ARCHIVES

OF

BIOCHEMISTRY

a-L-Fucosidases MIDORI

AND

BIOPHYSICS

(1977)

from Almond Emulsin: Characterization Enzymes with Different Specificities’

OGATA-ARAKAWA, Department

353-358

181,

of Biochemistry,

TAKASHI Kobe

University

Received

MURAMATSU, School

December

of Medicine,

AND

Zkuta-ku,

of the Two AKIRA Kobe,

KOBATA Japan

14, 1976

Almond emulsin contains two kinds of a+fucosidases, which could be separated by gel tiltration on Sephadex G-200. One enzyme hydrolyzed Fucc~l+IGlcNAc and Fucal-•3GlcNAc linkages in milk oligosaccharides, but did not hydrolyze Fucal+2Gal or Fucal+6GlcNAc linkages. The other enzyme hydrolyzed the Fucal+2Gal linkage in 2’-fucosyllactose, but did not appreciably hydrolyze other fucosyl linkages. Enzymological properties of the two a-L-fucosidases are described.

The substrate specificities of cy-L-fucosidases are different according to the origin of the enzymes. Thus, the enzymes from marine gastropods have extremely broad aglycon specificity (2), while the enzymes from Clostridium perfringens (31, Aspergillus niger (4), and Bacillus fulminans (5) cleave only the Fuccul+2Gal linkage. In this communication, we describe the substrate specificities of two a+fucosidases from almond emulsin. These enzymes were found by us during the course of studies on P-galactosidase from the source (6). MATERIALS

AND

General

IgG,

obtained

from

Pierce

Methods

Descending paper chromatography was carried out using ethylacetate-pyridine-water (12:5:4, v/v/ v) as a solvent. Paper electrophoresiz was performed at a potential of 73 V/cm for 1 h using pyridineacetic acid-water (3:1:387, v/v/v), pH 5.4, as a buffer. Radiochromatogram scanning was performed by a Packard Model 7201 radiochromatogram scanner. Liquid scintillation counting was performed by a Packard Model 3320 liquid scintillation spectrometer. Protein content was determined by the method of Lowry et al. (11) using bovine serum albumin as a standard.

Assay of the a-u-L-Fucosidase Lacto-N-Fucopentaitol II

immunoglobulin

Acting

on

Enzyme solution (10 ~1) was mixed with equal volumes of ~Hllacto-N-fucopentitol II (0.84 nmol, 1.2 x 10’ cpm) and 0.15 M citrate-phosphate buffer, pH 5.0, containing 10 mglml of galactono-l&+5-lactone and was incubated at 37°C for 60 min. ARer termination of the reaction by adding 0.1 ml of ethanol, the reaction mixture was applied to filter paper and chromatographed for 4 days and then the chromatogram was analyzed by radiochromatogram scanning (Fig. 2). The amount of the product (lactoN-tetraitol) released was determined by liquid scintillation counting. The enzymatic activity thus determined was proportional to the amount of enzyme up to 30% hydrolysis. One enzyme unit was defined as the amount of the enzyme required to hydrolyze 1 pmol of the substrate per minute under the conditions given.

data of this study were presented at meeting of the Japanese Biochemiused:

was

METHODS

Milk oligosaccharides were prepared in our laboratory as described previously (7). Structures of milk oligosaccharides used in the present study are summarized in Fig. 1. NaB3H4 reduction of the oligosaccharides was performed according to Takasaki and Kobata (8). 13H1Amino acid-labeled mouse myeloma IgG2 (91, 13Hlfucose-labeled mouse myeloma IgG glycopeptide (9), and [acetyl-14Clacetylatd bovine IgG glycopeptides (10) were prepared as described previously. @Galactosidase from jack bean meal (6) was prepared by S. Ogata in our laboratory. p-Nitrophenyl-P-gala&side, p-nitrophenyl-a-mannoside, and p-nitrophenyl-fl-N-acetylglucosaminide were purchased from Sigma Chemical Co. p-Nitro1 Preliminary the 47th annual cal Society (1). 2 Abbreviation

phenyl-a-L-fucoside Chemical Co.

G. 353

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003-9861

354

OGATA-ARAKAWA,

MURAMATSU,

AND KOBATA

2'-Pucosyllactose

Fucol*2Gal61*4Glc

3-Fucosyllactose

Fucul t 3 GalE1’4Glc

lacto-N-fucopentaose

I

Ga151'3G1cNAc61+3Ga161+4G1c 2 + FUCCll

I.acto-N-fucopentaose

II

GalE1’3GlcNAc81+3Gal6l*4Glc 4 + FUCUl

Lacto-N-fucopentaose

III

Gal61+4GlcNAcE1+3Gal6l*4GlC 3 +

FUCUl La&o-N-difucohexao&

I

Ga~61+3GlcNAc61+3Gal6l+4Glc 4 t

+ Fuca1

FUCUl

FIG. 1. Structure of milk oligosaccharides.

FIG. 2. Chromatographic analysis of the product of cu+fucosidase digestion. The reaction mixture was applied to a sheet of Whatman No. 1 paper and developed for 4 days in ethylacetam-pyridine-water (12:5:4). The results of radiochromatogram scanning are shown. Positions of standard markers are shown at the bottom of the figure. 1, lacto-N-fucopentaitol II; 2, lactoN-tetraitol; 3, la&-N-triitol II. A, [3H]lacto-N-fucopentitol II (0.64 nmol); B, [3Hllacto-Nfucopentaitol II (0.64 nmol) incubated with 0.0049 milliunit of the enzyme fraction I of Sephadex G-200 column chromatography under the assay conditions; C, the product in B was recovered from the chromatogram and was incubated with 0.14 unit of ;B-galactosidase in 45 ~1 of 0.05 M citrate-phosphate buffer, pH 3.5, at 37°C for 16 h with a small amount of toluene.

Assay of a-L-Fucosidase Acting on 2’-Fucosyllactitol

tive lactitol released was used for the determination of the enzyme activity.

The assay was performed as described above for the determination of another a-L-fucosidase activity, except~that 2’-fucosyllactitol (0.34 mnol, 1.2 X lo4 cpm) was used instead of la&o-N-fucopentaitol II, that galactono-l-+5-lactone was omitted from the reaction mixture, and that the duration of the chromatography was 2 days. The amount of the radioac-

Preparation of Two a--L-Fucosiduses from Almond Em&in All steps were carried out below 4°C. Step 1: Sephndex G-ZOO column chromatography. Almond emulsin (330 mg) was dissolved in 27 ml of 0.02 M acetate buffer, pH 5.0, containing 0.1 M NaCl

ALMOND

and the insoluble material was removed by centrifugation. The crude extract was concentrated by dialysis against polyethylene glycol 20,000 to 4.6 ml and was applied to a column of Sephadex G-206 (2.6 x 95 cm) equilibrated with 0.01 M acetate buffer, pH 5.0, containing 0.1 M NaCl. Fractions of 6 ml were collected. Major active fractions of the two a-cfucosidases were separately pooled (Fig. 3). Stop

2: Rechromatography

on Sephadex

355

WL-FUCOSIDASE

G-200.

Each enzyme fraction was concentrated to 1.5 ml by dialysis against ,polyethylene glycol 20,000 and was applied to the same column as that used for step 1. Fractions of 3 ml were collected. The major active fractions were pooled, concentrated, and stored at -20°C. Throughout the procedure, the enzyme acting on lacto-N-fucopentaitol II was purified Is-fold (Table I), and the enzyme acting on 2’-fucosyllactitol was purified 8fold (Table II).

TABLE PARTIAL

PURIFICATION Volume b-d)

Total actiyiv %c-

4.60 1.70

16.8 7.29

1.85

5.70

TABLE PARTIAL

PURIFICATION

1

I

1

1

I

I

0 FRACTION

NUMBER

FIG. 3. Sephadex G-200 column chromatography of cY+fucosidases of almond emulsin. The chromatographic conditions are described in the text. The arrow indicates the eluting position of blue dextran. Assays of cY+fucosidases were carried out as described in the text, except that the incubation time was 16 h. The enzyme fractions marked as I and II were pooled. O--O, cY-cfucosidase activity determined by using la&o-N-fucopentaitol II as a substrate; GO, a+fucosidase activity determined by using 2’-fucosyllactitol as a substrate.

Specific activity

(milliunita/mg

ALMOND SkP

Volume (ml)

Crude extract Sephadex G-200 (first) Sephadex G-200 (second)

0.0903 0.517 1.46

II

OF CY-L-FUCOSIDASE

II

FROM

EYULSIN Total activity (milli-

Specific

units)

activity (milliunitelmg of protein)

4.60 1.50

4.08 1.50

0.0218 0.127

0.65

1.01

0.186

from

The crude extract of almond emulsin hydrolyzed various fucosyl linkages found in milk oligosaccharides. However, these (YrJucosidases did not act on p-nitrophenyla-cfucoside. Thus, we determined their activities by using NaB3H,-reduced milk oligosaccharides as substrates. An example of such assays is shown in Figs. 2A and

FROM

of protein)

Crude extract Sephadex G-200 (first) Sephadex G-200 (second)

RESULTS

Separation of Two a+Fucosidases Almond Em,ulsin

I

EMULSIN

ALMOND SkP

I

OF (Y-L-FWOBIDASE

2B. Part of the radioactive lacto-N&copentaitol II was converted to radioactive lactoJV-tetraitol by the enzymatic action. The identity of the enzyme product as la&o-l\‘-tetraitol was confirmed by /3-galactosidase digestion, which converted the product to lacto-l\r-triitol II (Fig. 20. Therefore, the amount of radioactivity in the product region (Fig. 2B) represents the fucosidase activity. We found that, upon Sephadex G-200 column chromatography, the enzyme acting on the Fuccrl+4GlcNAc linkage of lacto-N-fucopentaitol II was separable from the enzyme acting on the Fuccul-+BGal linkage of 2’-fucosyllactitol (Fig. 3). The former enzyme was termed the a-L-fucosidase I, and the latter enzyme the cY-cfucosidase II. After rechromatography on Sephadex G-200, both enzymes were obtained in a state virtually free of the other fucosidase activity. The partially purified preparations were free from the following glycosidase activities when they were surveyed using p-nitrophenyl glycosides: cy-galactosidase, (Ymannosidase, p-mannosidase, and P-Nacetylglucosaminidase. However, the fucosidase preparations showed approxi-

356

OGATA-ARAKAWA,

MURAMATSU,

mately 0.2-0.4 unit/mg of protein of /3galactosidase activity as measured using p-nitrophenyl-&gala&side as a substrate. We decided to characterize the enzyme using the preparation at this stage, since various attempts to remove the pgalactosidase by ion exchangers and differential inactivation were unsuccessful, and the &galactosidase was completely inhibited by galactono-1+5lactone. The enzyme preparation was free from proteases when examined as described previously (6). Specificities of the a-u-L-Fucosiduses

cr-L-Fucosidase I hydrolyzed not only the Fuccul+4GlcNAc linkage in la&&-fucopentaitol II, but also the Fucal+3GlcNAc linkage in lacto-N-fucopentaitol III (Table III). Since both activities coincided upon Sephadex G-200 column* chromatography, it is likely that the two activities are due to a single enzyme. The enzyme also hydrolyzed the Fuccul+4GlcNAc linkage in

la&o-iV-difucohexaitol I, while the velocity of hydrolysis of the fucosyl linkage was slow, possibly due to the steric hindrance by the neighboring fucosyl residue. The enzyme preparation also hydrolyzed the Fuccul+3 sorbitol linkage with low velocity. The fucosidase did not hydrolyze the Fuccul+ZGal linkage in 2’fucosyllactitol and in lacto-N-fucopentaitol I. The Fuccul+GGlcNAc linkage in IgG glycopeptides was also not hydrolyzed. The specificity of a+fucosidase II was restricted. Among various substrates listed in Table III, the Fucal+2Gal linkage in 2’-fucosyllactitol has been the sole linkage hydrolyzed by the enzyme efficiently at the substrate concentration examined. Of course, the enzyme also hydrolyzed 2’-fucosyllactose. Enzymological Properties of the a-L-Fucosidases

a-L-Fucosidase I was most active at pH 5.5, while a-cfucosidase II exhibited

TABLE SPECIFICITIES

Substrate Lacto-N-fucopentaitol La&-N-fucopentaitol Lacto-N-fucopentaitol 2’-Fucosyllactitol 3-Fucosyllactitol La&o-N-difucohexaitl

OF THE

Fucosyl linkage I II III I

AND KOBATA

(FuccYl+2Gal) (Fucal+4GlcNAc) (Fuccxl+3GlcNAc) (Fucal-+2Gal) (Fuccul+BSorbitol) (Fucc~1-+4GlcNAc Fuccul+BGal) (Fuccul-+6GlcNAc)

III CY-L-FIJCCBIDASEP

Relative activity cr+Fucosidase 1 100 92.9 1.4 13.5 16.7b

I

cr-L-Fucosidase II 3.5 2.3 4.1 100 3.2 1

-c -c IgG glycopeptide and the derivative D The assays of a-L-fucosidases were carried out as described under Materials and Methods, except that the incubation time was 2 h. Linearities of enzymatic activities under the incubating conditions were confirmed. Results are expressed as percentages: 100 x (enzymatic activity toward a given substrate/ enzymatic activity toward the principal substrate). b That the susceptible linkage was Fucal-+4GlcNAc was confirmed by chromatographic analysis of the product. c [3H]Fucose-labeled mouse myeloma IgG glycopeptides (3000 cpm) were incubated with 0.018 milliunit of a-L-fucosidase I or 0.0077 milliunit of a-L-fucosidase II in 0.03 ml of 0.05 M citrate-phosphate buffer, pH 5.0, at 37°C for 15 h with a small amount of toluene. The reaction mixture was applied to filter paper and chromatographed as described in General Methods, and the radioactivity in the fucose region was determined by liquid scintillation counting. No release of fucose label was observed. In other experiments, Fucal-+6GlcNAc+N-[14C]acetyl-Asn-peptide (10) was prepared from N-[ocetyZ-14C]acetylated bovine IgG glycopeptide by endo+-Ar-acetylglucosaminidase digestion (8). The substrate (0.15 mnol) was incubated with 0.018 milliunit of a-L-fucosidase I or 0.0077 milliunit of cY-tfucosidase II in the standard reaction mixture at 37°C for 15 h with a small amount of toluene. No release of GlcNAc N-[‘Clacetyl-Asn-peptide was observed as examined by paper electrophoresis.

ALMOND

a-~-FUCOSIDASE

357

rather broad pH optima between pH 5 and 6.5 (Fig. 4). Various compounds did not show significant effects on the activities of the enzymes (Table IV). Kinetic constants of the a+fucosidases are summarized in Table V.

dase I, hydrolyzed Fuccul+4GlcNAc and Fucal+3GlcNAc linkages, but not Fucal+2Gal or Fuccul,~6GlcNAc linkages. cy+-Fucosidase with an apparent similar specificity has been reported to occur in Trichomonas foetw and has been called the Lea-destroying enzyme? since DISCUSSION the Fucal+4GlcNAc linkage is the deterTwo cr-L-fucosidases with different speci- minant of Lea blood group activity (13). ficities were isolated from almond emulHowever, the enzyme has not yet been sin. One enzyme, termed the 05L-fucosi- isolated or studied with respect to its properties. Thus, the enzyme from almond I I I I I I emulsin is expected to be a valuable tool for the studies of fucose-containing glycoconjugates. It has already been useful in structural studies of milk oligosaccharides (14, 15). Moreover, through these studies, it has been confrmed that the enzyme hydrolyzes Fucal+4GlcNAc and Fucal~3GlcNAc linkages in substrates in which the structures are much more complex than those used in the present study. 4 5 6 7 8 3 PH Another enzyme, a-L-fucosidase II, hydroFIG. 4. Effect of pH on enzyme activity. The as- lyzed the Fucal-2Gal linkage in 2’-fucosay was performed as described under Materials and syllactitol, but not the Fuccrl+4GlcNAc or Methods. The pH of the reaction mixture was Fucal+SGlcNAc linkage found in milk olchanged by the use of citrate-phosphate buffer (0.05 igosaccharides. In this respect, the enzyme pi final concentration) with different pH values. should be classified as Fuccrl-+BGal fucosiO-O, a+fucosidase I; O-O, o+fucosidase II. dase, such as those from C . perfringens (3) and A. niger (4). However, we observed that, at least at a dilute substrate concenTABLE IV tration, the velocity of hydrolysis of the EFFECTS OF SEVERAL COMPOUNDS ON ENZYMATIC fucosyl linkage in lacto-N-fucopentaitol I ACTIVITY was much slower than that of the linkage Compound ConcenRelative activity (%) tration in 2’-fucosyllactitol. The reason for the hubi) WL-FUa-L-FUcosidase I cosidase II great difference in the velocity of hydrolysis is unknown, while the difference in the None 100 100 linkage of the penultimate galactosyl resiEDTA 1 107 97.5 due might be a candidate to explain the M&l, 10 64.5 74.1 CaCl, 10 74.2 121.4 observation. We feel that substrate speciL-Cysteine 10 65.6 94.3 ficities of various Fuccwl+2Gal fucosip-chloromercuri1 71.3 88.4 dases, including that from almond emulphenylsulfonsin, should be reexamined by using varate ious model compounds such as milk oligoTABLE KINETIC

CONSTANTS

V

OF THE OI-L-FUCOSIDASES

Substrate

Enzyme

cY+Fucosidase I

La&o-N-fucopentaitol La&-N-fucopentaitol

cr+Fucosidase

2’-Fucosyllactitol

II

V (milliunits/ mg of protein) II III

Km (md

6.86 6.46

0.101 0.0950

4.64

0.675

356

OGATA-ARAKAWA,

MURAMATSU,

saccharides and fragments from blood group substances. Such study may afford valuable methods in defining the fine structure of blood group antigens (16). ACKNOWLEDGMENTS We thank Miss Miyoko Inohara for her expert secretarial assistance. This work was supported by grants from the Ministry of Education, Science, and Culture, Japan. REFERENCES 1. OGATA-ARAKAWA, M., MURAMATSU, T., AND KoBATA, A. (1974) Seiko&u 46, 663. 2. NISHIGAKI, M., MURAMATSU, T., KOBATA, A., AND MAEYAMA, K. (1974)J. Biochem. X5,509517. 3. AMINOFF, D., AND FIJRUICAWA, K. (197O)J. Biol. Chem. 245, 1659-1669. 4. BAHL, 0. P. (1970) J. Biol. Chem. 245, 299304. 5. KOCHIBE, N. (1973)5. B&hem. 74,1141-1149. 6. ARAKAWA, M., OGATA, S., MURAMATSU, T., AND KOBATA, A. (1974) J. Biochem. 75, 707-714.

AND KOBATA

7. KOBATA,

A. (1972) Methods Enzymol.

28, 262-

271. S., AND KOBATA, A. (1974) J. Biothem. 76, 783-789. 9. MURAMATSU, T. (1971) J. Biol. Chem. 246,55355537. 10. KOIDE, N., AND MURAMATSU, T. (1974) J. Biol. Chem. 249, 4897-4904. 11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193, 265-275. 12. TAI, T., ITO, S., YAMASHITA, K., MURAMATSU, T., AND KOBATA, A. (1975) B&hem. Biophys. Res. Commun. 65, 968-974. 13. WATKINS, W. M. (1972) in Glycoproteins (G&achalk, A., ed.), pp. 830-891, Elsevier, Amsterdam. 14. YAMASHITA, K., TACHIBANA, Y., AND KOBATA, A. (1976) Arch. B&hem. Biophys. 174, 562591. 15. YAMASHITA, K., TACHIBANA, Y., AND KOBATA, A. (1976) Biochemistry 15, 3950-3955. 16. HAKOMORI, S., AND KOBATA, A. (1974) in The Antigens (Sela, M., ed.), Vol. 2, pp. 79-140, Academic Press, New York. 8. TAKASAKI,