HUMAN STOMACH CATHEPSIN E ACTION ON HUMAN IMMUNOGLOBULINS
A. F. Kisseljov, S. V. Gulnik and N. I. Tarasova Moscow State University Department of Chemistry Moscow 119899 USSR
INTRODUCTION Human stomach mucosa contains four immunologically distinct aspartic proteinases pepsin A, gastricsin or pepsin C (in the form of zymogens), cathepsin D and cathepsin E. Enzymatic properties, primary structure, cellular origin, activation mechanism and physiological role of pepsin, gastric sin and cathepsin D are well characterized. Much less information is available about cathepsin E although the enzyme has drawn much attention in recent years. For comparison of enzymatic properties of stomach proteinases in hydrolysis of proteins we have undertaken a study of their action on human antibodies. Immunoglobulins were chosen as substrates because their primary and tertiary structures are well characterized and information about action of numerous proteinases on them is available. In addition, cathepsin E was found in lymphoid associated tissues and thus is believed to be involved in the immune response. Thus, information about the ability of cathepsin E to hydrolyze antibodies can contribute to our understanding of the physiological role of that enzyme as well.
ME1HODS Purified human immunoglobulins were incubated with pepsin, gastric sin and cathepsin E. Samples of reaction mixture were removed in definite time intervals, boiled with SDS-PAGE sample buffer and analyzed by SDS-PAGE. The concentration of three enzymes in the reaction mixtures were adjusted so that they had equal milk-clotting activities.
Structure and Functi01l of the Aspartic Proteinases Edited by B.M. Dunn, Plenum Press, New York, 1991
369
43.... 3 5.0. 5 No influence of the triphosphate was detected in the case of immunoglobulins cleavage at pH 4-5.5. This observation is consistent with the proposal that pH influences the reaction under investigation through the changes in immunoglobulin conformation mainly.
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We have not detected any alteration of milk clotting activity of cathepsin E at pH 5.3 in the presence of ATP either. So it is probable that cathepsin E displays ATP-dependence of action only in the case of low molecular weight substrates.
DISCUSSION Thus, cathepsin E is the least active gastric protease in immunoglobulin cleavage. At the same time the specific milk-clotting activity of cathepsin E was found to be approximately two times higher than that of pepsin and gastric sin. Kinetic parameters of peptide hydrolysis do not differ much for pepsin6 •7 and cathepsin E.B.9 The main structural difference of cathepsin E from other mammalian aspartic proteases is that cathepsin E exists in a dimeric form. Elucidation of cDNA structure of the enzyme lO and the data on molecular weight determination permit the suggestion that cathepsin E exists predominantly as a dimer consisting of two identical and catalytically active subunits. Nothing is known about the spatial arraignment of subunits, but one can assume that the dimerization leads to sterical hindrance in binding of large protein substrates. This may be a reason for lower catalytic activity against immunoglobulins, compared to monomeric aspartic proteases. However, this same argument does not yield a satisfactory explanation for the high milk-clotting activity of cathepsin E. The other reason for low rates of immunoglobulin hydrolysis by cathepsin E can be an unfavorable primary structure of the molecule that is a target for proteolytic action, i.e., of the hinge region of immunoglobulins. Unlike pepsins, the amino acid preference of cathepsin E is poorly characterized and the enzyme specificity awaits more careful study. Precise localization of cathepsin E hydrolysis sites in immunoglobulins is in progress now. REFERENCES R. Heimer, S. S. Schnoll and A. Primack, Biochemistry 6:127-134 (1967). H. Bennich and M. W. Turner, Biochim. Biophys. Acta 175:388-395 (1969). T. E. Michaelsen, B. Fangione and E. C. Franklin, J.l mmunol. 119:558-563 (1977). A. G. Pardo, E. S. Rosenwaser and B. Fangione, J. lmmunol. 121:1040-1044 (1978). D. J. Thomas, A. D. Richards, R. A. Jupp, E. Ueno, K. Yamamoto, I. M. Samloff, B. M. Dunn and J. Kay, FEBS Lett. 243:145-148 (1989). 6. B. M. Dunn, B. Parten, M. Jimenez, C. E. Rolph, M. J. Valier and J. Kay, in: "Aspartic Proteinases and their Inhibitors," V. Kostka, ed., 221-243, W. de Gruyeter, Berlin (1985). 7. B. M. Dunn, M. J. Valier, C. E. Rolph, S. I. Foundling, M. Jimenez and J. Kay, Biochim. Biophys. Acta 913:122-130 (1987). 8. I. M. Samloff, R. T. Taggart, T. Shiraishi, T. Branch, W. A. Reid, R. Heath, R. W. Lewis, M. J. Valier and J. Kay, Gastroenterology 93:77-84 (1987). 9. R. A. Jupp, A. D. Richards, J. Kay, B. M. Dunn, J. B. Wyckoff, I. M. Samloff and K. Yamamoto, Biochem. J. 254:895-898 (1988). 10. T. Azuma, G. Pals, T. K. Mohandas, J. M. Couvreur and R. T. Taggart, J. Bioi. Chem. 264:1674816753 (1989). 11. P. K. Ivanov, M. M. Chernaya, A. E. Gustchina, I. V. Pechik, S. V. Nikonov and N. I. Tarasova, Biochim. Biophys. Acta (in press) (1990). 12. N. I. Tarasova, B. Foltmann and P. Szecsi, Biochim. Biophys. Acta 869:96-100 (1986). 13. B. Foltmann, P. B. Szecsi and N. I. Tarasova, Anal. Biochem. 146:353-360 (1985). 1. 2. 3. 4. 5.
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A. F. Kisseljov, S. V. Gulnik and N. I. Tarasova Moscow State University Department of Chemistry Moscow 119899 USSR
INTRODUCTION Human stomach mucosa contains four immunologically distinct aspartic proteinases pepsin A, gastricsin or pepsin C (in the form of zymogens), cathepsin D and cathepsin E. Enzymatic properties, primary structure, cellular origin, activation mechanism and physiological role of pepsin, gastric sin and cathepsin D are well characterized. Much less information is available about cathepsin E although the enzyme has drawn much attention in recent years. For comparison of enzymatic properties of stomach proteinases in hydrolysis of proteins we have undertaken a study of their action on human antibodies. Immunoglobulins were chosen as substrates because their primary and tertiary structures are well characterized and information about action of numerous proteinases on them is available. In addition, cathepsin E was found in lymphoid associated tissues and thus is believed to be involved in the immune response. Thus, information about the ability of cathepsin E to hydrolyze antibodies can contribute to our understanding of the physiological role of that enzyme as well.
ME1HODS Purified human immunoglobulins were incubated with pepsin, gastric sin and cathepsin E. Samples of reaction mixture were removed in definite time intervals, boiled with SDS-PAGE sample buffer and analyzed by SDS-PAGE. The concentration of three enzymes in the reaction mixtures were adjusted so that they had equal milk-clotting activities.
Structure and Functi01l of the Aspartic Proteinases Edited by B.M. Dunn, Plenum Press, New York, 1991
369
43.... 3 5.0. 5 No influence of the triphosphate was detected in the case of immunoglobulins cleavage at pH 4-5.5. This observation is consistent with the proposal that pH influences the reaction under investigation through the changes in immunoglobulin conformation mainly.
370
We have not detected any alteration of milk clotting activity of cathepsin E at pH 5.3 in the presence of ATP either. So it is probable that cathepsin E displays ATP-dependence of action only in the case of low molecular weight substrates.
DISCUSSION Thus, cathepsin E is the least active gastric protease in immunoglobulin cleavage. At the same time the specific milk-clotting activity of cathepsin E was found to be approximately two times higher than that of pepsin and gastric sin. Kinetic parameters of peptide hydrolysis do not differ much for pepsin6 •7 and cathepsin E.B.9 The main structural difference of cathepsin E from other mammalian aspartic proteases is that cathepsin E exists in a dimeric form. Elucidation of cDNA structure of the enzyme lO and the data on molecular weight determination permit the suggestion that cathepsin E exists predominantly as a dimer consisting of two identical and catalytically active subunits. Nothing is known about the spatial arraignment of subunits, but one can assume that the dimerization leads to sterical hindrance in binding of large protein substrates. This may be a reason for lower catalytic activity against immunoglobulins, compared to monomeric aspartic proteases. However, this same argument does not yield a satisfactory explanation for the high milk-clotting activity of cathepsin E. The other reason for low rates of immunoglobulin hydrolysis by cathepsin E can be an unfavorable primary structure of the molecule that is a target for proteolytic action, i.e., of the hinge region of immunoglobulins. Unlike pepsins, the amino acid preference of cathepsin E is poorly characterized and the enzyme specificity awaits more careful study. Precise localization of cathepsin E hydrolysis sites in immunoglobulins is in progress now. REFERENCES R. Heimer, S. S. Schnoll and A. Primack, Biochemistry 6:127-134 (1967). H. Bennich and M. W. Turner, Biochim. Biophys. Acta 175:388-395 (1969). T. E. Michaelsen, B. Fangione and E. C. Franklin, J.l mmunol. 119:558-563 (1977). A. G. Pardo, E. S. Rosenwaser and B. Fangione, J. lmmunol. 121:1040-1044 (1978). D. J. Thomas, A. D. Richards, R. A. Jupp, E. Ueno, K. Yamamoto, I. M. Samloff, B. M. Dunn and J. Kay, FEBS Lett. 243:145-148 (1989). 6. B. M. Dunn, B. Parten, M. Jimenez, C. E. Rolph, M. J. Valier and J. Kay, in: "Aspartic Proteinases and their Inhibitors," V. Kostka, ed., 221-243, W. de Gruyeter, Berlin (1985). 7. B. M. Dunn, M. J. Valier, C. E. Rolph, S. I. Foundling, M. Jimenez and J. Kay, Biochim. Biophys. Acta 913:122-130 (1987). 8. I. M. Samloff, R. T. Taggart, T. Shiraishi, T. Branch, W. A. Reid, R. Heath, R. W. Lewis, M. J. Valier and J. Kay, Gastroenterology 93:77-84 (1987). 9. R. A. Jupp, A. D. Richards, J. Kay, B. M. Dunn, J. B. Wyckoff, I. M. Samloff and K. Yamamoto, Biochem. J. 254:895-898 (1988). 10. T. Azuma, G. Pals, T. K. Mohandas, J. M. Couvreur and R. T. Taggart, J. Bioi. Chem. 264:1674816753 (1989). 11. P. K. Ivanov, M. M. Chernaya, A. E. Gustchina, I. V. Pechik, S. V. Nikonov and N. I. Tarasova, Biochim. Biophys. Acta (in press) (1990). 12. N. I. Tarasova, B. Foltmann and P. Szecsi, Biochim. Biophys. Acta 869:96-100 (1986). 13. B. Foltmann, P. B. Szecsi and N. I. Tarasova, Anal. Biochem. 146:353-360 (1985). 1. 2. 3. 4. 5.
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