residues are involved in carbohydrate recognition. SDS-PAGE analysis of the other peptides resolved by HPLC showed a-14 kDa polypeptide in the fraction eluted at 77% (Fig. 2) and a mixture of 14 kDa and 17 kDa polypeptides in the fraction eluted at 83%. Separation of these two polypeptides was thus performed by a preparative PAGE-SDS and the purified 17 kDa polypeptide was checked for purity by analytical electrophoresis (Fig. 2). The N-terminal sequence of the 17 kDa and 14 kDa polypeptides for the three jacalins studied were similar and corresponded to the sequence of the a-chain recently determined [4] (Table III). This gives strong evidence that the 17 kDa polypeptide is a precursor of that a-chain. Moreover, both 14 kDa and 17 kDa polypeptides purified from A. tonkinensis jacalin, gave a similar peptidic profile upon HPLC of the CNBr induced fragments with the exception of a supplementary peptide derived from the 17 kDa polypeptide

( D220nm

A( N~

0,6

1

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,

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Fig. 2. Separation of the CNBr fragments of the 17 kDa (upper profile) and the 14 kDa (lower profile) polypeptides of the jacalin from A. tonkinensis. H P L C profile of the polypeptides on a C 4 reverse phase column in an acetonitrile gradient. The arrowed peak eluted at 60% acetonitrile from the 17 kDa polypeptide was N-terminally sequenced. The inserts show the SDS-PAGE profile of the two purified polypeptides. Arrows correspond to molecular weight standards of respectively 14000 and 20000.

eluted with 60% acetonitrile similarly to the elution of the /3-chain (Fig. 2). This peptide was N-terminally sequenced and the sequence corresponded to the Nterminal sequence of the /3-chain of A. tonkinensis (Table 2). This result unequivocally proofs that the

T A B L E II Sequences of the [3-chains of jacalins from three different species Species

Chain

Sequence

A. tonkinensis

[31 C N B r f r a g m e n t 10 [31

1 10 20 K Q R SS GGI E S Q N I I V G S w G A I K C s K Q R SS G IGE S Q . . N E Q SS GII G S Q T V I V G P W G AJQ V T

A. champeden

[32

A. integrifolia A. integrifolia a

[31 [31 132

[33 a

N E Q S SG IG, . N E Q S G IG N E Q S SG G N E Q S SG G D E Q S GII G

S Q T V I V G S Q T V I V G

S W

G AIK

V S

S W

G AIK

V S

S Q T V I V G

V S

S Q T V I

P W G AIQ V G P W G ArK

V

S

Sequences of the three [3-chains from Ref. 3. Boxed residues correspond to conserved aminoacids . . . . indicates where the sequence was stopped.

222 TABLE Ill

Acknowledgements

N-terminal sequences o[" the polypeptides o[ the jacalins from three Artocarpus species

Species

Polypeptide Sequence

A. tonkinensis 17 kDa 14 kDa A. champeden 14 kDa A. integrifi)lia 17 kDa ¢~-chain ~'

I 10 20 GKAFDDGAFTGIREINLSINKETAIGD GKAFDDGAFTGIREINLSINKETAIGD GKAFDDGAFTGIREINLSINKETAIGD GKAFDDGAFTGIREINLSINKETAIGD

We wish to thank Dr. Francis Gauthier for the hospitality with which he recieved one of us (D.N.L.) in his laboratory. Dr. Do Ngoc Lien was supported by a grant of the Minist6re des Affaires 6trangbres of France

(C.I.E.S.). References

GKAFDDGAFTGIREINLSINKETAIGD..

" N-terminal sequence according to Refs. 3 and 4.

/3-chains of jacalin originate from a proteolytic event at the C-terminal region yielding the 14 kDa a-chain. It suggests that the methionine yielding a C-terminal CNBr-cleaved tetrapeptide (Y-L-S-L) in A. integrifolia [4] is the C-terminal amino acid in A. tonkinensis at which the CNBr cleavage occur. The proteolytic Cterminal cleavage of a jacalin precursor to yield a heterodimer of the form c~/3 is very similar to what is found for the lectins of several Leguminosae [12,13]. The existence of a high variability on the /~-chains can have several practical implications. The fact that the commercially prepared jacalin is heterogeneous could be explained by the different Artocarpus species from which the seeds were collected. In view of the attempts to analyse the tertiary structure from jacalin crystals [14], it could be worthwhile to use jacalins purified from one species yielding a unique/3-chain. The Artocarpus species originate from Polynesia and reached only recently Eastern Malaysia and South America [15]. The variability of the /3-chain could be an excellent tool to follow the evolution of the breadfruit and jackfruit along its man made geographical migrations.

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