Indian Journal of Biochemistry & Biophys ics Vol. 38, June 200 I, pp. 193- 198

Characterization and biological activities of Chenopodium leaf hemagglutinin (CLH)

K N Suseelanl*, K B Saini s2 and R Mitral

iNuc lcar Agricu lturc and Biotechnology Division, Bhabha Atomic Rcsearch Ccn tre, Mumba i 400085, India

2Ccll Biology Division, Bhabha Atomic Rcsearch Ccnt rc, Mumbai 400085, India

Received 27 Ma rch 2000; revised 3 1 July 2000

A hcmagglut in in (CLI-I) hav ing nati vc molccular mass of 58 kDa and subunit molccu lar mass of 33 kDa had bccn pu ri fied fro m thc leavcs of ChenopodiulII alllarwll icolor. Thc protcin aggluti natcd rabbit eryth rocytcs and no agglutination was obscrved with any of thc groups A, B or 0 of hu man blood. Thc amino acid co mposition rcvea lcd that CL I-! was rich in aspartic ac id, glutam ic acid, glyc ine and phenylalanine and also signi ficant amou nt of meth ionine. Thc N-tcrmina l amino aci d scqucncc analysis showcd that CLI-! had no homology with any of the plan t hcmaggluti nins studied so fa r. It was in ac tivc towards hu man pcri pheral blood cc ll s but mitogcn ic for mousc splecn B-Iymphocytcs. CL I-! in hibitcd protcin sy nthcs is in rat thymocytes at high conccntra tio n. CLI-! did not inhi bit TMV infcc tion of Icaves indica ting abscnce of an tivira l propcrties.

Chenopodiulll (c. aJl lO ranticolor) is a uni versal loca l les ion host plant fo r many viruses. The les ion acts as a check for viruses spread by synthes izing pathogenesis-related (PR) protei n and other defense proteins ' . Earlier ex periments on the inducti on of PR proteins in Chenopodium leaves in response to tobacco mosa ic virus (TM V) revealed that they are presenting the intercellul ar space in the leaf ti ss ue2

.

Interestingly, among the several PR proteins, a hemagglutin in [ChenopodiulIl leaf agg lutinin (CLH)] was also detected whi ch was present in both normal as well as TMV infec ted leaves. On puri fi ca tion, the CLH was found to be a thermostable protein with activity at a broad range of pH 3-1 2 (ref. 3) . The role of thi s particul ar hemagglutinin in the pl ant is not yet understood. In this paper, we report the sequence and composition of this homodimeric protein bes ides its vari ous biolog ical acti vities.

Materials and Methods Leaves collected from 45-60 day old Chenopodium

plants were used as biological matcri al. All the chemicals and reagents used were of

analyti cal grade. Fine chemi cals and molecular weight markers for gel filtration were procured from Sigma Chemical Company, St Louis, Mo, USA. Liquid

* Author for corrcspondcncc Tclcphonc No.: 5592642. 5505050, Ex tn . 22642 Fax No.: 9 1-22-5505 15 1, 9 1-22-55196 13 E- mail: nad@magnum.barc.crnct.in

scintill ati on fl ui d (Coc ktai l W) was obtained from SISCO Research Laboratori es, Mumbai, India. [I~C!­Leucine and e I-l]-thy midine were produced from BRIT, Mumbai, Indi a.

Purification oj Chenopodiulll leaJ hemagglutinin The methodo logies used to puri fy ChenopodiulIl

leaf hemagglutinin (C LH ) have been menti oned in the earli er paper3.

Amino acid cOlllposition Amino ac id analysis was perfo rmed by the method

of Ashman and Bosserhoff in a Pharmacia H PLC instrument using dual pump binary grad ient system and C-1 8 column. The pro tein sample was hyd rolyzed in 1 ml of 6 N HCI at I 10° fo r 24 hr and the dried ac id-free hydrolysate was dissolved in 100 III of 0.25 M K-borate buffe r, pH 10.4 before derivati zatioll . The number of try ptophan residucs were determined by the spectrophotometri c method of Spande and Witkop5.

Amino acid sequence analysis The amino ac id sequence analysis was carried out

in the Indian Institute of Technology, Mumbai, India. The puri fied CLH showed single band of mo lecu lar weigh 33 kDa on SDS-PAG E. N-termin al sequence based on the Edman degradati on reaction was performed in a Shimadzu PPSQ gas phase protei n sequencer, using a C-1 8 co lu mn fo r reverse phase HPLC of phenyl thiohydantoin (PTH) amino acids,

194 INDI AN J. BIOCHEM. BIOPHYS. , VOL. 38, JUNE 200 1

and on-l ine PTH analyzer and a CR-7 A data processor. Edman degradati on reacti on was performed on the puri fied CLH electrob lotted onto a po lyv inylidine difluoride (PYDF) membrane.

Chem icallllodijication studies

Modification of histidine Chemi cal modification of purified hemagglutinin

was investigated in order to identi fy the amino ac ids in volved at the ac ti ve sites.

The modi fica tion of hi st idine was done by following the method of Melchior and Farnel. the protein (100 fl. g) was taken in eppendorf tubes in a final volume of 200 fl.1 with 20 mM Na- Phosphate buffe r, pH 7. 2. Diethyl pyrocarbonate (1 5 fl.1 ) was added to the solution. The modified sample was then dialysed against 20 mM Tris-HCI, pH 8.0, at 4°C and the agglutinati on assay performed.

Modification of lysine To 70 fl.1 (200 fl. g) CLH, 130 fl.1 0.1 M Tris-HCI

buffe r, pH 8.0 was added. To this IS fl.1 of citraconic anhydride was added with constant stirring and the mi xture was allowed to react for 30 min at room temperature. After the reaction the sample was dia lysed against 20 mM Tris-HCI buffer, p H 8.0 and the agglutinati on ac ti vity was estimated.

Modification of tryptophan Oxidation of CLH with NB S (N-bromo

succinimide) was carried out according to the method of Spande and Witkop5. The CLH sample of 70 fl.1 (200 fl. g) was taken in a I ml cuvette and to it, 930 fl.1 of 0.1 M Na-acetate buffe r, pH 5.0 was added. An aliquot of 5 fl.1 of 10 mM NBS in water was then added with rapid mixing. After 10 min , the absorbance at 280 nm was consecuti ve additi ons. The number of res idues ox idi zed was ca lcul ated.

Modification of carboxyl groups The hemagg lutinin solution of 70 fl.1 (200 fl. g) was

dissolved in 1 ml of I M a-aminobutyric acid methyl ester, pH 4.7. To it , 50 fl.1 of 1.1 M 1-ethyl-3-(3 -dimethyl aminopropyl)-carbodiimide hydrochl oride (EOC) was added twice at an interval of 30 min . The mixture was stirred constantl y, maintaining the pH at 4.7 by adding HC!. The reaction was carried out for 5 hr. the agglutinati on activity was estimated after dialyz ing the sample against 20 mM Tris-HCI, pH 8.0.

Biological activit ies of CLH

Antiviral acti vity The effec t of CLH on the mul tiplication of TMY

was assessed in Nicotiana glll/inosa and Nico/ianG tabacum (Xanthi NN). To assess the antivira l act ivity, TMY was mi xed with CLH and was inoc ul ated in such a manner that one-hal f of the leaf served as control (TMY onl y) and rhe other half was treated (TMY + CLH). Concentrati on of TMY was 21.6 fl. g/ml. The concentrati ons of CLH were 2.3 and 11.6 fl. g/ml in 10 mM phosphate buffer (PB) , p H 7.0. The molecul ar rati o of CLH to TM Y were 125 and 67 1 in the two experiments. In each set, three plants were se lected and eight leaves fro m each pl an t were used fo r half-lea f inocul ati on as per the protocol mentioned above. N. glutinosa was left at room te mperature, whereas Xanth i NN was left at 22°C. The number of lesions were counted on each half-leaf, two days after inoculati on.

Mitogenic p roperty The mitogenic property of hemagglutinin was

assessed by standard lymphocyte pro li fera ti on assay using spleen cells of C3H mi ce7

. Spleens were di ssected from 10- 12 week old female C3H mice and single cell suspension was prepared in RPM! 1640 ti ssue culture med ium (Cat. No. R4 130, Sigma Chemi ca ls Co., USA) containing 15 mM HEPES buffer, 2 mM L-glutamine and 10% CPSR 2 (controlled processed serum replacement, Cat. No. 9030, Sigma Chemica l Co., US A). Erythrocytes were lysed by treating with 0.83 M NH4C1. Cell s were incubated with diffe rent concentrations of hemagglutinin ranging from 0.43 to 37.5 fl. g/ml in a fin al vo lume of 200 fl.1 containing 2x l 05 cell s in a 96 well Numc microculture plate at 3rC for 72 hr in 5% CO2 atmosphere. At the end of incubat ion , cell s in each well were pulsed with I fl.Ci of e H]-thymidine (Speci fic Activity 6500 fl.Ci/mmol) fo r 16 hr. The cell s were harvested on glass fibre filter di scs and e H]-thymidine incoporati on was esti mated using a liquid scintillation counter.

For assessing the mitogenicity of hemagglutinin to T and B lymphocytes, spleni c lymphocytes were separated into T-cell rich (non-adherent) and B-cell rich (adherent) sub-populations by fractionation on ' nylon wool' columns essenti ally according to the method of Julius et a18

• The column was prepared by packing 300 mg wool (Leucopak, Feanwall Labs, USA) is a 5 ml steril e plastic syri nge and was equilibrated with RPM I 1640 med ium containing

SUSEELAN el at.: CHARACTERIZATION AND BIOLOGICAL ACT IVITIES OF CLH 195

10% foetal calf serum. Fifty million splenic lymphocytes were loaded on the column and it was incubated in 5% CO2 incubator at 37% for 30 min. The el ution was done with 2 volumes of prewarmed medium. After the collection of non-adherent fraction (T-cell rich), the column was washed with 10 volumes of the medium and adherent cells (B-cell rich) were obtained by repeated flushing of the co lumn with fresh medium. The separated T and B-cell rich fractions were incubated with hemagglutinin for mitogenic assay as described above. Con A and LPS (E.co li ) were used as positive controls for T-cell and B-cell mitogenesis, respectively .

Effect of CLH on in vivo protein synthesis in thYlI/ocytes Thymocytes were isolated from 5 weeks old rat.

The cells were suspended in RPM I 1640 medium. The density was estimated to be 155x l06 cells/ml. A pulse of 0.5 /1Ci of C4C]-leucine in a final volume of 1.5 ml was given (I ml cells,S /11 C4C]-leucine of 0.5/1Ci and 495 /11 PBS). The cells were incubated from 30- 180 min at an interval of 30 min . Two independent treatments were given Li sing 40 /1 g and 80 /1g of CLH. The reaction was stopped by adding TCA to a final concentration of 10%. The precipitated protein was adsorbed on Whatman 3 fi lter paper discs by suction. The di sc was washed thrice with 10% TCA followed by acetone. The di scs were then air dried and used for tadioactive counting.

Results

Properties of purified Chenopodium/eaf hell/agg/ ut ill in

Amillo acid composition ofCLH

Amino ac id composition of CLH is li sted in Table I. The CLH contained a large amou nt of acidic amino acids like aspartic acid, glutamic acid and glycine whi ch amount to be 48% of the total amino acid content. The hemagglutinin al so contained a higher amount of methionine as compared to other seed or lea f agglutinins. The phenylalanine content was fou nd to be considerably high.

AII/in o acid sequence analysis The sequence of 15 amino acids starti ng from N­

terminal of the purified CLH subun it (33 kDa ) was determined. The deduced sequence from the data was found to be Asn-Glu-Gln-Lys-Val-Tyr-Ile-Thr-Ile­Gly-Gly-Leu-Gly-Asp-Asp.

Table I-Amino ac id compositon of ChenopodiulIl leaf hemagglutinin (CLH)

Amino acid Aspartic acid

Glutamic acid

Serine

Histidine

Glycine

Threonine

Arginine

Alanine

Tyrosine

Methionine

Valine

Mole percentage 22.4

12.1

4.8

1. 1

13.4

5.3

1.9

1.5

0.3

4.3

5.8

Phenylalanine I 1.5

Isoleucine 4.1

Leuci ne 6.7

Lysine 1.5

Cysteine 3.2

Tryptophan* 3.7

*Number of tryptophan residues were calculated spectrophotometrical I y

Chemicalmodijicatioll studies of CLH More than 90% of agglutination activ ity was lost

when lysi ne res idue was modified with citraconic anhyd ride. Simi lar results were obtained when histidine was modified with diethyl pyrocarbonate. From the sequential oxidation of tryptophan which involved about 12 residues using NBS, about 90% lossof agglutination activity was observed (data not shown) . The resu lts reveal that lysine, hi stidine and tryptophan are involved at the active sites. However, CLH lost on ly 55% of its activity when carboxy l groups were modified with l-ethy l-3-(3-dimethyl ­aminopropy l) carbod iimide followed by coupling with cx-aminobutyric acid methyl es ter.

Mitogenicity ofCLH The hemagglutinin was mitogenic to mouse spleen

cells (Fig. 1a; Stimulation Index (S I) = 1.9 at 37 /1g/ml). Enrichment of spleen cells in B lymphocytes (Nylon adherent subpopul at ion) sign ificant improved their response to the--hemagglutinin (S I = 8.2 at 37 .5 /1 g/ml). On the other hand, enrichment in T-cells did not improve their response in comparison to that of un fractionated spleen cells. These data suggest that CLH was likely to be a polyclonal B-cell mitogen, though less potent than LPS (Fig. I a). It did not show any mitogenic activity towards human peripheral blood lymphocytes (Fig. Ib).

196 INDI AN J. BIOCHEM. BIOPHYS., VOL. 38, JUNE 200 1

Effec t of CLH 0 11 in vivo protein synthesis in thYll/ocytes

In order to understand the effect of CLH on protein sy nthesi s, the study of its acti on on rat thymocytes was undertaken. At two different concentrat ions of CLH used, there was no marked inhi bition of in vivo protein sy nthes is in the presence of CLH. However, at higher concentration of CLH (80 Jl g), 25 % inhibition was observed (Fig. 2).

Anti-viral activity of CLH Three plants were selected and eight leaves from

each plant were used for half-leaf inocul ation for each set of experiment. Number of lesions was counted

1. 55,580 1 (a I

1",162 13503°1 ! 66 . 588

20 ..---. 8 · C~ll~

t:r--t::. T - C9\lS 18 0---0 lot a l s plun COlliS

C - Only C~ll s .., 16 ,

0

)(

E 14 a. v

c 12 0 -ro '-0 10 a. '-0 v c

8 ()I

c ~ 6 E C >-~ 4 l-. I ..,

2

o 2·5 60 0 10 20 30

after 48 hr of inocul ation and the data are shown in Tab le 2. The result showed th at CLH did not inhibit TMV infection which reveals that it was not an anti­viral agent.

Discussion CLH is a powerful hemagg lutinin with a specific

ac ti vity of about 160 agg lutinati on unit per Jl g of protein. This hemagglutinin is synthes ized exclusive ly in the leaves. Chenopodillm seeds do not contain a hemagg lutinin whi ch either aggluti nate rabbit erythrocy tes or cross-react with CLH-antiboclies. The di ffere nces between hemagg lutinin in seeds and in other vegetat ive parts have been reported9

.

(b I

0---0 Human P9riph9ral Lymphocyhs

C· Only C~lls

40 02·5 10 20 30 40

ConA LPS Concentration 01 C LH Lug/mil

1 Concentration of CUi ConA (AJg/ml)

Fig. I- Miloge nic aCli vity assay of CLH by the method of 3[Hl -thymidinc incorporation. [(a): 3[H]-thymidine incorpora lion lIsing mi ce spl een cc lls. (c), Control (on ly cells); (0), total spleen ce lls; (. ), enriched B-cells; (t-.), enriched T-cells. (b): 3[Hl-thymid ine incorporation us ing human Iymphocytcs. (c), Control (on ly cell s); (0), human peripherallymphocytesl .

SUSEELAN et a/.: CHARACTER IZATION AND BIOLOG ICAL ACTIVITI ES OF CLH 197

10

M I

8 0

x

L 6 0... U

4

2

0 0 30 60

C

40.09

80.09

90 120 150 180

Time (min)

Fi g. 2-lnhibi tion of ill FiFO protein sy nthesis in rat thymoeytes . [(0) , Control; (e ), protein sy nthesis in presence of 40 J.l.g of CLH ; (t. ), protcin syn thcs is in prescnec of 80 J.l. g of CLH)l.

Table 2- Biologieal acti vity of Chellopodilllll leaf hemagg lutinin (CLH )

Speeics

N. gilltinosa

Xallfhi NN

Mcan numbcr of lesions (per half leaf) + SO (n=24 hal f Icaves)

(Experimcn t I) (Experimcnt 2) TMV CLH+TMV TMV CLH+TMV

67 ± 4

73 ± 6

CLH : TMV CLH :TMV

( I : 125) ( I : 671)

83 ± 14

76 ± 20

94 ±1

195 ± 78

87 ± 7

191 ± 83

The ami no acid composit ion revealed that CLH is an acidic protein wh ich holds some similarities with winged bean lectin L-I 10. However, CLH contains a hi gh level of pheny lalanine and a low amount of alanine. Unlike other leaf and seed phytohemag­gluti nins, CLH has a significant methionine content (Table j ). CLH does not seem to be a glycoprotein as peri odic acid-SchiFf's staining of po lyacrylamide gel has proved negati ve (da ta not shown).The chemical modificat ion studi es results have revealed that lysine, histidine and tryptophan are in volved at the active sites. The c~i rbox y l groups are on ly partially in vo lved.

The treatments with proteases like pronase, papain and proteinase K resulted in a sign ifica nt destruction of agg lutination activity of CLH. However, no loss of agglutination activity was found with trypsi n treatment (data not shown). Also, no protease inhibitor ac ti vity was observed when CLH was incubated with ei ther trypsin or He/icoverpa armigera gut extract (data not shown).

e H]-thym idine incorporation in enri ched mouse spleen B-ceils showed a considerab le st imulation with

optimal concentration of CLH (Fig. I ). Since isolated T-cell s did not show any st imulation, CLH can be considered as a B-cell mitogen. No mitogenic acti vity was observed towards human peripheral blood lymphocytes. No mitogen ic leaf hemagglutinin has been reported so fa r, fo r wh ich the hemagglutin in has got a complex carbohyd rate specificity. An earli er report showed that pokeweed root lectin , Pa-l was mitogenic for human and murine T and B-cells" -' 3.

The degree of mitogenecity of CLH was found to be comparable with that of Pa-l. CLH is a dimer and it has been suggested Waxdal and Basham l2 that B-cell act ivation requires the in teract ion of several cell receptors with multimeric stimulants. CLH is the first hemagg lu tinin whi ch has a complex carboh ydrate specificity and at the same time, it is mitogeni c spec ifi call y for mouse sp leen B-cells.

The studies on in vivo protein sy nthes is using rat thymocytes suggested that CLH is not a highly potent inhibitor of protein synthesis. However, at hi gher concentration of CLH (80 J1. g), 25 % inhibiti on protein synthesis was observed. The CLH aggluti nated thymocytes isolated from rat. Strong agglutination was observed with asci tes cell s from mice also (data not shown).

Several plant hemagglu ti nins are potent inhi bitors , of viruses which have glycoproteins in their viri onsl4. CLH, though found among the proteins present in the intercellular space of TMY infected leaf ti ssues, is not an anti -viral protein for TMY. Chenopodilll11 is reported to contai n an ant i- viral protein in its leaf. Li ke many other plant species , Chenopodilllll alllaranlic%r al so contains a single chain RIP (ri bosome inactivati ng protein) '5.' 6. The amino acid seq uence analysis CLH did not reveal signi ficant homology with any of the leaf or seed phytohemagglutinins so far described. The B-cell s spec ific CLH showed a un ique amino acid composition and amino ac id sequence.

Acknowledgement The authors wish to thank Dr. A. S. Bhagwat,

Molecular Biology and Agriculture Division, Bhabha Atomi c Research Centre, Mumbai, India, for hi s help in amino acid analysis using HPLC instrument.

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198 INDIAN J. BIOCI-IEM . 1310PHYS., VOL. 38, JUNE 2001

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