A lectin from the thigh muscle of Rana tigerina

Biochimica et BiophysicaActa, 991(1989)~5--469 Elsevier

465

BBA23129

A lectin from the thigh muscle of Rana tigerina Manjunath S. Shot * and M. Madaiah Biochemislryl~ivfJon, Departmentof Chemistry, Karnatak University,Dharwad(India)

(Received3 March1989)

Keywords: Lectinpurification;Affinitychromatography;~-Galactosidebinding;(Thighmuscle);(R. tigerina) I.¢¢tin activity has been detected in the. thigh muscle extracts of Rana tigerina, which was found to ~gglutinate both trypsinized and untrypsinized rabbit erylteecytes. The leetin has been purified to homogeneity by MEPBS (0.01 M phos#tat~buffered saline (pH 7.2) with 4 mM ~-mereaptoethanol) buffer extraction of the tissue and affinity chromatography on acid-treated Sepharose 613. The molecular weight (Mr ) of the purified lectin was determined by SDS-polyaerylamide gel electroplmresis and gel fdtration on Sephadex G-75, which gave values of 15 500 + 1000 and 32000 4-1000, respectively, suggesting that the lectin is a dimer. Amino acid composition data of the lectin has revealed that it contains a high proportion of glyciue and alanine, and low amounts of sulphur-containing amino acids. Hapten-inhibition study of this lectin has shown that it is galactose-specific. Hemagglutination activity of the leetin can also be inhibited by/~-galactoside containing oligosaccharides.

inU'ede~ion Lectins constitute a special group of sugar-binding proteins of non-immuue origin that agglutinate cells and precipitate glycoconjugates. They have been identi. fled in many types of organisms and display a wide variety of unique and interesting chemical mid biological properties. In recent years, there has been increasing interest in lectins from animal cells and tissues [1-3]. Animal lectins have been studied with increasing interest for their possible role in cell recognition [4,5], although these molecules have not been well characterised as plant lectins; however, some of them have been studied extensively [6,7]. Different lectins with different specificities have been isolated from eggs of various Rana species [8-12]. In the present study we report the purification of a #-galactoside specific lectin from the thigh muscle of Rana tigerina by affinity chromatography and some of its properties.

* Present address: MolecularBiophysicsUnit, Indian Instituteof Science,Bangalore.560012,India. Abbreviation:MEPBS,0.01 M phosphate-bufferedsaline(pH 7.2) with4 mM/~.mercaptoethanot. Correspondence:M. Mad.~'ah,Bi~h,z:nistryDivision,Departmentof Chemistry,KamatakUniversity,Dharwad-580003,India.

Materi~ls and Methods Fresh thigh muscles were removed from the sacrified animals and were rinsed with normal saline (0.15 M NaC1). The homogenate of the tissues was prepared in 0.01 M phosphate-buffered saline (pH 7.2) (PBS) containing 4 mM p-mercaptoethanol (MEPBS). The homogenate was kept stirred overnight in the cold and was sedimented at 5000 rpm for 30 rain in the cold. The clear supematant was used for the purification of the lectin. Different inhibitory sugars, Sepharose 6B, standard proteins used for M, estimation by SDS-PAGE and gel permeation chromatography and trypsin were obtained from Sigma (St. Louis, MO, U.S.A.). All other chemicals used were of the highest available purity. Hemagglutination assay Hemaggiutinating activity of the samples was determined with trypsinized and untrypsJnized rabbit erythrocytes by the serial 2-fold dilution in mierotiter plates as described previously [13,14]. Hemagglutinin inhibition study was performed using different sugars, with trypsinized rabbit erythrocytes by. serially diluting the sugar solutions. One unit of hemagglutinin activity was defined as the lowest concentration of lectin giving visible hemagglutination. Affinity chromatography on acid-treated Sepharose.6B The acid.treated Sepharose 6B was prepared accordhag to the method of Ersson et al. [!5]. The clear

0~104-4165/89/$03.50O 1989ElsevierSciencePublishersB.V.(BiomedicalDivision)

466

supernatant from the thigh muscle extract was applied to the affinity column (1.4 x 15 cm) containing acidtreated $epharose 6B, previously equilibrated with MEPBS (pH 7.2). The unadsorbed fraction was eluted from the column ~.th the starting buffer until no protein was detectable in the effluent (approx. 20 column vol.). The specifically bound fraction was then eluted with the equilibrating buffer (MEPBS) containing 0.1 M lactose. The flow rate was maintained at 12 ml/h and 3 ml fractions were collected. All column operations were carded out in the cold.

?rotein estimation The protein content of the samples was determined according to the method of Lowry et al. [16] using bovine serum albumin as standard. Polyacrylamide gel electrophoresis (PAGE) The purity of the lectin preparation was confirmed by PAGE, using 7.5% gels at pH 8.3 and was performed according to the method of Davis [17]. SDS-PAGE of the purified sample was carried out according to d,.e procedure of Laemmli [18] using 10% gels. Molecular weight (Mr) of the leetin was determined by SDS-PAGE in the presence and absence of jO-mercaptoethP,nol. The gels were calibrated using the following staadard proteins: bovine serum albumin (66000), ovalbumin (45000) pepsin (34700), ,8-1actoglobulir, (18400) and lysozyme (14300). Gels were stained with silver nitrate [19].

Gel filtration The molecular weight of the l ~ t i n was determined by gel permeation chromatography on a column (1.5 X 46 cm) of Sephadex G-75, eqy~ilibrated with MEPBS. The flow rate was maintained at 12 ml/h with a fraction size of 3.0 ml. Bovine serum albumin (68000), ovalbumin (45 000) pepsin (34 700) a-chymotrypsinogen (25400) and cytochrome c (13000) were used as calibration proteins. The/fir of the Iectin was calculated by constructing a calibration graph of log Mr vs. It=/V0. Amino acid analysis The amino acid ~o.=aly,~sof the purified lectin was performed on a LKB model a:nino acid analys,;r following the hydrolysis of the sample in 6 M HCI for 24 h at ll0°C.

TABLEI Purification of fl.galactoside.specificiectinfrom thigh muscleof Rana tigerina Preparative steps

Total

Total

protein acL

Spec.

Yield Purifica-

act.

(%)

tion

(rag) (H.U) (H.U/mg) (-fold) 186 1651.7 8.9 100 1

Crudeextract Affinity chromatography on acid-treated Sepharose6B 3.5 1827096.7516129.0

1.9 1106.2

~the lectin has been depicted in Table I, and the elution !profile is presented in Fig. 1. The unadsorbed fraction ehited as a major protein peak, is devoid of any hemag~utinin activity. The bound protein was eluted as a single minor peak fr(',m the matrix using 0.1 M lactose hi the elution buffer, a specific inhibitor of the lectin. A ll06-fold purificat~.on has been achieved in a single step. Typically 120/~g of lectin was recovered from 1 g of thigh muscle, which corresponds to 0.64% of the total soluble proteins in the extract. The extraction of the lectin was carried out in the presence and absence of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate and ~8-mercaptoethanol in the extraction buffer. We observed that the extraction of the lectin from the thigh muscle both in the presence or absence of PMSF gave similar products. Likewise, the amount of lectin extracted from the starting material in the presence and absence of the detergent (SDS) remains the same, which clearly implies that this lectin does not require detergent for solubilization. However, when we eliminated pmercaptoethanol from the homogenization buffer and the purification medium, very little lectin was recovered

°51

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~

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0.1 M Lac

i.

0.3-

I

-]128 N

o2-~

0.1-

32 16

Results

Purification of the lectin Among the different crude preparations of tissues from R. tigerina tested for hemagglutinating activity, the thigh muscle extract was found to contain maximum hemagglutinin activity, and hence this was used for the purification of the lectin(s). The purification steps of

20

3o

40

50

60

Co

Fr~'t ion numbers

Fig. I. Purificationof a g-galactoslde.specificlectin from the thigh n~uscleof K tigerina by affinitychromatographyon acid-treated Sephasose6B.Thecolumn(1.4x 15cm)wasequilibratedwithMEPBS at 4°C. Elution of adsorbedfraction was started at the point indicated by an arrow, with0,I M lactosein MEPBS.o, absorbanc¢at 280 ne*t;&, hemagglutininactivity.

467

.~_

~

OA

w I

1

I

.....

4.4 4.6 4.s s.o Log M r Fig. 3, Determination of molecular weight (Mr) of lectinby SDSPAGE, This was carriedout accordingto the method of Laemmli [18] using I0~ gels.The M r was calculatedby comparison with standard proteinsa, Bovine serum albumin (66000); b, ovalbumin (45000); c. pepsin (34700); d, trypsinogen (24000); e, B-lactoglobulin(I~,400); and f.lysozyme (14300). ,t.~

A B Fig. 2. Polyacrylamidegel electrophoreticpattern of the purified lectin from the thigh muscle extracts of It tigetina by affinity ch "omatographyon acid.treatedScpharose6B. (A) PAGE at pH 8.3 and (B) SDS-PAGE. with very low hemagglutinating activity. With these observations we concluded that this lectin requires a reducing agent (~-mercaptoethanol) to keep the protein in an active form. Similar findings have been made by Joubert et at. [5] and Cera et at. [20] in the case of rat soluble lectins (S-type) are assayed in the presence of free thiols, because oxidation ir~ibits thei~ carbohydrate-binding property.

Homogeneity and characterization of the lectin The homogeneity of the lectin preparation was confirmed by PAGE at pH 8.3 and SDS-PAGE. In both cases lectin gave a single protein band (Fig. 2) SDSPAGE carried out in the presence of ~-mercaptoethanol gave a protein band without a~y change in its mobility. The molecular weight of the lectin by SDS-PAGE was calculated to be M, 15 500 + 1000 (Fig. 3). Gel filtration on ~phadex G-75 of the purified lectin gave a single symmetrical protein/agglutinin peak (Fig. 4), which mrther confirms the homogeneity of the preparation, From gel filtration data the M, of the lectin was found to be 32000 + 1000 (Fig. 4), which indicates that the native lectin exists as dimer.

Carbohydrate specificity The carbohydrate-binding specificity of the lectin was determined by the hapten-inhibition study in microtiter plates and the data has been given in Tablo II. From the table, it is evident that lactose is the most potent inl~ibitor of the lectin. Among the different

a

b

c

d

llll

o2o

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1

256~

EE 0,15

~

0.1C

32

~ o.o~

i

4~ lO

20

30 40 rio Elution volume {ml)

60

Fig. 4. Gel filtrationof the purifiedlectinon SephadexG.75 column. The column(1.5×46 cm) was equilibratedwith MEPB$.The position of the arrowsindicatedifferentstandard proteinsusedfor the calibration. a, bovine serum albumin (68000); b, ovalbamin (450C0); c, pepsin(34700); d, a-chymotrypsinogen(25400); and e, cytochrome c (13000). TABLE I!

Hapten.inhibitionstudy offrog thigh musclelectin The followingsugars were tested and had no inhibitionat 100 raM; glucose, N-acetyl glucosa;nine, mannose, cellobiose, t..fucose, N-acetylgalactnsamineand p-nitrophenyl-a-D-galactosid¢. Carbohydrateused

Minimum concentration

D-Galactose Oalactosamine Methyl-a-~galactoside Methyl@D-galactoside p-Nitrophenyi-~B-D-galactoside

of carbohydraterequired for completeinhibition (raM) 50 12.5 50 6.25 3.17.

o-Nitrophenyl@D-galactosid~

3.12

Lactose

0.19

Melihiose Raffmose o-Nitrophenyl.a-D.galactoside

25 12.5

100

468 TABLE II!

Amino acid composition of frog thigh muscle lectin Tr, trace; n.d., not determined. Aminoa~d

Number of residuesper mole of lectin

Asp Thr Ser Giu Gly Ala Val Met lie Leu Tyr Phe His Lys Trp

9.05 10.10 16.03 8.78 27.32 21.76 10.10 Tr 7.56 10.20 2.30 3.12 2.56 6.25 n.d.

monosaccharides tested, methyl-fl-galactoside inhibits the hemagghtination, followed by melibiose, raffinose, methyl-a-galactoside and galactose. The glucopyranoside sugars are non-inhibitory to the lectin activity. Hence the carbohydrate-binding specificity of the lectin is directed towards fl-galactosides.

Amino acid composition The amino acid composition of the purified lectin (Table III) showed high amounts of glycine (13.7~), alanine (13.0~) and a low content of sulfur-containing amino acids. The amino acids proline and ar~nine were not detected in the hydrolysate. Discussion

In recent years, animal lectins have been studied for their role in cell-recognltiun. In vertebrates, only a few species have been screened for the distribution of hemagglutinating activities. Different vertebrate tissues were found to contain single or multiple forms of lectins [3,5]. This is the first report of a muscle lectin from Rana species. The extracts from different tissues like liver, brain and thigh muscle of R. tigerina were found to agglutinate both trypsinlzed and normal rabbit erythrocytes. (data not shown). The lectin from the thigh muscle of K tigerina has been purified to homogeneity in a single step, using acid-treated Sepharose 6B. This lectin appears to be similar to the other soluble lectins isolated from mammalian tissues, which do not require detergents for solubilization [22]. Based upon the available structural information on animal lectins, Drickamer [21] recently categorized these molecnles into different groups, such as CaZ+-dependent (C-type),

thiol-dependent (S-type), serum immunoglobulins, man. nose-phosphate receptors, viral hemagglutinins and serum amyloid proteins. This purified muscle lectin bdongs to the S-type which can be solubilized in the absence of detergents and is Ca2+-independent. Based upon the classification of the lectins as reported by Gallagher [23], the muscle lectin belongs to the group endolectin. The purified lectin is specific towards saccharides bearing the non-reducing terminal D-galactose linke~ it., a/]-configuration. The evidence for this conclusion is O-nitrophenyl-a-D-galactoside inhibits the hemagglutination at a higher concentration (100~ inhibition at 100 mM) followed by methyl-a-l)-galactoside (100% inhibition at 50 mM). D-Galactose and D-galactosamine were found to be inhibitors, while N-acetyl-I)-galactosamine was not, which clearly implies that either the free hydroxyl or amino group at C-2 position is necessary for the monosaccharides to inhibit the hemagglutination. With respect to mono- and disaccharides and their derivatives, the similar trend of carbohydrate-binding propensities have also been reported in other fl-galactoside-specific let,tins isolated from mammalian tissues [5,20,24-27]. In SDS-PAGE the purified lectin migrates as a single molecular species of a subunit of Mr 15 500 and on gel filtration gave an apparent Mr of 32000, which indicates the dimeric nature of the lectin. The similar type of dimeric form of lectins have been reported for other fl-galactoside-binding lectins from rat [20] and chicken tissues [27]. Similarly, on gel filtration in the presence of SDS buffer (0.1% in the equilibration buffer), the lectin eluted as a monomeric component with an apparent Mr of 16000. The evidence for this assumption is that this lectin does not require detergents for solubilizatinn, thereby distinguishing this lectin from membrane-associated lectins (C-type) [21]. Many reports on jg-galactoside binding lectins present in the extracts of different vertebrate tissues have shown that these molecules are externalized and may be associated with the extracellular matrix [3,24-28]. The exact functions of most vertebrate lectins remain unclear and are the subject of considerable speculation [28]. Recently, the significant possible clue about the functions of some S-type (thiol-dependent) lectins has come from work on their localization in developing rat brain [29]. This indicates that these molecules may be involved in the selective establishment of interneuronal contacts, which in turn suggests that these lectins are mediators of cell-celi or cell-matrix interactions in other situations as well. Very recently Clerch et al. [30] and others [31-33] have reported the primary structures for several S-type lectins. These sequence studies reveal that members of this class of lectins from diverse tissues and organisms are structurally similar to one another showing sequence

469

homology and form an evolutionarily related family [21]. Hence, further investigations on the purification of lectins from other tissues of this animal and t h ~ structure-function relationships may throw some insight on the biological roles and evolutionary relatedness of these soluble (S-type) lectins from Rana species and other mammalian tissues. Rde~nees 1 Simpson, D.L., Thome, D.IL and Lob, H.H. (1978) Life Sci. 22, 727-748. 2 Barondes,S.H. (19~1)Annu. Rev. Biochem.50, 307-331. 3 Baroodes,S.H. (1984) Science233,1259-1264. 4 Monsigny, M., Kieda, C. and Roche, A.C. (1979) Biol. Cell. 35, 289-300. 5 Joubert, R., Caron, M. and Bladier, D. (1986) Comp. Biochem. Physiol. 85B, 859-863. 6 Kitagaki, H., Natsumoto, I., Sasaki, H. and Seno, N. (1985) J. Biochem.98, 385-393. 7 Roof, C.F. and Wang, J.L (1983)£ Biol.Chem. 258,10657-10663. 8 Sakakibara, E, Takayanagi,G., Kawunchi, H., Watanabe, IL and Hakomod, S. (1976) Biochim.Biophys.Acta 444, 386-395. 9 Sakakibara, F., Takayanagi, G., Ise, H. and Kawauchi, H. (1977) Ya~-Za~hi 97, 855-862. 10 Yokota, M., Sakakibara, F. and Kawauchi, H. (1975) Yakugaku. Zasshi 95, 50-55. 11 Eawauehi, H. Sakakibara, F. and Watanabe, IC (1975) Experl. entia 31, 364-365. 12 Roberson, M.M. and Barondes, S.H. (1982) J. Biol. Chem. 257, 7520-7524. 13 Shet, M.S., Swamy,B.M. and Madaiah, M. (1985)Ind. J. Biochem. Biophys. 22, 313-315.

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