ANA1

t’T,CAl

RIOCHtMIS-TRY

92, 41-45

(1979)

Isolation of an Arabinogalactan Protein by Lectin Affinity Chromatography on Tridacnin-Sepharose 4B

An arabinogalactan one-step procedure lectin binding column buffer. proteins

from the small is calcium-ion

protein involving

the style canal of Gludiolus has been isolated affinity chromatography. using the galactose-binding

from lectin

giant clam. dependent.

‘7iitluc.n~ so that

in the presence of calcium This method has a general containing

P-linked

galactosyl

the

r~tr.rinttr. coupled arabinogalactan

can be eluted by washing utility for the purification

by

to Sepharose protein which

4B. The lectin is bound to the

the column with of polysaccharides

a calcium-free and glyco-

a

residues.

Macromolecules containing galact.ose are common constituents of plant tissues. Of these, the group of proteoglycans containing hydroxyproline and a carbohydrate ‘component containing arabinose and galactose as the major monosaccharides are currently attracting considerable interest ( l-6). Water-soluble arabinogalactan proteins of this type have been isolated from seeds of every taxonomic group (I), from many gums, mucilages, and other extracellular secretions (3), as well as from the culture filtrates of suspension-cultured callus cells (7). They have been implicated in functions such as adhesion, cellular recognition, nutrient supply, and response to fungal and bacterial infection (3). Isolation of the arabinogalactan proteins is a prerequisite to a study of their form and function. Classical purification based on solvent precipitation, gel filtration, and ionexchange procedures are tedious and do not generally resolve proteoglycans efficiently. The capacity of arabinogalactan proteins to interact specifically with the p-glycosyl artificial carbohydrate antigens described by Yariv (8) provides a useful means of isolating these proteoglycans from plant ex-

tracts, but is limited by the destructive procedures required to disrupt the precipitated complex (9). An alternative approach is to purify the arabinogalactan proteins by affinity chromatography using immobilized carbohydratebinding macromolecules. The lectin from the Tridrrcnu mrr.\-irrrtr clam (tridacnin) ( 10, 1 I), and the IgA mouse myeloma J 539 (12. 13) have been shown to form precipitates with arabinogalactan proteins from a number of sources ( 14) by virtue of their capacity to bind galactosyl residues. Recently the myeloma protein has been insolubilized and used t’o isolate an arabinogalactan protein from suspension cultures of Lolilrrtl Itirrlti,jlorum ( 15.16). We have previously shown that the Glrrtliollrs style contains an arabinogalactan protein which will interact with tridacnin and peanut agglutinin as well as the J 539 myeloma ( 17,18). Of these galactose-binding macromolecules, tridacnin was the most suitable for use in the purification of the G1ariiolrr.s style arabinogalactan protein because of its ready availability and specificity for p-galactosyl residues. In addition the binding interaction is calcium-ion depend41

0003.2697/79/010041-05$02.00/0 Copyright All npht\

1 197Y hy Academic Pm\\. Inc. of reproductmn in any form rercrved

42

GLEES0N.JERMYN.ANDCLARKI-I

ent (19). a fact we have exploited in the disruption of the lectin-carbohydrate complex. A rapid method is described for the isolation of arabinogalactan proteins by affinity chromatography using tridacnin coupled to Sepharose 4B. MATERIALS

AND METHODS

Puri$c.ution of the ~r~l~i~rosc~-hitltlitl~lettin (triducnin) from Triducnrr ttlclsittza. Specimens of Tridactw muximci were collected from the Great Barrier Reef, Queensland, Australia. and we thank the Queensland Fisheries Service for permission to collect these clams. The clams were transported frozen to the laboratory where they were thawed, opened, and the hemolymph (body fluid) collected. The hemolymph was dialyzed against distilled water and lyophilized. Tridacnin was purified by affinity chromatography on acid-treated Sepharose 6B according to Baldo (20). The purified lectin was dialyzed, lyophilized, and stored at 4°C. The yield was about 0.5 mg of purified lectini ml of hemolymph. Preparation of flze crfjniry supporf. Tridacnin (45 mg) was dissolved in 20 ml of 0.1 M NaHCO,, pH 8.0. containing 0.5 M NaCI, and added to 3 g of swollen, washed CNBr-activated Sepharose 4B (Pharmacia, Uppsala). The slurry was shaken on a rotary shaker for 2 h at room temperature. Measurement of the absorption at 280 nm indicated that about 75%) of the tridacnin coupled to the activated gel. Any remaining active groups in the gel were blocked by incubating the gel in 0.1 M Tris, pH 8.1, overnight at 4°C. The coupled gel was then washed four times on a sintered glass funnel alternately with 0.1 M sodium borate buffer, pH 8.0, containing 0.5 M NaCl, and 0.1 M sodium acetate buffer, pH 4.0, containing 0.5 M NaCl. The gel was then equilibrated with 0.15 M NaCl containing 0.01 M CaCl, and was ready for use. All chromatographic operations were performed at 4°C.

1rr.sStrtltlrr~,(,tl.si.\. Mature Gl~ctliolus styles were collected 24 h after flower opening (21) and were ground with 0.05 M Tris-HCI. 0.15 M NaCI, 0.001 M CaCI,, pH 7.4. at 4°C. centrifuged at 10.000~ for 30 min. and the supernatant dialyzed against distilled water and lyophilized. Yuri\’ ritztiRc,tl-hitlditzg LISSN~.The p-glucosyl Yariv antigen-binding assay for the detection of arabinogalactan proteins was performed according to Jermyn and Yeow ( 1). Protrin dt~rc~rttlinrrtiotl. Total protein was calculated from an amino acid analysis, performed by Dr. C. M. Roxburgh, CSIRO. Division of Protein Chemistry, on a hydrolysate obtained by refluxing the sample in 6 N HCl for 24 h under nitrogen. C~trhoh~drm~c~de~ertttinution. Total carbohydrate was determined, as galactose, by the phenolsulfuric acid method (22). Carhohydrcr~c~ crtttrlysis. A sample ( 1.65 mg) of the tridacnin-Sepharose 4B-bound fraction of Gladiolus style was hydrolyzed in 2 ml of 2.5 N trifluoracetic acid at 100°C for 2 h in a sealed tube under nitrogen. These conditions were found to be necessary for complete hydrolysis of the arabinogalactan protein. After hydrolysis the acid was removed on a rotary evaporator and the hydrolysate was then reduced with sodium borohydride. acetylated (23), and examined by gas-liquid chromatography (24). Gel j?ltratiotl otz Scphrrrosc~4B. The tridacnin-Sepharose 4B-bound fraction of Glndiolus style was examined by gel filtration using a column (91 x I .5 cm) of Sepharose 4B (Pharmacia) equilibrated with 0.02 M sodium phosphate, pH 7.0, containing 1% NaCl. Celhlosr acetate rlrc,rrophorc~sis. Electrophoresis on a cellulose acetate membrane was performed in Tris-barbital-sodium barbital buffer. I = 0.05, pH 8.8. using a Beckman Microzone electrophoresis apparatus. The membrane was stained with pglucosyl Yariv antigen (1 mg/ml in 1V NaCI) for 10 min and destained in 1%’ sodium chloride.

ARABINOGALACTAN

FIG. I. Elution of tridacnin-Scpharosc washed elution o-glucosyl

with with

profile the

same

calcium-free Yariv antigen

of affinity 4B. The solution solution binding

PROTEIN

chromatography column was to

remove

and assay

all

of G/nt/ir~//,v G,tyle extract equilibrated in 0.15 hl NaCl nonbound

material:

0. I M lactose. respectively. (- - --): Absorbance at 180

Cllrollrcrlo,~Jll.tr~~ll~~ 011 tridoct2it7 -Srphrrrose dB c.olu~urr. Crude Gltrdio1u.r si.yle extract ( IO mg), dissolved in 0. I.5 M NaCl containing 0.01 hl CaCI, (4 ml) was loaded directly onto the column (3.5 x I cm) and chromatographed as shown in Fig. I. The fractions were assayed for carbohydrate and their capacity to bind to ,!Sglucosyl Yariv antigen. All the ,%glucosyl Yariv antigen-

FIG. 3. Gel filtration ofthe tridacnin-Sepharose 48 column (91 x I.5 cm) in 0.02 M phosph:ate hydrate t---j and p-gluco~yl Yariv antigen-binding

43

ISOLATION

the

( IO mgl containing

arrows

were started. nm (------I.

indicate

on a column (3.5 0.01 hl CaCI, the

Carbohydrate

pwitions

7 I cm) and was at which

profile

(-);

binding material was retained on the column. This bound material was eluted specifically by washing the column with 0.15 ht NaCl in the absence of calcium ions as the capacity of tridacnin to bind to galactosecontaining macromolecules is calcium-ion dependent (19). With most lectin affinity systems. bound material is specifically eluted by competing ligand. eg.. monosac-

4B-bound fraction fromG/didrrv style (4.3 buffer. pH 7.0, I ? NaCI. Fractions were activity (- -).

mg) on a Sepharow assayed for carbo-

44

CiLEFSON.

01

igin

JERMYN.

-

+ FIG. 3. Cellulose dacnin-Sepharose style

in Tris-barbital-sodium

acetate 4B-bound

electrophoresis fraction barbital

for 45 min at 4 mA. The membrane /Sglucosyl Yariv antigen (I mg/ml min and destained in ICC NaCI.

from

of the triC;ln~/io/~.s

buffer. was stained in I’X NaCIJ

pH

8.8. with for IO

AND

CLARKE

shown in Fig. 3. Only one positively charged component is observed upon staining the membrane with j$glucosyl Yariv antigen. The electrophoretic and chromatographic behavior of the arabinogalactan protein indicates that it is a single component which has been isolated by the one-step procedure. The chemical analysis of the bound fraction is shown in Table I. The capacity of the affinity support was determined by overloading the column with crude style extract. The affinity support was found to bind 1 mg of Gltrttiolus style arabinogalactan protein per 3 mg of coupled tridacnin. The affinity support can be used many times and is regenerated by reequilibrating with 0. I5 M NaCl containing 0.01 M CaCl,. Although this galactose-binding lectin is coupled to a matrix which is itself composed of galactose it is unlikely that there is any interaction between the binding site of this lectin and the matrix support. In a control experiment no binding of tridacnin to a Sepharose 4B column to which BSA was coupled, was detected. Tridacnin is known to bind to other plant arabinogalactans and galactans (14.25), and terminal galactose residues (26) have been implicated in the binding reaction. This afTABLE

charides. In this case elution of the bound material was achieved simply by washing the column with a calcium-free buffer. Complete elution was achieved and no chelating agents were required. Subsequent elution of the column with 0.1 M lactose followed by dialysis and freeze-drying did not yield any further material. RESULTS The Gladic~lus style bound fraction elutes as a single peak when examined by gel filtration on Sepharose 4B (Fig. 2). The electrophoretic pattern of the bound fraction on a cellulose acetate membrane at pH 8.8 is

ANALYSIS

OF GLALUOLLIS

I

SI \r’~t

ARABINOCAI

4~ TAN

PROI-FIN ISOLATED BY AFFINIT) CHRO~~AT~~~RAPHY ON TRIDA~NIN-SEPHAROSE Percentage

(by

weight)

composition

Carbohydrate Protein Monosaccharide carbohydrate Galactose Glucose Arabinose Rhamnose

90.0 3.0 composition component

” The glucose content of the isolated from batch to batch, while the ratio arabinose remained constant.

of (r/r) 71.8 17.3” 10.9 Trace material varied of galactose to

ARABINOGALACTAN

PROTEIN

finity support should prove useful in purifying other galactose-containing polysaccharides and glycoproteins. Tridacnin is easily purified in substantial quantities and the method of elution of the bound macromolecule, without the use of a sugar competitor. is particularly suitable for the further analysis of the purified material.

I?.

Glaudemans. C. P. J.. Zissis. E.. and ( 1974) ~‘trrhrd~~d. Kc\. 40, 129.

13.

Potter.

2.

3.

Stone. B. A. A. E..ed.).pp?l-29. tralia.

14. 1. 16.

Andrew. 1. G.. and .I. in press.

17.

Gleeson. Knox.

irr AGP News Univ. ofMelbourne.

(Clarke. Aus-

18.

Glee\un. rd.).

19.

Baldo.

A. E.. and 53, 3-28.

.(. Delmer, Cell

D. Wall

8.

R. B. t 1978)

P.. and 1,amport. 0. Biochemistry (Solheim.

L.. eds.), pp X5-104. O%lO. Lamport. D. T. A. (1977) Phytochemistry V. C.. ed5.b.

7.

Knox.

York. Anderson. Knox.

Vol.

Recent

J. 85,

Advances

in

Gray.

K.. Uhlenbruck. I/,/,,iro~r)c h~,,?ri.\/r~

P. A.. Jermyn, A~r.s/. .I. P/trrl/ G. (1975)

Stone.

iti

S. P., and

Australia Pro