The Proteolytic Specificity of the Natural Enediyne-Containing Chromoproteins Is Unique to Each Chromoprotein

The proteolytic specificity of the natural enediyne-containing chromoproteins is unique to each chromoprotein

N Zein*, P Reiss, M Bernatowicz and M Bolgar

Bristol-Myers Squibb Company, Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA

Background: Enediyne chromoproteins are potent histone Hl. This model peptide was incubated individu- antitumor antibiotic agents. They consist of a labile ally with similar concentrations of the kedarcidin, neo- nine-membered enediyne chromophore non-covalently carzinostatin and maduropeptin chromoproteins as well associated with a stabilizing acidic polypeptide. Studies as the kedarcidin apoprotein.The reaction products were in vitro on three members of this superfamily of natural analyzed by electrospray liquid chromatography/mass products - kedarcidin, maduropeptin and neocarzino- spectrometry. Our results indicate that all proteins cleave statin - demonstrated that their chromophores cleave the peptide at selected backbone amides, and that these DNA at sites specific to each chromophore. Recently, sites vary according to the chromoprotein used. we showed that these chromoproteins possess prote- Moreover, the kedarcidin apoprotein appears to be less olytic activity against histones ia vitro, with histone HI specific than the kedarcidin chromoprotein complex. as a preferred substrate. Based on these results, we specu- Conclusions: The small size, unique architecture and lated that this selective proteolytic activity may be very acidic nature of the enediyne chromoproteins are important in vim in the delivery of the enediynes intact highly unusual. These natural products exhibit the dual to the DNA in chromatin. functionalities of specific DNA cleavage and selective Results: We show here that each chromoprotein gener- proteolytic activity. This observation adds to the fasci- ates a unique set of Hl fragments as revealed by gel nating properties of these molecules and suggests that it analyses of the Hl cleavage reaction products.To probe is possible not only to design small moieties to cleave the observed cleavage specificity, we synthesized a 24- DNA but also to conceive of small proteins to deliver amino-acid peptide representing a basic region of these moieties intact to defined areas of chromatin.

Chemistry & Biology July 1995, 2:451-455 Key words: antitumor chromoprotein, enediyne, histone Hl cleavage, protease activity

Introduction component of the complexes served to stabilize and Enediyne chromoproteins are unusually potent anti- regulate the availability of the labile chromophores. tumor antibiotics with a unique molecular architecture. Having observed a lo-fold higher potency for the They consist of a labile nine-membered enediyne-con- KDC chromoprotein than the KDC chromophore in taining chromophore, non-covalently associated with a cell-based studies, we compared the activity of purified highly acidic, stabilizing polypeptide. These chromopro- KDC apoprotein, the intact chromoprotein and the teins include kedarcidin (KDC), neocarzinostatin purified chromophore against DNA and several pro- (NCS), macromomycin, actinoxanthin, maduropeptin teins in vitro. In contrast to the chromophore and the (MDP) and Cl027 [l-16]. A comparison of the amino- chromoprotein, the apoprotein had no activity against acid sequences (Hans Marquardt, personal communica- DNA. The chromophore, however, did not exhibit any tion) shows that KDC shares 38.7 % identity with NCS cleavage activity against any of the proteins tested, in a Ill-residue overlap (four gaps), 36.0 % identity whereas the apoprotein and the chromoprotein both with macromomycin in a ill-residue overlap (two cleaved proteins selectively. Among the proteins tested, gaps) and 40.2 % identity with actinoxanthin in a 107- histones, the most opposite in net charge to the highly residue overlap (five gaps).The amino-acid sequences of acidic apoprotein, were cleaved most readily. Moreover, MDP and Cl027 have not yet been disclosed. histone Hl, richest in lysines, appeared to be the preferred substrate 120,211. Similar activity was noted for Mechanistic studies have been performed primarily on both MDP and NCS chromoproteins (their corre- the chromophores of NCS, KDC, Cl027 and MDP sponding apoproteins were not tested for availability [l-20]. These chromophores were shown to cleave reasons). Histones combine with DNA to form chro- DNA in vitro at sites specific to each chromophore. matin, the material that makes up chromosomes. Based This DNA damage was proposed to be responsible for on our results and on chromatin structure, we specu- the potent biological activities of the holoantibiotics. lated that the highly acidic apoproteins may provide Furthermore, it was suggested that the apoprotein the chromophores with targeted delivery in vim to the

*Corresponding author.

0 Current Biology Ltd ISSN 1074-5521 451 452 Chemistry & Biology 1995, Vol 2 No 7

Apoprotein~-

Unknown mechanism of uptake “: ‘,,‘:’

Fig. 1. The protein component of the chromoprotein assists in delivery of the chromophore to DNA. Proposed scheme for the way in which the highly acidic apoproteins may provide the chromophores with targeted delivery in vivo to the spacer DNA, by cleaving histone HI. The chromophore is shown in green.

spacer DNA, after cleaving HI (Fig.l).These observa- set of fragments unique to each chromoprotek regardless tions, along with the fact that different chromophores of chromoprotein concentration (Fig. 2). target different DNA sequences, led us to investigate whether these natural proteases exhibit specificity in Incubation of HZ with varying concentrations of the KDC their Hl cleavage. apoprotein (10 pg ml-’ to 1 mg m-l) and comparison of the resulting cleavage profiles with those obtained using Here we show that the chromoproteins described above cleave histone Hl to yield a set of fragments unique to each chromoprotein used.To probe the specificity exhib- (a) (b) ited by the chromoproteins, we synthesized a 24amino- 123456 $23456 7 acid peptide, representing a particularly basic region of - histone Hl 122-281. The peptide was incubated individually with similar concentrations of the KDC, NCS and MDP chromoproteins as well as the KDC apoprotein. The reaction products were then analyzed by electro- - 29 kDa spray liquid chromatography/ mass spectrometry (LC/MS) [29]. Our results indicate that each chromoprotein specifically cleaved the peptide bonds at different sites, and that the KDC apoprotein is less specific than - 14.3 kDa the KDC chromoprotein complex.

Results and discussion Cleavage of histone Hl by the KDC, NCS and MDP chromoproteins and by the KDC apoprotein Incubation of Hl with 10 Pg ml-’ (=50:1 molar ratio Hl:chromoprotein) of KDC, NCS and MDP chromoproteins results in cleavage profiles that vary according to the chromoprotein used as shown by SDS-PAGE (Fig. 2, NCS and MDP cleavage patterns shown). At this con- Fig. 2. SDS-PAGE of the reaction products of (a) NCS and (b) centration, MDP produces higher molecular weight frag- MDP with calf thymus histone Hl. Reaction conditions: ments than do NCS and KDC, suggesting that MDP 50 mM Tris-HCI (pH 7.5), 1 mg ml-’ Hl and varying concen- may be more selective in its HI cleavage. trations of NCS and MDP in a total volume of 10 ml, 37 “C overnight. The control reactions were carried out under identi- cal conditions except that the chromoproteins were replaced To ensure that the difference in Hl cleavage pattern is by pure water. Lane 1 : control reactions with HI. Lanes 2, 3, due to a difference in specificity among the three chro- 4, 5 and 6: reactions of HI with 10 tq ml-‘, 50 )*g ml-‘, moproteins and not to a difference in activity, Hl was 100 pg ml-‘, 500 wg ml-’ and 1 mg ml-‘, respectively, of incubated with several concentrations of the three chro- NCS (a) and MDP (b). Lane 7: protein size standards from 200 to 14.3 kDa. The band corresponding to NC5 protein seen in moproteins, ranging from 10 p,g ml-’ to 1 mg ml-’ .This (a) (lanes 5 and 6) migrates close to the 14.3-kDa marker and variation in concentration did not alter the above results, that corresponding to MDP protein in (b) (lanes 5 and 6) that is, the three chromoproteins cleaved Hl to yield a migrates close to the 29-kDa marker. Specific proteolysis by chromoproteins Zein et al. 453

Fig. 3. Cleavage of peptide substrate by apo/chromoprotein. (a) Peptide 1 with (a) ‘AEKTPVKKKA “AKKPAGAKRK “ASGP-NH, numbered residues. The asterisks in (b)-(e) (b) AEKTPVKKK*A AKKl’AGAR*R*K*A*SGP-NH indicate the observed cleavage sites caused by: (b) KDC apoprotein, (c) KDC (c) AEKTPVKKK*A AKKPA*GARRK ASGP-NH, chromoprotein, (d) NCS and (e) MDP. (d) AEKTPVKK*K*A AKKPAGARRK* ASGP-NH, (e) AEKTPVKKKA AKKPAGARRK* ASGP-NH, I the KDC chromoprotein suggested a slight difference in NCS are after LysX, Lys9 and Lys20. MIIP cleaves the their cleavage patterns (data not shown). However, the peptide at one major site, which is after Lys20. These observation that at equal concentrations there is a differ- cleavage sites resemble those found for proteases in that ence in activity between the apoKDC and the KDC their substrates have common PI sites (nomenclature of complex (211 makes it difficult to draw conclusions Schechter and Berger 1301) f or cleavage, specifically ly5ine about their difference in specificity.The specificity differ- and arginine (trypsin-like). Thus, the proteins appear to ence, however, becomes clearer in the study using the exhibit a sequence-dependent substrate specificity. peptide as substrate. To ensure that the difference in peptide cleavage is due Peptide cleavage by the KDC, NCS and MDP chromoproteins to a difference in specificity among the chromoproteins LC/MS analyses show that incubation of peptide 1 (Fig. and not to a difference in activity, poptide 1 was incu- 3a) with KDC, NCS and MDP chromoproteins at a bated with either 100 pg nil-’ of KIX or 100 pg nil-’ concentration of 10 kg ml-’ results in peptide bond of NCS. This lo-fold increase in protein concentration cleavage at specific sites. did not alter the cleavage data. MDP was not assayed at the higher concentration due to lack of material. The LC/MS results are summarized in Figure 3 and Table l.As expected from the Hl study described above, The results obtained in the peptide-cleavage reacrionc the cleavage Fites vary according to the chromoprotein mirror those observed in the Hl cleavage experiments. used. The KDC chromoprotein cleaves the peptide after Each of the protcolytic proteins gives a unique set of two major sites, Lys9 and Ala1 5. The points of attack by cleavage products. Although the active-site residues

Table 1. Cleavage sites of the chromoproteins.

Chromoprotein Retention time MW Sequence Cleavage site

KDC 14:45 897.9 16-24 GAKKKASGJ” A%’ 14:51 1028 IL9 AEKJPVKKK K*A 16:05 1594.6 l-l 5” AEKTPVKKKAAKKPA A*G 16:20 1464.4 1 O-24” AAKKPACARRKASGP K*A 17:06 2474.7 l-24”

NCS 4:51 329.2 21-24” ASCP K*A 1535 1028.1 l-9 AEKJPVKKK K*A 16:08 899.7 IL8 AEKJPVKK K*K 16:48 1592.7 9-24” KAAKKPAGARRKASGP K*K 16:52 1464.4 1 O-24” AAKKPAGARRKASGP K*A lb:57 2163.5 l-20 AEKTPVKKKAAKKPAGARRK K*A ord 2-21 EKTPVKKKAAKKPAGARRKA A*& 17:31 2475.0 I-24” -

MDP 4:45 329.3 21-24” ASGP K*A 16:45 2163.5 l-20 AEKTPVKKKAAKKPAGARRK K*A e 2-21 EKTPVKKKAikKPAGARRKA A*EyrA*S 17:17 2474.7 l-24”

“Unique sequence based on MW. hUnderlined portions of sequence were deduced by tandem mass spectrometry. ‘Asterisk indicates site of cleavage. dFor retention time (R.T.) 16:57, there are two possible sequences based on molecular weight; distinction by MS/MS was unsuccessful. However, since fragment 21-24 at R.T. 4:51 was observed, it is most likely that the fragment at R.T. 16:57 corresponds to l-20. %r R.T. 16:45, there exist two possible sequences based on molecular weight; distinction by MS/MS was unsuccessful. However, since fragment 21-24 at R.T. 4:45 was observed, it is most likely that the fragment at R.T. 16:45 corresponds to I-20. i 454 Chemistry & Biology 1995, Vol 2 No 7

Table 2. Cleavage sites of the KDC apoprotein.

Retention Time MW Sequence Cleavage Site

4:56 258.1 22-24” SGP A9 5:08 329.1 21-24 ASGP K*A 5:14 457.4 20-24a KASCP R*K 15:30 1028.1 l-9 AEKJPVKKKb K*A 16:47 2163.7 l-20 AEK-JPVKKKAAKKPAGARRK K*A 16:55 1879.1 l-18a AEKTPVKKKAAKKPACAR R*R 17:03 2035.1 I-IF AEKTPVKKKAAKKPAGARR R*K 17124 2474.7 1 -24a -

aUnique sequence based on MW. bunderlined portions of sequence were deduced by tandem mass spectrometry. CAsterisk indicates site of cleavage.

remain to be identified, our results are consistent with activity together with a knowledge of the chemi- the low degree of homology shared between the three stry of their corresponding enediyne chro- chromoproteins.The amino-acid sequences of KDC and mophores could be invaluable for the design of NCS share only 38.7 % sequence identity. In addition, targeted antitumor agents. preliminary data on the amino-acid sequence of MDP show that it shares no homology with KDC or NCS (H. Marquardt, personal communication). These obser- Materials and methods vations suggest that at least some of the amino acids Reagents involved in the protease active sites are different and KDC chromoprotein and chromophore, MDP and NCS chro- raise interesting questions regarding the evolutionary moproteins, prepared as described [2,12,21], were kindly pro- vided by J.E. Leet, D.R. Schroeder and J. Golik, respectively implications of the different specificities observed. (Division of Chemistry, Bristol-Myers Squibb, Wallingford, CT), The 24-amino-acid peptide was synthesized by the solid- Peptide cleavage by the KDC apoprotein phase method using Boc/Benzyl chemistry [28]. The chemicals Incubation of the KDC apoprotein (10 pg ml-l; 9 x and solvents used in peptide synthesis were purchased from 1O-7 M) with peptide 1 (9 x 1O-4 M) results in several Applied Biosystems. Calf thymus histone Hl was obtained major cleavage sites.As can be seen from Figure 3 and from Boehringer Mannheim. Table 2, these sites are after the following amino acids: Lys9, Arg18, Arg19, Lys20 and Ala21. These results indi- Cleavage of histone H 7 by the KDC, NCS and MDP cate that the apoprotein cleaves the peptide at more chromoproteins and by the KDC apoprotein sites than its corresponding chromoprotein. This differ- Histone Hl (4 x 10m5 M) was incubated separately with the ence in proteolytic spec&city between the apo and the KDC apoprotein, KDC, NCS and MDP chromoproteins chromokedarcidin is intriguing. In the absence of (IO p,g ml-’ = IO-’ M; 50 p,g ml-‘; 100 kg ml-l; 500 pg ml-‘; NMR data and a crystal structure of the KDC chromo- 1 mg ml-‘), in 50 mMTris-HCl buffer at pH 7.5.The incubation was carried out at 37 “C overnight at = 50:1 to 0.5:1 protein, it is difficult to offer a definitive interpretation molar ratio in histone: apo/chromoprotein in a total volume of of this result. An appealing explanation, however, is that 10 ~1. The samples were then heated in a denaturing dye for the chromophore induces a small conformational one minute and analyzed on a 17 % SDS polyacrylamide gel. change on the protein such that the structure of the The protein bands were visualized with Coomassie blue. active site of the chromoprotein is slightly different from that of the apoprotein. Reaction of the chromoproteins/apoprotein with the synthetic peptide Peptide 1 (9 x IO-” M) was reacted individually with the Significance KDC chromoprotein (10 kgml-’ = 8 x lo-‘M and Many antitumor antibiotics mediate their effects 100 kg ml- 1 = 8 x 10e6 M), the NCS chromoprotein by damaging DNA. Often, the efficacy of these (IO kg ml-l = 8 x IO-’ M and 100 pg ml-’ = 8 x 1O-6 M), the agents suffers from their poor solubility, low sta- MDP chromoprotein (IO mg ml-’ = 3 x IO-’ M) and the apokedarcidin (10 p,g ml-‘; 9 x lo-’ M) in 50 mM Tris-HCl bility and inefficient cellular uptake. The enediyne buffer at pH 7.5 at 37 “C overnight. chromoproteins seem to have naturally overcome these problems, thanks to their apoprotein com- Purity of the chromoproteins studied ponent. The apoprotein serves multiple purposes: HPLC analyses and/or visualization by gel-staining with it solubilizes the chromophore, shields it from Coomassie blue showed single bands corresponding to the deactivation and apparently facilitates its deli+ery chromoproteins. Different preparations of the chromopro- to the DNA in the nucleus. A more thorough teins were tested in the histone HI cleavage assay [21] and understanding of the substrate specificity of these the activity was consistent from batch to batch. As an addi- proteins and their mechanisms of proteolytic tional check that the proteolytic activity is associated with Specific proteolysis by chromoproteins Zein et al. 455 the chromoprotein, KDC was chromatographed using a size 1. Antibiot. (Tokyo) 21, 44-49. exclusion column and fractions were collected throughout 9. Khoklov, A.S., et a/., & Fomina, I.P. (19691. Physico-chemical and biological studies on actinoxanthin, an antibiotic from Actinomyces the entire run. Each fraction was then assayed for Hl cleav- globisporus 1131. /. Antibiot. (Tokyo) 22, 541-544. age activity [21]. The fraction m which the chromoprotein 10. Khoklov, A.S., Reshetov, P.D., Chupova, L.A., Cherches, B.Z., Zhigis, eluted contained essentially all of the activity placed on the L.S. & Stoyachenko, I.A. (1976). Chemical studies on actinoxanthin. column (D.W. Phillipson. unpublished results). I. Antibiot. (Tokyo) 29, 1026-l 034. 11. Hanada M., et al., & Forenza, S. (1991). Maduropeptin, a complex of new macromolecular antitumor antibiotics. /. Antibiot. (Tokyo) Electrospray liquid chromatography/mass spectrometry 44, 403-414. (LUMS) analyses of the reaction mixtures 12. Schroeder, D.R., et al., & Doyle, T.W. (1994). Isolation, structure The reaction mixtures were analyzed by LC/MS [29]. All determination, and proposed mechanism of action for artifacts of maduropeptin chromophore. /. Am. Chem. Sot. 116, 9351-9352. LC/MS data was obtained on a Finnigan TSQ700 (San Jose, 13. 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