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Chapter 16

Presence of Catalytic Activity of the Antibody Light Chain Raised against Complementarity Determining Region Peptide of Super Catalytic Antibody

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Y . Zhou, E. Hifumi, H. Kondo, and T . Uda* Department of Biosciences, Hiroshima Prefectural University, Shobara City, Hiroshima 727-0023, Japan *Corresponding author: Phone: 81-824-74-1756; fax: 81-824-74-0191; email: [email protected]

A monoclonal antibody i41SL1-2 raised against the peptide of complementarity determining region-1 (CDRL-1) of super catalytic antibody, 41S-2-L, which is capable of emzymatically destoroying the gp41 molecule of H I V - 1 envelope, was prepared. The light chain, i41SL1-2-L, catalytically decomposed the CDRL-1 peptide through the successive reaction. Based on the molecular modeling, i41SLl-2 possesses a catalytic triad composed of Ser, His, Asp, whose positions are identical to those of the catalytic antibody, VIPase.

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Introduction Some interesting natural antibodies possessing high catalytic efficiency have been reported so far (1-5). The characteristic ones are the autoantibodies to vasoactive intestinal polypeptide (VIP) by Paul et al. (1) and to D N A by Gabibov et al. (2). Authors have recently found a novel natural catalytic antibody (referred to as "super catalytic antibody") (6-9). The light chain (41S2-L) of the antibody could enzymatically decompose the antigenic gp41 peptide (19 mer peptide: a highly conserved region in many HIV-1 strains) as well as the intact gp41 molecule which plays an important role for the entry of human immunodeficiency virus (HIV) into human T cells (10-11). It is revealed that the complementarity determining region-1 (CDRL-1; R S S K S L L Y S N G N T Y L Y ) of the 41S-2-L substantially concerns with the antigen recognition (12). In this paper, we will describe that the light chain of the antibody (J41SL1-2) raised against the C D R 1 of 41S-2-L has the ability to enzymatically decompose the antigenic CDRL-1 peptide. The molecular modeling suggests the presence of a serine protease-like catalytic site on the surface of the light chain of i41SLl-2.

Experimental The i41SLl-2 mAb was raised against the synthetic peptide of CDRL-1-Cys (Cys was introduced at the C-terminus in order to conjugate with K L H ) by immunizing Balb/c mice. The peptides used in this study were synthesized by the Fmoc solid-phase method using an automated peptide synthesizer (Applied Biosystems 431 A , C A , USA). The resultant peptides were purified by reversedphase H P L C (RP-HPLC; Waters 490E, Waters m B O N D A S P H E R E C i column; Waters, N Y , U S A ) and the purities were confirmed to be over 99% (data not shown). The peptide identification was established using an ion-spray type mass spectrometer (API-in, Perkin-Elmer Sciex, Ontario, Canada; orifice voltage = 85 volts). Light and heavy chains of the antibody were isolated, purified and refolded according to the procedures described in the preceding references (6-8). Prior to carrying out the degradation reaction of CDRL-1 by the light chain of i41SLl-2 mAb (i41SLl-2-L), most glassware, plastic ware and buffer solution were sterilized by heating (180 °C, 2 hr), autoclaving (121 °C, 20 min) or passing through a 0.2 pm sterilized filter Manipulations in the experiment were mostly performed in a safety cabinet to avoid contamination from the ah. The degradation reaction was carried out in 7.5 m M phosphate containing 5% D M S O , 9 m M HEPES (pH 7.1) at 25 °C. For monitoring of the reaction, 20 pi of the reacting solution was injected into the reversed phase H P L C (Jasco, 8

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202 Tokyo, Japan) under the isocratic condition with the column temperature of 40 °C. Messenger R N A was isolated from the hybridoma secreting i41SLl-2 mAb using a m R N A purification kit (Amersham Pharmacia Biotech U K Ltd, U K ) . Then the c D N A s of the heavy and light chain were synthesized by first-strand c D N A Synthesis K i t (Life Science Inc., F L , U S A ) . Sequencing was carried out using the Auto Read Sequencing K i t (Amersham Pharmacia Biotech) and an automated D N A sequencer (ALF n, Amersham Pharmacia Biotech). The computational analysis was performed by a work station (Silicon Graphics Inc., P A , U S A ) and the software A b M (Oxford Molecular Ltd., Oxford, U K ) which is for building up the three dimensional molecule. The resultant P D B data was applied to minimize the total energy by using the software Quanta 96 (Molecular Simulations Inc., C A , USA). This software uses the algorithm C H A R M M for the energy minimization of a molecule (13). For the graphics of the structure, the software Protein Adviser Ver. 3.5 (FQS Ltd., Fukuoka, Japan) was employed.

Results and Discussion The established i41SLl-2 mAb could specifically bind to the antigenic peptide (CDRL-1-Cys) but not cross-react with other peptides (gp41 peptide, gpl20 V 3 loop peptide of fflV-1 and VIP) and proteins (HSA, O V A , B S A , p24 of HTV-1). The apparent affinity constants of the intact i41SLl-2 mAb, its heavy and light chain for the antigenic peptide were evaluated by using E L I S A . The values were 3.6 X 1 0 , 2 . 7 X 1 0 , 1.8 X 1 0 / M , respectively. The affinity of the heavy chain was lower than the intact antibody by about one hundred fold. That of the light chain was also lower than the heavy chain by about ten fold. These are normal values as observed when the subunits were isolated from the intact antibody (8). 9

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Sequencing of the cDNAs of variable region of the heavy and light chain of i41SLl-2 mAb was performed and the amino acid sequences were deduced as presented in Figure 1. Sequence of the light chain showed the highest score to germ line bd2. Molecular modeling was carried out utilizing the deduced amino acid sequences. Figure 2 shows the three-dimensional structure of the variable region of i41SLl-2 mAb. A potential catalytic triad composed of Asp, Ser, and His, which are present as a catalytic site of many serine proteases, is generated in the structure. Interestingly, the three amino acid residues (Asp in FR-1, Ser in CDR1, H i s in CDR3) are located in the identical positions as reported by Paul for VIP cleaving catalytic antibody light chain (VIPase) (14). Figure 3 shows the comparison of the amino acid sequences of the light chains between i41SLl-2 and VTP cleaving antibody. This coincidence with respect to the positions of three residues implies that the light chain of i41SLl-2 mAb has an ability to catalytically cleave the antigenic peptide, C D R L - 1 . 1

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a) heavy chain 10 Q V Q L Q H S G A E L V R P G S S V C A G GTT C A A CTG C A G CAC TCT GGG GCT G A G CTG GTG A G G CCT GGG TCT TCA GTG 20 31 35 K V S C K A L G Y T S T D Y E I H W A A G GTG TCC TGC A A G GCT TTG GGC TAC ACA TCT ACT GAC TAT G A A ATA CAC TGG CDR1 40 50 52 A 53 V K Q T P V R G L E W I G A I H P G G T G A A G C A G A C A CCT GTG CGT GGC CTG G A A TGG ATT GGA GCT ATT CAT CCA GGA 60 65 70 S D V I V Y N Q K F K G T A T L T A AGT GAT GTT ATT GTC TAC A A T C A G A A G TTC A A G GGC A C G GCC A C A CTG ACT GCA CDR2 80 82 A B C 83 D K S S S T A Y M E L R S L T S E D GAC A A A TCC TCC AGC ACA GCC TAC A T G GAG CTC A G A AGT CTG A C A TCT G A G GAC 90 95 102 S A V Y Y C T R E G G S V D Y V W G TCT GCT GTC TAT TAC TGT ACA AGA G A G GGG G G A TCT GTT GAC TAC GTT T G G GGC CDR3 110 Q G T L V T V S C A A GGG ACT CTG GTC ACT GTC TCT

b) light chain D V V M T Q (from amino acid sequence analysis) D I V M T Q T Q L T L T I N I G Q P GAC ATT GTG A T G ACC CAG ACT C A A CTC ACT TTG A C G A T T AAC ATT GGA CAA CCA 20 24 27A B C D E 28 A S I S C K S S Q S L L D S D G K T GCC TCC ATC TCT TGC A A G TCA AGT C A G AGC CTC TTA GAT AGT GAT GGA A A G A C A CDR1 34 Y L N W L F Q R P G Q S P K R L I Y TAT TTG AAT T G G T T G TTC C A G A G G CCA GGC C A G TCT CCA A A G CGC CTA ATC TAT 50 56 60 L V S K L D S G V P D R F T G S G S CTG GTG TCT A A A CTG GAC TCT GGA GTC CCT GAC A G G TTC ACT GGC AGT GGA TCA CDR2 70 80 G T D F T L K I S R V E A E D L G V GGG A C A GAT TTC A C A CTG A A A ATC AGC A G A GTG G A G GCT GAG GAT TTG GGA GTT 89 97 100 Y Y C W Q G T H F P L T F G A G T K TAT TAT TGC TGG C A A GGT A C A CAT TTT CCT CTC A C G TTC GGT GCT GGG ACC A A G CDR3 107 L E L R C T G G A G CTG A G A

Figure I. The nucleotide sequences of heavy and light chain variable region of 141SL1-2 and fie deduced amino acid sequences. Underline (=) is fie region of the primer. In the light chain, six amino acid residues at N-terminus were determined by amino acid sequence analysis as indicated with bold letters.

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Figure 2. Structure of the variable region of 141SL1-2 by molecular modeling. A catalytic triad composed ofAsp , Ser , His is presented in the structure of light chain of 141SL1-2 variable region. The positions of the three amino acid residues are identical to those of the VIP cleaving light chain reported by Paul etal. (14). 1

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In accordance with the above assumption, the degradation reaction of the C D R L - 1 peptide was carried out by using i41SLl-2-L. As shown in Figure 4, the degradation of the CDRL-1 peptide initiated after mixing of 141SL1-2-L and C D R L - 1 . The CDRL-1 peptide completely disappeared at about 47 hr. In contrast, the CDRL-1 peptide failed to be degraded without i41SLl-2-L. No induction period was observed in this case during the degradation reaction, which is different from the case of super catalytic antibody 41S-2-L (6-8). The H P L C chromatogram is presented in Figure 5. A new peak appeared at the retention time of 19.4 min gradually increased, then reached the maximum and decreased as the reaction time elapsed. The peak was collected and submitted to the analysis of mass spectroscopy, showing m/z ([M+H] ) = 1709.8 and m/z ([M+2H] ) = 855.7. These signals indicate that the molecular weight is 4

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1709.1(±0.4). This is coincident with that of S S K S L L Y S N G N T Y L Y , whichis the fragmented peptide of CDRL-1 ( R S S K S L L Y S N G N T Y L Y ) . Consequently, it is suggested that the peptidic bond between Arg^Ser of the C D R L - 1 was cleaved by i41SLl-2-L. The selective scission for R - X bond is a characteristic feature of serine protease. This fact agrees with the feature of i41SLl-2-L possessing a catalytic triad composed of Asp, His, and Ser. Though 141SL1-2-L did not cross-react with VTP, the germ line is considered to be the same (bd2) as the VIP cleaving antibody light chain. From the viewpoint of natural catalytic antibody, it is very important to clarify how the germ line which produces the antibody generating a catalytic triad, is recruited. 2

Marten et al.; Biological Systems Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

Marten et al.; Biological Systems Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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1 10 141SL1-2 light chain D W M T Q T Q L T L T M

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Figure 4. Time course of the degradation of CDRL-1 peptide by the antibody light chain, 141SL1-2-L CDRL-1, 50 pM; 141SL1-2-L, 0.4 pM; reaction temperature, 25 °C; reaction buffer, 7.5 mMphosphate containing 5% DMSO, 9 mM HEPES (pH 7.1). The CDRL-1 antigenic peptide was immediately degraded by mixing with 141SL1-2-L (0), and completely disappeared at 47 hr. TheCDRL-1 antigenic peptide failed to be degraded without i41SLl-2-L (A). No induction period was observed in this degradation reaction unlike the case of super catalytic antibody 41S-2-L.

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Reaction time (hr)

FigureS. HPLC chromatogram for the degradation of CDRL-1 peptide. Column, PuresilCis, Waters; HPLC, Jasco corporation Eluent, 21% acetonitrile including 0.065% TFA under 40 °C at 0.5 ml/min. CDRL-1 peptide at the retention time of 14 min decreased after the mixing with i41SLl-2-L (arrows) andfinallydisappeared at 47 hr. On the other hand, a new peak at 19.4 min was observed at the reaction time of 24.8 hr, which increased, then reached a maximum and decreased The peak was identified as being the fragmented peptide SSKSLL YSNGNTYL Y by mass spectroscopy.

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NLISNLYKI^AMQKRIA^INDTWADSH RSSKS L L Y S N G N T Y L Y case 1

VIP : CDRL-1 peptide :

NLISNLYKIO^AMQ^i^RTY^DTFVADSH RSSKS L L Y S N G N T Y L Y case 2

Figure6. Comparison of the amino acid sequences between CDRL-1 peptide and VIP. Underline (=) is a part of the consensus sequence. Casel and 2 are the comparisons of the sequences at the different positions.

Figure 6 compares the sequences of VIP with C D R L - 1 . Three amino acid residues are coincident in case 1 or 2. However, a clear conclusion which kind of feature of immunized antigen recruits bd2 germline cannot be drawn at present time. Conclusively, this study strongly suggests the way how to explore the natural catalytic antibody by searching the location of Asp, Ser, His residues in light chain after establishing mAbs raised against peptides.

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Acknowledgements This study has been supported in part by the Grant-in-Aid (111793007, 12035222, 09217249 and 10180225) for Scientific Research of the Ministry of Education, the Yazaki Memorial Foundation for Science & Technology, the Extensive Research Program of Hiroshima Prefectural Government, and The Association for the Progress of New Chemistry.

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References 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14.

Paul, S.; Voile, D. J.; Beach, C. M.; Johnson, D. R.; Powell, M. J.; Massey, R. J. Science 1989, 244 , 1158-1162. Shuter, A . M.; Gololobov, G.V.; Kvashuk, O. K . ; Bogomolova, A . E.; Smirnov, I. V . ; Gabibov, A . G. Science 1992, 256 , 665-667. Sun, M.; Gao, Q. S.; Li, L.; Paul, S. J. Immunology 1994, 153, 5121-5126. Takagi, M.; Kohda, K . ; Hamuro, T.; Harada, A . ; Yamaguchi, H.; Kamachi, M . ; Imanaka, T. FEBS Letter 1995, 375 , 273 -276. Matsuura, K.; Ikoma, S.; Yoshida, K.; Shinohara, H. Biochem. Biophy. Res. Commun. 1998, 243, 719-721. Uda, T.; Hifumi, E.; Okamoto, Y.; Zhou, Y.; Ishimaru, M. Clinical Science, Supplement 2 1998, 429-433. The 12th World AIDS Conference, Geneva (Switzerland). Hifumi, E.; Okamoto, Y.; Uda, T. J. Biosci. Bioeng. 1999, 88, 323-327. Hifumi, E.; Okamoto, Y.; Uda, T. Appl. Biochem. Biotech. 2000, 83 , 209220. Uda, T.; Hifumi, E.; Ohara, K . Chem. Immunol. 2000, 77, 18-32. Vella, C.; Ferguson, M.; Dunn, G.; Meloen, R.; Langedijk, H.; Minor, P. D . ; Gen. J. Virol. 1993 , 74 , 2603-2607. Kennedy, R. C.; Henkel, R. D.; Pauletti, D.; Allan, J. S.; Lee, T. H.; Essex, M . ; Dreesman, G. R. Science 1986, 231, 1556-1559. Hifumi, E.; Sakata, H . ; Nango, M.; Uda, T. J. Mol. Catal. A: Chemical 2000, 155, 209-218. Anchin, J. M.; Mandal, C.; Culberson, C.; Subramaniam, S.; Linthicum, D . S. J. Mol. Graphics 1994, 12 , 257-266. Gao, Q. S.; Sun, M ; Tyutyulkova, S.; Webster, D.; Rees, A . ; Tramontano, A . ; Massey, R. J.;Paul, S. J. Biol. Chem. 1994, 269, 32389-32393.

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