An unusual case of mannosidosis with severe deficiency of acid mannosidase in leukocytes and high residual enzymatic activity in skin fibroblasts

37

Clinica Chimica Acta, 107 (1980) 37-42 @ Elsevier/North-Holland Biomedical Press

CCA 1498

AN UNUSUAL CASE OF MANNOSIDOSIS WITH SEVERE DEFICIENCY OF ACID MANNOSIDASE IN LEUKOCYTES AND HIGH RESIDUAL ENZYMATIC ACTIVITY IN SKIN FIBROBLASTS

INNA V. TSVETKOVA,

EUGENIA

L. ROSENFELD

Institute of Biological and Medical Chemistry, Academy Filatou Children’s Hospital N 13, Moscow (U.S.S.R.)

(Received

January

and IRINA of Medical

G. PRIGOZINA Sciences

of U.S.S.R.,

31st, 1980)

Summary A case of mannosidosis with high residual activity of fibroblast acid mannosidase is described. The mutant enzyme was thermostable and has an almost normal K, value. The patient had mild clinical manifestations, but excretion of mannose-rich oligosaccharides was very high. This case of mannosiddsis is compared with those reported in literature.

Introduction Mannosidosis is an inherited storage disease first described by Ackerman [ 11. This disorder is a result of acid cy-mannosidase deficiency in various tissues [ 2,3]. Clinically, mannosidosis is characterized by coarse facies, hepatosplenomegaly, skeletal changes, psychomotor retardation and other signs typical of the mucopolysaccharidoses. However, unlike the mucopolysaccharidoses, in mannosidosis the excretion of mannose-rich glycosylated compounds, instead of acid glycosaminoglycans is observed [4,5]. Clinical diagnosis of mannosidosis must be confirmed by determination of acid mannosidase deficiency in leukocytes and cultured skin fibroblasts and decreased enzymatic activity in the patient’s parents, which are obligate heterozygotes. In some cases of mannosidosis altered properties of the mutant enzyme, namely an increased were found [6,7]. Mannosidosis, like many K, and decreased thermostability other storage diseases, is a very heterogeneous disorder in both its phenotypic signs and the degree of the deficiency of the enzyme. In this paper, a case of mannosidosis is described with a severe deficiency of acid mannosidase in

Address U.S.S.R.

for correspondence: Dr. Inna V. Tsvetkova, Institute of Biological Academy of Medical Sciences, Pogodina 10. 119121 Moscow. U.S.S.R.

and Medical Chemistry.

38

leukocytes, and a high residual activity of the enzyme blasts with a very small increase in the K, value.

in cultured

skin fibro-

Case report A boy, P.A., was brought to the Children’s Hospital when aged 2.5 years with a diagnosis of mucopolysaccharidosis. The child was the product of a normal pregnancy and delivery, and was considered to be normal at birth. From the age of 6 months he had frequent upper respiratory infections and during the first year gradual motor and mental retardation. The common features included coarse facies, a flat nasal bridge and hepatosplenomegaly. Corneal opacities and skeletal anomalities were not found. A urine spot test for mucopolysaccharides was negative. Materials and methods

Leukocytes were prepared from freshly drawn blood as previously described [8], Cultured skin fibroblasts were obtained from the child, his parents and normal control subjects. The cells were grown in nutrient mixture 199 supplemented with 20% fetal calf serum in an atmosphere of 95% air and 5% COZ. Ten days after passage the cells were harvested mechanically. Extracts of leukocytes and fibroblasts were prepared by centrifugation of water homogenates after addition of Triton X-100 up to a concentration of 2 g/l. The activity of a-mannosidase was assayed using 2.5 mmol/l 4-methylumbelliferyl-cu-D-mannoside (Koch-Light Co., U.K.) in 0.1 mol/l phosphate-citrate buffer, pH 4.0 and 5.5 for the acid and neutral enzyme respectively. In some experiments acetate buffer and a varied concentration of substrate were used (see legends for figures and tables). The activity of other lysosomal hydrolases were determined using 4-methylumbelliferyl-glycosides as previously described

[91* For thin-layer chromatography of urinary oligosaccharides, 20 ~1 of untreated urine were applied to a silica gel plate (Merck or Sillufol). After chromatography for 2 h in n-butanol-acetic acid-water (2 : 1 : 1, by volume), the mannose-rich oligosaccharides were located with aniline-diphenylamine or orcine reagent. Results

Acid mannosidase activity in the child’s leukocytes was less than 1% of normal (Table I). This value was significantly lower than those in leukocytes of two children with mannosidosis from another family described by us previously [lo]. The mannosidase activity in the father’s leukocytes was about half normal (52.2%) and that in mother’s leukocytes was only 25.8% of normal. The activities of some other lysosomal glycosidases in leukocytes of the child and his parents were within normal limits. A study of pH-dependence of the a-mannosidase activity of the leukocytes showed that at pH 3-4, the activity was negligible, and at pH 5.5-6.0, it was nearly normal (Fig. 1). These results allowed us to diagnose this patient as having mannosidosis.

39 TABLE I ACID HYDROLASE

ACTIVITIES

IN LEUKOCYTES

(nmol

.

mg-’ protein.

h-l)

ff-D-mannosidase

P-D-hexosaminidase

a-L-fucosidase

P-D-ghwuronidase

117 f 42 (79-201)

1140 f 124 (5’72-1894)

38 f 12 (13-65)

309 f 98 (142-350)

Controls (n = 10) mean f S.D. (range) Patient P.A.

0.8

1446

55.6

318

Father

62.0

1905

36.0

481

Mother

30.0

2400

78.4

586

Surprisingly the acid mannosidase activity in cultured skin fibroblasts from the child was rather high (25% of normal; Table II). This value was significantly higher than that found in most cases of mannosidosis reported by other authors [6,11,13]. The apparent K, of acid cu-mannosidase in the patient’s fibroblasts was only slightly increased (2 mmol/l as compared to 0.8 mmol/l in control). Moreover the acid cu-mannosidase in the patient’s fibroblasts was rather thermostable (Fig. 2), unlike the enzyme in mannosidosis described by Beaudet [6]. In our case, after 30 min of pre-incubation at 56°C in acetate buffer, pH 4.0, only a small decrease in enzyme activity in both fibroblasts and leukocytes was observed. The mutant enzyme in mannosidosis described by Beaudet [6] ‘was almost completely inactivated after 10 min pre-incubation at 56°C. The acid mannosidase activity of the patient’s cell extracts was increased at 15 mmol/l substrate compared to 2.5 mmol/l (Table III). Thus the enzyme activity of the fibroblasts was increased by about 40%. Unlike the patient’s enzyme, the mannosidase activity of control fibroblasts was hardly activated

\

o?

10

l-

3.5

d”

4.0

4.5

0

\

Control

0 Patient

”

5.0

5.5

”

6.0

6.5

’

7.0

PH Fig. 1. pH-dependence of a-D-mannosidase activity in leukocytes (acetate buffer. 0.1 mol/l).

40

Leukocytes

01

Fibroblosts

0 Control

. Control

0 Potlent

t2 Patient

30

15 Minutes at 56 “C

Fig.

2.

Heat

nosidase

inactivation

activity

of

acid

in the patient’s

a-D-mannosidase leukocytes

at 15 mmol/l substrate. The acid increased from 0.8 to 6.0 nmol only 5% of that in the control. patient’s and normal leukocytes. was found only in the patient.

TABLE

at 56’C

was determined

ACTIVITY

Specific Controls

226

? S.D.

pH

4.0).

of 15

The

man-

mmol/l.

mannosidase of the patient’s leukocytes was . mg-’ - h-l, bu t this value was nevertheless Zn2+ activated the acid mannosidase both in In fibroblasts noticeable activation by Zn2+

IN

CULTURED

SKIN

FIBROBLASTS

% of normal

activity

(nmol

mg-’

Protein

. he1

control

f 84

57.0

25

Father

97.0

42

Mother _

170.0

15

P.A.

III

EFFECT

OF

INCREASED

ACTIVITY

(nmol

SUBSTRATE

. mg-l

protein

CONCENTRATION

2.5

mmol/l

2.5

mmol/l mmol/I

+ 1.0

mmoI/I

Zn2+

AND

Zn”

ON

THE

ACID

MANNOSI-

. h-I)

Patient

15.0 -

mol/l,

(122-354)

(range)

DASE

0.1

concentration

(n = 12)

Mean

TABLE

buffer

II

ACID+D-MANNOSIDASE

Patient

(acetate

at a substrate

Control

leukocytes

fibroblasts

leukocytes

0.8

35.3

1.5

50.0

112.0

165.0

6.0

47.8

101.0

178.0

74.0

fibroblasts 159

41

Thin-layer chromatography showed some bands of oligosaccharides with a high degree of polymerization as the major abnormality in the urine of the child. The amount of mannose in the acid hydrolysates of these oligosaccharides was increased. Discussion Our case of mannosidosis differed in several ways from those described previously. Firstly, there was a high residual activity of acid mannosidase in cultured skin fibroblasts from our patient. It was 25% of normal control values, whereas the enzyme activity in previously described cases did not exceed 10% of the controls [6,7,11 J. There was, however, a severe deficiency of acid mannosidase activity in the child’s leukocytes. Secondly, the K, value of fibroblast mannosidase exceeded the normal value only 2-fold, whereas the mutant enzymes described by Beaudet [6] were characterized by at least a 25fold increase in K, values. Furthermore, the acid mannosidase of our patient was thermostable unlike the ‘enzyme in some other cases of mannosidosis [6,7]. Similar properties of acid mannosidase (high residual activity, thermostability) have been reported by Buch [12]. The latter author described two cases with only 4-&fold increase in K, value for acid mannosidase in fibroblasts. Such a small increase in K, value resembled the K,,, in our case rather than that reported by Beaudet [6]. In Beaudet’s patient the acid mannosidase was characterized by a 25-fold increase in K,. Like the enzyme described by Bach [ 121, the acid mannosidase of our patient was activated by high substrate concentration and by Zn’+. However, contrary to Bach’s results, the enzyme activity in our experiments did not reach that of the control. Such results must be taken into account in the clinical enzymatic diagnosis of mannosidosis, as already pointed out by other authors [ 6,121. Whereas the leukocyte activity of the patients described by Bach [12] was 5-6% of a normal control, that of our child was very low. Nevertheless, the case reported by us can be considered as a variant of the mannosidosis described by Baeh [ 121. In his cases as well as in ours, some properties of the defective enzyme were different from those of the mutant enzymes previously described. The clinical manifestations of the disease in this child were milder than in two other patients described by us [lo]. It is possible that the acid mannosidase activity in the tissues of the child was high enough. On the other hand, the excretion of mannose-rich oligosaccharides was significantly higher at the age of 3 years than at 2.5 years. This fact indicates that the residual activity of acid mannosidase in the child’s tissues is insufficient for normal utilization of mannose-containing compounds and therefore the disease is bound to develop. References 1 &kerman, P.A. (1967) A generalized storage disorder resembling Hurler’s syndrome. Lancet 2. 239 2 Carrel, M., Dance. N., Masson, P.K.. Robinson, D. and Winchester. B.G. (1972) Human mannosidosis -the enzymic defect. Biochem. Biophys. Res. Comm. 49, 579 3 Hultberg, B. (1970) Properties of cu-mannosidase in mannosidosis. &and. J. Clin. Lab. Invest. 26, 155 4 Norden, N.E.. Lundblad. A., Svensson, S., &kennan. P.A. and Autio, S. (1973) A mannose-containing trisaccharide isolated from urine of three patients with mannosidosis. J. Biol. Chem. 248. 6210

42 5 Strecker, G.. Foumet, B.. Bouquelet, S., Montreuil. J.. Dhondt, J.L. and Farriaux, J.P. (1976) Etude chimique des mannosides urinaires excretes au cows de la mannosidose. Biochimie 58. 579 6 Beaudet, A.L. and Nichols, B.L. (1976) Residual altered a-mannosidase in human mannosidosis. Biothem. Biophys. Res. Comm. 68, 292 7 Taylor, H.A., Thomas, G.H., Aylsworth. A., Stevenson. R.E. and Reynolds, L.W. (1975) Mannosidosis: Deficiency of a specific o-mannosidase component in cultured fibrablasts. Clln. Chim. Acta 59, 93 8 Tsvetkova, I.V. and Kozina. A.B. (1974) Detection of Tay-Sachs disease carriers by determination of the N-acetylhexosaminidase A activity in blood. Vopr. Med. Chim. 6.631 9 Tsyetkova. I.V.. Zolotuchina, T.V.. Rosenfeld, EL. and Rozovsky, I.S. (1979) Glycosidase activity in cell culture of human amniotic fluid. Vopr. Med. Chim. 2. 214 10 Tsvetkova. I.V.. Novlkova, I.M., Rosenfeld, EL., Barishnev, IJy.1. and Prigozina. I.G. (1980) Biochemical diagnosis of mannosidosis in two families. Vopr. Med. Chim. 3 11 Milla. P.J.. Black, I.E., Patrick, A.D., Hugh-Jones, K. and Oberholzer. V. (1977) Mannosidosis. Clinical and Biochemical study. Arch. Dis. Child. 52,937 12 Bach. G., Cohn, G.. Lasch, E.E., Massrl, N., Sekeles. E., Omey, A., Legum, C. and Cohen. M.M. (1978) A new variant of mannosidosis with increased residual enzymatic activity and mild clinical manifestation. Pediatr. Res. 12. 1010 13 Booth, C.W., Chen, K.K. and Nadler, H.L. (1976) Mannosidosis: Clinical and biochemical studies in a family of affected adolescents and adults. J. Pediatr. 88, 821