Communication
`
`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`Vol. 260 No. 20 Issue of Se tember 15 pp. 10905-10908,1985
`0 198$ by The‘ American fmiety of Biological Chemists, Inc.
`Printed in U.S.A.
`
`Disulfide Spacer between
`Methotrexate and Poly@-lysine)
`
`A PROBE FOR EXPLORING THE REDUCTIVE
`PROCESS IN ENDOCYTOSIS*
`(Received for publication, June 17, 1985)
`Wei-Chiang Shenz, Hugues J.-P. Ryser, and
`Laura LaManna
`From the Department of Pathology, Boston University
`School of Medicine, Boston, Massachusetts 02118
`
`in the
`considered to be the first step
`reduction has been
`sequential degradation of the proteins. Cells which are unable
`to reduce the disulfide bond will accumulate cystine inside
`be the major
`lysosomes (7). This accumulation appears to
`cause of the lysosomal storage disease known as cystinosis
`(8). In spite of its importance, the exact nature of this reducing
`reaction as well as its intracellular location are still largely
`unknown.
`One of the difficulties in studying the cleavage of intramo-
`lecular disulfide bonds of endocytosed proteins is that it does
`not alter significantly the physicochemical properties of the
`macromolecules. Consequently, conventional methods that
`rely on the release of labeled amino acids, or on changes in
`POly(D-lySine) is taken up avidly by cultured cells
`either molecular weight or isoelectric point, are unable to
`as a
`through adsorptive endocytosis and can serve
`detect a difference in the redox state of the cysteinyl residues.
`carrier to increase cellular uptake of other molecules.
`Furthermore, reduction or oxidation of isolated proteins can
`While direct conjugation of methotrexate to poly(D-
`occur as an artifact during cell homogenization.
`lysine) yields a conjugate devoid of cytotoxic effects
`In this communication, we use a conjugate in which meth-
`because poly(D-lysine) is not digested in lysosomes, the
`otrexate (MTX’) is linked to
`poly(D-lysine) (poly(~-Lys))
`indirect conjugation using a triglycine spacer or a di-
`through a disulfide spacer as a probe to study the reducing
`sulfide spacer strongly inhibits the growth of both the
`reaction in cultured CHO cells. Previously we have shown
`wild type and the methotrexate transport-defective
`
`that direct linkage of MTX to PO~Y(D-LYS) gives a conjugate
`Chinese hamster ovary cells. Cell treatment with 3 mM
`that is totally devoid of cytotoxic effect, even
`though it is
`NH,Cl or 50 pg/ml leupeptin prevents the effect of
`taken up
`equally well as poly(L-lysine) by cultured cells
`conjugate with the triglycine spacer, but not of conju-
`through endocytosis (9). MTX-poly(~-Lys) lacks cytotoxicity
`gate with the disulfide spacer.
`On the other hand,
`because, unlike poly(L-lysine), it can not be degraded intra-
`preincubation with 2-mercaptoethanol abolishes the
`cellularly and thus does not generate small molecular active
`effect of the drug-disulfide conjugate in the methotrex-
`drugs (9). We have demonstrated that the introduction
`of
`ate transport-defective mutant, but not the effect of
`the drug-triglycine conjugate. The disulfide conjugate
`appropriate spacers between MTX and poly(~-Ly~) will ren-
`shows an identical cytotoxic effect in a-minimal essen-
`der the conjugates cytotoxic. These spacers include a small
`tial medium and RPMI 1640 media, even though cells
`peptide that can be digested by lysosomal proteases (10) and
`grown in the latter have only
`half the glutathione
`an acid-sensitive linkage that hydrolyzes spontaneously at the
`content as cells grown in the former medium. We con-
`acid pH of endosomes-lysosomes (11). Poly(D-lysine) there-
`clude that the reductive process through which meth-
`fore offers a tool to study specific drug-carrier linkages that
`otrexate is released from the disulfide spacer (a) occurs
`will be cleaved under different intracellular conditions. The
`inside cells and not at the cell surface, ( b ) requires
`drug-disulfide linkage described in this report offers a model
`neither acid pH nor lysosomal enzymes, and ( c ) is not
`for the study of the reducing process in endocytosis because
`mediated by a glutathione-disulfide exchange reaction
`the cleavage of the disulfide bond can be easily assessed by
`requiring high glutathione concentrations. Although
`the growth inhibition due to the releasing of a cytotoxic drug
`
`the cellular compartment in which this reductive proc-
`in the intact cells.
`ess occurs is not yet identified,
`there are reasons to
`assume that it is prelysosomal.
`
`MATERIALS AND METHODS
`MTX was supplied by Lederle Laboratories. Poly(D-lysine) hy-
`drobromide ( M , 60,000), triglycine, l-ethyl-3-(3-dimethylaminopro-
`py1)carbodiimide (EDC), leupeptin, and 3-(2-pyridyldithio)propionic
`acid N-hydroxylsuccinimide ester (SPDP) were obtained from Sigma.
`The tissue culture products were from GIBCO, Grand Island, NY.
`The CHO cell line CHOPro-3 MtxRII 5-3, characterized as MTX-
`resistant due to transport defect (12), was given to us by Dr. W. F.
`Flintoff, University of Western Ontario, London, Ontario, Canada.
`Unless specified, these cells were grown in tu-MEM medium with 10%
`fetal bovine serum. The preparation of MTX-poly(~-Lys) has been
`
`The intracellular processing of endocytosed macromole-
`cules involves several biochemical events in the course of a
`multistep transport sequence. For example, proteins can be
`partially degraded in prelysosomal vesicles (l), can undergo
`conformational changes (2) and dissociate from their recep-
`tors (3) in acidic endosomes (4), and can be totally hydrolyzed
`to amino acids in lysosomes ( 5 ) . Another general reaction in
`the degradation of proteins is the reductive cleavage of the
`disulfide bonds. In cases such as insulin degradation (6), this
`
`* This work was supported by National Institutes of Health Grants
`CA 34798 (W.-C. S.) and CA 14551 (H. J.-P. R.). The costs of
`publication of this article were defrayed in part by the payment of
`page charges. This article must therefore be hereby marked “aduer-
`tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
`this fact.
`$ Recipient of a Cancer Research Scholar award from the American
`Cancer Society, Massachusetts Division.
`
`The abbreviations used are: MTX, methotrexate; POIY(D-LYS),
`poly(D-lysine); CHO, Chinese hamster
`ovary; EDC, 1-ethyl-3-(3-
`dimethylaminopropy1)carbodiimide; SPDP, 3-(2-pyridyldithio)pro-
`pionic acid N-hydroxylsuccinimide ester; a-MEM, cy-minimal essen-
`7; MTX-GGG,
`tial medium; PBS, phosphate-buffered saline, pH
`MTX-triglycine conjugate; MTX-GGG-~OIY(D-L~S), MTX-poly(~-
`
`Lys) conjugate with a triglycine spacer; M T X - S S - ~ ~ ~ ~ ( D - L ~ S ) , MTX-
`a 3-(aminoethy1dithio)propionic acid
`POIY(D-LYS) conjugate with
`
`spacer; MTX-poly(~-Lys), MTX-poly(~-lys) conjugate with a direct
`linkage.
`10905
`
`IMMUNOGEN 2092, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`10906
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`Reductive Cleavage of Disulfide Bonds in Endocytosis
`
`extracted five times each with 2 ml of ether. The final solutions after
`incubation at 37 "C for 10 min
`to remove the ether were used to
`measure glutathione concentrations by Ellman's reagent as described
`by Tietze (14).
`
`described (9). The concentrations of poly(D-Lys) in various conjugates
`were determined by the trypan blue precipitation method (13).
`Preparation of MTX-GGG-Poly(o-Lys)-To
`a solution containing
`10 mg of MTX and 5 mg of triglycine in 1 ml of H20, pH 7, 10 mg of
`EDC was added. After 2 h, the reaction was diluted to 2 ml by H20,
`cooled in ice, and adjusted by 1 N HCI to pH 4.5. The precipitate was
`RESULTS
`collected by centrifugation and redissolved in 2 ml of H 2 0 by slowly
`adding 1 N NaOH. The acid precipitation was repeated once, and the
`The growth inhibition in both the wild type and the MTX
`final product of MTX-GGG was dissolved in 1 ml of H,O at pH 7. RF
`transport-defective CHO cells by MTX and its poly(~-Ly~)
`values of thin-layer chromatography (methano1:acetone:acetic acid =
`conjugates is shown in Fig. 1. In the wild type cells, the growth
`1O:lO:l) were 0.25 and 0.04 for MTX and MTX-GGG, respectively.
`is inhibited by MTX and by the two spacer-linking conjugates,
`The ratio of glutamic acid and glycine in the 6 N HCl hydrolysate
`i.e. MTX-GGG-~O~Y(D-LYS) and MTX-SS-~O~Y(D-LYS), but
`was 1 to 3.3, indicating a 1:l.l ratio of MTX to triglycine in the final
`is not influenced by MTX-poly(~-Lys) at concentrations up
`product.
`A 0.5-ml aliquot of MTX-GGG solution (10 mg/ml) was added to
`to 0.3 PM. In the MTX transport-defective cells, which are
`an equal volume of POIY(D-LYS) solution (20 mg/ml). After mixed
`resistant to both free MTX and the poly(~-Ly~) conjugate,
`thoroughly, 10 mg of EDC was added. The reaction was kept at room
`the growth is also inhibited by the two conjugates with the
`temperature for 2 h with occasional mixing and was then separated
`triglycine and disulfide spacers. The ID5,, values for MTX-
`by a Sephadex G-50 column equilibrated with PBS. The conjugate
`
`GGG-poly(~-Lys) and MTX-SS-~O~~(D-L~S) are almost iden-
`which emerged from the column at the void volume was collected and
`tical in the two cell lines, i.e. 43 and 42 nM in the wild type
`had an average of 5.5 mol of MTX/each mol of POIY(D-LYS).
`cells,
`and 36 and 54 nM in the MTX transport-defective
`Preparation of MTX-ss-Poly(~-Lys)-To a solution of 45 mg of
`MTX and 30 mg of cystamine in 1.5 ml of H 2 0 at pH 7, 30 mg of
`respectively. The growth inhibitory effects of the two conju-
`EDC was added. Heavy precipitation formed during the mixing, and
`gates are due to the toxicity of MTX rather than that of the
`the reaction was allowed to proceed for 0.5 h on ice and then 0.5 h at
`spacers or p o l y ( ~ - L y ~ ) moieties, because Leucovorin, a MTX
`room temperature. MTX-cystamine precipitate was isolated by cen-
`antagonist, can completely protect the cells from the cytotox-
`trifugation, washed twice with 2 ml of cold H20, and then converted
`icity of the conjugates (Table I).
`to MTX-thioethylamide (MTX-cysteamine) by heating for 5 min
`
`When M T X - S S - ~ O ~ ~ ( D - L ~ S ) was preincubated with 2-mer-
`with 2 ml of 10% 2-mercaptoethanol in a boiling water bath. During
`the reduction of the disulfide bond, the yellow solid of MTX-cysta-
`captoethanol before the addition to the cultures, the inhibi-
`mine was slowly dissolved. The small amount of insoluble material
`tory effect of this conjugate in the transport-defective cells
`in the final solution was removed by centrifugation, and the super-
`was abolished (Table I). This pretreatment did not change
`natant solution containing MTX-thioethylamide, 2-mercaptoethanol,
`
`the effect of M T X - G G G - ~ O ~ ~ ( D - L ~ S ) (Table I). Treatments
`and cysteamine was kept in
`a freezer. MTX-thioethylamide was
`with either leupeptin
`(50 Fg/ml) or NH&l (3 mM) were
`purified by repeating precipitation in pH 4 for several times to remove
`effective to protect cells from the cytotoxicity of MTX-GGG-
`the excess of 2-mercaptoethanol before coupling to SPDP-modified
`poly(~-Lys). KF values from
`thin-layer chromatography (metha-
`p o l y ( ~ - L y ~ ) , but had no effect on the growth inhibition by
`no1:acetone:acetic acid = 1O:lO:l) were 0.25, 0.00, and 0.60 for MTX,
`
`M T X - S S - ~ O ~ ~ ( D - L ~ S ) (Table I).
`MTX-cystamine, and MTX-thioethylamide, respectively.
`The intracellular levels of glutathione in the MTX trans-
`SPDP-modified POIY(D-LYS) was prepared by mixing 1.5 ml of
`port-defective cells grown in either a-MEM or RPMI 1640
`poly(~-Lys) solution (20 mg/ml in phosphate buffer saline, pH 8)
`medium were compared. When cells were cultured in a-MEM
`with 0.15 ml of SPDP (2 mg/ml in absolute alcohol). After 2 h
`at
`medium, the intracellular level of the reduced glutathione was
`room temperature, the modified POIY(D-LYS) was purified by extensive
`dialysis in PBS, pH 7, at 4 "C.
`19.0 nmol/mg of cell protein. This quantity was significantly
`To prepare MTX-SS-~OIY(D-LYS), 5 mg of fresh purified MTX-
`thioethylamide was added to a solution containing 20 mg of SPDP-
`modified POIY(D-LYS) in 1 ml of PBS. The reaction was allowed to
`stand overnight at 4 "C and was centrifuged to remove any precipitate.
`The conjugate was purified on a Sephadex G-50 column. The final
`had an average of 6.9 mol of MTX/
`product of MTX-SS-~OIY(D-L~S)
`each mol of POIY(D-LYS).
`Growth Inhibitory Effects of MTX-GGG-PO~~(D-L~S) and MTX-SS-
`Poly(n-Lys)-Wild
`type of CHO cells or their MTX transport-defec-
`tive mutants were seeded in 25-cm2 culture flasks with 5 ml of growth
`medium at a density of 5 X lo4 cells/flask. After 24 h of incubation,
`various amounts of MTX conjugates were added to each flask. Other
`After a 3-day
`agents, if required, were added at the same time.
`incubation, cells were fed with drug-free growth medium. One or 2
`days after
`feeding, when cells
`in the control flasks had
`reached
`confluency, the cell number in each flask was counted with a Coulter
`counter. The numbers were compared with that of the untreated cells
`in control flasks.
`In the pretreatment of 2-mercaptoethanol, a solution containing
`
`0.1 mM MTX-SS-~OIY(D-L~S) and 3 mM 2-mercaptoethanol in PBS
`was incubated at 37 "C for 2 h. Five p1 of this incubated solution was
`added to the culture flasks to
`give a final concentration of 0.1 pM
`MTX and 3 p~ 2-mercaptoethanol in the 5 ml of medium. The cells
`in the flasks were processed as described above to determine growth
`inhibitory effects.
`Intracellular Glutathione Determination-The MTX transport-de-
`fective mutant cells were grown in 25-cm2 flasks and were fed 1 day
`before the glutathione measurement. Cells from four confluent mon-
`olayers were combined after the trypsinization and washed twice each
`with 5 ml of PBS. Cell pellets after centrifugation were lysed in 1 ml
`of 10 mM phosphate buffer, pH 7, with 5 mM EDTA, and the proteins
`in the cell lysates were precipitated with ice-cold 5% trichloroacetic
`acid solution. After centrifugation, the supernatant solutions were
`
`[MTX]. p M
`FIG. 1. Growth inhibition of the wild type and the MTX
`transport-defective cells by increasing concentrations of MTX
`
`given as free drug or as p ~ l y ( ~ - L y ~ ) conjugates. Both the wild
`CHO cells were
`type (- - -) and the MTX transport-defective (-)
`seeded at 5 X IO4 cells/flask in a-MEM medium with 10% fetal bovine
`serum. After 24 h, the sparse monolayers were
`treated with MTX
`(A), MTX-poly(~-Lys) (A), M T X - G G G - ~ ~ I Y ( D - L ~ S )
`(O), and MTX-
`SS-POIY(D-LYS) (0). Cells were harvested and counted after 5 days as
`described under "Materials and Methods." The cell number in control
`flasks was 5.4 X lo6 cells/flask for the wild type and 4.0 X lo6 cells/
`flask for the MTX transport-defective mutants.
`
`IMMUNOGEN 2092, pg. 2
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`IPR2014-00676
`
`

`
`10907
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`14.0
`
`Reductive Cleavage of Disulfide Bonds in Endocytosis
`TABLE I
`decades, insulin degradation has been a subject of extensive
`study because of its potential involvement in the hormone
`Effects of Leucovorin, 2-mercaptoethanol, leupeptin, and NH4C1 on
`function and regulation (6). Nevertheless, the intracellular
`the cytotoxicity of M T X given either as MTX-GGG-poly(DLysi or as
`MTX-SS-poly(~-Lysi to transport-defective CHO cells
`location of the enzyme, glutathione-insulin transhydrogenase,
`which cleaves the disulfide linkage between
`the A and B
`Number of survival cells"
`chains of insulin, is still uncertain.
`Various localizations,
`MTX-GGG-
`MTX-SS-
`including the plasma membranes (15), the endoplasmic retic-
`POIY(O-LYS)
`poly(0-Lys)
`ulum (16), and the cytosol (17), have been suggested. Besides
`0.3 p M
`0.1 p M
`0.3 p M
`0.1 p M
`its role in the protein catabolism, the reductive cleavage of
`% of control
`disulfide bonds is required for the intracellular activation of
`3.2
`3.9
`16.9
`No addition
`several functional proteins, including enzymes (18) and toxins
`109.6
`96.1
`Leucovorin (3 PM)
`
`(19). In protein toxins such as diphtheria toxin, the reduction
`103.2
`2.9
`2-Mercaptoethanolb
`of the disulfide linkage between
`the A and B fragments is
`3.4
`11.2
`46.4
`84.3
`Leupeptin (50 pg/ml)
`NHXI (3 mM)
`considered essential for the A fragment to acquire an active
`17.6
`102.6
`34.0
`4.0
`conformation (19) and to
`escape from
`intracellular vesicles
`' Number of cells in control flasks was 5 X lo6 cells/flask (25 cm2)
`before it is degraded by lysosomal enzymes (20). This suggests
`set at 100%.
`Conjugates were pretreated with 2-mercaptoethanol as described
`that the disulfide bonds in proteins can be reduced at anearly
`under "Materials and Methods."
`stage in the endocytosis process.
`to be
`Because several oxidoreductases have been found
`associated with plasma membrane (15, 21) and because glu-
`tathione can be transported through plasma membranes to
`reach the outside of the cells (22), it is possible that some
`reduction of disulfide bonds may occur at the cell surfaces.
`That such a surface reaction is not a major factor in the effect
`
`of M T X - S S - ~ O ~ ~ ( D - L ~ S ) is indicated by the fact that the
`conjugate is as effective in wild type CHO cells as in their
`MTX transport-defective mutants. Since the mutant cells are
`not inhibited by either free MTX or MTX-thioethylamide,
`these two compounds would be equally ineffective if released
`by a reductive process at the cell surface. It could be argued
`that MTX-thioethylamide released at the cell surface could
`cross-link to sulfhydryl groups of membrane proteins and be
`carried with them across the cell membrane. We found, how-
`
`ever, that pretreatment of M T X - S S - ~ O ~ ~ ( D - L ~ S ) by 2-mer-
`IMTX].# M
`captoethanol abolishes its cytotoxicity. It does not appear
`FIG. 2. Growth inhibitory effects of M T X - C G G - ~ O ~ ~ ( D -
`likely that the same compound would be transported differ-
`
`Lys) and MTX-SS-~O~Y(D-LYS) on the MTX transport-defec-
`tive CHO cells cultured in either a-MEM or RPMI 1640
`
`ently when released from M T X - S S - ~ O ~ ~ ( D - L ~ S ) a t the cell
`medium. Cells were treated with M T X - G G G - ~ O I ~ ( D - L ~ S )
`(0) and
`surface or during 2-mercaptoethanol pretreatment. MTX-
`(0) as described in the legend to Fig. 1, except
`M T X - S S - ~ O ~ ~ ( D - L ~ S )
`GGG-poly(~-Lys) also has comparable effects on the wild
`or RPMI
`that the experiments were also carried out in (u-MEM (-)
`type and the mutant CHO cells, indicating that the two cell
`1640 medium (- - -). The cell number in control flasks was 5.6 X lo6
`cells/flask in wMEM medium and 4.5 X lo6 cells/flask in RPMI
`lines do not
`differ in
`the endocytosis of the p o l y ( ~ - L y ~ )
`1640 medium.
`carrier. These considerations all suggest that the growth in-
`
`hibitory effect of M T X - S S - ~ O ~ ~ ( D - L ~ S ) requires endocytosis
`of the conjugate and intracellular cleavage of its disulfide
`bond.
`An intracellular cleavage of M T X - S S - ~ O ~ ~ ( D - L ~ S ) could be
`
`either enzymatic or nonenzymatic. In the latter instance, it
`would be expected that the cellular level of glutathione, the
`major known intracellular reducing agent in mammalian cells
`(22), would influence the efficiency of the disulfide cleavage.
`We have shown that a 2-fold difference in the level of intra-
`DISCUSSION
`cellular glutathione can be achieved by growing cells in media
`that differ in their cysteine content. When grown in a-MEM
`This paper demonstrates the
`effectiveness of two drug
`medium, which contains 0.63 mM cysteine ( 2 3 ) , the cellular
`
`conjugates in which MTX is linked to p o l y ( ~ - L y ~ ) through
`level of glutathione was 19.0 nmol/mg of cell protein. When
`two different types of spacer. The first conjugate, MTX-GGG-
`grown in RPMI 1640 medium, which contains only 0.24 mM
`
`p ~ l y ( ~ - L y ~ ) , as we reported previously, is effective in inhib-
`cystine but no cysteine (24), the cellular level of glutathione
`iting the growth of MTX transport-defective CHO mutant
`was 9.7 nmol/mg of cell protein. Since the cells grown in these
`
`cells (10). We confirm here that the triglycine spacer is indeed
`
`two media responded similarly to MTX-SS-~O~Y(D-LYS), we
`degraded by the lysosomal proteases. The second conjugate,
`conclude that intracellular cleavage of the conjugate does not
`
`MTX-SS-~OIY(D-LYS), is not susceptible to the
`lysosomal
`proteolysis and therefore must be cleaved by a reduction on
`require high levels of glutathione and it is not likely to be due
`the disulfide linkage.
`to a nonenzymatic glutathione-disulfide exchange reaction.
`The molecular mechanism and the intracellular location of
`Cysteamine has also been suggested as the physiological
`this reductive reaction in the endocytotic pathway are largely
`hydrogen donor for the nonenzymatic reduction of disulfide
`undetermined. This reaction, however, is generally regarded
`bonds inside the lysosomes (25). Intralysosomal levels of
`as an important step in protein degradation. During the last
`cysteamine can be changed by treating cells with lysosomo-
`
`higher than that in the cells cultured in RPMI 1640 medium,
`i.e. 9.7 nmol/mg of cell protein. This difference in intracellular
`glutathione contents, however, was not reflected in the cyto-
`
`toxicity of M T X - S S - ~ O ~ ~ ( D - L ~ S ) , since both MTX-GGG-
`
`p o l y ( ~ - L y ~ ) a n d M T X - S S - ~ O ~ ~ ( D - L ~ S ) produced an identical
`growth inhibition when the MTX transport-defective
`cells
`were cultured in either medium (Fig. 2).
`
`IMMUNOGEN 2092, pg. 3
`Phigenix v. Immunogen
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`
`

`
`Reductive Cleavage of Disulfide Bonds
`
`I.
`
`in Endocytosis
`
`10908
`tropic amines like NH,Cl. Such treatment is known to in-
`3. Geuze, H. J., Slot, J. W., Strous, G. J. A. M., Lodish, H. F., and
`Schwartz, A. L. (1983) Cell 3 2 , 277-287
`crease the lysosomal pH beyond the values required for opti-
`4. Tycko, B., and Maxfield, F. (1982) Cell 28,643-651
`mal proteolysis and thus to inhibit lysosomal proteolysis (26),
`5. Goldstein, J. L., and Brown, M. S. (1974) J . Biol. Chem. 2 4 9 ,
`as evidenced in this paper by a decrease
`in the cleavage of
`5153-5162
`
`MTX-GGG-~O~Y(D-LYS). Increasing the lysosomal pH would
`6. Goldstein, B. J., and Livingston, J. N. (1981) Metab. Clin. Exp.
`have two effects relevant to the cleavage of disulfide bonds,
`3 0 , 825-835
`7. Thoene, J. G., and Lemons, R. (1980) Pediatr. Res. 1 4 , 785-787
`namely to decrease lysosomal levels of cysteamine by decreas-
`8. Thoene, J. G., Oshima, R. G., Ritchie, D. G., and Schneider, J.
`ing its lysosomotropic capture (27) and to raise the lysosomal
`A. (1977) Proc. Natl. Acad. Sci. U. S. A. 7 4 , 4505-4507
`pH beyond the values
`required for an optimal activity
`of
`9. Shen, W.-C., and Ryser, H. J.-P. (1979) Mol. Phurmacol. 16,
`lysosomal thio1:protein disulfide oxidoreductase
`(28, 29).
`614-622
`Since cell treatment with NH4C1 did not influence the cleav-
`0. Shen, W.-C., and Ryser, H. J.-P. (1981) Fed. Proc. 4 0 , 6 4 2
`1'
`age of MTX from its disulfide linkage, it can be inferred that
`1. Shen, W.-C., and Ryser, H. J.-P. (1981) Biochem. Biophys. Res.
`1
`neither lysosomal cysteamine nor lysosomal oxidoreductases
`Commun. 1 0 2 , 1048-1054
`2. Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976)
`1
`play major roles
`in the cleavage of M T X - S S - ~ O ~ ~ ( D - L ~ S ) .
`Somatic Cell Genet. 2 , 245-261
`Although our results cannot rule out the possibility that the
`3. Shen, W.-C.. Yam, D.. and Rvser, H. J.-P. (1984) Anal. Biochem.
`1
`disulfide spacer in the conjugate is cleaved in lysosomes by a
`.
`-
`.
`
`142,521-524
`nonenzymatic and pH-independent reduction with a hydrogen
`14. Tietze. F. (1969) Anal. Biochem. 27. 502-522
`donor other than glutathione and cysteamine, we think more
`15. Varandani, P. T . (1973) Biochem. biophys. Res. Commun. 5 5 ,
`likely that this reaction is enzymatic in nature and is taking
`689-696
`16. Ibbetson, A. L., and Freedman, R. B. (1976) Biochem. J. 1 5 9 ,
`place in some nonlysosomal compartments. Knowing that the
`377-384
`interchain disulfide bonds in diphtheria toxin are
`reduced
`17. Goldstein, B. J., and Livingston, J . N. (1980) Biochem. J. 1 8 6 ,
`before it reaches
`lysosomes, we suggest, by analogy, that
`351-360
`
`M T X - S S - ~ O ~ ~ ( D - L ~ S ) undergoes enzymatic reduction
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`
`Acknowledgments-We are grateful to Dr. Richard A. Laursen for
`F. Flintoff (University of
`the amino acid analysis, to Dr. Wayne
`Western Ontario) for making available to us the methotrexate-resist-
`ant cell line, and to Lederle Laboratories for supplying to us metho-
`trexate in this investigation.
`
`IMMUNOGEN 2092, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676