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J. Biol. Chem., Vol. 276, Issue 18, 15177-15184, May 4, 2001
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From the The cytotoxic lymphocyte serine
proteinase granzyme B induces apoptosis of abnormal cells by cleaving
intracellular proteins at sites similar to those cleaved by caspases.
Understanding the substrate specificity of granzyme B will help to
identify natural targets and develop better inhibitors or substrates.
Here we have used the interaction of human granzyme B with a cognate
serpin, proteinase inhibitor 9 (PI-9), to examine its substrate
sequence requirements. Cleavage and sequencing experiments demonstrated that Glu340 is the P1 residue in the PI-9 RCL,
consistent with the preference of granzyme B for acidic P1 residues.
Ala-scanning mutagenesis demonstrated that the P4-P4' region of the
PI-9 RCL is important for interaction with granzyme B, and that the P4'
residue (Glu344) is required for efficient
serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9
RCL sequence and containing either P1 Glu or P1 Asp were cleaved by
granzyme B (kcat/Km 9.5 × 103 and 1.2 × 105
s
Importance of the P4' Residue in Human Granzyme B
Inhibitors and Substrates Revealed by Scanning Mutagenesis of the
Proteinase Inhibitor 9 Reactive Center Loop*
,,,
, and**
Department of Biochemistry and Molecular
Biology, Monash University 3800, Melbourne, Victoria, Australia,
§ Biomedicinal Chemistry Group, School of Pharmacy, The
Queen's University of Belfast, Belfast BT9 7BL, United Kingdom, the
¶ Australian Centre for Blood Diseases, Box Hill Hospital, Box
Hill 3128, Melbourne, Victoria, Australia, and the Baker Medical
Research Institute, St. Kilda Central
8008, Melbourne, Victoria, Australia
1 M1, respectively) but were
not recognized by caspases. A substrate containing P1 Asp but lacking
P4' Glu was cleaved less efficiently (kcat/Km 5.3 × 104 s1 M1). An
idealized substrate comprising the previously described optimal P4-P1
sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was
efficiently cleaved by granzyme B
(kcat/Km 7.5 × 105 s1 M1) and was
also recognized by caspases. This contrasts with the literature value
for a tetrapeptide comprising the same P4-P1 sequence
(kcat/Km 6.7 × 104 s1 M1) and
confirms that P' residues promote efficient interaction of granzyme B
with substrates. Finally, molecular modeling predicted that PI-9
Glu344 forms a salt bridge with Lys27 of
granzyme B, and we showed that a K27A mutant of granzyme B binds
less efficiently to PI-9 and to substrates containing a P4' Glu. We
conclude that granzyme B requires an extended substrate sequence for
specific and efficient binding and propose that an acidic P4' substrate
residue allows discrimination between early (high affinity) and late
(lower affinity) targets during the induction of apoptosis.
*
The work was supported by grants from the National Health
and Medical Research Council of Australia.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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