Proc. Nat. Acad. Sci. USA Vol. 73, No. 3, pp. 837-841, March 1976 Cell Biology
Effect of concanavalin A on expression of cell surface sialyltransferase activity of mouse thymocytes (glycosyltransferases/mitogens/plasma membrane)
RICHARD G. PAINTER AND ABRAHAM WHITE Department of Cell Biology, Institute of Biological Sciences, Syntex Research, Palo Alto, California 94304
Contributed by Abraham White, December 12, 1975
ABSTRACT Incubation of mouse thymocytes with mitogenic concentrations of concanavalin A causes a 2-fold increase in cell-surface-associated (but not total cell) sialyltransferase activity (ectosialyltransferase, CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) as judged by incorporation of ["4C]sialic acid into endogenous cell acceptors and into added desialylated fetuin acceptor. The concanavalin-A-induced enhancement of enzymic activity is essentially complete within 1 hr after addition of mitogen and remains at elevated levels for 12 hr, declining rapidly thereafter. Intact cells labeled previously with ["Clsialic acid and then incubated briefly with hydrolytic enzymes, including neuraminidase and insoluble trypsin, released 43-66% of total cell-associated radioactivity without appreciably changing cell viability. Alterations in sialyltransferase activity due to concanavalin A treatment could not be explained b a mitogen-mediated (a) uptake of radioactive precursors, ( cell death, (c) increased product catabolism, or (d) activation of sialyltransferase by mitogen binding to the enzyme. Furthermore, the process does not require active protein synthesis. The results are consistent with a rapid concanavalin-A-induced exposure of potential enzymic activity that was previously inaccessible to substrate.
sequent to exposure to mitogens. For example, mitogen-induced increases in insulin receptors (10), Na+,K+-dependent ATPase (11), ouabain-binding sites (12), and alterations in surface antigens (13, 14) have been reported. A number of additional techniques are now available, including methods applicable to the radioactive labeling of both galactose (15) and N-acetylneuraminic acid (NANA) (16) localized specifically at the cell surface. One of these (16) involves specific labeling of surface glycoproteins (or glycolipids) by utilizing an endogenous sialyltransferase (ectosialyltransferase, CMP-N-acetylneuraminate:D-galactosyl-glycoprotein Nacetylneuraminyltransferase EC 2.4.99.1),* apparently localized at the cell surface (16-18), to catalyze the transfer of [14C]NANA from CMP-[14C]NANA, which does not enter cells, into membrane acceptors or, alternatively, into exogenously added macromolecular acceptors. This communication reports the successful application of these techniques to measure the kinetics of early (90% as judged by trypan blue exclusion) thymocytes were preincubated at 370 with and without mitogenic concentrations of Con A. Cultures were harvested after 1 hr by centrifugation, and washed twice with cold 0.12 M NaCl0.01 M potassium phosphate (pH 6.5)-i mM MgCl2.
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FIG. 1. Incorporation of [14CJNANA into trichloroacetic acidinsoluble (circles) and acid-soluble (squares) fractions of intact thymocytes incubated with 8.56 gM CMP-_[4C]NANA after treatment with (closed symbols) and withoat (open symbols) 5 ,g/ml of Con A for 1 hr prior to assay. At various times duplicate aliquots containing 1.3 X 107 cells (0.138 mg of cell protein) were withdrawn and measurements were made of radioactivity incorporated into acid-soluble and -insoluble fractions.
Washed cell pellets were assayed for ability to incorporate [14C]NANA into endogenous cell acceptors as a function of time at 37'. Fig. 1 indicates that control thymocytes incorporate radioactivity into trichloroacetic acid-insoluble cell acceptors at a relatively constant rate for a period of 3 hr. The rate of incorporation is equivalent to 0.53 pmol of [14C]NANA/mg of cell protein per hr. Preincubation of thymocytes with mitogenic concentrations of Con A results in a doubling in rate of ['4C]NANA incorporation to a value of 1.28 pmol/mg of cell protein per hr. As in the control, the rate of incorporation is approximately linear with time. On the other hand, incorporation of radioactivity into acid-soluble fractions is quite low, as might be expected from the known impermeability of cells to the nucleotide sugar. In any case, there is no detectable response of this parameter to the presence of Con A. When desialylated fetuin acceptor was included in the assay medium, a similar response was observed, resulting in rates of incorporation equivalent to 1.0 and 2.0 pmol/mg of cell protein per hr for control and Con-A-pretreated cells, respectively (data not shown). In the above experiments, there was no significant loss of cell viability during the incubation period. The relationship between the Con A concentration used in the 1 hr preincubation period and sialyltransferase levels is depicted in Fig. 2. The rates of incorporation of radioactivity into both cellular acceptors and fetuin acceptor increase with increasing Con A concentration, reaching a maximum response at 5-10 ,ug/ml. The dose response of enzyme after 1 hr of culture parallels that of the mitogenic response as assessed by cellular incorporation of [3H]thymidine into DNA after 48 hr of culture. Assay of cells for sialyltransferase activity as a function of preincubation time following the addition of mitogen (Fig. 3) revealed that the magnitude of ,he response to Con A, when compared to that in control cells, reaches a maximum within the first few hours of culture with regard to both incorporation into endogenous acceptors (Fig. 3A) and in the presence of exogenous acceptors (Fig. 3B). The respective enzymic levels remain relatively constant for 12 hr in ConA-treated cultures, declining rather abruptly between 12
Cell Biology: Painter and White
Proc. Nat. Acad. Sci. USA 73 (1976) -
839
IE 1500
Ir
I
3
30
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_
Effect of concanavalin A on expression of cell surface sialyltransferase activity of mouse thymocytes (glycosyltransferases/mitogens/plasma membrane)
RICHARD G. PAINTER AND ABRAHAM WHITE Department of Cell Biology, Institute of Biological Sciences, Syntex Research, Palo Alto, California 94304
Contributed by Abraham White, December 12, 1975
ABSTRACT Incubation of mouse thymocytes with mitogenic concentrations of concanavalin A causes a 2-fold increase in cell-surface-associated (but not total cell) sialyltransferase activity (ectosialyltransferase, CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) as judged by incorporation of ["4C]sialic acid into endogenous cell acceptors and into added desialylated fetuin acceptor. The concanavalin-A-induced enhancement of enzymic activity is essentially complete within 1 hr after addition of mitogen and remains at elevated levels for 12 hr, declining rapidly thereafter. Intact cells labeled previously with ["Clsialic acid and then incubated briefly with hydrolytic enzymes, including neuraminidase and insoluble trypsin, released 43-66% of total cell-associated radioactivity without appreciably changing cell viability. Alterations in sialyltransferase activity due to concanavalin A treatment could not be explained b a mitogen-mediated (a) uptake of radioactive precursors, ( cell death, (c) increased product catabolism, or (d) activation of sialyltransferase by mitogen binding to the enzyme. Furthermore, the process does not require active protein synthesis. The results are consistent with a rapid concanavalin-A-induced exposure of potential enzymic activity that was previously inaccessible to substrate.
sequent to exposure to mitogens. For example, mitogen-induced increases in insulin receptors (10), Na+,K+-dependent ATPase (11), ouabain-binding sites (12), and alterations in surface antigens (13, 14) have been reported. A number of additional techniques are now available, including methods applicable to the radioactive labeling of both galactose (15) and N-acetylneuraminic acid (NANA) (16) localized specifically at the cell surface. One of these (16) involves specific labeling of surface glycoproteins (or glycolipids) by utilizing an endogenous sialyltransferase (ectosialyltransferase, CMP-N-acetylneuraminate:D-galactosyl-glycoprotein Nacetylneuraminyltransferase EC 2.4.99.1),* apparently localized at the cell surface (16-18), to catalyze the transfer of [14C]NANA from CMP-[14C]NANA, which does not enter cells, into membrane acceptors or, alternatively, into exogenously added macromolecular acceptors. This communication reports the successful application of these techniques to measure the kinetics of early (90% as judged by trypan blue exclusion) thymocytes were preincubated at 370 with and without mitogenic concentrations of Con A. Cultures were harvested after 1 hr by centrifugation, and washed twice with cold 0.12 M NaCl0.01 M potassium phosphate (pH 6.5)-i mM MgCl2.
a W
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MINUTES
FIG. 1. Incorporation of [14CJNANA into trichloroacetic acidinsoluble (circles) and acid-soluble (squares) fractions of intact thymocytes incubated with 8.56 gM CMP-_[4C]NANA after treatment with (closed symbols) and withoat (open symbols) 5 ,g/ml of Con A for 1 hr prior to assay. At various times duplicate aliquots containing 1.3 X 107 cells (0.138 mg of cell protein) were withdrawn and measurements were made of radioactivity incorporated into acid-soluble and -insoluble fractions.
Washed cell pellets were assayed for ability to incorporate [14C]NANA into endogenous cell acceptors as a function of time at 37'. Fig. 1 indicates that control thymocytes incorporate radioactivity into trichloroacetic acid-insoluble cell acceptors at a relatively constant rate for a period of 3 hr. The rate of incorporation is equivalent to 0.53 pmol of [14C]NANA/mg of cell protein per hr. Preincubation of thymocytes with mitogenic concentrations of Con A results in a doubling in rate of ['4C]NANA incorporation to a value of 1.28 pmol/mg of cell protein per hr. As in the control, the rate of incorporation is approximately linear with time. On the other hand, incorporation of radioactivity into acid-soluble fractions is quite low, as might be expected from the known impermeability of cells to the nucleotide sugar. In any case, there is no detectable response of this parameter to the presence of Con A. When desialylated fetuin acceptor was included in the assay medium, a similar response was observed, resulting in rates of incorporation equivalent to 1.0 and 2.0 pmol/mg of cell protein per hr for control and Con-A-pretreated cells, respectively (data not shown). In the above experiments, there was no significant loss of cell viability during the incubation period. The relationship between the Con A concentration used in the 1 hr preincubation period and sialyltransferase levels is depicted in Fig. 2. The rates of incorporation of radioactivity into both cellular acceptors and fetuin acceptor increase with increasing Con A concentration, reaching a maximum response at 5-10 ,ug/ml. The dose response of enzyme after 1 hr of culture parallels that of the mitogenic response as assessed by cellular incorporation of [3H]thymidine into DNA after 48 hr of culture. Assay of cells for sialyltransferase activity as a function of preincubation time following the addition of mitogen (Fig. 3) revealed that the magnitude of ,he response to Con A, when compared to that in control cells, reaches a maximum within the first few hours of culture with regard to both incorporation into endogenous acceptors (Fig. 3A) and in the presence of exogenous acceptors (Fig. 3B). The respective enzymic levels remain relatively constant for 12 hr in ConA-treated cultures, declining rather abruptly between 12
Cell Biology: Painter and White
Proc. Nat. Acad. Sci. USA 73 (1976) -
839
IE 1500
Ir
I
3
30
0_w
W
cL
-4i
z
Im
_
