Characteristics of an aminohydrolase distinct from adenosine deaminase in cultured human lymphoblasts

280

Bmeh~mtca et Bmphys~ca Acta, 658 (1981) 280--290 © Elsewer/North-Holland Biomedical Press

BBA 69232

CHARACTERISTICS OF AN AMINOHYDROLASE DISTINCT FROM ADENOSINE DEAMINASE IN CULTURED HUMAN LYMPHOBLASTS

PETER E DADDONA and WILLIAM N KELLEY Departments o f Internal Medicine and Bmlog~cal Chemistry, Human Purme Research Center, The Unwerszty o f Mwh#gan Medzcal School Ann Arbor, MI 48109 ( U S A ) (Received September 29th, 1980)

Key words Adenosine deammase, ery thro-9-(2-Hydroxy-3-nonyl)adenme (Human B-lymphoblast cell)

Summary An m h e n t e d deficiency of adenosine deammase (adenosine arnmohydrolase, EC 3.5.4.4) is associated with an autosomal recessive form of severe combined lmmunodeflelency disease. Affected patmnts exhibit markedly reduced or absent adenosine deammatlng aetlwty m various tissues. In thin study we have demonstrated the presence of a low level ammohydrolase activity in 11 different normal and adenosme deamlnase-deflclent lymphoblast cell hnes which IS apparently distract from normal adenosine deammase. Based on enzymatac, physical and lmmunoreaetlve properties, this lymphoblast ammohydrolase does n o t appear to be related to adenosine deammase and is most hkely coded for by a different gene locus. In future mvestlgatmns designed to eharactenze m u t a n t forms of adenosine deamlnase, it will be important to dlstmgmsh this lymphoblast amlnohydrolase actlwty from putative products of the adenosine deammase gene locus.

Introduchon Adenosine deammase (adenosine ammohydrolase, EC 3.5.4.4) catalyzes the conversmn of either adenosine or deoxyadenoslne to produce lnoslne and deoxymosme, respectively, with the hberatlon of ammoma. The discovery of a deficmncy of adenosme deamlnase m some patmnts with an autosomal recessive form of severe combined lmmunodeflcmncy disease has prowded an Important clue to the pathogenems of immune dysfunction at the molecular level [ 1--6].

Abbrew.attons

EHN A, ery thro-9-(2-hydroxy-3-nonyl)adenme

281 A characterization of the residual adenosine deammase activity in patients with adenosine deammase deficiency and severe combined lmmunodeflclency disease has been difficult due to the extremely limited amount of enzyme activity expressed in vamous tmsues of these patients. Further, any interpretation of the nature of the genetic heterogeneity of adenosine deammase in these patients could be complicated without a complete understanding of the molecular and electrophoretm heterogeneity of the enzyme expressed m normal human tissues [7--10]. Recent studies have shown that human adenosine deamlnase exists either as a 'particulate' species or as one of t w o soluble molecular forms, designated small form (Mr 38 000) and large form (Mr 298 000), which are lnterconvertlble [10]. The small form of the enzyme from human erythrocytes is a single polypeptlde exhibiting multiple electrophoretm forms arising from post-translational modification of a single gene product. In part, this heterogeneity may be due to sulfhydryl group modification on the enzyme [11--15]. The large molecular form of adenosine deammase (Mr 298 000) is composed of t w o molecules of small-form adenosine deammase (Mr 38 000) and one molecule of a specific adenosine deammase-bmdmg protein (Mr 213 000) (also termed conversion factor or complexmg protein) [16--18]. The large form of the enzyme also exists as 'tissue specific' electrophoretm variants. These variants appear to be generated by heterogeneity in the carbohydrate portion of the binding protein and are n o t due to additional variation in the small-form deammase present in the complex [19]. Early studies of adenosine deammase molecular heterogeneity mdmated the presence of a low level of an 'intermediate" form of the enzyme (Mr 110 000) in a variety of human tissues [10]. In addition, analysis of splenic tissue from a patient with adenosine deammase deficiency and severe c o m b m e d lmmunodeficiency disease revealed that the molecular form of adenosine deammase was exclusively the 'mtermedlate' species [20]. Subsequently, Schrader et al. [21] demonstrated that an 'ammohydrolase' intermediate m molecular weight between large- and small-form adenosine deammase existed m normal spleen as well as in splenic tissue of another patient with adenosine deammase deficiency and kmmune dysfunction. This splenic 'ammohydrolase' appeared to be different from normal adenosine deammase based on Km value, lmmunoreactlvity, pH o p t i m u m and msensitlvity to the potent adenosine deammase inhibitor, EHNA. It has n o t been established, however, whether this low level of 'ammohydrolase' activity studied m these two subjects (a) arose from adenosine deammase degradation during post-mortem tissue autolysis, (b) represented a modified product of the adenosine deammase gene locus, or (c) was a universal flndmg in viable human cells. In this study, we have demonstrated the presence of an ammohydrolase in 11 different normal and adenosine deammase
282

Materials and Methods [8-14C]Adenosme (59 mCl/mmol) and [U-14C]deoxyadenosine (500 mC1/ retool) were obtained from Amersham/Searle erythro-9-(2-Hydroxy-3-nonyl) adenine ( E H N A ) was a generous giftfrom Burroughs WeUcome, while 2'-deoxycoformycm was generously provided by Parke Davis, A Division of Warner/ Lambert. The adenosine deammase-bindmg protein was purified to homogenelty from h u m a n kidney as previously described [17]. All other chemicals and reagents used were of the highest quahty commercially available. Normal B-lymphoblast cell hnes ( G M 130, G M 131, G M 333, G M 621, G M 1078 and MGL-8) and adenosine deammase-deficient B-lymphoblast cellhnes ( G M 2471, G M 2606, G M 2294, G M 2445 and G M 2756 transformed from lymphocytes of patients wlth adenosine deaminase deficiency) were obtmned from the Hum a n Genetic Mutant Cell Repository, Camden, NJ. All lymphoblast celllines were cultured m R P M I m e d i u m supplemented wlth 1 0 % adenosine deamlnase
283 cell extracts was quantified using prewously described radmlmmunoassay techmques [22,24]. Protein concentratmn was determined by the method of Lowry et al. [25] using bovine serum albumin as standard. Results Extracts from a normal B-lymphoblast cell hne (MGL-8) and an adenosine deammase
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Fig 1 Sucrose denslty ultracentnfugation of adenosine deammatmg ac~vlty m human B-lymphoblast cell extracts Cell extracts were prepared by freeze-thawing cells five tlmes followed by centnfugatlon at 100 0 0 0 X g The soluble preparation was then apphed to an isokmetlc sucrose grachent (10--28,2%) and centrifuged a t 4 ° C f o r 4 0 h at 3 4 0 0 0 rev / r a m w l t h a S p m c o S W 4 1 r o t o r i n a B e c k m a n m o d e l L 5 - 5 0 ultracentrifuge Fractions were collected and assayed for adenosine deammating acUvlty using [14C]adenosine (2 mCl/mmol, 4 r a M ) r e a c t i o n m l x t u r e (o o) a n d r e a c U o n m z x t u r e c o n t a t m n g 2 0 0 # M E H N A (e e) A N o r m a l B - l y m p h o b l a s t cell extract, MGL-8 B Adenosine deammase-deflclent B-lymphoblast cell extract, GM 2445

284 TABLE I A D E N O S I N E D E A M I N A T I N G A C T I V I T Y IN N O R M A L A N D A D E N O S I N E D E A M I N A S E - D E F I C I E N T B-LYMPHOBLAST CELL LINES N u m b e r of c o n t r o l cell h n e s undlcated m p a r e n t h e s i s Mean ,+ 1 S D b a s e d o n a t least t h r e e d e t e r r n i n a t m n s of e a c h cell h n e T o t a l a d e n o s i n e d e a m m a t m g a c t i v i t y ( A D A ) In l y m p h o b l a s t cell e x t r a c t s was a s s a y e d using [ 1 4 C ] a d e n o ~ n e (2 m C H m m o l ) at a final c o n c e n t r a t m n of 4 m M E H N A - m s e n a i t v e a d e n o s i n e d e a m l n a t m g a c t i v i t y was d e t e r m i n e d using 4 m M [ 1 4 C ] a d e n o s m e (2 m C l / m m o l ) w i t h 2 0 0 / a M E H N A A d e n o s i n e d e a m m a s e a c t l w t y was c o n s i d e r e d to r e p r e s e n t the d i f f e r e n c e o f t h e t o t a l a c t i v i t y a n d t h e E H N A - m s e n satire a m m o h y d r o l a s e a c t i v i t y L y m p h o b l a s t cell h n e s

N o r m a l (6) ADA-dehclent GM2294 GM 2 7 5 6 GM 2 4 7 1 GM 2 6 0 6 GM 2 4 4 5

A d e n o s i n e d e a m m a t i n g a c t i v i t y ( n m o l / m m per m g ) Total

EHNA-msensltlve ammohydrolase

Adenosine d e a m m a s e (Total minus E H N A Insensltlve actlvlty)

72 69 + 15 48

O 26 + 0 17

72 43

25 30-+ 053,+ 040-+ 0 33 +200,+

058,+002 049 +002 029,+001 0 32-+ 0 0 1 1 93-+005

24 72 004 011 0Ol 007

1 14 002 001 002 0 10

ultracentnfugatlon, dialyzed and reapphed to a further sucrose gradient, the enzyme actawty was invariably present as a single peak with an S2o.w of 7.4 S. This EHNA-msensltlve ammohydrolase actlwty showed no evidence of dissociation to a species of s20.w 3.8 S. Addltmnal cultured B-lymphoblast cell hnes from normal and adenosine deammase~leflcmnt subjects were analyzed for adenosine deammatmg actwlty. As shown m Table I, the level of total adenosine deammatmg activity m adenosine deammase-deflcmnt B4ymphoblast cell hnes varied from 0 5 to 35% of normal. In four of the five m u t a n t cell hnes studmd, the vast m a j o n t y of the adenosine deammatmg actlwty present was the EHNA-msenmtlve ammohydrolase. In addition, the level of EHNA-msensltlve ammohydrolase actlwty m four of the five m u t a n t cell lines overlapped the range noted in six cell lines derived from adenosine deamlnase positive mdlwduals. Normal lymphoblast adenosine deammase (small-form, S2o.w 3 8 S) isolated from sucrose gradients was incubated with a 100-fold molar excess of adenosme deammase-bmdmg protein, and subjected to sucrose gradient ultracentnfugatlon. The resulting enzyme activity profile revealed complete conversion of the small-form adenosine deammase to the large molecular form of the enzyme (s20,w 10.0 S) (Fig. 2A). Under the conditions of this experiment, no detectable EHNA-msensltive ammohydrolase activity was observed. Using the same experimental conditions, lymphoblast EHNA-msenslttve ammohydrolase (s20.w 7 4 S) isolated from sucrose gradients was also incubated with a 100-fold excess of adenosine deammse-bmdmg protein. The EHNA-msensltlve ammohydrolase molated from either normal or adenosme deammase-deflcmnt lymphoblasts could not be converted to a species of hw,her s20,w as observed with normal small-form adenosine deammase (Fig. 2B).

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Fig 2 I n t e r a c t i o n of a d e n o s i n e d e a m m a s e and the EHNA-msenssUve a m m o h y d r o l a s e w l t h a d e n o s i n e d e a m m a s e - b m d m g p r o t e i n A L y m p h o b l a s t a d e n o s l n c d e a m i n a s e i s o l a t e d f r o m sucrose grachents ( 3 2 0 0 n m o l / m m ) was i n c u b a t e d for 2 0 n u n at 3 7 ° C w l t h 0 5 m g b o v l n e s e r u m a l b u m i n (e -') o r w l t h 0 5 m g p u n f l e d a d e n o s i n e d e a m m a s e - b m d m g p r o t e i n p r e p a r e d as d e s c r i b e d m M e t h o d s (o o) l ~ n f l e d a d e n o s i n e d e a m m a s e - b m d m g p r o t e i n was i n c u b a t e d a l o n e as a b o v e (~ A) B L y m p h o b l a s t E H N A insenslttve a m m o h y d r o l a s e I s o l a t e d f r o m s u c r o s e g r a d i e n t s (35 n m o l / m m ) was i n c u b a t e d as a b o v e w l t h 0 5 mg bovine serum albumin (e e) or w i t h 0 5 m g a d e n o s i n e d e a m m a s e - b m d m g p r o t e i n (o o) Purified a d e n o s i n e d e a m m a s e - b m d m g p r o t e i n was i n c u b a t e d a l o n e (~ ~) All i n c u b a t i o n m l x t u r e s w e r e c e n t r i f u g e d i n t o s e p a r a t e s u c r o s e grachents

The physical and kinetm properties of the EHNAoinsensltwe amlnohydrolase isolated from normal and adenosine deaminase~leflcmnt lymphoblast cell lines and normal small-form lymphoblast adenosine deammase are summarized in Table II. The EHNA-msensltave ammohydrolase has an s20,w of 7.46 -+ 0.18 S (mean -+S.D., 11 determinations) with a calculated mean molecular weight of 110 000, while small-form adenosme deammase has an s20,w of 3.8 -+ 0.02 S {mean -+S.D., five determmatmns) with a calculated mean molecular weight of 38 000. The EHNA-msensltlve amlnohydrolase has; (a) a sharp pH o p t i m u m of 5.5--6.0 as compared to the higher broad pH o p t i m u m of adenosine deamlnase, T A B L E II CHARACTERISTICS OF THE MOLECULAR IN L Y M P H O B L A S T S

FORMS OF ADENOSINE DEAMINATING ACTIVITY

E x t r a c t s f r o m n o r m a l l y m p h o b l a s t s a n d a d e n o ~ n e d e a m i n a s e - d e h c ~ e n t l y m p h o b l a s t s were s u b j e c t e d t o sucrose g r a d i e n t u l t r a c e n t r d u g a t l o n P e a k f r a c t i o n s c o r r e s p o n d i n g to the E H N A - m s e n s l t w e a m m o h y d r o l a s e were i s o l a t e d as d e s c r i b e d m M e t h o d s A d e n o s i n e d e a m m a s e was i s o l a t e d f r o m n o r m a l l y m p h o b l a s t s m a ~ m f l a r m a n n e r N u m b e r s m p a r e n t h e s i s i n d i c a t e the n u m b e r of d e t e r m i n a t i o n s All o t h e r values r e p o r t e d r e p r e s e n t t h e average of t w o d e t e r m m a t m n s Property

EHNA-msensltwe ammohydrolase

Adenosine deammase

S e d l m e n t e t l o n c o e f h c l e n t (1 10 -13 S) E s t i m a t e d m o l e c u l a r wesght ( f r o m S 2 0 , w ) pH Optimum K m value adenosine deoxyadenosme Relative substrate specificity deoxyadenosme/adenosme

746-+018(11) 1 I0 000 5 5--6 0

38-+002(5) 38 0 0 0 6--8

3 06 mM 0 72raM 01

0 065 mM 0076mM 10

286 (b) a markedly elevated Km value for adenosine (3.06 mM) and deoxyadenosme (0.72 mM) as compared to the Km value of adenosine deaminase with these t w o substrates (0.065 and 0.075 mM, respectively), and (c) a lower rate of catalytac actlwty with the substrate deoxyadenoslne, as compared to that with adenosme, than noted with adenosme deammase. The heat stability of adenosine deammatmg actlwty Is shown In Fig. 3. Under the conditions of our study, adenosine deamlnase exhibited a loganthmm decay of enzyme actlwty at 68°C with a t~/2 of approx. 30 mln, while the EHNA-msenmtlve aminohydrolase actlwty was reduced by less than 5% over the 90 mm incubation penod. Dunng the time course of adenosine deammase heat inactivation, no EHNA-msensitIve aminohydrolase a c b w t y was generated. The effect of divalent cations on adenosine deaminatmg catalytic activity is shown m Table III. Of the divalent cations tested, only Iron and nickel selectively Inhibited normal adenosine deaminase. The relative substrate specificity of the EHNA-msensltlve aminohydrolase was compared to adenosine deamInase as shown In Table IV. The potential substrates, AMP, dAMP, ADP, dADP, ATP, dATP, cytldme, cytosine, deoxycytadine, CMP, dCMP, CDP, dCDP, CTP, dCTP, guanine, guanosine, deoxyguanosine, u n d m e and d e o x y u n d i n e , did not compete with the conversion of adenosine to inosme whether catalyzed by adenosine deammase or the EHNA-msensitire ammohydrolase under the conditions of our study. However as shown m Table IV, adenine, 6-chloropunne and 2,6~hammopunne reduced the deammatmn of adenosine to mosme when catalyzed by the EHNA-msensltlve ammohydrolase while N-carbamyl-~-alanlne and 6-methylammopunne nboslde reduced the deammatmn of adenosme when catalyzed by adenosine deammase. A further differential effect was observed with the competitive adenosme deammase mhlbitors. While the EHNA-msensitlve ammohydrolase was relatively msensltlve to the adenosme deammase inhibitor, EHNA, it was completely mhlblted by 2 ' < l e o x y c o f o r m y c m similar to adenosine deammase. Using

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F | g 3 H e a t s t a b t h t y o f a d e n o s i n e d e a m m a t l n g actlvlty L y m p h o b l a s t eel] l y s a t e s ( p r o t e m e o n c e n ~ a t l o n , 2 m g / m l ) w e r e i n c u b a t e d at 6 8 ° C m b u f f e r A for varlous tames E x t r a c t o f M g L - 8 c o n t m n m g n o r m a l adenosine deammase (e -') E x t r a c t o f G M 2 4 4 5 c o n t a i n i n g e x c l u s l v e l y E H N A 4 n s e n s l t l v e a m m o hydrolase acttwty (o o) (see Flg 1B)

287 T A B L E III S E N S I T I V I T Y O F L Y M P H O B L A S T A D E N O S I N E D E A M I N A T I N G A C T I V I T Y T O I N H I B I T I O N BY DIVALENT CATIONS All d i v a l e n t c a t i o n s w e r e u s e d as t h e i r c h l o r i d e salts a t a final c o n c e n t r a t i o n of 2 5 m M T h e E H N A - m s e n sitlve a m m o h y d r o l a s e a n d a d e n o s i n e d e a m m a s e w e r e isolated f r o m sucrose g r a d i e n t s a n d w e r e a s s a y e d using K m c o n c e n t r a t i o n s of a d e n o s i n e m t h e p r e s e n c e of d i v a l e n t c a t i o n s T h e E H N A - m s e n m t i v e a m m o h y d r o l a s e a n d a d e n o s i n e d e a m m a s e w e r e a s s a y e d using [ 8 - 1 4 C ] a d e n o s m e ( 2 / ~ C f f ~ m o l ) a t final a d e n o s i n e c o n c e n t r a t i o n s of 4 m M a n d 0 1 raM, r e s p e c t i v e l y R e s u l t s are e x p r e s s e d as a p e r c e n t a g e of i n h i b i t i o n c o m p a r e d to the control wtth only buffer added Catton

Iron Nickel Magnesium Manganese Calcium MercUry Cadmium Cobalt Copper Barlum Zinc

P e r c e n t m h l b i t l o n of a d e n o s m e d e a m m a t m g a c t l w t y EHN A-msensltlve an~ m o h y d r o l a s e

Adenosine deammase

0 4 16 23 25 94 76 30 96 14 93

71 76 10 48 18 97 96 56 98 5 98

T A B L E IV APPARENT TIVITY

SUBSTRATE

SPECIFICITY

OF

LYMPHOBLAST

ADENOSINE

DEAMINATING

AC-

T h e f m a l c o n c e n t r a t i o n of e a c h s u b s t r a t e t e s t e d was 2 5 m M w t t h t h e e x c e p t i o n of E H N A a n d 2 ' - d e o x y c o f o r m y c m w h i c h w e r e used a t final c o n c e n t r a t i o n s of 2 0 0 /JM a n d 20 /~M, r e s p e c t i v e l y All s o l u t t o n s w e r e p r e p a r e d m 50 m M Trls HCI, p H 7 4, o n t h e d a y o f t h e s t u d y T h e E H N A - m s e n s l t l v e a m m o h y d r o l a s e a n d a d e n o s i n e d e a m m a s e w e r e i s o l a t e d f r o m sucrose g r a d i e n t s a n d w e r e a s s a y e d using K m c o n c e n t r a t i o n s of a d e n o s a n e m t h e p r e s e n c e of v a r t o u s c o m p o u n d s T h e E H N A - m s e n ~ t i v e a m m o h y d r o l a s e a n d a d e n o s i n e d e a m m a s e w e r e a s s a y e d using [8 - 1 4 C ] a d e n o s m e (2 /JCl//Jmol) a t final a d e n o ~ n e c o n c e n t r a t t o n s o f 4 m M a n d 0 I raM, r e s p e c t i v e l y R e s u l t s a r e e x p r e s s e d as a p e r c e n t a g e o f m l ~ b i t i o n c o m p a r e d to t h e c o n t r o l with only buffer added Compound

Adenine N-Carbamyl-~-alamne 6 - M e t h y l a m m o p u r m e rtboslde 4-Ammo-5-tmldazole carboxamlde-HCl 6-Chloropurme nbostde 6-Chloropynne 6-Methyl m e r c a p t o p u r l n e rlbostde Cordycepm (3'-deoxyadenosme) 2,6-Dtammopurme 5-Adenosyl homocysteme Homocysteme thlolactone EHNA 2'-Deoxycoformycm

P e r c e n t m h l b l t l o n of d e a m m a t m g a c t l w t y EHNA-msensltlve ammohydrolase

Adenosine deammase

75 0 42 47 18 70 25 51 99 12 9 8 99

51 #8 88 42 24 15 55 90 8 8 7 98 99

288 specific radmactlve-labeled substrates and estabhshed radiochemmal assay procedures, neither the EHNA-msensltlve ammohydrolase nor adenosine deamlnase were able to catalyze the deammatmn of guanine, adenine, AMP or cytadme. The possibility that this EHNA-msensltlve ammohydrolase activity reflected an aggregated or denatured form of adenosme deammase was examined further by a radmlmmunochemlcal assay. Eqmvalent achwtaes of lymphoblast adenosine deammase and EHNA-msensltlve ammohydrolase, 10, 5.0 and 2.5 nmol/ mm per ml of each respectively, were assayed vnth a prewously described competitive adenosme deammase radmlmmunoassay [24]. Under the condltmns of this assay, the least detectable quantity of adenosine deammase was eqmvalent to 0.17 nmol/mm per ml or 0.3 ng/ml. Usmg the radmlmmunoassay, the dflutmns of lymphoblast adenosine deammase showed a normal competltmn for antibody bmchng similar to the adenosine deammase standard used m the assay (slope --1.0) and were found to be completely cross-reactive (greater than 99%). When the same dflutmns of EHNA-msenmtlve ammohydrolase activity were assayed, vkrtually undetectable levels of adenosme deammase protein (Le., less than 1% nnmunoreactlve material relative to normal adenosine deammase) were observed. In a similar manner both lymphoblast adenosine deammase and the EHNA-msensltlve ammohydrolase were assayed for the presence of the adenosine deammase-bmdmg protein using a prewously described radmlmmunoassay techmque [22]. In both cases the level of adenosme deammasebmdmg protem was vkrtually undetectable (less than 0.5 ng/ml). These data suggested that the EHNA-msensltlve ammohydrolase did n o t share any antigenre determmants m c o m m o n vnth normal adenosine deammase and further was not associated or complexed with the adenosine deammase-bmdmg protein m lymphoblast cell hnes. Discussion

In this study we have demonstrated the presence of an EHNA-msenmtlve ammohydrolase m both normal and adenosme deammase
289 deficient as well as the normal cell lines analyzed. These data suggest that the level of adenosine deammase act]vlty does not apparently regulate the level of the EHNA-insenslt]ve ammohydrolase act]vlty m lymphoblasts. The EHNA-insenslt]ve arnmohydrolase present In both normal and adenosme deammase
290 The presence of this low level of EHNA-msensltlve ammohydrolase act~wty described m lymphoblasts may be a umversal fmdmg m a variety of normal human tissues. Therefore, m future lnvestlgataons designed to characterize the low level of residual adenosine deammase in patmnts with adenosine deflcmncy and severe combined immunodeflclency dLsease, it will be important to either exclude the presence of this EHNA-lnsensltlve amlnohydrolase a c h w t y or be able to dLstmguLsh It from a putative altered product of the adenosine deamlnase gene locus. In our study we have presented several dlstmguLshmg charactenstms of the EHNA-lnsensmve ammohydrolase and adenosine deammase for this purpose.

Acknowledgements The authors would hke to express their appreciatmn to Susan B. Stnkwerda for her free techmcal assistance. This research was supported by the Nataonal F o u n d a t m n March of Dimes Research Grant, 1--393, Nataonal Institute of Health Grant, AM 19045, and the National Cancer Institute Grant, CA 26284.

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