FEMS Microbiology Letters 128 (1995) 9-14

The enhancement of the human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells by trypsin treatment is ascribed to the binding of trypsin to the Fc

fragment

Yoshihiko Sawa, Ken-ichiro Shibata, Tsuguo Watanabe *

Department of Oral Bacteriology, Hokkaido University School of Dentistry, Kita 13, Nkhi 7, Kita-ku, Sapporo 040, Hokkaido, Japan

Received 9 January 1995; revised 8 February 1995; accepted 10 February 1995

Human immunoglobulin G Fc fragment-binding activity of Mycopiastna salivariutn cells was remarkably enhanced by trypsin treatment of the cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of proteins of the cells treated with trypsin was the same as that of the cells treated with pronase, although pronase treatment had been shown to reduce the activity in our previous study @EMS Microbial. L.&t. 123,305-310, 1994). This contradiction was clarified by the finding that trypsin bound the Fc fragment more strongly than the cells, and a small amount of trypsin remained in the cells treated with trypsin and washed well. On the basis of these results, it was concluded that the enhancement of cell activity by trypsin treatment was ascribed to binding of the Fc fragment to trypain remaining in the trypsin-treat4 cells.

Keywords: Mycoplasma saliva&m; Fc fragment-binding activity; Trypsin

1. introduction

Mycoplasma salivarium cells were demonstrated to bind the Fc fragment of human immunoglobulin (Ig) G, and the binding capacity was shown to be enhanced by trypsin treatment of the cells 111. Re- cently, it was shown that the capacity was signifi- cantly reduced by pronase treatment of the cells and that membrane proteins with molecular masses of 88, 90 and 150 kDa were specifically digested 121.

l Corresponding author. Tel: +81 (11) 716-2111; Fax: +81 (11) 75681%.

This study was designed to clarify the mechanism involved in the enhancement of the human IgG Fc fragment-binding activity by trypsin treatment of iU. salivarizun cells.

2. Materials and methods

2.1. Chemicals

Pronase E and trypsin were purchased from Merck (Darmstadt, Germany); alkaline phosphatase CAP)_ conjugated Fc fragment of human IgG (AP-Fc) from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA); peroxidase-conjugated Fc fragment of

0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDfO378-1097(95)00073-9

10 Y Sawa et al. / FEMS Microbiology Letters 128 (1995) 9-14

human IgG (Per-Fc) from Rockland (Gilbertsville, PA); peroxidase and AP substrate kits from Bio-Rad

Laboratories (Harbour Way South, Richmond, CA); and AP and p-nitrophenyl phosphate from Sigma Chemical Co., Ltd. (St. Louis, MO). Other chemicals were from commercial sources and were of analyti- cal or reagent grade.

2.2. Mycoplasma cells

M. salivarium ATCC 23064 was grown in PPLO broth (Difco Laboratories, Detroit, MI) supple- mented with 1% (v/v) PPLO serum fraction (Difco), the pH of which was adjusted to 6.8. When the pH

of the culture was 7.4, the cells were harvested by centrifugation at 15 000 X g for 30 min and washed twice with 0.25 M NaCl.

2.3. Trypsin treatment of the cells

The cells were suspended in 10 mM Tris-HCl (pH 7.4) containing 0.25 M NaCl (TBS). One part of the

cell suspension (2 mg of cell protein/ml) was treated at 37” C for 1 h with one part of TBS or trypsin solution at a concentration of 2, 10 or 20 mg/ml. Similarly, the cells were treated with trypsin inacti- vated with N “-tosyl-L-lysyl-chloromethylketone (TLCK) as follows and also TLCK by itself. After washing three times with 0.25 M NaCl, the cells were used in the following experiments.

The mixture consisting of 0.5 ml of 0.4 mM trypsin solution, 0.1 ml of 100 mM TLCK and 0.3 ml of TBS was incubated at 37” C for 30 min.

Trypsin treated with TLCK (T-trypsin) by this proce- dure was confirmed to be completely inactivated in preliminary experiments.

2.4. Lytic effect of trypsin on M. salivarium cells

The percent lysis of the cells treated with trypsin was determined by the method described previously

[21.

2.5. Determination of Fc binding activity

The cells were suspended in TBS supplemented with 6% (w/v) bovine serum albumin (BSA-TBS). A 5-~1 volume of AP-Fc (3 pg of protein) or Per-Fc

(8 pug of protein) was added to 1 ml of the cell suspension (1 mg of cell protein/ml). After incuba-

tion at 20” C for 1 h with shaking, the cells were washed by centrifugation (15000 rpm for 10 min), using a microcentrifuge (HIMAC centrifuge CRlSB,

Hitachi Ltd., Tokyo, Japan).

2.6. AP-Fc binding activity

The cells incubated with AP-Fc were solubilized by suspension in 0.1 M carbonate buffer (pH 9.6)

and incubated at 37” C for 1 h with shaking. A O.l-ml volume of the cell lysate was placed into

three wells of a microtitration plate, followed by the addition of 0.1 ml of a p-nitrophenyl phosphate solution (2 mg/ml). The plate was scanned and the absorbance at 405 nm was read with a Microplate

Reader (Bio-Rad).

2.7. Per-Fe binding activity

The cells incubated with Per-Fc were solubilized by suspension in Solution A (for color development)

of peroxidase substrate kit (Bio-Rad). The cell lysate (90 ~1) was incubated with 10 ~1 of Solution B (substrate) of the kit in wells of a microtitration plate as mentioned above, and the absorbance at 415 nm

was read.

2.8. Determination of Al-‘-Fc binding activity of

trypsin

A 0.1 or 0.05-ml volume of a solution (1.25 mg/ml) of trypsin was applied onto nitrocellulose sheets by use of a Slot-blot kit (Bio-Rad). The

nitrocellulose filter blots were treated at 37” C for 2 h with BSA-TBS, and then incubated at 37” C for 3 h with AP-Fc solution (6 pg of protein/ml) or a solution of AP (Sigma) in BSA-TBS (10 pg of protein/ml) as controls. The filter blots were washed and then treated for 1 to 2 min with substrate of AP substrate kit (Bio-Rad) to allow color development at the sites of AP-conjugate binding. Reflexion at 500 nm was measured in a Shimadzu Dual-Wave Length

TLC Scanner CS-930 (Shimadzu Co., Kyoto, Japan). For comparative purposes, the activities of T-

trypsin, TLCK and pronase were also determined.

Y. Sawa et al. / FEMS Microbiology Letters 128 (1995) 9-14 11

2.9. Determination of the amount of trypsin in the trypsin-treated M. salivarium cells

The amount of the enzyme remaining in the trypsin-treated cells washed well was determined on the basis of the benzoyl+arginine p-nitroanilide (BAPA)-hydrolysing activity of the cells. The cali- bration curve was made in advance by plotting the initial velocity of the BAPA-hydrolysing activity of trypsin against the amount of the enzyme used.

One part of the cell suspension (2 mg of cell protein/ml) was incubated with one part of trypsin solution (20 mg/ml) at 37” C for 1 h, washed two or three times with 0.25 M NaCl and resuspended in 1 ml of TBS. A 0.2-ml volume of the mixture consist- ing of 0.1 ml of 10 mM BAPA in 100 mM Tris-HCl (pH 7.4), 0.8 ml of distilled water, and 0.1 ml of the trypsin-treated cell suspension was placed into three wells of a microtitration plate. The plate was incu- bated at 37” C and scanned at timed intervals at 405 nm to determine the BAPA-hydrolysing activity of the cells. The amount of trypsin or pronase was determined by interpolating the initial velocity of the activity from the calibration curve.

For comparative purposes, the amount of pronase in the pronase-treated cells was also determined.

2.10. Protein determination

Protein concentrations were determined by the method of Lowry et al. [3].

2.11. Polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (SDS-PAGE) was performed in 10% gels by the method of Laemmli 141.

3. Results and discussion

We recently reported that pronase treatment re- duced the Fc fragment-binding activity of M. sali- varium cells, accompanied by the degradation of the three membrane proteins with molecular masses of 88 kDa (88 kp), 90 kDa (90 kp) and 150 kDa (150 kp) [2]. Contrarily, trypsin treatment enhanced the activity drastically in a dose-dependent manner (Fig.

0 20 40 60 m Unto (mln)

Fig. 1. The lytic effect of trypsin on M. saliuarium cells. One part of the cell suspension (2 mg of protein/ml) was incubated with one part of trypsin solution (20 mg/ml) (U) at 37” C for 1 h. As controls, the cell suspension diluted 1:2 with TBS was incubated for 1 h on ice (01, at 20” C (0) or at 37” C (0). Percent lysis of the cells was determined by the method described before 121.

1). We speculated that membrane proteins responsi- ble for the activity were exposed by trypsin treat- ment. However, this speculation was not supported because the SDS-PAGE profile of proteins of the trypsin-treated cells were indistinguishable from that of the pronase-treated cells (Fig. 2). The Fc frag- ment-binding activity of the cells treated with T- trypsin was approximately 50% of that of the cells treated with trypsin, but still much higher than that of the non-treated cells (Table 1). There was no detectable difference in SDS-PAGE profiles of pro- teins between the cells treated with T-trypsin and those not treated (Fig. 2). The Fc fragment-binding activity was not affected with TICK (Table 1). More than 90% of the cells were not disrupted by trypsin treatment (Fig. 3), suggesting that the enhancement of the activity was not caused by the disruption of the cells.

The above results suggested that the enhancement of the activity was due to the binding of the Fc fragment to trypsin remaining in the trypsin-treated cells. In fact, it was confirmed that the Fc fragment bound trypsin and T-trypsin but did not bind pronase and TLCK (Table 2). In addition, the cells contained trypsin and pronase despite being washed well after treatment by the enzymes (Table 3). Furthermore, Slot-blot analysis showed that AP failed to bind trypsin (data not shown), and AP-Fc binding activity paralleled Per-Fc binding activity (Fig. 1).

12 Y. Sawa et al. /FEMS Microbiology Letters 128 (199.5) 9-14

ABCD ,w

97.4

66.2

31.0

Fig. 2. SDS-PAGE of proteins of M. saliuarium cells treated with

trypsin, pronase and T-trypsin. One part of the cell suspension (2

mg of cell protein/ml) was treated at 3P C for 1 h without (lane

A) or with one part of trypsin solution (20 mg/ml) (lane B),

pronase solution (10 mg/ml) (lane C) or T-trypsin solution (20

mg/ml) (lane D). SDS-PAGE was performed in 10% gels and

stained by use of a Silver stain kit (Bio-Rad).

On the basis of these results, it was concluded that enhancement of the Fc fragment-binding activity of the cells by trypsin treatment was ascribed to the binding of the Fc fragment (but not AP) of AF’-Fc to trypsin remaining in the treated cells.

Mammalian cell cultures are widely used for a

variety of purposes such as studies into the effect of pathogenic bacteria on the biological and physiologi- cal activities or the signal transduction system of host cells. Trypsin is often used for dispersion of cells grown in sheets or monolayers. The possibility suggested by this study that trypsin remains in the cells that are dispersed with trypsin, and washed well, and binds IgG contained in the culture medium, should be taken into consideration when cells dis-

“1 6

1

cant 1:l 5:l 1O:l

Fig. 3. The effect of trypsin on the Fc fragment-binding activity of

M. saliuarium cells. One part of the cell suspension (2 mg of cell

protein/ml) was treated at 37” C for 1 h with one part of

TBS-BSA (cant) or trypsin solution at a concentration of 2 (l:l),

10 (51) or 20 mg/ml (l&l). The cells treated with trypsin were

examined for AP-Fc (shaded) or Per-Fc (0) binding activity by

the method described in the text. Each value, expressed as ab-

sorbance change at 405 nm for AP-Fc binding activity or 415 nm

for Per-Fc binding activity per min per mg of cell protein,

represents the mean and standard deviation of five determinations.

Table 1

Effect of trypsin on AP-Fc binding activity of M. saliuarium cells

Cells treated with AP-Fc binding activity (OD/min/mg)

Mean (n = 5) SD. I’ *

None 0.212 0.023 -

Trypsin 1.501 0.032 < 0.001

T-trypsin * l 0.7987 0.036 < 0.001

TLCK 0.221 0.015 NS

* Analysed by Student’s t-test. l * TLCK-inactivated trypsin. NS:

Not significant. T: P < 0.001 compared with AP-Fc binding

activity of the cells with trypsin.

Table 2

AP-Fc binding activity of proteases

Proteases AP-Fc binding activity at the indicated

amounts ( pg of protein) of proteases

62.5 125

Trypsin 0.12 * 0.28

T-trypsin * * 0.05 0.16

Pronase 0.00 0.00

TLCK 0.00 0.00

* Reflection at 500 nm. Values are means of 3 determinations.

* * TLCK-inactivated trypsin.

Y. Sawa et aL / FEMS Microbiology Letters 128 (1995) 9-14 13

Table 3 Amounts of protease in the protease-treated M. saliuarium cells

Proteases Amounts of protease in the protease-treated cells washed

Twice Three-fold

Ministry of Education, Science and Culture of Japan and by a grant from Yakuruto Co. Ltd., Japan.

Pronase

TrYpsin

2.94 * 2.95 1.79 0.76

l pg/mg of the cell protein. Values are means of 3 determina- tions.

persed with trypsin are used for studies on effects of bacteria or chemicals on mammalian cells. Other- wise, the results obtained may lead to serious errors.

Acknowledgements

This work was partly supported by a Grant-in-Aid for Scientific Research (C) (No. 06671802) from the

[II Watanabe, T. (1993) Binding of Fc fragment of IgG from human and seven animal species to Mycoplasma saliuarium cells. Microbios 76,77-84.

(21 Shibata, K-I., Sawa, Y., Inoue, S., Noda, M. and Watanabe, T. (19941 Membrane proteins with molecular masses of 88,90 and 150 kDa are responsible for binding of human im- munoglobulin G Fc fragment to the native cells of My- cophsma salivarium. FEMS Microbial. L&t. 123, 305-310.

131 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (19511 Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

[41 Laemmli, U.K. (19701 Cleavage of structural proteins during the asxmbly of the head of bacteriophage T4. Nature 227, 680-685.