Extended-Spectrum Cephalosporinase in. Acinetobacter baumannii

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1 AAC Accepts, published online ahead of print on 1 June 0 Antimicrob. Agents Chemother. doi:./aac Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 REVISED AAC Extended-Spectrum Cephalosporinase in Acinetobacter baumannii José-Manuel Rodríguez-Martínez, 1 Patrice Nordmann, 1 * Esthel Ronco, and Laurent Poirel 1 Service de Bactériologie-Virologie, INSERM U1 Emerging Resistance to Antibiotics, Assist. Publique/Hôp. de Paris, Faculté de Médecine and Université Paris-Sud, Hôpital de Bicêtre, K.-Bicêtre, 1 and Service de Microbiologie, Hôpital Raymond Poincaré, Assist. Publique/Hôp. de Paris, Faculté de Médecine Paris-Ouest, 0 Garches, France Keywords: Extended-Spectrum AmpC, A. baumannii, cefepime Running title: ESAC in A. baumannii * Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, rue du Général Leclerc, Le Kremlin-Bicêtre Cedex, France. Phone: Fax: nordmann.patrice@bct.aphp.fr Downloaded from on May, 01 by guest 1

2 An AmpC-type β-lactamase conferring high level resistance to expanded- spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C ß-lactamase (named ADC-) possessed a ProArg substitution together with a duplication of an Ala residue at position 1 (inside the Ω-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC- hydrolyzed ceftazidime, cefepime and aztreonam at high level, that allows to classify this enzyme as an Extended- Spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property. Downloaded from on May, 01 by guest

3 Acinetobacter baumannii is commonly associated with serious nosocomial infections (,, ). The emergence of multidrug-resistant A. baumannii strains, and specifically those resistant to carbapenems, has created severe therapeutic challenges. A growing number of β-lactamases conferring resistance to expanded-spectrum cephalosporins have been identified in Acinetobacter spp. Resistance to oxyiminocephalosporins (ceftazidime and cefotaxime) is usually related to the overproduction of the resident AmpC-type β-lactamase (1,, ) encoded by the gene bla ampc. The overexpression of that latter gene has been associated to the insertion sequence ISAba1 providing a strong promoter (, ). Recently, the AmpC variants have been named according to a nomenclature specific to A. baumannii, being ADC for Acinetobacter- Derived Cephalosporinases (). Most AmpC-type β-lactamases naturally produced by Gram-negatives hydrolyze amino- and ureido-penicillins, cephamycins (cefoxitin and cefotetan) and at a lower Downloaded from on May, 01 by guest 1 level oxyimino-cephalosporins, such as ceftazidime, cefotaxime and ceftriaxone, and 1 monobactams such as aztreonam (). Zwitteronic cephalosporins (cefepime and 1 cefpirome) together with carbapenems are usually excluded from the spectrum of 1 activity of AmpC β-lactamases (). However, natural cephalosporinases possessing

4 broadened substrate activity have been reported in Enterobacteriaceae and Pseudomonas aeruginosa (1, 1, ). These Extended-Spectrum AmpCs (ESAC) confer reduced susceptibility to all cephalosporins (1, 1, ). They differ from regular cephalosporinases by amino acid substitutions or insertions/deletions in four specific regions that are all located in the vicinity of the active site: the Ω loop, the H- helix, the H- helix and the C-terminal extremity of the protein (1, 1, ). Here, we describe the first ESAC-type β-lactamase from A. baumannii with a broadened substrate activity toward expanded-spectrum cephalosporins and monobactams. A. baumannii KI clinical isolate was obtained from a rectal screening of a patient who had been transferred from the Guadeloupe Islands (French Caribbean Islands) and admitted at the Raymond Poincaré hospital (Garches, Paris, France) in January 00. This patient did not receive any antibiotic treatment. A. baumannii KI was selected for further study on the basis of its uncommon pattern of resistance to β-lactam antibiotics, Downloaded from on May, 01 by guest 1 including high level resistance to expanded-spectrum cephalosporins and reduced 1 susceptibility to carbapenems. In addition, it was resistant to cotrimoxazole and 1 fluoroquinolones, and susceptible to aminoglycosides.

5 Susceptibility testing were performed by Etest (1) and interpreted according to the CLSI guidelines (). It showed that A. baumannii KI was resistant to amino-, carboxy- and ureido-penicillins, to expanded-spectrum cephalosporins including ceftazidime, cefotaxime, ceftriaxone, cefepime, and cefpirome, to aztreonam, and had reduced susceptibility to imipenem and meropenem (Table ). Using cloxacillincontaining plates as described (), the susceptibility to ceftazidime and cefepime were restored, suggesting i) overproduction of the AmpC β-lactamase, and ii) the likelihood for this AmpC to possess ESAC properties. Mating-out assays and electrotransformation were performed as described (0), but failed to transfer any cephalosporin resistance marker from A. baumannii KI to A. baumannii BM or to E. coli TOP recipient strains, suggesting that this resistance to expanded-spectrum cephalosporins was chromosomally-encoded. Whole-cell DNA of A. baumannii KI was extracted as described previously (). Primers PreAmpC-PISAba1 and PreAmpC-Ab1 were used in combination with primer PreAmpC-Ab to amplify 1,1-bp and 1,-bp Downloaded from on May, 01 by guest 1 fragments, respectively, encompassing the entire bla ampc gene with or without the 1 P ISAba1 promoter, respectively (Table 1). All the inserts containing the P ISAba1 promoter 1 will be named accordingly with P+. The amplification products were cloned into E.

6 coli Top using the ZeroBlunt TOPO PCR cloning kit (Invitrogen, Cergy-Pontoise, France) followed by selection on plates containing 0 µg/ml of amoxicillin and 0 µg/ml of kanamycin. A. baumannii strains AYE and CIP0 reference strains were used to clone two regular bla ampc β-lactamase genes (, 1). Strains AYE corresponds to a multidrug resistant isolate from France (1) whose complete genome sequence was determined (, ). DNA sequence analysis showed that β-lactamase ADC- had and amino acid changes compared to regular β-lactamases ADC-0 and ADC-, corresponding to the ADC ß-lactamases of strains CIP0 and AYE, respectively (Fig. 1). No amino acid change was identified among the conserved motifs SVSK, YSN or KTG, neither in the helix H- or helix H-, previously associated to the extended activity of ESACs (1, 1, ). However, a single amino acid substitution, ProArg, associated with a duplication of the Ala residue at position 1, both of them located inside the Ω-loop, were identified. Thus, the molecular basis of the extended-spectrum hydrolysis profile of ADC- might be related to those specific substitutions. The Downloaded from on May, 01 by guest 1 presence of the insertion sequence ISAba1 providing strong promoter sequences (- 1 [TTAGAA] and - [TTATTT]) immediately upstream of the bla ampc gene was noticed, 1 as previously observed when the bla ampc gene is overexpressed ().

7 MICs of all β-lactams for the different clones were higher in the presence of the P ISAba1 promoter (Table ). E. coli TOP (padc--p+) was resistant to most β- lactams except to cefepime, cefpirome, and carbapenems. E. coli TOP (padc-- P+), producing a regular cephalosporinase, was resistant to amoxicillin and to narrow spectrum cephalosporins and remained susceptible to the expanded-spectrum cephalosporins, including ceftazidime, cefotaxime, cefepime, cefpirome and aztreonam (Table ). The recombinant E. coli strain harboring the bla ADC- gene showed a broadened hydrolytic activity against ceftazidime, cefotaxime, cefepime, cefpirome and aztreonam compared to the regular variant, increasing the resistance rates significantly (from to 1-fold), suggesting that ADC- might be considered as an ESAC enzyme. In order to better define the clinical impact of this ADC- variant, recombinant plasmids padc--p+ and padc--p+ were additionnally electro-transformed into E. coli HB (lacking OmpC and OmpF porins) (1). By using E. coli HB, our aim was to mirror the intrinsic weak permeability of A. baumannii and therefore better appreciating Downloaded from on May, 01 by guest 1 the impact of the ESAC enzymes. MICs of cefepime, cefpirome and aztreonam for the 1 ESAC and regular variants were, 1, > µg/ml and,, 1 µg/ml, respectively 1 (data not shown). Those MIC values were higher than those observed of the wild-type

8 E. coli strain, as expected. The significantly different impact on the resistance level shown by the ESAC and non-esac AmpC variants in a porin-deficient E. coli strain would also likely apply to A. baumannii. Isoelectric focusing analysis performed as described (1) using crude culture extracts of A. baumannii KI and of the different clones obtained by sonication gave a single β-lactamase signal for all of them, corresponding to a pi value of. consistent with the production of an AmpC β-lactamase. β-lactamases ADC- and ADC- were purified to near homogeneity (>%) as previously described (0). The molecular mass of those proteins determined by SDS- PAGE analysis was kda. The specific activities, determined with 0 µm of benzylpenicillin as substrate, were and µmol/min/mg of protein for ADC- and ADC-, respectively. Purified β-lactamases ADC- and ADC- were used for kinetic measurements (K m and k cat ) calculated as described (0). The kinetic parameters for penicillins and Downloaded from on May, 01 by guest 1 carbapenems for both AmpC enzymes were similar whereas the catalytic efficiency of 1 the purified β-lactamase ADC- against cephalosporins was higher than that of ADC- 1 (Table ). This higher catalytic efficiency observed for ADC- was noticeable in

9 particular for ceftazidime, cefotaxime, cefepime, cefpirome, and aztreonam (Table ). Those data confirmed the ESAC property of ADC-, that associates two specific features being i) an higher ability to hydrolyze ceftazidime, cefotaxime and aztreonam compared to a regular AmpC, and ii) an extended spectrum activity toward cefepime and cefpirome which are substrates usually weakly hydrolyzed by regular AmpCs. No significant difference of IC 0 values for clavulanic acid was observed between both enzymes (1. and.1 mm, for ADC- and ADC-, respectively). Compared to the regular ADC- β-lactamase, ADC- contained several amino acid substitutions, including the substitution ProArg together with a duplication of the Ala residue at position 1 located inside the Ω-loop. A site-directed mutagenesis strategy was used with a protocole described by the manufacturer (Quick change sitedirected mutagenesis kit; Stratagene) and as reported (1) for evaluating the amino acid changes that could be involved in the extended-spectrum resistance spectrum of ADC-. Using recombinant plasmids padc--p+ and padc--p+ as templates, and Downloaded from on May, 01 by guest 1 primers KI-A1-DelF/KI-A1-DelR and Aye-A1-InsF/Aye-A1-InsR, deletion or 1 insertion of Ala1 residue were respectively performed (Table 1). This allowed to 1 obtain recombinant plasmids padc--p+(a1del) (with a single A1 residue) and

10 padc--p+(a1ins) (with two A1 residues). Then the ArgPro and ProArg substitutions were generated by using primers KI-RP-F/KI-RP-R and Aye- PR-F/Aye-PR-R, respectively (Table 1) obtaining other recombinant strains in a second step as indicated in Table. Sequence analysis of the different inserts confirmed the presence of the expected amino acid changes. Duplication of the Ala1 residue in β-lactamase ADC- resulted in an increased resistance pattern toward expanded-spectrum cephalosporins (Table ), that was however not sufficient to explain MIC differences observed between E. coli (padc--p+) and E. coli (padc--p+). Thus, as a second step, the ProArg substitution was generated allowing to achieve an identical ESAC phenotype as compared to ADC-. Similarly, the reverse modifications performed with ADC- allowed the recovery of the regular AmpC phenotype, supporting the hypothesis that both amino acid modifications (Ala1 duplication and ProArg substitution) were responsible for the extended-spectrum profile of ADC- (Table ). Whether the other Downloaded from on May, 01 by guest 1 amino acid differences identified between ADC- and ADC- (Fig. 1) could play a 1 specific role in the functionality or stability of ß-lactamase ADC- remains unclear.

11 Extension of the Ω-loop for AmpC β-lactamase from Enterobacter cloacae has been shown to broaden its hydrolysis spectrum (1, 1). In that latter case, the mutant enzyme exhibited an increased opening of the entrance of the substrate-binding pocket (). The Ala1 duplication that is observed here in AmpC ADC- might have similar consequences for its hydrolysis spectrum. We describe here the first ESAC conferring resistance to expanded-spectrum cephalosporins in A. baumannii after those reported in Enterobacteriaceae and recently in P. aeruginosa. Noticeably, ADC- expression did not have any impact on carbapenem resistance. Clinical implications and spread of this resistance trait shall be now evaluated with A. baumannii isolates from worldwide origin. Funding This work was partially funded by a grant from the INSERM, the Ministère de l'education Nationale et de la Recherche (UPRES-EA), Université Paris XI, France Downloaded from on May, 01 by guest 1 and mostly by a grant from the European Community (TROCAR, HEALTH-F ). J-M. R-M. is funded by a postdoctoral grant from the Ministerio de Educacion 1 y Ciencia (00/0). Partially Supported by Ministerio de Sanidad y Consumo,

12 Instituto de Salud Carlos III - FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD0/000). Acknowledgments We thank R. Bonomo for managing the ADC nomenclature and providing us with ADC names. Downloaded from on May, 01 by guest 1

13 REFERENCES 1.Bou, G. and J. Martinez-Beltran Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC β-lactamase in Acinetobacter baumannii. Antimicrob. Agents Chemother. :-..Bratu, S., D. Landman, D. A. Martin, C. Georgescu, and J. Quale. 00. Correlation of antimicrobial resistance with β-lactamases, the OmpA-like porin, and efflux pumps in clinical isolates of Acinetobacter baumannii endemic to New York City. Antimicrob. Agents Chemother. :-00..Bush, K., G. A. Jacoby, and A. A. Medeiros. 1. A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. :1-1..Cisneros, J. M. and J. Rodriguez-Baño. 00. Nosocomial bacteremia due to Downloaded from on May, 01 by guest 1 Acinetobacter baumannii: epidemiology, clinical features and treatment. Clin. 1 Microbiol. Infect. :-. 1

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15 . Hujer, K. M., N. S. Hamza, A. M. Hujer, F. Perez, M. S. Helfand, C. R. Bethel, J. M. Thomson, V. E. Anderson, M. Barlow, L. B. Rice, F. C. Tenover, and R. A. Bonomo. 00. Identification of a new allelic variant of the Acinetobacter baumannii cephalosporinase, ADC- β-lactamase: defining a 1 1 unique family of class C enzymes. Antimicrob. Agents Chemother. :1-.. Levin, A. S., C. E. Levy, A. E. Manrique, E. A. Medeiros, and S. F. Costa. 00. Severe nosocomial infections with imipenem-resistant Acinetobacter baumannii treated with ampicillin/sulbactam. Int. J. Antimicrob. Agents 1:-. 1. Mammeri, H. and P. Nordmann. 00. Extended-spectrum cephalosporinases in Enterobacteriaceae. Anti-Infect. Agents Med. Chem. : Mammeri, H., P. Nordmann, A. Berkani, and F. Eb. 00. Contribution of Downloaded from on May, 01 by guest 1 extended-spectrum AmpC (ESAC) β-lactamases to carbapenem resistance in 1 Escherichia coli. FEMS Microbiol. Lett. :-0. 1

16 1. Nordmann, P. and H. Mammeri. 00. Extended-spectrum cephalosporinases: structure, detection and epidemiology. Future Microbiol. : Nukaga, M., S. Haruta, K. Tanimoto, K. Kogure, K. Taniguchi, M. Tamaki, and T. Sawai. 1. Molecular evolution of a class C β-lactamase extending its 1 substrate specificity. J. Biol. Chem. 0:-. 1. Nukaga, M., K. Taniguchi, Y. Washio, and T. Sawai. 1. Effect of an amino acid insertion into the omega loop region of a class C β-lactamase on its substrate specificity. Biochemistry : Philippon, L. N., T. Naas, A. T. Bouthors, V. Barakett, and P. Nordmann. 1. OXA-1, a class D clavulanic acid-inhibited extended-spectrum β- lactamase from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1:1-1. Downloaded from on May, 01 by guest 1 1. Poirel, L., S. Marqué, C. Héritier, C. Segonds, G. Chabanon, and P. 1 Nordmann. 00. OXA-, a novel class D β-lactamase involved in resistance 1 to carbapenems in Acinetobacter baumannii. Antimicrob. Agents Chemother. 1 :0-0. 1

17 1. Poirel L, Naas T, Guibert M, Chaibi EB, Labia R, Nordmann P. 1. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum ß-lactamase encoded by an Escherichia coli integron gene. Antimicrob. Agents Chemother. : Potron, A., L. Poirel, J. Croizé, V. Chanteperdrix, and P. Nordmann. 00. Genetic and biochemical characterization of the first extended-spectrum CARBtype β-lactamase, RTG-, from Acinetobacter baumannii. Antimicrob. Agents Chemother. : Rodriguez-Martinez, J. M., P. Nordmann, N. Fortineau, and L. Poirel. 0. VIM-1, a metallo-β-lactamase with increased carbapenemase activity from Escherichia coli and Klebsiella pneumoniae. Antimicrob. Agents Chemother. :1-.. Rodriguez-Martinez, J. M., L. Poirel, and P. Nordmann. 00. Extended- Downloaded from on May, 01 by guest 1 spectrum cephalosporinases in Pseudomonas aeruginosa. Antimicrob. Agents 1 Chemother. :1-. 1

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19 Table 1. Primers used in this work. Primer Sequence ( to ) PCR product size (bp) Purpose PreAmpC-Ab1 GAGCTAATCATGCGATTTAAA 1, Cloning AmpC CDS region PreAmpC-Ab GCTTAGGATATGTTTGGTTCTT PreAmpC-PISAba1 GACCTGCAAAGAAGCGCTGC 1,1 (with PreAmC-Ab) Cloning AmpC plus P ISAba1 AmpC-Ab1 GGAAAGGTTGTGGCTTTGTCT AmpC Sequencing Site directed mutagenesis KI-A1-DelF GGC CCA CTC GAT GCC CCA GCA TAT GGC Deletion of Ala1 KI-A1-DelR GCC ATA TGC TGG GGC ATC GAG TGG GCC Aye-A1-InsF GGC CCA CTC GAT GCC GCC CCA GCA TAT GGC* Insertion of Ala1 Aye-A1-InsR GCC ATA TGC TGG GGC GGC ATC GAG TGG GCC Aye-PR-F ATT CGA GTT AAC CGC GGC CCA CTC GAT GCC Substitution ProArg Aye-PR-R GGC ATC GAG TGG GCC GCG GTT AAC TCG AAT KI-RP-F ATT CGA GTT AAC CCC GGC CCA CTC GAT GCC Substitution ArgPro KI-RP-R GGC ATC GAG TGG GCC GGG GTT AAC TCG AAT *Modified bp are underlined. 1 Downloaded from on May, 01 by guest

20 Table. MICs of β-lactams for A. baumannii KI, A. baumannii AYE expressing the ESBL VEB-1, reference strain A. baumannii CIP0, E. coli TOP strains harbouring recombinant plasmid padc-, padc-, padc-0, padc--p+, padc--p+, padc--p+ (A1Del), padc--p+ (A1Ins), padc--p+ (A1Del+RP), padc--p+ (A1Ins+PR), and E. coli TOP reference strain. β-lactam (s) a A. baumannii KI A. baumannii CIP0. A. baumannii AYE E. coli TOP (padc-) E. coli TOP (padc-0) E. coli TOP (padc-) E. coli TOP (padc--p+) b E. coli TOP (padc--p+) E. coli TOP (padc--p+) E. coli TOP (padc--p+) E. coli TOP (padc--p+) E. coli TOP (padc--p+) E. coli TOP (ptopo) A1Del c A1Ins d A1Del+RP A1Ins+PR Amoxicillin > > > > > > > > > > Amoxicillin+ CLA 1 1 > > > > > Ticarcillin > > > 1 > 1 > Ticarcillin + CLA 1 > 1 > 1 > Piperacillin > 1 > 1 > > > > > > 1 Piperacillin + TZB > Cefuroxime > > > 1 > > > > > > Ceftazidime > > > > 1 > > 0.1 Cefotaxime > > > 1 > 0.0 Cefepime 1 > Downloaded from on May, 01 by guest

21 Cefpirome > > Aztreonam > Imipenem Meropenem a CLA, clavulanic acid at a fixed concentration of µg/ml; TZB, tazobactam at a fixed concentration of µg/ml. b P+ correspond to entire blaampc gene including PISAba1 promoter. c A1Del correspond to the deletion of this Alanine residue at position 1. d A1Ins correspond to the insertion of this Alanine residue at position 1. 1 Downloaded from on May, 01 by guest

22 1 1 Table. Kinetic parameters of ß-lactamases ADC- (ESAC) and ADC- of A. baumannii ADC- ADC- β-lactam Km kcat kcat/km Km kcat kcat/km kcat/km (µm - 1.s -1 ) (µm) (s -1 ) (µm -1 s -1 ) (µm) (s -1 ) (µm -1 s -1 ) for ADC-/ADC- Benzylpenicillin Ampicillin Piperacillin Cephaloridine Cephalothin Cefoxitin b Cefotaxime b Downloaded from Ceftazidime Cefepime 1, , Cefpirome , Aztreonam b ND a ND -- Imipenem a ND, no detectable hydrolysis (< 0.01 s -1 ), for a maximum amount of µg of purified enzyme, and up to 00 nmol of substrate. on May, 01 by guest

23 b For β-lactams with a Km value less than µm, Ki values were determined instead of Km values, with cephalothin used as the substrate. Data are the means of three independent experiments. Standard deviations were within 1% of the means. Downloaded from on May, 01 by guest

24 Legend to Figure. Amino acid sequence alignment including ADC- as an ESAC, ADC-0 and ADC- as regular AmpCs, and ADC- taken as reference sequence as published (). Amino acid identities are indicated by dashes. The typical AmpC β-lactamase domains (SVSK, YSN, and KTG) are underlined. The helix H- and H- are boxed in grey. The Ω-loop is boxed in grey and double underlined. Differences observed inside the Ω- loop are boldened. The vertical arrow indicates the position of the +1 amino acid (cleavage site for signal peptide). Numbering is according to the sequence of the mature protein. Downloaded from on May, 01 by guest

25 ADC- ADC-0 ADC- ADC MRFKKISCLLLSPLFIFSTSIYAGNTPKDQEIKKLVDQNFKPLLEKYDVPGMAVGVIQNNKKYEMYYGLQSVQDKKAVNSSTIFELGSVSKLFTATAGGYAKNKGKISFDDTPGKYWKELKNTPIDQV F R D N ADC- NLLQLATYTSGNLALQFPDEVKTDQQVLTFFKDWKPKNSIGEYRQYSNPSIGLFGKVVALSMNKPFDQVLEKTIFPALGLKHSYVNVPKTQMQNYAFGYNQENQPIRVNRGPLDAAPAYGVKSTLPDM ADC Q P A P ADC Q Q---P P ADC Q P P ADC- ADC-0 ADC- ADC LSFIHANLNPQKYPADIQRAINETHQGRYQVNTMYQALGWEEFSYPATLQTLLDSNSEQIVMKPNKVTAISKEPSVKMYHKTGSTNGFGTYVVFIPKENIGLVMLTNKRIPNEERIKAAYAVLNAIKK F TP F D T S V Downloaded from on May, 01 by guest