Isolation and characterization of an inhibitory glycopeptide against guinea pig lymphotoxin from the surface of L cells

YOSHIRO

ECOBAYASHI,

JUN-ICHI

and TOSHIAKI Division

of Chemical

SAWADA

OSAWA

Toxicology and Immunochemistry. Faculty of Pharmaceutical University of Tokyo. Bunkyo-ku. Tokyo I13. Japan

Scicnccs.

which has inhibitory activity against guinea pig lymphotoxin (CLTi was of L cells. It has an approximate mol. wt of 1600 (gel filtration) and contatns galactose. mannose. fucose and N-acetylglucosamine in a molar ratio of 2:2: i :3. /i-Galactosidase treatment of the glycopeptide significantly diminished its inhibitory activity against GLT. Furthermore. GLT was found to be inhibited by the lectins (Kicintrs cownur~i.s haemagglutinin. Concanavalm A. and Pkasrol~r.s rdyrrri.r haemagglutinin) which bind preferentially to sugar chains like those of serum glycoproteins (serum glqcoprotein-type sugar chains) on the cell surface. These data indicate that swum glycoprotein-type sugar chains on the cell surface of target cells scrvc as receptors for GLT and that the /Q&ctosyl residue at the non-reducing terminal of the sugar chain is an import~~nt part of the receptor. Abstract-A

glycopeptide

isolated from the cell surfaces

MATERIALS AND METHODS

II\;TRODL’CTIOh

The iIn~ort~~nt role of cell surface ~~~rhohydr~tes in the interaction of certain ~ymphokines with their target cells has recently been suggested by Remold (1973) in work on the guinea pig migration inhibitory factor (MIF)t and by Rocklin (1976) in work on the human MIF and human leukocyte inhibitory factor. Hessinger ef al. (1973) and Tsoukas rr crl. (1976) have demonstrated that ~~urn~~n lymphotoxin exerts its effect via interaction with specific cellular receptors which are sensitive to proteolytic digestion. In previous papers (Sawada ef ctl.. 1976. 1977h). WC reported that the sensitivity of target cells to guinea pig lymphotoxin (GLT) is significantly lowered by the trypsin treatment of the cells and. furthermore. several sugars having terminal /I-galactosyl residue show definite inhibitory, activity against GLT. These results suggest that the cytotoxicity of GLT is triggered by the hmding of GLT to the cell surface receptor sites of a glycoprotcin nature having terminal /I-galactosyl residues. In this paper. we describe the isolation of a glycopeptidc which can effectively inhibit the illter~ction of GLT with target cells from L cells.

LP3 cells which had been adapted to grow in a protein and lipid-free synthetic medium (DM 130: Kyokuto Selyaku Co.. Tokyo. Japan) were used ‘as target cells and malntaincd as descrihcd previously (Sawada & Osawa. 1977).

~~~~Is~,~~~I~.~ r&wis ll~{enlagglutinin (PHA-P) was purchased from Difco (Detroit. Mich. U.S.A.). Concanavalin A (Con A) from jack bean meal (Sigma Chemical Co.. St. Louis, MO.. U.S.A.) was purified according to the method of Agrawal and Goldstein ( 1967). Rici,llr\ wwtn~~ti.s haemagglutinin (RCA) and Btrtrhi~rirr pw~wc~~~ hacmagglutinin (BpH) were purified according to the methods previously described (Tomita pr ui.. 1971: frimura & Osawn. 1972).

GLT was partially purified as described previously (Sawada c’t LI/.. 1975). The GLT used in this study was Fraction C after DEAE-Sephadex column chromatography (Sawada ~‘t ~1.. 1975) and stored frozen at ~ 10 C.

CJLT activity was assayed according to the morphoiogical micro-assay method previously descrihcd (Sahada & Osawa. 1977). 96 well microplates and lids (Microtest II No. 3040: Falcon Plastics Co.. Oxnard. Calif,. U.S.A.) were used for assaying GLT activity. 50 ICI of the L.P3 cell suspension in DM IX wcrc dropped into each well of the microplates. The cells wcrc precultured for X&25 hr in a CO2 incubator (S”,, CO, 95”,, air) at 37 ‘C and allowed to form micromonoiayers. Then. SO ~tl of l-fold serial dilutions of GLT in DM-120 wcrc added to the wells. At an appropriate time. 100 111ol’ S”,, glutaraldehyde in 0.1 ;2/1Na phosphate buffer (pH 7.2) was added to fix the ccl1 morphology and cooled at 4’C at least for I hr before cell counting was performed. Numbers of morphologically

* This investigation was supported b) research grants from the Ministry of Education. Science and Culture of Japan. 1- Abbrevi~itions used: MIF. rnigr~~tio~l inhibitory factor: GLT. guinea pig ~ymph~~toxin: PHA-P. ~‘/~~~.~~,~~I~s dqaris haemagglutinin; Con A. Concanavalin A; RCA, Rkntts conuwmi.s haemagglutinin ; BpH. Balthi,licr ~wporrrr haemagglutinin: DM-H. DM-I20 in 25 ml14 HEPES-NaOH. pH 7.0: PBS. IO ml14 Na phosphate buffered saline, pH 5.9: MEM, Eagle’s minimal essential medium containing 2 mM glutamine and 60 &ml of kanamqcin. hi

61

YOSHIRO

KOBAYASHI,

JUN-ICHI

interted mickoscopc (IO0x ). The survival ratio (s) was caculated as follows: .t = N’,Vc. where I\’ and ,V, arc the numbers of intact cells 24 hr after the treatment in the GLT-containmg medium and in the control medium. respcctivelq. One unit per ml of GLT activity was detincd as the concentration of GLT which gives Y = 0.5 after 24 hr of incuhatlon when the initial number of target cells was 355~mm (corresponding to IO.000 cells per well) and the culture volume was IO0 jtl.

(a) Hlrh Icc~tit1.s.To avoid the cqtotoxiclty of Iectins alicl prolonged incubation with target cellh. a Irapid assay method for GLTqtotoxicity using puromycin (Sawada CI ~(1.. 19770) was employed. L,P? cells were allowed to form a monolayer (I .S x IO4 cells/50 /&well) on each well of a mlcrotitcr plate. Then. 25 ~tl of medium was removed l’ollowed by the addition of 25 111of GLT. 25 ~tl of lectin solution and 25 111 of 0.4 m&1 puromycin solution The cells humidified air for 5ht were incubated in 5”,, C02-95”,, at 37 ‘C and fixed with glutaraldehydc to stop the reaction. The survival ratio corresponding to a giLen inhibitor cotlcentration was calculated from the number or intact cells in the cultures treated with GLT-contmning or control media in the prescncc ol’a given concentration of inhihito-. (h) W’ith (/l!,cop(,/‘rit/~,.s. The media of prcculturcd monola\crs of L.P3 cells wel-e replaced with 50 111of cooled DM 120 in 25mXf HFPES NaOH (pH 7.0; DM-H). Then. 75 /II of inhihitol- in DM-H and 2.5 jtl ol’ GLT-containing or control medium (DM-H) were added to each well. Alter incubation for I hr at 4 C‘. the monolaycl-s \\ crc washed twice with DM-H, and the washed monolaycra MCI-C‘ further incubated at 37 c‘. After 24 hr. the cells wcrc fixed with glutaraldch>dc and the
(a) Gtr/r~c/o.\c o\-rtltr.~~NaB’H, mct/lo[/. The method dcvcloped hy Hunt and Brown (1974) was used with a slight modification. Monolayer cultures of L cells in Roux bottles wcrc harvested by scraping and suspended in IO m!L1 Na phosphate buffered saline (PBS; pH 5.9) containing bovine serum albumin (recrystallized. Miles Laboratories. Kankakee. III.. [J.S.A.: 1 m&ml). This suspension was then with C/o.~trlr/ilr/n pwfi.imqcll,\ neuraminidasc treated (0.7 mU ‘IO” cells: Bochringer Mannhcim. Germany) for IOmin at 37 C. This treatment rclcascd i5”<, of the total cellular sialic acids as rc\calcd by the pcriodatc resorcinol method (Jourdinn
SAWADA

and TOSHlAKl

OSAWA

Monolayer cultures of L cells (IO’ cells) in Roux bottles or on 1OOmm Falcon platic tissue culture plates wcrc washed with IO ml of PBS (pH 7.0) five times to remove the medium completely. and then treated with I ml of tripsin solution in PBS (lO,qlml; Code TRL. Worthington Biochemical. Freehold. N.J.. U.S.A.) for 30 min at 37 C‘. At this stage. the viability of the cells was found to bc greater than 95”,, by the Trypan Blue dye exclusion tat. After ccntrifugntion, the supernatant was kept fro/en at 20 C. Pooled supcrnatnnts were centrifuged for 70 min at 49.000 y to remove insoluble materials and then scparntcd into Fr. A (higher mol. wt fraction) and Fr. B (lowet mol. u t fraction) by ultrafiltration throu_eh the collodion msmhrane (Sartorius Memhrwne Filter GmBH. GGttingen. Germany).

L cells wcrc precultured on 60-mm Falcon plaptic tissue culture plates for 20 25 hr to form a monolayer (2.5 x IO“ cclls:plate). The medium was decanted and then the monola)cr with trypsin (IO&ml in 0.5 ml of MEM-25 mtLI HEPES NaOH. pH 7.3) for 30 mm at 37 C follo\vcd by the addition of calf strum (final concentration: IO”,,) to stop the reaction After incubntlon for various intervals. GLT (3.5 I!. ml) was ahsorbcd with the washed monolayer for 1 hr at 37 C. The supernatant was ccntrifugcd for IO min at 300 y to rcmovc cells and debris. The residual activity of GLT was a?saycd and cypressed in U, ml.

(a) Rr~tliotrc~~ir~I//!,c,~,/“‘/)titlc.\. Paper clcctrophorcsia wab cnrricd out on Whatman No. I paper in pyridinc:acetic acid: water (I : 10:X9. pH 3.6) at 35 V.cm for 7 hr. After drying. the paper was cut (I cm width) and cwtracted with I ml or \Latcr in il counting vial. The radioactivity was dctcrmincd with a Beckman liquid scintillation counter using toluenc-Triton X-100 (2: I). (h) I;oJ. j>rc/>trarlio/t. Paper clcctrophorcsis was carried out under the same conditions as above. After drying. the locations of the glycopcptides wcrc determined from a standard guide strip which was stamed with O.Z”,, ninhydrin in 95” 0 ethanol. The areas containmg glycopeptidcs were cut out and extracted with distilled water.

The carbohydrate composition of the glycopeptides was detcrmincd h> the method of Takasaki and Kohata (1974) with ;I few modifications. This method is based on quantitativc radioisotope labelling of sugars with NaB3H, and separation of the resulting radioactive sugar alcohols by paper chromatography l’ollowcd by borate paper electrophorosis. (a) Preparation of -3H-lobclled standard sugar alcohols: NnB’H, (271 mCi:mmolc) was dissolved in l’reshlq dlstillcd anhydrous dimethylformamide at a COINccntration of 32 m&1 and stored in a tuhc with an airtight scrc\\ cap at -20 C. Alknlinc NaBAH, solution was prcpnrcd by mving 40 111ol” the NaB”H, solution prcparcd ahovc with I ml of 0.05 ,‘L’NaOH. 5 ~tl of each standard sugar solution (I m,ZI) were mixed with 20 /Al 01‘ alkalme NaB’H, solution. and incubated for 4 hr at 30 C followed b> the adchtion of I00 /tl of glacial acetic acid to stop the reaction. The mixture was then evaporated to dryness and the residue dissolved in ?“,, acetic acid in methanol and agam cvaporntcd to dryness. This evaporation procedure was repeated tike times. The resultmp residue was then dissolved in water. and evaporation rcpcatcd SIX timca and tinally to dryness. Under thcyc conditions. the specific activit! 01‘ “H-sorbitol was I.3 x 105cp~i;nmole and the actlvltles of the other sugar alcohols were almost the same. (h) Estimation of sugar composition: The glycopcptidc (0. I I 113)\\as hydrolyred in 6 AI tritluoracctic acid for X hl at IO0 C‘ irl ~LICUO.The hqdrolysatcs \vcrc cwporatcd to

An Inhibitory

Glycopeptide

against

dryness. and dissolved in water followed by repeated cvaporations (five times). -‘H-labelled sugar alcohols were obtained in the same manner as described above. The residue was dissolved in water and separated into neutral and amino sugar alcohols hy descending paper chromatography in ethyl acetate:acetic acid:formic acid:water (1X:3: I :4. v.;v).The radioactive sugar alcohols, which were cluted from the filter paper with water. were spotted on Whatman No. I paper and sub,jccted to paper elcctrophorcsis. Paper clcctrophorcsis was performed in 0.06 M borate huffer (pH9.5) at 40 V:cm (for neutral sugars) and 66 V:cm (for amino su?irs) for 3 hr. After drying. the paper was cut out (0.5 cm wtdth), extracted with 0.5 ml of water in a counting vial and the radioactivity determined with a Beckman liquid scintillation counter using 4.5 ml of toluenc Triton X-100 (2:lm v/v). The identification of radioactive regions was performed by comparing their mobilities with those of standard sugar alcohols. The amount of radioacti\itv was found to be proportional to the number of moles of monos~lcchar~des when a standard sugar mixture R:~S trratcd with NaR3H, and then subjected to paper clrctrophoretic separation in the same way.

Amino acid was determined after hydrolysis for 24 hr in constant boiling HCI (5.7 N) at 110C‘ in a sealed, evacuated tube using a Hitachi 034 amino acid analyzer according to the method of Spackman er rzi. (195X).

To

it

glycopeptldc

(pH 4.0) containing 0.5 ,M NaCI was added tosidase (0.3 units; Chmmio /rmpu.c). The reaction at

/I-galacmixture

37 C for 24 hr.

REWLTS

AND DISCUSSION

As shown in Fig. I. more than 5 x 10’L cells/ml were required to detect changes in GLT activity after absorption of 4.3 U/ml of GLT. The absorption reached a plateau after I hr at 37-C (data not shown). Then we examined the absorption of GLT with trypsinized L cells. and observed that trypsinized L cells lost their capacity to absorb GLT. but then their capacity was restored again to the level of non-treated

1 4 --*--A*

3 1

l\

2

/ 0.24

0 48

63

Pig Lymphotoxin

0

i Time

after

trypsinizofion, hr

Fig. 2. Absorption of GLT with trypsinircd L cells. GLT (3.5 U/ml) was absorbed for I hr with a trypsinized monolayer of L cells which had been incubated for various times after trypsinization. Experimental details are given in the text. The dotted line shows the original activity of GI_1. residual GLT activity was As a control experiment, measured after absorption with an untrypsinized monolayer of L cells (0).

L cells after incubation for 3 hr (Fig. 2). These observations are in good agreement with those of Hessinger et d. (1973). and indicate that the receptor molecules for GLT on the plasma membr~~ne of L cells. if they exist. are sensitive to trypsin treatment, but that they can regenerate quickly.

in 0.1 hl Na phosphate

(3 nmole)

buffer

was incubated

Guinea

0.72

1.2

Ceil number /unit GLT, ~lO~~cells/un~t

Fig. I. Dependency of GLT absorption with a L cell monolayer on the cell number per unit GLT. Various volumes of GLT-containing medium (4.3 units/ml) were absorbed with a L cell monolayer (5 x 10h cells) at 37’C for 2 hr. The residual GLT activity was assayed as described in the text.

Remold (1973) reported that a Ic-L-fucose residue is a part of the receptor for guinea pig migration inhibitory factor on the macropha~e plasma membrane. In order to test that carbohydrate moieties play an important role as ceil surface receptors for GLT also, inhibitory activities of various &tins (Con A, PHA-P, RCA and BpH) against GLT were assayed. To avoid the cytotoxic effect resulting from a long exposure of the Iectin to target cells. a rapid assay method for GLT-cytotosicity was employed as described in Materials and Methods. From the results shown in Fig. 3, the concentrations of RCA, Con A, PHA-P and BpH required for 50”, inhibition of GLT-cytotoxicity was found to be 5. 10, 10 and 95 icgiml, respectively. Previous studies (Kawaguchi cr r/l.. 1974; Irimura rt ~1.. 1975: Kaifu rt ai., 1975: Kaifu & Osawa. 1976: Kawaguchi & Osawa, 1976) indicated that RCA recognizes the u-~~-D-galactopyranosyi-( I--+ 4)-2-acetamido-2-deoxy-D-glucose sugar sequence. Con A recognizes the 0-(2-acetamido-2-deoxy-/I-I)-glucoI --t 2)-m pyranosyl)-( I --+ 2)-0-r-D-mannopyranosyl-( mannose sugar sequence and PHA-P recognizes the ~-~-~-gaiact~~pyranosyl-( I -+4)-0-(2-acctamido-2deoxy-~-I~-glllcopyranosyl)-(I --+ 2~-~-rn~lnnose sugat sequence M sugar chains like those of serum glycoproteins(serumglycoprotein-type sugar chains). whereas BpH recognizes the 0-P-n-galactopyranosyl(I --+ 3)-2-acetamido-2-deoxy-rl-galactose sugar sequence in sugar chains like those of mucins (micin-type sugar chains]. Coyne ct a/. (1973) reported that GLT behaves as a protein in CsCl density gradient and. furthermore, no direct interaction of GLT with various lectins was observed when GLT was applied to columns of RCA-Sepharose 4B, Con A-Sepharose 4B, BpH-Sepharose 4B and wheat germ agglutininSepharose 4B and its activity was fully recovered from the columns without any retardation (data not

64

YOSHIRO

KOBAYASHI.

JUN-ICHI

SAWADA

and TOSHIAKI

OSAWA

Fig. 3. Inhibitory activities of various lectins against GLT. The inhibition assays were carried out as described in the text. The logarithms of the survival ratios (ordinate) \vcre plotted against the final concentration of inhibitor. (a) PHA-P; (b) Con A; (c) RCA: (d) BpH. ( l -_) 0.XL’ml of GLT; (~ 0-10.4 U;ml of GLT. Vertical arrows indicate the conccntl-ation \\hcre SO”,, inhibition of GLT-qto-

shown). The fact that RCA. Con A and PHA-P can exert strong inhibitory activity against GLT, therefore, suggests that serum glycoprotein-type sugar chains serve as cell surface receptors for GLT. Thcsc results are in good agreement with our previous results (Sawada rt (I/., lY77h) that galactose and N-acetyllactosamine arc inhibitors against GLT.

In order to isolate small mol. wt glycopeptides which can serve as cell surface receptors for GLT. fractionation of Fr. B obtained bq ultrafiltration ol trypsin fragments of L cells was carried out on a column of Sephadex G-25 as shown in Fig. 4. Inhibitory activity against GLT of the fractions was tested by the method described in Materials and Methods and the results are shown in Fig. 5. If a fraction possesses

inhibitory

activity.

it can

bc expected

to in-

crease the survival ratio of the cells after 24 ht depending on its concentration. because the effect of

B-I

---t

B-2

B-3

o.51

B-4

n

0 20

30

40

Fraction

number

50

60

Fig. 4. Sephadcx G-25 gel-filtration of Fr B. Fr B obtained by ultrafiltratlon of trgpsln fragment5 of L cells (4 x IO”cells) wab dissolved in a small amount of distilled water and applied to a column of Scphndcx G-25 (I .2 x -lX cm). Elution was pcrformcd with distilled \\atcr and fractions of I.? ml wcrc collected at a How rate of 5 ml per hr. reaction (Dubois PI ( *~) A?“,, ( O- ) phenol-H2S0,

Ul.. IYShl.

d

0

100

200

0

100

200

0

Concentration,

IO

20

0

5

IO

pM

Fig. 5. Inhibitory activities of various fractions against GLT. The inhibltion assuyh wcrc carried out as described in the text. The logarithms of the surviwl ratios (ordinate) here plotted against the final concentration of inhibitor which was oxpressed as ~1.v of neutral sugar determined by the phenol H2S0, reaction (Dubois er trl.. 1956) using I>-glucose as a standard. (a) 0. Fr. B-I ; x. Fr. B-2: l . Fr. B-3: A. Fr. B-4. (h) 0. Fr. C-l: 0. Fr. C-2. (c) 0. Fr. D-l: 0. Fr. D-2: x. Fr. D-3; a. Fr. D-4. (d) 0. Fr. E; 0. P-galactosidase-trc3ted Fr. E. The preparation of B-fractions is shown in Fig. 4. <‘- in Fig. 7. D- in Fig. 8. and E- in Fig. 9.

An

Inhibitory

Glycopeptidc

against Guinea

b

Pig Lymphotoxin

1

1

40

Fraction number Fig. 6. Seph;~~cx G-25 gel filtration of Fr. B isolated from ~radiolnhelled L cells. (a) Fr. B from L cells labelled by the gnlactose ouidasc--NnB”H, method. (b) Fr. B from L cells labelled hq the pyridoxal phosphate-NaB3H, method. (c) Fr. B from L cells labclled b> I,-[‘Hl-glucosamine hydrochloride. The location of Fr. B-? IS indicated by 11 vcrtiwl ZIITOLV.

the pulse treatment of the’ cells with GLT at 4 C employed in this assay is assumed to reflect the binding of GLT to target cells as described previously (%wada c’f t/l.. 1977h). From Fig. 5. it can be seen that only Fr. B-3 has the inhibitory activity. To asccrtain that Fr. B-3 was derived from ccl1 surface &coproteins, we labelled L cells by the gal:lctosc-NaB-3H, method and also by the pyridoxal phosphate NaB”H, method. and isolated Fr. B-3 from the labelled cells. By both methods of cell labelling. Fr. B-3 was found to bc labcllcd showing that this fraction most probably origintttcd from ccl1 surface glycoproteins (Fig. 6). FLlrthcrmorc. siucc radioactive Fr. B-3 could be isolated from the cells which were labcllcd by incubation with I,-[‘HI-glucosamine (Fig. 6). this fraction was not derived from the contaminating medium. Then, Fr. B-3 ~‘21s further fractionated into Fr. C-1 and Fr. C-7 b> gel filtration on a column of S&adeu G-IO its shown in Fig. 7. Among thcsc

fractions. only Fr. C-Z was found to be inhibitory against GLT (Fig. 5). Approximately 78”,, of the neutral sugars of Fr. B-3 was recovered in Fr. C-2 (Table I ). However. when Fr. C-2 was subjected to paper clcctrophoresis. three ninhydrin positive spots were dctccted i[~dic~~tin~ that Fr. C-2 was still a mixture of’ three peptides. Since Fr. C-2 contained N-acetylglucosamine and ,r\;-,cetylgalactosamine in a molar ratio of approx 3: I, the sugar moieties of this fraction wcrc assumed to be made up of mucin-type sugar chains and serum glycoprotein-type sugar chains. In order to separate inhibitory glycopcptide which bear a scrttm ~l~c~~prl?teil~-t~pe sugar chains, Fr. C-2 w;bs treated with alkaline NaBH, to remove alkali-lahilc mucin-type sugar chains and the reaction mixture sul>jected to gel filtration on a column of Biogel P-1. As shown in Fig. 8. three peptides were clearly separatcd after the alkaline NaBH, treatment. and the Iupest pcptide. Fr. D-l. was found to be inhibitor!-

Fr. B-3 Fr. C-2 F’r. D-l Fr. E Fraction number Fig. 7. Sephadex G-IO gel-liltration of Fr. B-3. Fr. B-3 (300 mg) was dissolxd in :I small amount of distilled water and applied to a column of Sephadex G-IO (I.1 x I IOcm). Elation was carried out with distilled water and fractions of 2.3 ml were collcctcd at :I Row rate of 5 ml per hr.

78 (3.74)

100 100

6 (0.27) 5 (0.24)

IO IO

loo(4.xl)

“ Neutral sugars were ~ictcrmirled hq the phenol HLSOJ renction (Dubois ~‘t rii.. 1956) using o-giucose as a standard. and are expressed taking the amount of neutral sugars in Fr. B-3 as 100. “The concentration of glycopcptides. expressed as the concentration of neutral sug;lrs. which is required for 50”,, inhibition of GLT activity (see Fig. 5). Espcrimental details

arc in the tat.

YOSHIRO

KOBAYASHI,

JUN-ICHI

SAWADA

0.3

0.2

0. I

I 30

I

20 Fraction

number

Fig. X. Biogel P-2 gel-filtration of alkaline NaBH,-treated Fr. C-2. Fr. C-2 prepared from 3 x 10” L cells was dissolved in 0.2 M NaOH-0.1 M NaBH,. and the solution incubated for 24 hr at 37-C in a sealed tube 111vacua. The excess borohydride was destroyed by careful addition of 2 !‘vf acetic acid to pH 6.0, and an aliquot of the mixture was applied to a column of Biogel P-2 (1.6 x 54 cm). Elution was carried out with 0.1 !2/1triethylamine bicarhonatc (pH 7.0) and fractions of 2.4ml were collcctcd at a flou rate of 10 ml per hr. ( l--) AZso. The clution volumes of Blue Dextran and v-glucose arc mdicatcd by vertical arrows. against GLT (Fig. 5). Paper electrophoresis of Fr. D-l obtained from ~-[3H]-glucosamine-labelled cells is shown in Fig. 9. Fr. D-l was further purified by preparative paper electrophorcsis to obtain a purified peptide (Fr. E) which corresponds to the major radioactive peak in Fig. 9. The data pertaining to the purification of the inhibitory glycopeptide are summarized in Table I. Purrial (./1uI.(l(.tl’Ti=utiojl

Fukuda & Egami. 1971). Since the amount of glycopeptide available for chemical analyses was limited. the sugar components liberated by acidic hydrolysis wcrc labelled with NaB”H, and the labellcd sugar alcohols separated by borate electrophoresis according to the methods of Takasaki and Kobata (1974). as described in Materials and Methods. From the results. Fr. E was found to contain mannose, galactosc. fucose and R’-acctylglucosamine in a molar ratio of 2:1: I :3 (average values of the l’our experiments taking fucosc as 1). When Fr. E was treated with P-galactosidase. it lost all of its galactose and 75”,, of its inhibitory activity against GLT (Fig. 5). The amino acid analysis of Fr. E revealed that this glycopeptide contains aspartic acid and glutaminic acid in a molar ratio of I :2 taking aspartic acid as 1. Thcsc results strongly suggest that strum glycoprotein-type sugar chains on the cell surface scrvc as rcccptors for GLT and that a /&I,-galactosyl residue at the non-reducing terminal of the sugar chain is an important part of the receptor. This finding is the starting point of our study on the mechanism of the cytotoxic activity of GLT.

REFEREhCES

Agrawal B. B. L. & Goldstein I. J. (1967) Biochim. hiophys. .Acttr 147. 262. Coyne J. A.. Remold H. G., Rosenberg S. A. & David J. R. (1973) .I. ~/)I/IIIVI.110. 1630. Dubois M.. Gilles K. A.. Hamilton J. IS.. Rebers P. A. & Smith F. (1956) Alltrlj,r. Cllr/n. 28. 350. Fukuda M. & tgami F. (1971) Biochl. J. 123. 407. Hessingcr D. A.. Dayncs R. A. & Granger G. A. (1973) Proc. 1111111. ./lcutl. sci., L:.s.il. 70, 3082 Hunt R. C‘. B Brown J. C. (1974) Biochcwi.stry 13. 22. Irimura T.. Kauaguchi T., Ter;to T. & Osawa T. (1975) Ctr/+oh~~d. xc\. 39. 3 17. Irlmur;i T. & Osaua T. (1972) Ir,c,h\ Biohw Biopll)‘.s. Jourdlan G. W.. Dean C‘/I~WI. 246. 430.

I

IO Distance

I

I

20

30

1

L. & Roseman

S. (1971) J. hiol.

Kaifu R. & Osnwa T. (1976) Crrrhoi~ytl.Rcs.

52. 179.

Kaifu R.. Osawa T. & Jeanlor R. W. (1975) C‘trrhoh~d. Rcs. 40. II 1. Kawnguchi T.. Matsumoto I. & Osaw~~ T. (1974) Biodlc~,,li.srrr 13. 3 169. Kawaguchi T. & Onawn T. (lY76) Biocllc,vii,stry IS. 4SXI. Remold H. G. (1973) J. cup hfrd. 138. 1065. Rocklin R. E. (1Y76) J. 1mmu1~ 116. 816. Sawada J.. Kishic T. & Osawa T. (1977tr) Microhid InSawada

I

OSAWA

151. 475.

qf Fr. E

The approximate mol. wt of the inhibitory glycopeptide thus isolated (Fr. E) was estimated to be 1600 by means of gel filtration on a Biogel P-2 column, employing II-glucose. raffinose. stachyose and Unit A glycopeptide of porcine thyroglobuhn (mol. wt 1800;

0

and TOSHIAKI

J.. Koha\;ishi

Y. & Osawa

T.

t 1977h) Jrrn J. CY,I.

J. & Osawa T. (1977) Jap. J. <‘\,I. .tfd. J.. Shioiri-Nakano K. & Osaw:~ T. (1975) />/n/lrllfiorl 19. 33.5. Sawada J.. Shioil-i-Nakano K. & Osnwa T. (1976) CY,I. .Zfcc/. 46. 263. Spackman D. H.. Stein W. H. & Moore S. (1958) Cllcrrl. 30. I I YO.

Sa\vada

47. X7.

Sawadn

Tvu,lsJtrp. J. Arlulxt.

from origin, cm

Fig. 9. Paper clectrophoresis of radiolabelled Fr. D-l. Fr. D-l obtained from u-[‘Hlglucosamine-labelled L cells (4 x IO’cells) was subjected to paper electrophoresis under the conditions described in the text.

Tomita M., Kurokawa T.. Onoraki K.. Ichlki N.. Osawa T. & Ukita T. (1972) E\-pericwricr28. 84. Tsoukas C. D., Rosenau W. & Baxter J. D. (1976) J. Ivmu,~. 116, 184.