Proc. Natl. Acad. Sci. USAVol. 93, pp. 8225-8229, August 1996Biochemistry

Engineering actin-resistant human DNase I for treatment ofcystic fibrosis

(DNA enzymology/mutagenesis/protein engineering/actin inhibition/sputum viscoelasticity)

JANA S. ULMER*, ANDREA HERZKA*, KAREN J. Toyt, DANA L. BAKERt, ANTHONY H. DODGEt,DOMINICK SINICROPIt, STEVEN SHAKt, AND ROBERT A. LAzARuS*§Departments of *Protein Engineering, tBioAnalytical Technology, and *Pulmonary Research, Genentech, Inc., 460 Point San Bruno Boulevard,South San Francisco, CA 94080

Communicated by James A. Spudich, Stanford University School of Medicine, Stanford, CA, April 10, 1996 (received for review February 7, 1996)

ABSTRACT Human deoxyribonuclease I (DNase I), anenzyme recently approved for treatment of cystic fibrosis(CF), has been engineered to create two classes of mutants:actin-resistant variants, which still catalyze DNA hydrolysisbut are no longer inhibited by globular actin (G-actin) andactive site variants, which no longer catalyze DNA hydrolysisbut still bind G-actin. Actin-resistant variants with the leastaffinity for actin, as measured by an actin binding ELISA andactin inhibition of [33P] DNA hydrolysis, resulted from theintroduction of charged, aliphatic, or aromatic residues atAla-114 or charged residues on the central hydrophobic actinbinding interface at Tyr-65 or Val-67. In CF sputum, theactin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelas-ticity as determined in sputum compaction assays. The re-duced viscoelasticity correlated with reduced DNA length asmeasured by pulsed-field gel electrophoresis. In contrast, theactive site variants H252A or H134A had no effect on alteringeither viscoelasticity or DNA length in CF sputum. The datafrom both the active site and actin-resistant variants demon-strate that the reduction of viscoelasticity by DNase I resultsfrom DNA hydrolysis and not from depolymerization offilamentous actin (F-actin). The increased potency of theactin-resistant variants indicates that G-actin is a significantinhibitor of DNase I in CF sputum. These results furthersuggest that actin-resistant DNase I variants may have im-proved efficacy in CF patients.

action proposed, wherein DNase I reduces CF sputum vis-coelasticity due to the depolymerization of F-actin (7). Puri-fied actin filaments art known to be depolymerized by DNaseI (14, 15). In addition, F-actin is in equilibrium with itsmonomeric globular form (G-actin), which interacts with alarge number of actin binding proteins including DNase I (16,17). G-actin binds DNase I with high affinity and is a potentinhibitor (Ki 1 nM) of DNA hydrolytic activity (18-20),potentially influencing the effectiveness of DNase I in vivo.Thus actin in its different forms may have multiple roles withrespect to DNase I efficacy in CF sputum.We sought to elucidate both the basic mechanism of action

of human DNase I in CF sputum as well as to assess thesignificance of G-actin as an inhibitor of DNase I in CFsputum. Our approach was to generate and characterize twoclasses of mutants: actin-resistant variants that catalyze DNAhydrolysis comparable to wild type but are no longer inhibitedby actin, and active site variants which bind actin with affinitycomparable to wild type but no longer catalyze DNA hydro-lysis. The protein engineering strategy pursued in this paperdemonstrates that the reduction of viscoelasticity by DNase Iin CF sputum results from DNA hydrolysis and not fromdepolymerization of F-actin. Furthermore we show that G-actin is a significant inhibitor of DNase I in CF sputum,suggesting that actin-resistant variants may have improvedclinical benefit for CF patients.

Respiratory symptoms, recurrent infectious exacerbations, andprogressive lung destruction in cystic fibrosis (CF) can be beascribed to several contributing factors, including a defectiveCF transmembrane conductance regulator gene, abnormalelectrolyte transport, bacterial persistence, and the accumu-lation of viscous secretions in the airway (1-3). In response tothese infections leukocytes infiltrate the airways and, uponlysis, release their contents which include DNA and filamen-tous actin (F-actin)-two macromolecules that contribute tothe viscoelastic nature of CF sputum. DNA comprises about4-10% of the total dry weight of purulent CF sputum (4, 5);concentrations up to 20 mg/ml have been observed (D.S.,unpublished results). F-actin is an abundant protein in leuko-cytes (6); its concentration in CF sputum is reported to rangefrom 0.1 to 5 mg/ml (7).Recombinant human deoxyribonuclease I (DNase I) re-

duces the viscoelasticity of CF sputum in vitro and improveslung function and reduces respiratory exacerbations in CFpatients (8-12). The ability ofDNase I to reduce the viscoelas-ticity of pulmonary secretions has been attributed to theenzymatic hydrolysis ofDNA (8, 13). However, this notion hasrecently been questioned and an additional mechanism of

MATERIALS AND METHODS

Mutagenesis and Expression. Site-directed mutagenesis wascarried out by the method of Kunkel et al. (21); mutations wereverified by dideoxy sequencing (22). Variant DNA was purifiedfrom 500 ml cultures of transformed Escherichia coli XL1 BlueMRF' (Stratagene) using Qiagen tip-500 columns (Qiagen,Chatsworth, CA). Human embryonic kidney 293 cells (ATCCCRL 1573) were grown in serum containing media in 150 mmplastic Petri dishes and log phase cells were transiently co-transfected with 22.5 ,ug DNase variant DNA and 17 jigadenovirus DNA (23). About 16 h after transfection, cells werewashed with phosphate-buffered saline (PBS) and the mediumwas changed to serum free media; cell culture supernatant washarvested after 96 h. Harvests were concentrated - 10-foldusing Centriprep 10 concentrators and generally containedfrom 10 to 100 ,g of DNase I variant.DNase Activity Assays. The methyl green assay was used to

measure DNA hydrolytic activity of DNase I (24). DNase Iconcentrations were determined by ELISA by using a goatanti-DNase I polyclonal antibody coat and detecting with arabbit anti-DNase I polyclonal antibody conjugated to horse-radish peroxidase. In both assays, multiple sample dilutions

Abbreviations: CF, cyctic fibrosis; F-actin, filamentous actin; G-actin,globular actin.§To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 93 (1996)

were compared with standard curves of purified recombinanthuman DNase I (Pulmozyme; Genentech) to determine con-centrations. The relative specific activity is defined as theDNase I concentration determined in the methyl green assaydivided by the DNase I concentration determined in theDNase I ELISA; the data are normalized to wild-type DNaseI.

Actin Binding ELISA. MaxiSorp plates (Nunc) were coatedwith 100 ,ul human Gc globulin (Calbiochem) at 10 ,ug/ml in25 mM Hepes (pH 7.2), 4 mM MgCl2, and 4 mM CaCl2 at 4°Cfor 16-24 h. After discarding the Gc globulin, excess reactivesites were blocked with 200 ,ul buffer A (25 mM Hepes, pH7.5/4 mM CaCl2/4 mM MgCl2/0.1% BSA/0.5 mM ATP/0.01% thimerosal/0.05% Tween 20). Buffer A was used in alldilution steps unless otherwise noted; incubations were atroom temperature for 1 h. The wash buffer was PBS containing0.05% Tween 20. Rabbit skeletal muscle G-actin [1 mg/ml;prepared by the method of Pardee and Spudich (25) orobtained from Sigma] was dialyzed overnight at 4°C against 5mM Hepes (pH 7.2), 0.2 mM CaCl2, 0.5 mM ATP, and 0.5 mM2-mercaptoethanol. After centrifugation at 13,000 x g for 5min, the amount of G-actin-ATP was quantitated by measuringthe absorbance at 290 nm, using 290 = 28.3 mM-1'cm-1 (19).Following the addition of 100 gl of 50 ,ug of G-actin per ml inbuffer A, the plates were incubated and washed, and 100 ,lI ofcell culture harvest at various dilutions was added. Afterincubation and washing, 100 pul of anti-human DNase I rabbitpolyclonal antibody-horseradish peroxidase conjugate (19 ng/ml) was added. Following incubation and washing, colordevelopment was initiated by adding 100 plI per well of SigmaFast o-phenylenediamine and urea/H202 reagent (preparedaccording to Sigma) and stopped by adding 100 ,ul per well 4.5M H2SO4. The A492 was measured and plotted versus theDNase I concentration. The resultant sigmoidal curves were fitto a four parameter equation by nonlinear regression analysis(26); the EC5o value is the DNase I concentration that pro-duced a half-maximal signal.

Actin Inhibition Assays. Varying concentrations of G-actinwere preincubated in duplicate for 15 min at room temperaturewith 0.54 nM DNase I variant in buffer A containing 0.5 mM2-mercaptoethanol and 150 mM NaCl. Reactions were initi-ated by the addition of 33P-labeled M13 DNA and salmontestes DNA (Sigma) to a final concentration of 4.1 ,ug/ml,incubated at room temperature for 2 h, and quenched with 25mM EDTA and cold TCA (6.7% final concentration). After 10min on ice samples were centrifuged at 9300 x g for 5 min at4°C and the acid-soluble counts determined. Plots of thefractional activity (cpm inhibited/cpm uninhibited) versusactin concentration were fit by nonlinear regression analysisusing KALEIDAGRAPH version 3.0.1 (Synergy Software, Read-ing, PA) to the following equation to determine the Ki value.

Fractional activity =

[EO] + [IO] + Ki - V([Eo] + [Io] + KJ)2 - (4. [E] * [j)2 * [EO]]

where [Eo] is the DNase I concentration and [I,] is the totalG-actin concentration.CF Sputum Compaction Assays. Assays using CF sputum

collected from four patients and stored at -80°C prior to usewere carried out as described (27); sputa were incubated intriplicate with DNase I samples at various concentrations for20 min at room temperature.

Pulsed-Field Gel Electrophoresis. Pulsed-field gel electro-phoresis was carried out in 1% agarose gels; after electro-phoresis, gels were stained with ethidium bromide and imagedin a Molecular Dynamics Fluorimager.

RESULTS AND DISCUSSIONProtein Engineering. Selected mutations were made based

on the x-ray crystal structures of bovine DNase I complexedwith either G-actin or DNA (28, 29). A model of a ternarycomplex of DNase I, actin, and DNA, constructed by super-imposing the DNase I structures (28-30), shows that the DNAand actin binding sites are adjacent yet distinct; the inhibitionof DNase I by actin due to steric hindrance is apparent (Fig.1). Bovine pancreatic DNase I shares 78% identity and super-imposes with an overall rms deviation for main chain atoms of0.56 A with the human enzyme, recently solved at 2.2-Aresolution (30). The contact residues at the actin bindinginterface as well as those at active site are conserved. Bothhuman and bovine DNase I have been found to reduce theviscosity of purulent sputum (8, 13).The binding of DNase I to actin involves hydrophobic,

electrostatic, and hydrogen bonding interactions over a buriedsurface area of 1849 A2 (17, 28); the actin binding interface isshown in Fig. 2. Main-chain interactions result from theparallel ,B-strands formed by Tyr-65, Val-66, and Val-67 ofDNase I and Gly-42, Val-43, and Met-44 of actin. The DNaseI side-chains forming the core of the interface are hydrophobicand include Tyr-65, Val-67, and Ala-114; interactions periph-eral to this central hydrophobic region are polar in nature andinvolve His-44, Asp-53, and Glu-69. Initially, alanine substi-tutions for these residues were made to assess their relativecontribution to actin binding. Further mutagenesis lead toactin-resistant variants that were created by introducingcharge, charge reversals, or steric bulk. Active site variantswere created by alanine substitution at residues His-134,

FIG. 1. Model of a ternary complex of human DNase I with G-actinand a DNA oligonucleotide. Ribbon diagram of the DNase I-actincomplex superimposed with the DNase I-d(GGTATACC)2 complex[Brookhaven Protein Data Bank 1ATN (28) and 1DNK (29), respec-tively] and human DNase I (30). Residues at the active site (His-134,Glu-78, His-252, and Asp-212) are in yellow and those at the actinbinding interface (His-44, Asp-53, Tyr-65, Val-67, Glu-69, and Ala-114) are in pink. The figure was made by using MIDASPLUS (31).

8226 Biochemistry: Ulmer et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 8227

FIG. 2. Structure of DNase I complexed with G-actin. The struc-ture (1ATN) was taken from the Brookhaven Protein Data Bank (28);DNase I is represented in grey and actin is depicted in blue. Residuesat the actin binding interface that were altered are highlighted in pink.The figure was made by using MIDASPLUS (31).

His-252, Asp-212, and Glu-78; these residues have been im-plicated in the general acid-base catalysis mechanism thatoccurs at the scissile phosphate bond (29, 32). In addition, themore conservative substitution of Gln for His at position 134or 252 was also tested.DNA Hydrolysis and Actin Binding Assays. The relative

specific activity of DNase I variants was assessed by comparingthe specific DNA catalytic activity of the variant to that of wildtype. DNase I concentration was determined by ELISA;reaction with the anti-DNase I antibodies also implied that themutants were correctly folded. None of the active site variantscatalyzed DNA hydrolysis, confirming the critical role of theseresidues in the enzyme mechanism. In contrast, the actinbinding site variants all had similar specific activity compared

Active Site Variants

A

3 1.0_

Z ._co

wO^ ) 'v'

with wild type, demonstrating that changes at the actin bindinginterface do not affect the active site (Fig. 3 A and C).The binding affinity of DNase I variants for G-actin was

assessed by an ELISA where actin was bound to immobilizedGc globulin, a protein whose affinity for actin is unaffected byDNase I (33-35); the EC5o values are plotted in Fig. 3 B andD. The active site variants bound with the same high affinityto G-actin as wild-type DNase I, again confirming the inde-pendent nature of these two sites. However, a wide range ofreduced affinities for actin was found for the actin binding sitevariants (Fig. 3D).

Selected variants were also assayed for actin inhibition ofDNase I catalyzed hydrolysis of 33P-labeled DNA in solution.The DNase I variants and their respective Ki values arereported in Table 1. The inhibition of DNase enzymaticactivity by actin in solution correlated well with the relativeactin binding affinity determined in the solid-phase actinbinding assay (Fig. 3D and Table 1).DNase I Variants at the Actin Binding Interface. Single

point mutations introducing charged, aliphatic, or aromaticresidues at Ala-114 resulted in actin-resistant variants thatwere reduced in actin binding by over 10,000-fold relative towild-type DNase I (Fig. 3D). This large effect is likely due tosteric hindrance, since Ala-114 resides on an internal 3-strandand lies at the bottom of a pocket with its side chain in van derWaals contact with that of Val-45 of actin (Fig. 2). Theintroduction of charge on the central hydrophobic interface atTyr-65 and Val-67 (Y65R, V67K, V67D) also greatly impairedactin binding (Figs. 2 and 3D). The fact that Y65R and V67Kare much more actin resistant than Y65W or V67M implicatescharge rather than steric hindrance as the critical factor.Moderate reductions in actin binding were noted upon re-moval of the aromatic side-chain at Tyr-65 (Y65A) andintroduction of a charge reversal at Asp-53 (D53R), whichshould disrupt the salt bridge formed with Arg-39 and thehydrogen bonds to His-40 of actin (Fig. 3D and Table 1).Mutations at His-44, which interacts with Thr-203 and Glu-207in actin, and Glu-69, which forms a salt bridge with Lys-61 of

Actin Binding Site Variants

--

o o

I m

< a)Z 1Q <u

c 0 1.0 r-.o

.2, i3 D-o._ 0.5 .- -o

0wt A A Q A A Q

E78 H134 D212 H252

DNase I Variant

1.0

0.5

0

10410310210

1wt ADY AMRY ARW ADKM AKMR EMRY

H44 D53 Y65 V67 E69 A114

DNase I Variant

FIG. 3. Relative specific activity and actin binding affinity of active site (A and B) and actin binding site (C and D) DNase I variants. The relativespecific activity for DNA hydrolysis, as defined in Materials and Methods, is normalized to wild type. The fold reduction in actin binding affinity[EC5o (variant)/EC5o (wild type); mean ± SD, n > 2] was determined by the actin binding ELISA and is normalized to wild type. The relativeactin binding affinity is plotted on a logarithmic scale for the actin binding site variants (D); bars labeled with an asterisk (*) represent a lowerlimit due to protein expression.

Biochemistry: Ulmer et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

Table 1. Inhibition constants (K1) for G-actin inhibition of33P-labeled DNA-catalyzed hydrolysis by DNase I variants

DNase I variant Ki, nMWild-type DNase I 1.3H44A 3.9D53A 24D53R 47Y65A 36Y65R >1000V67K 161E69R 3.5A114R >10,000

The inhibition constants (Ki) were calculated using the knownconcentrations of DNase I variant and G-actin, the measured frac-tional activity, and fitting the data by nonlinear regression analysis tothe equation for tight binding reversible inhibition as described inMaterials and Methods.

actin, had minimal effects (Fig. 3D and Table 1), suggestingthat their contributions to the binding energy of the complexare negligible.

Activity of DNase I Variants in CF Sputum. Sputum com-paction assays were used to measure the relative viscoelasticityof CF sputum after incubation with wild-type and selectedDNase I variants (27); the percent change in compaction isshown in Fig. 4. The active site variants (H134A, H252A),which no longer catalyze DNA hydrolysis but still bind G-actinand could therefore depolymerize F-actin, did not reducesputum viscoelasticity. However, both wild type and the actin-resistant variants tested (D53R, Y65A, Y65R, V67K) de-creased viscoelasticity in a dose-dependent manner. The datafrom both sets of mutants demonstrate that the reduction insputum viscoelasticity by DNase I results from DNA hydrolysisand not actin depolymerization. The hydrolysis ofDNA in CFsputum and subsequent reduction in DNA length by wild typeand the actin-resistant variant V67K was confirmed usingpulsed-field gel electrophoresis (Fig. 5); no reduction in DNAlength was seen with the active site variant H134A (D.S.,unpublished results).Most importantly, the actin-resistant variants were about 10

to 50-fold more potent in both reducing sputum viscoelasticityand in reducing DNA length compared with wild-type DNaseI (Fig. 4 and 5). Based on these results, we conclude thatG-actin is a significant inhibitor of DNase I in CF sputum, andthat the actin-resistant variants are not subject to this inhibi-

Qa

2._Cb0 -

0

CDCD.= ._

0Cu>

20

10

0

-10

-20

-30

-40

-50

0.01 0.1 1 10

DNase Variant Concentration (,ug/mi)

FIG. 4. Effect of DNase I variants on CF sputum compaction. Thepercent compaction of the sputum (mean ± SEM), which correlateswith sputum viscoelasticity, is plotted versus the variant concentration;wild type (-); active site variants: H134A (A) and H252A (0); andactin-resistant variants: D53R (-), Y65A (*), Y65R (A), and V67K(v).

wildtype DNase Ijg/ml

V67Klig/mi

I I I IMW C 4.83 1.60 0.53 0.18 0.06 0.02 0.53 0.18 0.06 0.02

FIG. 5. DNA length in CF sputum treated with wild-type DNaseand the actin-resistant variant V67K. DNase concentrations and DNAmolecular mass markers are indicated; C refers to a control sputum inthe absence of DNase. Pulsed-field gel electrophoresis was carried outas described.

tion. The actin-resistant variants tested, which had from about30 to >1000-fold reduced affinity for actin, were equivalent inthe compaction assay, suggesting that they were all uninhibitedby actin. Using the equation for tight binding reversibleinhibition in the materials and methods as well as assumingthat free G-actin is present minimally at its critical concen-

tration of 100 nM (6, 16) and a Ki of 1.3 nM, we calculate that99, 98, and 37% of wild-type DNase is inhibited at 0.1, 1, and10 ,ug/ml DNase, respectively, consistent with the data in Fig.4.

Biological Significance and Clinical Implications. The bi-ological significance of DNase I inhibition by actin is not wellunderstood; a potential role during mitosis and apoptosis hasbeen recently proposed (36). DNase I from mammaliansources is highly conserved (37); however, not all mammalianDNases are inhibited by actin (38, 39), suggesting that actininhibition of DNase I is not- essential for cellular function. Infact, a gene encoding a novel human DNase, termed LS-DNase, which is expressed predominantly in the liver andspleen and has 46% amino acid sequence identity to humanDNase I, has recently been cloned and expressed (W. F. Baron,C. Q. Pan, R.A.L., and K. P. Baker, unpublished data). Ofparticular interest is the fact that LS-DNase has DNA hydro-lytic activity that is not inhibited by G-actin. The finding thata family of DNase I-like enzymes exists within the humangenome raises additional questions as to the cellular biologyand biochemistry of DNase as well as the role of actin.The results presented here suggest that actin is a significant

inhibitor of DNase I in CF sputum. F-actin is present over a

range of concentrations in a limited number of CF sputumstudied to date (7); however, the precise amount of actincapable of inhibiting DNase I is unknown. Although theconcentration of actin, whether it exists as G-actin, F-actin, or

actin in complex with actin binding proteins, in sputumthroughout the CF patient population is not well defined, our

results suggest that actin-resistant variants may have improvedclinical efficacy, especially in patients with high sputum actinlevels.

We thank C. Eigenbrot and C. Schiffer for helpful discussions

.. .......f j ......I.. f.

Actin resistantvariants

.I...

8228 Biochemistry: Ulmer et aL

Proc. Natl. Acad. Sci. USA 93 (1996) 8229

regarding DNase structure; A. Spudich for helpful discussions on actin;Genentech's Assay Services Group for technical assistance; L.O'Connell for tissue culture expertise; M. Vasser, P. Jhurani, and P.Ng for oligonucleotide synthesis; and K. Andow and D. Wood forgraphics.

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Biochemistry: Ulmer et al.