THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 266, No. 1. Issue of January 5 pp. g a l 0 3 1991
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
hinted in
ir~.~.
Human Cytotoxic Lymphocyte GranzymeB
ITS PURIFICATION FROM GRANULES AND THE CHARACTERIZATION OF SUBSTRATE AND
INHIBITORSPECIFICITY*
(Received for publication, May 29, 1990)
Martin Poe, Joseph T. Blake, David A. Boulton, Maureen Gammon, Nolan H. Sigal, Joseph K. Wu,
and Hans J. Zweerink
From Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065
Granzyme B hasbeen purified to homogeneity from the formation of membrane lesions on target cells caused by
the granules of a human cytolytic lymphocyte line, cytoplasmic granules from cytolytic lymphocytes (8, 9). Pas631, in an enzymatically active form by a three-step ternak and Eisen (10) discovered that CTL granules contain
procedure. 631 granzyme Bhydrolyzed Nu-t-butylox- an active serine protease; subsequent studies have demonycarbonyl-L-alanyl-L-alanyl-L-aspartyl
(Boc-Ala-Ala- strated seven serine proteases in murine CTL (11-17) and
Asp) thiobenzyl ester with a katof 11 f 5 mol/s/mol two or three serine
proteases (18,19) in human CTLgranules.
enzyme and catalytic efficiency kCat/K,of 76,000 f A direct role for these proteases in cytolysis has not yet been
44,000 M” s-’. The hydrolysis of Boc-Ala-Ala-Asp
identified (20) and has not been universally accepted (21-23),
thiobenzyl ester by crude Q31 Percoll fractions paraldespite the fact that a number of protease inhibitors can block
leled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated CTL-mediated lysis (24-27).
The human analogs of the mouse CTL serine proteases HF
with granules. When chromatographed on Sephacryl
(28) and CCPl (29), originally identified by subtractive hy5-300, Q31 granzyme B eluted in two broad bands
corresponding to dimer and monomer, both of which bridization methods as highly expressed mRNAspecies in
electrophoresed at 35 kDA in reducing NaDodSOl CTL, have been identified. H F (30), also known as granzyme
polyacrylamide, and both of which showed a lag phase A (18), granzyme 1 (19), tryptase (31), and HuTSP (32), has
in assays. The lag phase in assays could be extended been purified to homogeneity by us (31) from a human CTL
with 0.03 mM pepstatin. Upon elution from ion-ex- line (Q31). We report here a similar purification for granzyme
change chromatography Q31 granzyme B electropho- B, also known as granzyme 2 (19), CCP2 (29), HLP (33), HSE
resed at 32 kDa in reducing NaDodS04 polyacryl- 26.1 (34, 35), and SECT (36), from Q31. Granzyme B is the
amide and did not have a lag phase in assays. The granzyme most specifically found in CTL (37-39). This puamino-terminal sequence of the 32-kDa Q31 granzymerification differed from earlier purifications of human CTL
B was identical to four other human cytotoxic T-lym- granzyme B (18,19)in minor details, but differed importantly
phocyte granzymes B in18 of 18 positions sequenced. in producing an enzymatically active granzyme B, and thus
Purified Q 3 l granzyme B hada preference for sub- demonstrated that granzyme B truly is an enzyme. Q31granstrates with Glu or Asp as the residue amino-terminal zyme B had significant activity with Boc-Ala-Ala-Asp thioto thescissile bond; little or no activity wasnoted with benzyl ester, whichwas synthesized (40) to evaluate the
oligopeptidesubstrates for trypsin-like,
chymotrypsin- prediction by Murphy et al. (41) that granzyme B would
like, and elastase-like proteases. Human plasma a1- hydrolyze substrates with aspartyl residues at PI.’ The isolaprotease inhibitor, human plasma az-protease macro- tion of granzyme B in an enzymatically active form should
globulin, soybean and lima-bean trypsininhibitors,
bovine aprotinin, phosphoramidon, and chymostatin advance studies of the role of this enzyme in cytolysis.
inhibited Q31granzyme B. The inhibition by al-proMATERIALSANDMETHODSANDRESULTS3
tease inhibitorwas rapid enough to be of physiological
significance.
DISCUSSION
Cell-mediated killing by cytotoxic T-lymphocyte (CTL)’ is
an important immunologic defense against tumor cell proliferation, viral infection, and transplanted tissue (for reviews,
see Refs. 1-7). CTL-mediated killing i s often associated with
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The abbreviations used are: CTL, cytotoxic T-lymphocytes;
BAADT, Boc-Ala-Ala-Asp-SBzl,N“-t-butyloxycarbonyl-L-alanyl-Lalanyl-L-aspartyl-thiobenzylester; Buffer A, 0.3 M NaCI, 0.1 M
NaHepes, pH 7.0, 1 mM Na2-EDTA, 0.05% (v/v) Triton X-100;
PIPES, piperazine-N,N’-bis (2-ethanesulfonic acid); EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; SBzl, thiobenzyl ester;
Succ, Ne-succinylamide;pNA, p-nitroanilide; MeOSucc, Nu-methoxysuccinylamide; TLCK, N“-tosyl-L-lysylchloromethyl ketone.
Human Q31 CTL granzyme B was purified to homogeneity
as a32-kDa protein in reducing NaDodSOr electrophoresis in
an enzymatically active form by a three-stepprocedure. Granzyme B previously had been purified to homogeneity from
human cytotoxic T-lymphocytes by Krahenbuhl et al. (18)
and Hameed et al. (19). It was unclear whether these forms
of granzyme B were enzymatically active, since they were not
tested with appropriate substrates. It was significant to find
Q31 granzyme B to be enzymatically active, since leukocyte
granules have been shown to containproteins similar in
‘The terminology of the reactive-site residue positions is from
Schechter and Berger (42).
Portions of this paper (including “Materials and Methods,” “Results,’’ Tables 1-5, and Figs. 1-7) are presented in miniprint at the
end of this paper. Miniprint is easily read with the aid of a standard
magnifying glass. Full size photocopies are included in the microfilm
edition of the Journal thatis available from Waverly Press.
98
Human
Lymphocyte
Granzyme
Cytotoxic
B
99
Q31 granzyme B was active at acidic pH (see Fig. 7), in
sequence to serine proteases but devoid of proteolytic activity,
e.g. the antibacterial protein azurocidin from the azurophil contrast to Q31 tryptase whose BLT esterase activity disapgranules of human neutrophils (43). Purified 631 granzyme peared below pH7 (31). Thus, at the acidic pH of lytic
B hydrolyzed Boc-Ala-Ala-Asp-SBzlwith a katof 680 & 280 granules (about 6.1, 52), granzyme B would be active, which
mol hydrolyzed/min/mol enzyme and a catalytic efficiency may be the reason that it is stored as an inactive precursor.
(kcat/&) of 76,000 f 44,000 M-' s-'. While this ratewas much The subsite specificity of the 32-kDa form of purified Q31
lower than the hydrolysis of Cbz-Lys-SBzl by Q31 granzyme granzyme B was investigated using the 33 oligopeptide subA (tryptase kat/&= 225,000 M" s-' (31)), it was near the strates listed in Table 4.
The inhibition of the ~-glutamyl-2-naphthylamide
hydrorates of hydrolysis of N-acetyltryptophanyl estersby chymolase activity of Q31 granzyme B by the 18 compounds listed
trypsin, for which kcat/& was 4,600, 4,900, and 254,000
s-' for ethyl-, methyl- and 4'-nitrophenyl-esters, respectively in Table 5 confirmed the substrate preference data in Table
A pref- 4. The lack of inhibition by benzamidine, leupeptin, and
(44). It also hydrolyzed ~-glutamyl-2-naphthylamide.
erence of granzyme B for substrates with Asp or Glu at PI, phenylmethylsulfonyl fluoride showed a lack of trypsin-like
the residue whose a-carboxyl peptide linkage was hydrolyzed, character, while the lack of inhibition by Nu-Cbz-L-phenylwas predicted by Murphy et al. (38). While a preference for alanyl chloromethyl ketone and phenylmethylsulfonyl fluoAsp/Glu at PI was unusual for a protease, a few other pro- ride showed a difference from chymotrypsin-like enzymes.
teases have shown this preference, Staphylococcus aureus V8 The lack of inhibition by elastatinal showed that Q31 granprotease (45) and the mammalian multicatalytic proteinase zyme B is not elastase-like. The inability of EDTA and EGTA
(46,47). Goetzl et al. (48) found that cultured T-lymphocytes to inhibit Q31 granzyme B showed that itis not ametalloprohave a major cleavage of vasoactive intestinal peptide car- teinase, and the lack of inhibition by pepstatin showed that
boxyl-terminal to Asp-3. This cleavage is quite sensitive to Q31 granzyme B is not an aspartic proteinase like cathepsin
phosphoramidon, but insensitive to phenylmethylsulfonyl flu- D.
In contrast to Q31 tryptase (31), Q31 granzyme B is not
oride and leupeptin, like granzyme B.
The Boc-Ala-Ala-Asp-SBzlesterase activity of Q31 CTL significantly inhibited by human plasma antithrombin 111.
appeared to associate almost exclusively with granzyme B. As The inhibition of Q31 granzyme B by human plasma a1shown in Fig. 1, Boc-Ala-Ala-Asp-SBzl esterase activity in protease inhibitor is rapid and almost complete at 0.01 mg/
Percoll fractions paralleled the BLTesterase (tryptase)activ- ml. Ifthe reaction were 100-130-foldfaster andmore complete
ity, except for the bottom layer where a technical problem at the normal human plasma level of1-1.3 mg/ml for a'masked the parallelism. Since tryptase activity has been protease inhibitor (53), the inhibition would be fast enough
shown to be a marker for lytic granules (49-51), the parallel- to be of physiological significance by the criteriaof Bieth (54,
ism suggested that granzyme B is also associated with lytic 55).
granules. The other substrate of granzyme B, ~-glutamyl-2REFERENCES
naphthylamide, was appreciably hydrolyzed by the superna1. Tschopp, J., and Jongeneel, C. V. (1988) Biochemistry 27,2641tant (top layer) of the Percoll gradient, as well as by the
2646
fractions that hydrolyzed Boc-Ala-Ala-Asp-SBzl, which
2. Podack, E. R. (1985) Immunology Today 6, 21-27
showed that Q31 CTL has more than one enzyme which
3. Young, J. D., and Cohn, Z. A. (1986) Cell 46,641-642
Since the supernahydrolyzes ~-glutamyl-2-naphthylamide.
4. Henkart, P. A. (1985) Annu. Rev. Imrnunol. 3 , 31-58
tant corresponded to thecytoplasmic fraction, it was possible
5. Cerottini, J., and MacDonald, H. R. (1987) Ann. Znst. Pasteur
that a Q31 cytoplasmic multicatalytic proteinase (46, 47) is
Zmmunol. 138,289-342
6. Young, J. D. (1989) Physiol. Rev. 69, 250-314
responsible for the excess ~-glutamyl-2-naphthylamide
hy7. Khavari, P. (1987) Yale J. Biol. Med. 60,409-419
drolysis by the Percoll supernatant fraction above the hy8. Nabholz, M., and Tschopp, J. (1989) Zmmurwl. Lett. 20,179-180
drolysis that was in proportion to the tryptase; the latter
9. Joag, S., Zychlinsky, A., and Young, J. D. (1989) J. Cell. Biochem.
activity probably was due to broken granules.
39,239-252
Q31 granzyme B appeared to be stored in lytic granules in 10. Pasternak, M. S., and Eisen, H. N. (1985) Nature 314, 743-745
a 35-kDa, relatively inactive form. After solubilization in high 11. Masson, D., Nabholz, M., Estrade, C., and Tschopp, J. (1986)
EMBO J. 5,1595-1600
salt andafter molecular sieve chromatography at pH4.5, the
fractions that would yield granzyme B upon ion-exchange 12. Pasternak, M. S., Verret, C. R., Liu, M. A., and Eisen, H. N.
(1986) Nature 322, 740-743
chromatography at pH 6.0 electrophoresed in reducing Na- 13. Young, J. D., Leong, L. G. Liu, C., Damiano, A., Wall, D. A,, and
DodSOawith principal bands a t 28 kDa (tryptase) and35 kDa
Cohn, 2. A. (1986) Cell 47,183-194
(see Fig. 5). When assayed for ~-glutamyl-2-naphthylamide14. Simon, M.M., Hoschutzky, H., Fruth, U., Simon, H.G., and
Kramer, M. D. (1986) EMBO J. 5, 3267-3274
hydrolysis, the molecular sieve chromatography fractions had
a substantial lag phase before a steady enzymatic rate was 15. Masson, D., and Tschopp, J. (1987) Cell 49,679-685
D., Rey, C., Haefliger, J., Qiao, B., Groscurth, P., and
seen, as for the crude homogenate in Fig. 4. The lag was also 16. Jenne,
Tschopp, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4814seen with Boc-Ala-Ala-Asp-SBzl hydrolysis but was less
4818
clearly seen because of the appreciable nonenzymatic rate of 17. Jenne, D. E., Masson, D., Zimmer, M., Haefliger, J., Li, W., and
Tschopp, J. (1989) Biochemistry 28, 7953-7961
Boc-Ala-Ala-Asp-SBzlhydrolysis.
The amino-terminal sequence of the purified 32-kDa form 18. Krahenbuhl, O., Rey, C., Jenne, D., Lanzavecchia, A., Groscurth,
P., Carrel, S., and Tschopp, J. (1988) J . Zrnmunol. 141, 3471of Q31 granzyme B was identical to the sequence predicted
3477
from the cDNA sequence for humanTSP granzyme B (33) as 19. Hameed, A., Lowrey, D. M., Lichtenheld, M., and Podack, E. R.
well as to thehuman granzymes B purified by Krahenbuhl et
(1988) J. Zmmurwl. 141,3142-3147
al. (18) and Hameed et al. (19). It differed from mouse gran- 20. Kramer, M. D., and Simon, M. M. (1987) Zmmunol. Today 8,
140-142
zyme B (11-17) at 2 of 18 positions and Q31 tryptase at 5 of
18positions (31). In thepreparation of purified 6 3 1 granzyme 21. Henkart, P. A., Berrebi, G. A., Takayama, H., Munger, W. E.,
and Sitkovsky, M. V. (1987) J. Zmrnunol. 139,2398-2405
B for sequenator analysis, it was discovered that when the 32- 22. Berke,
G . (1989) Imrnunol. Lett. 20, 169-178
kDa form was incubated at pH 6 theprotease was autodiges- 23. Ostergaard, H.L., and Clark, W. L. (1989) J. Zmmunol. 143,
tive with several sites of cleavage (data not shown).
2120-2126
"'
100
Human Cytotoxic LJ/mphocyte GranzymeB
24. Chang, T. W., and Eisen, H. E. (1980) Nature 1 2 4 , 1028-1033
25. Redelman, D., and Hudig, D. (1980) J. Immunol. 124,870-878
26. Hudig,D.,Gregg,N.
J., Kam, C., and Powers, J. C. (1987)
Biochem. Biophys. Res. Commun. 149,882-888
27. Hudig, D., Allison, N. J., Kam, C., and Powers, J. C. (1989) Mol.
Immunol. 26,793-798
28. Gershenfeld, H.K., and Weissman, I. L. (1986) Science 232,
854-858
29. Lobe, C. G., Finlay, B. B., Poranchych, W., Paetkau, V. H., and
Bleackley, R. C. (1986) Science 232,858-861
30. Gershenfeld, H. K., Hershberger, R. J., Shows, T. B., and Weissman, I. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1184-1188
31. Poe, M., Bennett, C. D., Biddison, W. E., Blake, J. T., Norton,
G. P., Rodkey, J. A., Turner, R. V., Wu, J. K., and Zweerink,
H. J. (1988) J. Biol. Chem. 263,13215-13222
32. Fruth, U., Sinigaglia, F., Schlesier, M., Kilgus, J. Kramer, M. D.,
and Simon, M. M. (1987) Eur. J. Immunol. 17,1625-1633
33. Schmid, C., and Weissman, C. (1987) J.Zmmunol. 1 3 9 , 250-256
34. Klein, J. L., Shows, T. B., Dupont, B., and Trapani, J. A. (1989)
Genomics 5, 110-117
35. Trapani, J. A., Klein, J. L., White, P. C., and DuPont, B. (1988)
Proc. Natl. Acad. Sci. U. S. A. 8 5 , 6924-6928
36. Caputo, A., Fahey, D., Lloyd, C., Vozab, R., McCairns, E., and
Rowe, P. B. (1988) J. Biol. Chem. 263,6363-6369
37. Redmond, M. J., Letellier, M., Parker, J . M. R., Lobe, C., Havele,
C., Paetkau, V., and Bleackley, R. C. (1987) J. Zmmunol. 1 3 9 ,
3184-3188
38. Bleackley, R. C., Duggan, B., Ehrman, N., and Lobe, C. G. (1988)
FEBS Lett. 234,153-159
39. Lobe, C. G., Shaw, J., Fregeau, C., Duggan, B., Meier, M., Brewer,
A., Upton, C., McFadden, G., Patient, R. K., Paetkau, V., and
Bleackley, R. C. (1989) Nucleic Acids Res. 17,5765-5779
40. Kam, C.-M., Odake, S., Narasimhan, L., Poe, M., Blake, J . T.,
Krahenbuhl, O., Tschopp, J., andPowers, J . C. (1990) Biochemistry, in press
41. Murphy, M. E. P., Moult, J. Bleackley, R.C., Gershenfeld, H.,
Weissman, I. L., and James, M. N.G. (1988) Proteins 4, 190204
42. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27,157-162
43. Wilde, C. G., Snable, J . L., Griffith, J. E., and Scott, R. W. (1990)
J.Biol. Chem. 265,2038-2041
44. Bender, M., and Kezdy, F. (1965) Annu. Reu. Biochem. 3 4 , 4976
45. Drapeau, G. R. (1976) Methods Enzymol. 45, 469-475
46. Wilk, S., and Orlowski, M. (1983) J.Neurochem. 40,842-849
47. Dahlmann, B., Rutschmann, M., Kuehn, L., and Reinauer, H.
(1985) Biochem. J. 228, 161-170
48. Goetzl, E. J., Kodama, K. T., Turck, C. W., Schiogolev, S. A.,
and Sreedharan, S. P. (1989) Immunology 66, 554-558
49. Garcia-Sanz, J. A., Velotti, F., MacDonald, H. R., Masson, D.,
Tschopp, J., andNabholz, M. (1988) Immunology 64,129-134
50. Henkart, P. A., Berrebi, G. A., Takayama, H., Munger, W. E.,
and Sitkovsky, M. V. (1987) J. Zmmunol. 1 3 9 , 2398-2405
51. Takayama, H., Trenn, G., Humphrey, W., Bluestone, J. A.,
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
Henkart, P. A., and Sitkovsky, M. V. (1987) J. Immunol. 1 3 8 ,
566-569
Henkart, P. A., Yue, Ch. C., Yang,J., and Rosenberg, S. A. (1986)
J.Immunol. 137,2611-2617
Travis, J., and Salveson, G. S. (1983) Annu. Reu. Biochem. 5 2 ,
655-709
Bieth, J. G. (1980) Bull. Eur. Physiopathol. Respir. 1 6 , (suppl.)
183-195
Bieth, J. G. (1984) Biochem. Med. 32, 387-397
Biddison, W.E., Rao, P. E., Talle, M.A., Goldstein, G., and
Shaw, S. (1984) J. Exp. Med. 1 5 9 , 783-797
Manyak, C.L., Norton, G. P., Lobe,C. G., Bleackley, R. C.,
Gershenfeld, H. K., Weissman, I. L., Kumar, V., Sigal, N. H.,
and Koo, G. C. (1989) J. Zmmunol. 142,3707-3713
Podack, E. R., and Konigsberg, P. J. (1984) J. Exp. Med. 160,
695-710
Weber, K., and Osborn, M. (1969) J.Biol. Chem. 244,4406-4412
Poe, M., Breeze, A. S., Wu, J. K., Short, C. R., Jr. andHoogsteen,
K. (1978) J. Biol. Chem. 2 5 4 , 1799-1805
Poe, M., Greenfield, N. J., Hirshfield, J. M., Williams, M. N.,
and Hoogsteen, K. (1972) Biochemistry 11,1023-1030
Poe, M., Bergstrom, A. R., Wu, J . K., Bennett, C. D., Rodkey, J.
A., and Hoogsteen, K. (1984) J. Biol. Chem. 259,8358-8362
Ellman, G. L. (1959) Arch. Biochem. Biophys. 8 2 , 70-77
Ellman, G. L. (1958) Arch. Biochem. Biophys. 74,443-450
Lottenberg, R.L., Hall, J. A., Blinder, M., Binder, E. P., and
Jackson, C. M. (1983) Biochim. Biophys. Acta742,539-557
Lottenberg, R. L., and Jackson, C.M. (1983) Biochim. Biophys.
Acta 742,558-564
Aoyagi, T., Kunimoto, S., Morishima, H., Takeuchi, T., and
Umezawa, H. (1971) J.Antibiot. (Tokyo) 2 4 , 687-694
Umezawa, H. (1982) Annu. Reu. Microbiol. 36,75-99
Rich, D. H. (1986) in Proteinase Inhibitors (Barrett, A. J., and
Salvesen, G., eds) pp. 179-217, Elsevier Science Publishers,
Amsterdam
Knight, C.G., and Barrett, A. J. (1976) Biochem. J. 1 5 5 , 117125
Clausbruch, U.C.V.,
and Tschesche, H. (1988) Biol. Chem.
Hoppe-Seyler 369,683-691
Ishikawa, I., and Cimasoni, G. (1977) Biochem.Biophys.Acta
480,228-240
Peters, P. J., Geuze,H. J., Vanderdonk, H. A., Slot, J. W.,
Griffith, J . M., Stam, N. J., Clevers, H. C., and Borst, J. (1989)
Eur. J. Zmmunol. 1 9 , 1469-1475
Barrett, A. J. (1986) in Proteinase Inhibitors (Barrett, A. J., and
Salvesen, G., eds) pp. 3-22, Elsevier Science Publishers BV,
Amsterdam
Van Noort, J. M., and Van der Drift, A.C.M. (1989) J. Biol.
Chem. 2 6 4 , 14159-14164
Irvine, G. B., Blumson, N.L., and Elmore, D. T. (1983) Biochem.
J. 2 11,237-242
Vanderslice, P., Craik, C. S., Nadel, J . A., and Caughey, G. H.
(1989) Biochemistry 28,4148-4155
Hendrick, J. P., Hodges, P. E., and Rosenberg, L. E. (1989) Proc.
Natl. Acad. Sci. U. S. A. 86,4056-4060
Human Cytotoxic Lymphocyte GranzymeB
Supplem?ntal M a t e r i a l t o
101
Human Cytotoxic Lymphocyte Granzyme B
102
hmmQ3IgrmZymB
humanTSP g n n z y m 8
mwse CCPl gram8
human HF
25 16
20
llGGHEAKPHSR
I I G G H E A I: P H 5 R
I I G GH E V K P H 5 R
IIOGNEVTPHSR
16
20
25
P
P
P
P
30
VlAYL
1 MA I L
V R ALL
VRYLL
30
-
"Activation. f a r Q31 granzym 8 Yhen eluted frm Sephacryl 1300. Q31 granzme B had a
signif7cmt lag time in enzymtic assays. The lag before f u l l a c t i v i t y was attained can be
Seen more c l e a r l y i n h y d r o l y s i s of L-glYtMy~-t-naphthylmide than f o r 8MOT (See Fig. 4).
sincethena-enzymaticrate
of hydrolysis was m c h smaller than for BMOT. I t twl: 12 nin
f o r t h e enzymatic r a t e t o reach a steadyvalve i n trace A. Addition of 0.0DeDStltlnA
g r e a t l y extended the activation tin? (trace 81. dlthovghDeDstdtinA d i d nor i n h i b i t t h e
activated g r m z w B (see Table 5 ) .
I
Supernale Top Middle Lower Bottom
Layer Band Band Band Layer
Enzymatic a c t i r i t i e s o f
human 031 CTL Percoll fractions.
The cmss-natchedbars
represent the BMOT esterase activities of the whole fractions.
with the supernate and botto~l
l a y e r s i n h i b i t i n g the non-enzymatic 8MOT esterase a l i t t l e . and the Open b a l l IHlreSent BLT
esterase IctIvitieS.
were chmnutographed a t 4C
.
A f t e r s o l u b i l i z a t i o n i n a I.%NaCl the Percoll fractions
on SeDhdCryl S ~ W
i n IM N~CI a t p~ 4.5. a h the fractions containing BARLIT esterase a c t i v i t y
were d i l u t e d w i t h 4 volums Hz0 and chmmtographed on nonos as described i n the Methods
section(seeTable
2 for yields).
A t y p i c a l e l u t i o n o f f M o d I s s ~ m a r l z e din Fig. 2 .
showing a clear seoaration of Q31 granzym 8 (mainly i n f r a c t i o n 37) f r m Q31 tryptase
( f r a c t i o n s 35 and 36). Sincethe purified enzym had an N-terminal sequence (see Table 3)
I d e n t i c a l t o hman gram8 ( I S , 19. 33. 35. 36) (see Fig. 3) we propose t o call t h i s
p r o t e i n 931 grmzyme 8.
+30pM Pepstatin
0-
The lag phase cwld be g r e a t l y extended by i n c u b a t i m w i t h 0.020~44p w s t a t l n A. a potent and
s p e c i f i c I n h i b i t a r of aspartic proteinases (67-69).
PepstacinA was sham by Knight and
8 a r r e t t ( 7 0 ) t o be a ~ p O . I n n i n h l b i t o r of cathepsin 0; c a t h e w i n 0 has been found i n h a a n
IynQhOCyteS(71.72)
and CTL l y t i c granules (73). C a t h e p ~ l n0 has been s h o n t o prefer
Cleavage between two bulky hydrwhobic residues (74) i n M P h i p l t h i C alDha-hellCeS (75) an4 10
e x h i b i t a lag phase with certain Substrates (76). Haever.thepreprc-peptide
of human
g r a n z m 8 i s predicted t o be terminated by a L-glutmyl residue (30.33). as has a l s o been
predicted for the Drepm-DeDtideS of Fat mast cell chymase.human neutlmhil elastase and
cathepsin G. and mouse IynQhacyte CCP-I and mouse granzyms C.0.E and F (77). so c a t h e w i n 0
would not be expected t o d i r e c t l y activate proprc-granum 8. It was posslble that catheusln
0 served t o enhance the s e l f - a c t i v a t i o n of g n n z w 8. rime the pepstatin-inhibited
incubation i n Fig. 4 d i d eventually s h a s m L-glut~lyl-2-naphrhylMlide hydrolase a c t i v i t y .
Atwc-step p m t e o l y t i c cleavage has been f w n d In a c t i v a t i m of eight m1tahmdrial proteins
(781. The f i r s t DrOteOlYtiC c l e a v w e in the mitcchonerlal proteins *a5 SDeCifiC f o r Arq
FRllCTlW NUMBER
F i "re 2 E l u t i o n p r o f i l e f o r Q3lgranzynr 8 f r m ~0116.
The upper trace. lr$lled
A(280).
&V
monitortrace.
The s o l i d l i n e l a b e l l e d NaCl i s the NaCl cmcentratlm i n the
eluate. m e l w e r tracesrepresentenzymatic
a c t i v i t y : I ) BLT esterase(tryptaseactivity).
wen c i r c l e r and dashed lines; 2 ) BMOT esterase (granzym 8 a c t i v i t y ) , c l o s e d c i r c l e s
and
s o l i dl i n e r .
Yield of ~ o c A ~ & ~ I & ~ sesterase
D S ~ Z ~a c t i v i t y upon p u r i f i c a t i o n
of Q31 granrym 8.
Material
fractlm
Crude hmogenate
Percell
Sephacryl S3W eluate
w a n d eluate
Volume
-
3onL
Z M
l%WL
Id
rate
S p e c i f i c esterase Total
esterase
rate
u.4lmlnl20 L
3
2 7W
31W
0.345
24
I200
W '
QP
3
4
5
7
8
9
IO
v
23
25
23
8.3
21
22
12
13
3.5
11
12
13
14
15
16
17
6.2
2.8
I8
9.0
6.9
7.6
9.0
*I
*,
W '
4
43
31
4 21.5
4 14.4
4
F i w e 5 E l e c t m p m r e s i s on polyacrylamide gels of hnun Q31 CTL g l a n z m B i n 1% UaOWSO4
b r c a p t o e t h m o l . The lane second f p m l e f t represented a portion of a Sephacryl 5 3 ~
f r a c t i o n h i g h i n granzym 8 activity; the l a w second fmn right f r a c t i o n 37 from Fig. 2.
The l e f t m s t and r i g h t m s t lanes were lr markers for the two l e f t and two r i g h t IawS.
respectively.
Human Cytotoxic Lymphocyte Granzyme B
103
K i n e t l cc o n s t a n t sf o r
BAADT h y d r o l y s i s a t H7
a
Sumnanzed i n Fig. 6 are 1 7 m a w r m n t l of t h e net r a t e of BAAD& i % l y n r , w i t h
t h e o r e t i c a l curve (The I o l i d l i n e i n t h e F i g u r e ) f o r
Vmax of 6.93 n4lmin and Km of 0 . 1 5 d .
N m - l i n e a rl e a s t - s q u a r e ra n a l y s i s
of t h e d a t a r h a e d ,V.
t o be 6.93+ 1.62 mRlnin.which
corresponded t o k c p of 11
5 maleshydrolyzed per rec per male enz- e. and Km t o be 0.15:
of 76,000 44.0W M-l
0.06IM With C l t l l y t l Ee f f i c i e n c yk c a t l K m
WCr-
f
1
2
3
4
I
4t
I
5
6
*i
4-
7
8
9
10
11
12
13
14
15
16
11
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
BENZOYL
H
H
H
ILE
GLU(0R) ARG
GLV
LYSLEU
D-VAL
D-VAl
LEU ARG
D-ILE ARG PRO
D-PRO ARG PHE
ARGPRO
ARG
PHE
D-VAL
PHE
LEU PnE
PHE ALA ALA
LEU PHE
PnE
PHE
n
PYROGLU
H
succ
succ
succ
MeOSUCC
BENZOYL
CBZ
. ...
TYP
TYR
CBZ
succ
succ
IPP
PRO
PRO
ALA ALA
ALA ALA
GLUTARYL
H
succ
PHE GLY GLY
MeOSUCC
MET
ALA
ALA ALA ALA
ACETYL
8DC ALA PRO ALA ALA
VAL PRO
ALA
ALA
8oc
succ
ALA PRO ALA ALA
ACETYL
UETPROALA ALA
MeOSUCC
ASN ALA
BDC
BOC
ALA
Boc
SER ALA
Boc
ASP ALA
VAL PRO ALA ALA
MeOSUCC
MeOSUCC
8DC
PHE
PHE
PHE
LEU
ALA
ALA
ALA
ALA
12
0.7
11 + 9.5
21 f D.7
2.6 + 1.0
16 +-3.3
13 T 3.3
-7.3 + 1.1
-4.6
2.9
-0.1 f 5.5
-13 + 8.1
-128-+ 2.9
-34 *-14
-7.5-:
18
-6.5 + 9.8
-24 1-1.1
-3.9-+ 3.0
-4.2 7 1.6
1.3 i 5 . 2
9.8
5.9
-5.5 + 4 . 9
-16 -'7.5
2.9 + 1.3
6.5 I 5 . 9
-0.3
5.2
9.8 + 5.2
13
-17
8.1 * 12
30 +-I. 9
185-+75
:
UET
PHF
ALA
z
-21 T 45
-22 7 4.4
-13
f
8.1
0.05
0.15
0.10
BOC-ALA-ALA-ASP-SEA. rnM
EQependenceof
t h er a t e
'
of h y d r o l y s i s of B a A l a A l d S p S B z l by 931 g r a n l y n r B
Upon
concentration of 8 a A l a A l d r p S B z l .
A11dyO were done ~n b u f f e r A a t 21 C w i t h 1.2
0.4 nM
g r a n 2 y m 8. The net r a t e of h y d r o l y s i s i s d e f i n e d
as t h e Observed r a t e mmnur t h er a t eo f
h y d r o l y s i sw i t hs u b s t r a t ea l o n e( n a n - e n z y m l t ? cr a t e ) .
The t h e o r e t i c a l curve was Calculated
of 6.93 n 4 l m i n .w i t h a M i c h a e l i s
f o r a maximal v e l a i t v a t i n f i n i t e s u b s t r a t e c o n c e n t r a t i o n
c m s t a n t( c o n c e n t r a t i i of o rh a l f - m a x m a lv e l a i t y )
Of 0 . 1 M .
HEPES
1M EDTA, t h e n e t r a t e
of
{H depenaeny - Y i t h 0 . 1 Rn 8AADT i n D.3M NaCl 0.lM Na
BAA01 Ydro Y I l S y 031 granzyme 8 minus thenonemymatic rate between pH 5.5 and 9.5 i s
I h a n i n Fig. 1. The pH a t which 031 g r a n z p e B most r a p i d l yh y d r o l y z e d0 . l M
BAADT appeared
t o be between pH 7 and 1.5. It was n o t e w o r t h yt h a tt h e r e *as n g n i f i c a n t a c t i v i t y It pH6.
I n h i b i t i o n of L-91ut~.yl-Z-naphthylanridare
- The l n h i b i t i o n d a t a a l l e n b l e d i n T a b l e
5
c l e a r l y c m f i r n e d t h e sequence data I30.331, h i c h r h a e d 031 granzyme B t o be a s e r i n e
protease i n h i b i t o r . The lack of
p r o t e a r e ,i nt h ea p p r e c i a b l ei n h i b i t i o n
by human
m-1
r i m - d e p e n d e n c e r e p o r t e d w I t h m-1 DrOteale I n h i b i t o r meant t h a t any translent5 *ere CLnplete
i n 10sec. The O l i g q e p t i d e i n h i b i t o r TLCK S h a d a time-dependent i n h i b i t i m of 031 granzyme
8. A l e m i l o g a r i t m i c p l o t of enzymatic r a t e versus t i m e a f t e r TLCK a d d i t l o n rhowed t h a t a t
rate t o d e c l i n e t o l l e Of t h e
0.05 q l m L (0.087 M) TLCK t h e t i m e f o r t h e e n z m a t i c
d i f f e r e n c e between t h e u n i n h i b i t e d and s t e a d y - s t a t ei n h i b i t e d rate was 270 + 135 SeC. This
I n h l b i t i o n of L-glutanryl-2-naphthylanidare a c t i v i t y of human 031 CTL granzyme by
s e l e c t e dC m e r c i a l l y - a v a i l a b l ep r o t e a s ei n h i b i t o r s .A r r a y s
were d m e with 1 . W M
L-glUtMlyl-2-n~phthylamide i n D.3oW NaCl 0 . l W NIHEPES pH 7.0 1 M EDTA, except assays w i t h
EDTA and EGTA. Negative I n h i b i t i o n valuesCOTrelpMlded t o S t i m l a t i o n of enzyme a c t i v i t y .
Inhibitor
1. Time-independent i n h i b i t o r s
A. M a c r a n o l e c u l a r i n h i b i t a s
A1 Limabean t r y p s i n i n h l b i t w
A2 Soy bean t r y p s i n i n h i b i t o r
protease i n h i b i t m
A3
Human
m]
A4 B o v i n e a p r o t i n i n ( T r a l y l o l )
A5
Human
a n t i t h r m b i n 111
A6 Human aZ+acroglobulin
F i ure 7 Dependence upon pH o f h y d m l y l i l O f BocAl&41&4rpS&,l by 031 granryrae 8 . H y d m l y r i r
h e r e done i n b u f f e r A a t 21'C w i t h 0.1Dm4 B 0 ~ A l a A l d ~ l p S 8and
~ 1 U i t h 1.2nM 031 granzmne
8 . and were c o r r e c t e d f o r t h e h y d r o l y l l l i n t h e
absence O f e n l y e (nonenzymaticrate) t o g i v e
t h e net rate.
-
H y d m l y s i s of c h m m p h o r i c o l i g o p e p t i d e r
The rater Of h y d m l y s i s of 33 a l i g w e p t i d e
p - m t r o a m l i d e and D l i g e p e p t l d e t h i o b e n z y l esters by P u r l f i e d 031 g r a n z y m 8 are summarized
i n Table 4. The r a t e s were each lerr Than 203: O f t h e r a t e o f
BAADT h y d r o l y s i s (CoDpoYnd 301,
and molt were not S i g n i f i C m t l y d i f f w e n t fmn zero. Many o ft h es u b s t r a t e s
*ere p m r l y
s o t u b l e i t h i s was r e f l e c t e d i n n e g a t i v e ratel and largestandarddeviations.
8. 0 1 i g q e p t i d e i n h i b i t o r s
81 A n t l p l i n
82 Chymostatin
83 E l a s t a t i n &
84 Leupeptin
85 P e p s t a t i n
86 PhosphoPmidon
87 ZPCK IN--Cbr-L-Phe-CH2Cl)
C. L a mOleCUlar-welght i n h i b i t o r s
C1 Benzamidine
C2 EDTA
C3 EGTA
C4
PMSF
::
C oI n hc iebni t ri oa nt i o n
50 * 3
52 f 14
85
13
40'19
-3
6
66
2
0.25 mglmt
0.25 mglnrt
:
0.01 mg/mL
0.25 qld
5
1 glmL
0701 mg1mt
f
0.05
0.05
0.05
0.05
0.03
0.05
0.05
f
i
rqlk
35
11
*
T3
nqlk
1
'
M
875
mglk
65-+ 5
16 f 12
wlk
36
D.lM
lnw
I n w
Inw
2 . Time dependent I n h i b i t o r s
D. D l i g w e p t i d e i n h i b i t o r s
D l TLCK (N-~-Toryl-L-Lyr-CH2CI)
18
+
42 7 16
mglmt
mglk
kobrli
0.05 m g i k
40
:20 rec
© Copyright 2024 ExpyDoc
