Comp. Biochem. Physiol. Vol. 76B, No. 2, pp. 277-281, 1983 0305-0491/83 $03.00 + 0.00 Printed in Great Britain © 1983 Pergamon Press Ltd

f l -N-ACETYLHEXOSAMINIDASE A N D ~-D-MANNOSIDASE FROM THE EPIGONAL

LYMPHOMYELOID ORGAN OF THE NURSE SHARK (GINGLYMOSTOMA CIRRA TUM)

GUNNAR LUNDBLAD, RAGNAR F~tNGE* and KERSTIN SLETTENGREN Department of Chemistry, National Bacteriological Laboratory, S-10521 Stockholm, Sweden and

*Department of Zoophysiology, University of G6teborg, P.O. Box 25059, S-40031, G6teborg, Sweden

(Received 28 February 1983)

Abstract--l. Lymphomyeloid (lymphohaemopoietic) tissues are rich in glycosidases probably contained within leucocytes. The glycosidases ~-N-acetylhexosaminidase and ~t-n-mannosidase found in the epigonal organ--a prominent lymphomyeloid tissue of the nurse shark Ginglymostoma cirratum--were purified and preliminary characterized.

2. The enzyme fl-N-acetylhexosaminidase was assayed as N-acetyl-fl-D-glucosaminidase but was shown to have also N-acetyl-fl-D-galactosaminidase activity. The pH optima were 4.5 and 4.2 for these two substrates. The enzyme was purified 40-fold by gel filtration. By isoelectric focusing the enzyme showed multiple forms with pI's = 6.0, 6.5, 6.7 and 6.9. The molecular weight was 144,000 + 13,000 (SD) estimated by gel filtration.

3. Ct-D-mannosidase was purified 30-fold by gel filtration. Isoelectric focusing in a narrow pH range gave the pl values 7.3, 7.6 and 7.85. The main pH optima were at 3.3, 3.7 and 4.5. The molecular weight was 275,000 ___ 19,000 (SD).

INTRODUCTION

In a previous investigation (Fringe et al. 1980) high activities of a series of glycosidases were found in lymphomyeloid tissues of some marine fish species. The tropical elasmobranch fish Ginglymostoma cir- ratum (nurse shark) possesses a remarkably well developed lymphomyeloid (lympohaemopoietic) tis- sue called the epigonal organ, which is associated with the gonads. The epigonal organ of Gin- glymostoma cirratum was found to contain all the fourteen glycosidases studied by Fringe et al. (1980). N-acetyl-fl-glucosaminidase and Ct-D-mannosidase showed especially strong activities. These two en- zymes are of great importance in the glycoprotein metabolism, as it is known that the carbohydrate moieties of glycoproteins to a high extent are formed of N-acetylglucosamine and mannose (Kornfield and Kornfield, 1976; Flowers and Sharon, 1979). A study of these two enzymes from the nurse shark epigonal organ therefore seemed relevant. A separation of the enzymes and a preliminary characterization were performed. A more extensive purification was impos- sible due to paucity of material.

MATERIALS AND METHODS

Biological material The nurse shark Ginglymostoma cirratum was captured in

Puerto Rico in 1979 and dissected immediately after it was killed. Pieces of the epigonal organ were stored in closed vessels at -20°C after addition of a few drops of 1-butanol.

Preparation of the crude extracts Parts of the frozen material were weighed and disin-

tegrated and homogenized in 0.9% NaC1 and 0.1% Triton

X-100 solution in a Virtis "45" disintegrator and a Dounce homogenizer respectively and then centrifuged at 10,000g for 8 min. All operations were carried out at 4°C.

Chemicals The chromogenic substrates p-nitrophenyl (p-NP)-N-

acetyl-fl -o-glucosamide, p-NP-N-acetyl-[3-o-galactosamin- ide and p-NP-fl-D-mannoside were purchased from Koch- Light Laboratory Ltd., Colnbrook, UK.

Enzyme assay

The glycosidase activity was determined according to Verpoorte (1972). The standard assay contained 25 #l en- zyme made up to 2.0 ml 2.0 mM p-nitrophenyl derivatives in 0.05 M Na-citrate buffer, pH 4.5, or at the desired pH in citrate-phosphate buffer. The reaction was stopped after 30min at 37°C by adding 2.0ml 0.5 M glycine-NaOH buffer, pH 10.5. For samples with low activities the incu- bation period was prolonged to 2 or 4 hr. The release of p-nitrophenol was read at 400 nm. One unit (U) of enzyme activity was defined as the amount releasing 1 #mol/hr.

Gel filtration chromatography Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala,

Sweden) was equilibrated in 0.1 M Tris-HC1 buffer, pH 8.0, 0.15 M NaC1 with 1% 1-butanol and chromatography was performed in a column (19 mm x 1.41 m) and eluted with the same buffer at 12.5ml/hr. The columns were run at +6°C, the effluents collected in a LKB Ultrorac and the absorbances of fractions controlled by a Uvicord Monitor (LKB) and measured at 280nm in a Beckman spec- trophotometer model DB-G.

Isoelectric focusing This was performed using equipment and supplies from

LKB Instruments. A 110 ml (LKB 8100-1) capacity column was used with a linear sorbitol gradient between 0 and 50% (w/v) containing 1% of the used ampholyte. Isoelectric

277

278 GUNNAR LUNDBLAD etal.

3 E

o

c 2 0

e o

J~

20

0 0 35 40 /.5 50 55 60

Frocti0n number

1 5 _

3 > ,

>

1o 0

e~

E

c W

Fig. 1. Separation of N-acetyl-fl-o-glucosaminidase and -D-mannosidase from Ginglymostoma cirratum by gel chro-

matography. A 5 ml sample of centrifuged homogenate from the epigonal organ was applied to Sephadex G-200 column as described in Methods. The effluent was collected in 4.8ml fractions at a flow rate of 12.5ml/hr at 6°C. Absorbance at 280nm ( ); activity of N-acetyl-fl- D-glucosaminidase (© O) and activity of

~-D-mannosidase (O O) in units (U) per ml.

focusing was carried out for 45 hr at +4°C and 480-1100 V. The anode was at the top. Fractions of 3.1 ml volume were collected for pH measurements at +4°C and assayed for glycosidase activity after dialysis against 0.9% NaCI.

Protein determination

The protein content was determined by the micro Kjel- dahl method modified by the use of a coulimetric analyser (LKB-Beckman Instruments AB, Bromma, Sweden).

Enzymes involved in the study fl-N-Acetylhexosaminidase (E.C. 3.2.1.53); N-acetyl-fl-

o-glycosaminidase (E.C. 3.2.1.30); N-acetyl-fl-o-galactos- aminidase (E.C. 3.2.1.53); ~-D-mannosidase (E.C. 3.2.1.24).

RESULTS

Centrifuged crude extracts of the epigonal organ of G. cirratum were purified by gel filtration on Sephadex G-200 and the fractions were assayed for ~-D-mannosidase and N-acetyl-fl-D-glucosaminidase activity (Fig. 1). The procedure resulted in a 30-fold purification of the first enzyme (fraction 47) and a 40-fold of the second (fraction 55). The yields were 100 and 88~ respectively.

Pools of dialyzed fractions of N-acetyl-fl- D-glucosaminidase with 30-fold purification were run in an isoelectric focusing column in a narrow pH range. These procedures resulted in the separation of 4 isoenzymes with pI values of 6.9, 6.7, 6.5 and 6.0. One of these runnings is shown in Fig. 2. The total yield of the enzyme was 77~. The active fractions were tested for the effect of pH upon the hydrolysis of N-acetyl-fl-D-galactosaminide. The pH activity curves (Fig. 3) show great similarities but have a slight displacement. The pH optima were 4.5 for N-acetyl-fl-D-glucosaminidase and 4.2 for N-acetyl- fl-D-galactosaminidase respectively. These enzymatic activities therefore most probably can be attributed to one single enzyme e.g. fl-N-acetylhexosaminidase.

Figure 4 shows a pH activity curve of ~-D-mannosidase 30-fold purified by means of Sephadex G-200 and then submitted to isoelectric focusing with an ampholyte carrier pH 3.5-10 as seen in the same figure. The pH curve from top fraction 13 was complex with pH optima at 3.3, 3.7 and 4.5. No separation of isoenzymes was obtained in the isoelectric focusing. However, isoelectric focusing in a narrow pH range of a pool of 28-fold purified ~-D-mannosidase gave after dialysis against 1~ gly- cine, 3 isoenzymes with the pH values 7.85, 7.6 and 7.3 as shown in Fig. 5. The total yield of the enzyme was 82~. The pH activity curve of purified

~I0 ~ 10

0 ..... c~ . ' 0 10 20 30

Froct ion number

Fig. 2. Isoelectric focusing of purified N-acetyl-fl-D-glucosaminidase. The pooled fractions 51-58 from the gel filtration shown in Fig. l were dialyzed against 1~ glycine and l ~ 1-butanol for 20 hr at 4°C and 34 ml thereof was applied to the column and run in ampholyte carriers in the pH ranges 3.5-10 (0.4 ml)

and 6.0-8.0 (2.3 ml).

0

Q

_= 0

(Z:

279

100

75

50

25

i

2

/ 3

i I I

4 5 6 pH

Epigonal lymphomyeloid organ of the nurse shark

Fig. 3. Effect of pH on the activity of purified fl-N-acetyl- hexosaminidase. (O O) N-acetyl-fl-o-glucosaminidase

and (S O) N-acetyl-fl-D-galactosaminidase.

8 :1 : ~oo ~ 6t f 1 ~ 6 =

2I t g ,,o°.2o >, 75

o

~. 50 2 (z;

p H

Fig. 4. Effect of pH on the activity of e-D-mannosidase from fraction 13 of an isoelectric focusing with an ampho-

lyte cartier in the pH range 3.5-10.

-D-mannosidase from the main peak Fig. 6 (fraction 19) shows an irregular course with a pH optimum at 4.5.

By the use of a Sephadex G-200 column ( 1 9 m m x l . 4 1 m ) in 0.1M Tris-HC1 pH 8.0+ 0.15 M NaC1 + 19/o 1-butanol the molecular weights for the two purified enzymes were calculated accord- ing to Andrews (1965). The molecular weights were 144,000 + 13,000 (SD) for fl-N-acetylhexosaminidase and 275,000+ 19,000 (SD) for CC-D-mannosidase (Fig. 7). The marker substances were: blue dextran 2000, 2,000,000 (Pharmacia, Uppsala), catalase, 244,000 (Miles, code 36-106, batch 4261), aldolase 161,000 (Worthington, 1123 ALD, 37E854, IgG, 150,000 (Antitetanus IgG, Dr L.-G. Falksveden, 264-700924 SBL), albumin 68,000 (Kabi, Stockholm,

1746 from normal plasma), ovalbumin, 45,000 (Schwarz-Mann, 2x cryst., lot No. X 1302) and cytochrome C, 12,400 (Sigma, horse heart).

D I S C U S S I O N

Multiple forms of glycosidases are rather frequent. For fl-N-acetylhexosaminidase (N-acetyl-fl-n-glu- cosidase with N-acetyl-fl-D-galactosidase activity), multiple forms are known from different sources of the enzyme (Robinson and Sterling, 1968; Hultberg et al., 1974; Christomanou et al., 1977; Calvo et al., 1978, Lundblad et al., 1981).

In contrast to the symmetrical pH activity curves of the 2 substrates used for fl-N-acetylhexosamini- dase assay (Fig. 3) the ~-n-mannosidase curve

6

i s

>

N

0 0 10 20 30

Fraction number

10

8

4

2

0

Fig. 5. Isoelectric focusing of purified ~,-D-mannosidase. The pooled ~-D-mannosidase active top fractions from a gel filtration on Sephadex G-200 were after dialysis subjected to isoelectric focusing with an ampholyte carrier pH 3.5-10, after which the obtained homogenous a-o-mannosidase re#on was pooled and dialyzed as described in Fig. 2 and subjected to a second isoelectric focusing in the ampholine interval

pH 7-9. ( ) pH gradient; (O O) ~-n-mannosidase activity.

C.ILP, 76/21~-e

280 (IUNNAR LUNDBLAD et al.

100

2

"~.. 50

0

25

I I I I

3 4 5 6 pH

Fig. 6. Effect of pH on the activity of c(-I)-mannosidase from fraction 19 in Fig. 5.

showed at least 3 optima (Figs 4 and 6). This is well known from the literature. ~t-D-Mannosidase from human liver (Minami et al., 1979) had pH optima at 4.0 and 6.5. In human plasma Hirani and Winchester (1979) found 2 forms of the enzyme with pH optima at 4.2 and 5.75-6.0. Foenaru and Dreyfus (1979) reported that Ct-D-mannosidase with one pH opti- mum at 5.5-6.0 in human red cells differed from lysosomal ~t-t)-mannosidases in liver with maxima at 4.0 and 6.5 and in leucocytes at 4.0. The isoelectric point, however, was the same, 5.8-6.0 for the different material.

Glycosidases are hydrolytic enzymes generally of lysosomal origin, although recent studies have shown that acid ~t-D-mannosidases and fl-D-glucuronidase may derive from different structures of the cell

(Dewald and Touster, 1973; Shoup and Touster, 1976; Tulsiani et al., 1977; Touster, 1978). C(-D-Mannosidase from rat liver is localized in lysosomes (L), Golgi membranes (G) and cytosol (S) with pH optima at 4.5, 5.5 and 6.5 respectively. The S-form, however, was extremely unstable and the G-form was difficult to get into solution with 0.1~ Triton X-100 which is recommended for hom- ogenization--extraction of glycosidases. Therefore, with the technique applied in the present study, the ~-D-mannosidase forms probably are of lysosomal origin.

The epigonal organ of the nurse shark contains enormous numbers of leucocytes, above all granu- locytes. These cells may be regarded as secretory cells capable of secreting enzyme granules by exocytosis (Wright, 1982). The glycosidases of the epigonal organ probably are mainly of leucocytic origin. The leucocytic glycosidases may be of indirect immu- nological importance by modifying the carbohydrate- containing surface antigens of cells involved in im- mune responses. Certain leucocytic glycosidases like lysozyme and chitinase could be of importance in defence against bacteria or parasites (Fringe et al., 1980). For structural aspects see Fringe and Mat- tisson (1981).

Acknowledgements--The authors wish to thank Ms A.-K. Tegnemo for skilful technical assistance and Ms Turppa for drawing the figures. The animal material was obtained by one of the authors (R.F.) at the Laboratory of Neuro- biology, San Juan, Puerto Rico. Thanks are due to the Director, Dr J. de Castillo.

REFERENCES

Andrews P. 1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606.

Calvo P., Reglero A. and Cabezas J. A. (1978) Purification and properties of fl-N-acetyl-hexosaminidase from the

2.0 ~ o m e C

~ albumin

o ~ . ~ m i n ~> 1.5

[~-N-acet yl.q L ucosa m i n i dase ---~ q ~ e-Aldotase o~Catntase

. . . . . . I g G - ~ o

o °ii°i °i ' I I i i i

xl0 t, xl05 xl06 Moteeutar Weight

Fig. 7. Estimation of molecular weights of purified enzymes by Sephadex G-200 gel filtration. Two or three determinations were made.

Epigonal lymphomyeloid organ of the nurse shark 281

mollusc Helicella ericetorum Mfiller. Blochem. J. 175, 743-750.

Christomanou H., (~.p C and Sandhoff K. (1977) Isoelectric focusing pattern of acid hydrolases in cultured fibroblasts, leucocytes and cell-free amniotic fluid. Neuropiidiatrie 8, 238-252.

Dewald B. and Touster O. (1973) A new ~t-D-mannosidase occurring in Golgi membranes. J. biol. Chem. 248, 7223-7233.

F~inge R. and Mattisson A. (1981) The lymphomyeloid (hematoopoietic) system of the Atlantic nurse shark, Gin- glymostoma cirratum. Biol. Bull. 160, 241)-249.

F~nge R., Lundblad G., Slettengren K. and Lind J. (1980) Glycosidases in lymphomyeloid (hematopoietic) tissues of elasmobranch fish. Comp. Biochem. Physiol. 67B, 527-532.

Flowers H. M. and Sharon N. (1979) Glycosidases-- properties and application to the study of complex carbo- hydrates and cell surface. Adv. Enzym. 48, 29-95.

Foenaru L. and Dreyfus J. C. (1979) ct-Mannosidase in human red cells. Biochim. biophys. Acta 556, 67-71.

Hirani S. and Winchester B. (1979) The multiple forms of ct-o-mannosidase in human plasma. Biochem. J. 179, 583-592.

Hultberg B., Ockerman P. A. and Nord6n N. E. (1974) Isoenzymes of four acid hydrolases in human kidney and urine. Clinica chim. Acta. 52, 239-243.

Kornfeld R. and Kornfeld S. (1976) Comparative aspects of glycoprotein structure. A. Rev. Biochem. 45, 217-237.

Lundblad G., Huldt G., Elander M., Lind J. and Slettengren K. (1981) fl-N-acetylglucosaminidase from Entamoeba histolytica. Comp. Biochem. Physiol. 68B, 71-76.

Minami R., Sato S., Kudoh T., Oyanagi K. and Nekao T. (1979) Age-dependent variations of lysosomal enzymes in human liver. Tohoku J. exp. Med. 129, 65-70.

Robinson D. and Sterling J. L. (1968) N-acetyl-fl- o-glucosaminidase in human spleen. Biochem. J. 107, 321-327.

Robinson D., Jordan T. W. and Horsburgh T. (1972) The N-aeetyl-fl-D-hexosaminidases of calf and human brain. J. Neurochem. 19, 1975-1985.

Shoup V. A. and Touster O. (1976) Purificatiofl and charac- terization of the ct-o-mannosidase of rat liver cytosol. J. biol. Chem. 251, 3845-3852.

Touster O. (1978) The chemistry and turnover of lysosomal enzymes. In Protein Turnover and Lysosome Function, pp. 231-250. Academic Press, New York.

Tulsiani D. R. P., Opheim D. J. and Touster O. (1977) Purification and characterization of ~-o-mannosidase from rat liver Golgi membranes. J. biol. Chem. 252, 3227-3233.

Verpoorte J. A. (1972) Purification of the two fl-N-aeetyl-glucosaminidases from beef spleen. J. biol. Chem. 247, 4787-4793.

Wright D. G. (1982) The neutrophil as a secretory organ of host defense. In Advances in Host Defense Mechanisms (Edited by Gallin J. I. and Fauci A. S.), pp. 75-110. Raven Press, New York.