Significance of glycosidases and phosphatases in Cercopithecus and Macaca cells

hr. J. Biochem. Printed in Great

Vol.

17, No. IO, pp. 1061-1066, Britain

1985

0020-71 IX/85 f3.00 + 0.00

Pergamon Press Ltd

SIGNIFICANCE OF GLYCOSIDASES AND PHOSPHATASES IN CERCOPITHECUS AND MACACA CELLS ULF BJARE, GUNNAR LUNDBLAD and INGER R~~BB National Bacteriological Laboratory, S-105 21 Stockholm, Sweden [Tel. 46%7300080] (Received 3 January 1985) Abstract-l. When selected ratios of different glycosidases and phosphatases from primary monkey kidney cells or from monkey kidney cell lines are presented graphically, characteristic patterns do evolve. 2. Three different subtypes of Vero cells show similar glycosidase patterns. 3. The Vero subtypes tested show glycosidase patterns that are closely similar to those of primary cells of Cercopithecus aethiops. 4. Glycosidase patterns of BS-C-I and CV-I cells are less similar to those of primary Cercopithecus cells than are those of Vero cells. 5. Primary kidney cells from Macaca cynomolgus show significantly different glycosidase patterns compared with those of different Cercopithecus cells. 6. The distinct glycosidase patterns can be used to classify the tested cell lines in relation to each other.

INTRODUmION It is well known that the surfaces of animal cells contain glycoproteins and glycolipids, which are of importance in different functions as in receptor specificity and in cellular recognition and for the expression of cellular immunogenicity and tumorogencicity (Andersson et al., 1977; 1978; Nilsson et al., 1977; 1980; Gahmberg et al., 1979; Gahmberg, 1981; Gahmberg and Andersson, 1982; Santer et al., 1984; for a review see Pollack et al., 1984). Glycosidic-

groups play a central role in determining the characteristics of biological structures such as glycoproteins and glycolipids, thus it is essential to gather information concerning glycosidases that are central in the synthesis and catabolism of protein-linked glycosidic units. Glycosidic groups are linked to proteins either through a N-glycosidic linkage to asparagine or through a 0-glycosidic linkage to serine, threonine, hydroxylysine or hydroxyproline. Glycosidases are crucial in determining the final shape of glycosidic side chains when trimming the precursor oligosaccharide molecules (Kornfeld and Kornfeld, 1976; Elting et al., 1980; Sharon and Lis, 1982; Marzella and Glaumann, 1983b; Schachter, 1984). Recently it has been demonstrated that glycosidases in lymphoid cells show distinct patterns that can be used to characterize different cell lines (Lundblad et al., 1984; Bjare et al., 1985). In this paper we will demonstrate that the same method can also be used to distinguish different monkey kidney cells. One aim is to use this procedure as an additional method to fulfill the obligatory requirement for vaccine production. Vero cells (Yasumura and Kawalika, 1963) are getting increasing interest as a substrate for vaccine production and there is a need for methods for identification of these cells and possibly even monitor their physiological condition during large scale cultivation. We have studied glycosidase and phosphatase activities of primary monkey kidney cells from

Cercopithecus aethiops and Macaca cynomolgus and compared these with those from a series of cell lines derived from Cercopithecus kidney cells, among others Vero cells. MATERIALS

AND METHODS

C&S The Vero cell subtypes I, 2 and 3 and GMK I and 2 were obtained from different laboratories that had been propagating them for extended periods independently of each other. BS-C-I (Hopps et aI., 1963) and CV-I were received from the American Type Culture Collection. Primary monkey kidney cells were taken from imported Cercopithecus aethiops and Macaca cynomolgus monkeys. Media and culture conditions

Cells were cultivated in stationary roux flasks or roller bottle flasks in Eagle’s MEM with 5% fetal calf serum or newborn calf serum, 100 IU/ml Penicillin and 100yg/ml Streptomycin. Preparation of crude homogenates

Cells were harvested by use of a disposable cell scraper to avoid the possible effect of trypsin on the enzyme activities. After suspension in PBS, cells were centrifuged at 200~ for 20 min at 20°C. The cell sediment was suspended in 0.1% Triton X-100 in PBS (without Ca and ME) and frozen at -20°C. After thawing; the material was disintegrated in a Dounce homogenizer and centrifuged at 7000g for IO min at 4°C. The supernatants after these operations were used as enzyme samples. Chemicals and enzyme assays The chromogenic substrates (p-nitrophenylglycosides) were purchased from Koch-Light Lab., Colnbrook, U.K. The pH values in the assay of the different enzymes are given in Table I. Glycosidase activity was determined according to Verpoorte (1972). The standard assay containing 25 pl enzyme in 0.5 ml 0.05 M citric acid/O.05 N sodium citrate buffer was added to 0.5 ml 4.0 mM p-nitrophenylglycoside in the same buffer, pH 4.5 or at the desired pH value. For alkaline phosphat&e 25ml 0.2 M glycine/l6ml 0.2 N NaOH, pH IO was used. The reaction was stopped after the appropriate time at 37°C by addition of l.Oml 0.5 M

1061

ULF BJARE er al

1062

Table I. Enzyme numbers, optimal pH and enzyme ratios EC Number 3.2.1.-

pH

Enzvme-ratios

r-N-acetyl-D-galactosaminidase

49

4.5

I -3.1.3.2 53

b-N-acetyl-D-galactosaminidase x-N-acetyl-D-glucosaminidase 8.n-acetyl-D-gtucosaminidase

53 50 30

4.5 4.5 4.5

/J-D-fucosidase

38

5.5

E-L-fucosidase p-L-fucosidase u-D-galactosidase b-D-gatactosidase 8.D-glucuronidase a-D-glucosidase B-D-glucosidase a-D-Mannosidase 8.D-Mannosidase Acid phosphatase Alkaline phosphatase

51

5.5 5.5 4.5 4.5 5.5 4.5 5.0 4.5 4.5 5.0 10.0

Enzyme

22 23 31 20 21 24 25 3.1.3.2 3.1.3.1

II = 30125 ,I, = 3?$

A= B= c = D= E= F= G=

30153 23/31 51/31 53151 53123 25123 25153

Note. The combination of both letters and reman numerals for indentification of enzyme ratios is used to make the presented data directly comparable with the earlier results published about lymphoid cells. Ratios A-G are identical with those previously published. The ratios I-III have been added to the left side of the diagram, and not to break the alphabetic order have been given different symbols.

glycine/O.SN NaOH buffer, pH 10.5. The release of pnitrophenol (p-NP) was read at 400nm in a Pye-Unicam SP8-100 spectrophotometer. One unit (U) of enzyme activity was defined as the amount which releases 1nmol pNP/hr. Protein derermination

The protein content was determined by the micro Kjeldahl method by the use of a Coulometric Analyzer (LKBBeckman Instrument AB, Bromma, Sweden). RESULTS

The absolute values of glycosidase activities from primary monkey kidney cells lines derived from Cercopithecus uethiops are presented in Tables 2(a) and (b). Enzyme values are calculated in relation to milligrams of cellular protein. As the separate enzyme activities show a highly complex pattern it is difficult to reveal any significant characteristics, except from the data of the acid and alkaline phosphatases in Vero subtypes (Mizuno et al., 1983). Yet there are significant differences for some of the glycosidases too. If, however the absolute enzyme values are presented as selected quotients (Q) of these numbers (Table l), charcteristic patterns evolve. In Fig. 1 the glycosidases in different Cercopithecus cells are presented as selected ratios of eight different enzyme activities expressed in relation to the amount of cellular protein. When plotted in the presented way many cell lines show a characteristic pattern distinct from that of other cell lines tested. Cells from Vero subtypes and primary monkey kidney cells from Cercopithecus uethiops have similar enzyme patterns as presented in Fig. 1. The two Vero subtypes Vero- 1 and Vero-2 show highly similar patterns, while Vero3 is somewhat more different. Some samples (4/9) of Vero-2 show a very high Q I which gives a large standard deviation. The same type of variation is found in O/9 Vero- 1 and l/10

Vero-3. Characteristically D I E for Vero-1 and Vero-3 while D > E for Vero-2. Thus the enzyme ratio 51/23 (E/D) varies characteristically from one subtype of Vero to another. Vero lines have G approximately about 1 and E about 2. Data from the individual samples of primary Cercopithecus cells show a relatively large II when B is small and inversely. (Values for individual samples not presented). In Fig. 2 the glycosidase patterns are presented for four other Cercopithecus cell lines. BS-C- 1 and CV- 1 reveal significantly different patterns from those of primary Cercopithecus and Vero cells. Some BS-C- 1 samples have very high D and for the individual BS-C-l samples D is never below 6. C on the other hand is always below 1. The latter is also true for CV-1 where all Q:s are below 7 and the following relationships are characteristic for single samples: D>E, F>E, BIG, and G> 1. The two GMK-subtypes on the other hand are fairly similar in between each other but are radically distinct from primary kidney cells from Cercopithecus and distinct from the other tested lines. GMK-1 have high II and highly variable B and C. GMK-2 generally have a very high II (16-26) while III and B are rather variable. For the individual samples the following characteristics hold true for both GMK-1 and GMK-2: C > D, F < 1, G < 1 and G < F. Two populations of Macaca cynomolgus, derived from separate geographical origins show different enzyme ratios-Fig. 3 Cynomolgus-2 characteristically have Q:s II > 10, B > 10 and C > 10 and G<< 1. While Cynomolgus- 1 have much lower B and C. This demonstrates that different populations of Cynomolgus have distinct glycosidase patterns. This is also evident from the absolute enzyme activities in Table 2(a). In Table 3 the Q:s presented in Figs. 1 and 2 are

24 25 3.1.3.2 3.1.3.1

49 53 50 30 38 51 22 23 31 :

17.3 132 270 1356 10.0

145 1380 4.7 9287 40.3 404 12.2 723 59.5 61.1 9.3 89.2 iO4 384 74.0

42 408 3.00 2486 13.0 92.0 6.5 262 38.0 30.5

monkey (Cyno(n = 9) * SD 20.4 116 3.8 139 200 129 1.5 46.7 34.3 9.9 216 123 65.8 484 123

62.6 422 3.8 2778 156 281 5.5 213 106 25.5 164 164 304 1818 126

Normal monkey kidney (Cercopithem) (n = 9) Mean &-SD

Enzyme activity in liberated p-NP in nmol/hr/mg

protein.

517 93.4 75.0 8.1 57.4 394 2982 2.4

5: ;A

21 24 25 3. I .3.2 3.1.3.1

8074 46.5 500

30 38 51

g-N-acelyl-o-ghrcosaminidase )!?-o-Fucosidase a-r;Fuwsidase a-o_GaIactosidase g-o-Galactosidase g-o-Clucuronidase a-o-Ghtcosidase /?-o-Giucosidase a-D-Mannosidase g+Mannosidase Acid phosphatase Alkaline phosphatase

79.8 1291 6.9

49 53 50

103 1405 10050 68.5 492 436 157 117 9.1 65.0 380 3553 7.8

1718 10.9 213 203 44.3 28.8 2.9 51.9 153 1391 3.1

GMK-2 (n = IO) Mean f SD 40.2 595 5.2

GMK-1 (n = II) Mean + SD

a-N-acelyl-o-galaclosaminidase /3-N-acetyf-o-gakrctosaminidase a-N-acelyl-o-glucosaminidase

Enve

EC Number 3.2.1.-

9.0 11.5 188 217 292

40.3 225 9.0 I.516 25.0 116 6.3 110 63.3 32.5 30.6 16.9 568 271 2209

10.8 44.9 140 149 352

21.5 151 6.7 194 22.3 14.1 0.9 103 28.1 16.5

Vera-2 (n = 9) Mean & SD

83.7 4.2 21.4 413 3006 6.2

14.7 439 1.4 3104 6.8 164 4.8 242 248

122 43.0 45.6 3.3 42.2 260 890 5.4

3900 37.5 225

55. I 510

177 190 52.5 23.3 105 588 2556 92.2

4260 23.6 79.6

213 613 2.2

BS-C-I (n=ll) Mean + SD

140 91.9 37.3 16.7 58.0 161 650 36.6

1390 4.1 33.9

72.9 206 -

17.1 265 1860 10.4 61.0 88.6 74.1 52.0 9.5 58.5 411 617 11.0

120 482 3087 14.1 91.4 211 136 78.2 16.9 63.9 1051 2140 19.4

lx-1 (n = 9) Mean f SD

10.5 127 581 5.2

115 213 35.8

30.0 275 1807 72.4

Vera-3 (n = 9) Mean it: SD

cell lines Vero-1, Vero-2 and Vero-3

119 493 5.75 2956 54.4 369 5.9 260 174 69.1

non-tumorogenic

cell lines GMK-I, GMK-2, BS-C-1 and CV-I from normal monkey-kidney

10.0 28.8 248 I688 253

65.9 301 6.8 1880 37.8 218 6.3 138 105 43.4

Vero-I (a = 9) Mean + SD

and from the transformed,

Table 2(b). Glycosidase and phosphatase aclivileis of cells from the transformed, non-tumorogenic (Cercopithecur)

fl-o_Giucosidase a-tr-Mannosidase g-o-hlannosidase Acid phosphatase Alkaline phosphatase

a-N-acelyl-D-galaclosaminidase ~-N-a~tyl-~~lact~~nida~ a-N-acetyl-~ghmosaminidase /?-N-acelyl-o-glucosaminidase g-u-Fucosidase a-L-Fucosidaae a-u-Galaclosidase g-o-Galactosidase g-o-Glucuronidase a+-Glucosidase

Eruyme

EC Number 3.2.1.-

Normal kidney molgus Mean

Table 2(a). Glycosidase and phosphalase activities of cells from normal monkey-kidney

E

ULF BJARE et

IO64

Vero

Cercopithecus

1

al.

1

(n = 9)

(l7=9)

cv-1

(fl=9) T

1

h Vero 3 tn =I01

Vero 2 (n=9)

GMK-1

~

0

IIIIUABCDEFG

IIIIUABCDEFG

Fig. 1. Glycosidase and phosphatase ratios of primary monkey kidney cells (Cercopithecus) and different sublines of a Cercopithecus cell line (Vero).

into five classes depending upon the heights of the respective columns. The number of columns within the same size range when comparing different combinations of cell lines are presented in Table 4. This type of presentation gives a simpler comparison of similarities and differences of the separate cell lines. Vero-1, Vero-2 and Vero-3 have 7, 8 or 9 Q:s (of 9) in common which indicates that the different clones are closely related or identical. Cercopithecus aethiops have 8 Q:s in common with Vero-1 and Vero-2 while 7 Q:s in common with Vero-3. This corresponds to the morphological characteristics, as Vero-1 and Vero-2 are more similar to primary Cercopithecus cells than Vero-3 is. CV-1 on the other hand only have 4 and BS-C-I 5 Q:s corresponding to those of Cercopithecus primary kidney cells. GMK being derived from Cercopithecus aethiops have 7 Q:s in the same size class. Macaca cynomolgus have significantly fewer Q:s in the same range as most of the other strains except Cercopithecus and GMK with which it has more similarity. All presented strains have a Q-A in the order of 6-8 which can be used as an internal standard as the enzyme activities assayed against these two separate substrates most likely are located in one and the same enzyme (Frohwein, 1967; Lundblad et al., 1983a).

grouped

IIIIIIABCDEFG

We have earlier discussed the significance of glycosidases in lymphoid cells (Lundblad et al., 1983b,

IlllmABCDEFG

Fig. 2. Glycosidase and phosphatase ratios of different Cercopithecus cell lines.

Bjare et al., 1985). Significant enzyme patterns could be used for characterization of different Epstein-Barr virus (EBV)-transformed lymphoid cells. Blymphocytes transformed during infectious mononucleosis showed a consistent enzyme pattern when comparing cell lines derived from different patients. EBV-transformed Burkitt’s lymphoma cells on the other hand showed distinct patterns for separate cell lines (Bjare et al., 1985). Testing the same method for characterization of different monkey kidney cells we have now demonstrated the nossible usage of this nrocedure. This has been done with two aims in mind.

M cynomolgus

1

M.cynomolgus

2

(n=4)

2

Ll c 4

i DISCUSSION

GMK-2 (n=lO)

1-1 ‘“=‘I)

IUIUABCDEFG

I IL

Fig. 3. Glycosidase and phosphatase ratios of primary monkey kidney cells from two populations of Macaca qvnomolgus of different geographical origin.

1065

Glycosidases and phosphatases in monkey cells Table 3. Distribution Size range

Vero- 1

cl 1-4

9, C, D,

48

E. F 1,II, A

a-12 > 12

III -

G

of Q:s within specified ranges for diRerent cell lines

Vero-3 -

BS-C- 1

B. C, D,

9, C, D,

B

E. F, G I, II, A III, -

E, F, G II, III, A I -

Vero-2

C,G I, II, A III, E, F D -

cv-1

Cer

GMK

Cyn

C III, B E, G I, II, A D,F -

G B, C, D, E, F 1, III, A

F, G I, B. C,

F, G I, D, E.

A

III, A

II -

II, III -

II. 9. C

D, E

Table 4. Number of Q:s that are within the same size range when comparing different cell lines from Table 3

1

Vera-2

Vero-3

BS-C- I

CV-I

Cer

GMK

4

4

4

5

a 5 5 7

a

5 1

2 3 4

5 7 -

6

I

3 3

6 5 9

5 4 -

VeraCYn GMK Cer cv-1 BS-C- I Vera-3 Vero-2

5 6

Cyn -

-

-

-

a

Cell bound glycosidases reflect specific metabolic activities of the cell, which are significant for different cell lines, glycosidases play a central role in the synthesis of specific structures and in cell trqnsformation with alterations of specific structures and control mechanisms (Allison and Sandelin, 1963; Bosman, 1972; Martinez et al., 1984; Jessup et al., 1984). Glycosidases and other lysosomal enzymes have been thoroughly investigated in cultured fibroblasts (Bosman, 1969; Heukels-Dully and Niermeijer, 1976; Houben and Remacle, 1978) lymphocytes and cultured lymphoid cell lines (Glade and Beratis, 1974; Minami et al., 1978; Dreyfus ef al., 1980; Andersen et al., 1982; Lundblad et al., 1983b; Bjare et al., 1985). Acidic glycosidases are involved in the lysosomal catabolism of glycoproteins which is probably initiated by the action of lysosomal proteases (Bosman et al., 1976; Winchester, 1984). Knowledge of enzymes responsible for these cellular functions is of obvious importance for evaluation and interpretation of results from studies of such cells. Further on there is a more practical aim to and structuralcomplement morphological with a cell characterization immunological functional-enzymatic methodology. There are absolute requirements for cellular identification of cell lines used for vaccine production for human usage (WHO, 1982). Analysis of glycosidases in different monkey kidney cell lines show that it is possible to use these data as a means of identification of a cell culture. Absolute numbers of enzymatic activity have limited value for this type of identification because of considerable variations of enzymatic activity between different preparations of a particular cell line. These variations are partly neutralized by the use of enzyme ratios, as the quantitative relationships between different enzymes are relatively stable. Selected ratios have been chosen among the high number of possible combinations of 15 enzymes. Those enzymes showing low activities and pronounced standard deviation have not been used. In this paper we have used the presented method to test it on ten different cell types and have de-

monstated that they can be distinguished from each other as long as they are not subtypes of the same cell line, as for Vero subtypes. Primary cell cultures from two different Macaca cynomolgus populations of different geographical origin can be distinguished from each other. When the number of Q:s within a specific height range are compared as in Table 4 the close relationship of Vero subtypes with Cercopithecus primary cells are evident. This corresponds with morphological data and growth characteristics. When seven or more Q:s out of nine are within the same range there is a close relationship or identity between the different cells tested. Four to six Q:s show an intermediary degree of relationship and three of fewer Q:s indicates a somewhat more distant relationship for the cells tested. The interrelated variations of glycosidases that give characteristic ratios points to the close connection on the regulatory level for synthesis of glycosidases. Acknowfedgemenfs-The authors wish to thank Mr J. Lind, Mrs A.-K. Tegnemo and Mrs C. Wising for excellent technical assistance.

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