Molecular form of adenosine deaminase in severe combined immunodeficiency

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MOLECULAR FORMOF ADENOSINE DEAMINASE IN SEVERECOMBINED IMMlJNODEFICIENCY1

Martin B. van der Weyden, Rebecca H. Buckley, and William N. Kelley' Departments of Medicine and Pediatrics, Duke University Medical Center, Durham, North Carolina 27710 Received

January

15,1974

SUMMARY. The specific activity of adenosine deaminase was reduced to appromy 0.5% of normal in splenic tissue obtained from a patient with severe combined immunodeficiency. Sedimentation analysis of splenic homogenate from this patient revealed a major peak of adenosine deaminase activity which corresponded with respect to the sedimentation coefficient of one of three molecular species observed in control spleens but had markedly reduced activity. These findings suggest that the molecular heterogeneity of humanadenosine deaminase is under the control of a single genetic locus and that the deficiency of adenosine deaminase activity in severe combined immunodeficiency is not due to a genetic deletion.

INTRODUCTION . A deficiency drolyase,

E.C.3.5.4.4)

activity

of adenosine deaminase (adenosine aminohyhas recently

been reported in a group of patients

with severe combined immunodeficiency (1,2,3). first

association of a specific

dysfunction,

enzyme defect with congenital T and B cell

the nature of this association

is of special interest.

present time, however, there is insufficient whether the deficiency

function,

which also alters

deletion,

which also involves possibility

Based on differences

(b) a reflection

immunefunction, loci critical

has been cited

At the

data to allow one to determine

of adenosine deaminase activity

to the immunologic dysfunction,

for the latter

Since this represents the

is

(a) causally related

of an aberration

or (c) the result

of cell

of a genetic

to the immuneresponse.

Support

(1).

in net charge and/or molecular size, multiple

of adenosine deaminase have been described in normal humantissue

forms

(4,5,6).

'This research supported in part by U.S.P.H.S. Research Grant AM 14362. Dr. van der Weydenis a Merck, Sharp and DohmeInternational Fellow in Clinical 2 Pharmacology. Address reprint requests to Dr. Kelley. Abbreviated in the text: S2Dw= sedimentation coefficient. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

590

Vol. 57, No. 3, 1974

There

has,

however,

adenosine

in tissues

and reduced our

findings

in splenic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

been no attempt

deaminase

deficiency report

BIOCHEMICAL

to characterize

from patients

activity

of this

on the activity

homogenate

obtained

the molecular

with

enzyme.

from a patient

combined

severe In this

and molecular

form

with

form of immuno -

communication

we

of adenosine

severe

deaminase

combined

immuno-

deficiency. METHODS --Enzyme assay. [S-14C]

adenosine

standard (0.16

(New England

Nuclear

deaminase

mM, 50.4 )ICi/umole (3-15

pg protein)

alcohol.

chromatography from the product 6000 v)

aliquot with

in a Packard formation

Tri-carb

by high

buffer,

on the paper

with

voltage

electrophoresis

light

scintillation

was linear

with

was spotted

containing

of the

at

substrate

(250 ma, 4000 to

(2.54 run),

and protein

of 50 ~1 of

on Whatman 3 MM

1 mM EDTA.

spectrometer time

at 37OC

by centrifugation

Separation

pH 8.5,

and

incubation

by the addition

was removed

The

adenosine pH 7.4,

After

carriers.

ultraviolet

liquid

of inosine

50 mM Tris,

of

inosine.

[S-14C]

of 100 ~1.

protein

appropriate

[S-14C]

contained

of the supernatant

was achieved

in 50 mM borate

identified

to

was terminated

The precipitated

paper

mixture

volume

the reaction

by the conversion

Corporation)

reaction

in a final

X g and a 20~1

was assayed

or 0.4 mM, 2 &i/umole),

15 to 120 minutes,

absolute 1,000

deaminase

adenosine

enzyme for

Adenosine

cut

Inosine out

was

and counted

at 63% efficiency. concentration

The

under

these

conditions. Enzyme preparation. and from one subject necropsy and that of time

.

with

The tissue

from

of two control

of tissue

at 6,600

X g for

Protein Lowry --et al

with

combined

the patient

bovine

was obtained

splenic

from

5 control

immunodeficiency

with

had been

3 to 4 volumes

30 minutes

with

severe

Crude

determination. (7)

tissue

subjects

(30 to 36 months).

one part

Splenic

severe stored

at the time

combined at -7O’C

homogenates of 10 mM Tris,

subjects

immunodeficiency for

a comparable

were prepared pH 7.4,

was determined

serum albumin 591

as standard.

using

and centrifuging

the method

length

by homogenizing

at 4OC. Protein

of

of

Vol. 57, No. 3, 1974

Sucrose

gradients.

in 10 mM Tris, splenic

BIOCHEMICAL

centrifuged

4.1)

RPM for

(9).

and catalase

11.3)

when subjected deaminase 10.4.

In contrast,

immunodeficiency

a S20w of 7.4.

with

the total control

activity

and that

from

of this

activity

The cause human tissues was that

not

result

homogenate

has not

been

of adenosine

has been unclear.

in the reduction

the

species

deaminase the

to

combined deaminase 2.2% of

in the

activity

inununodeficiency.

at a low level

control

preparation The nature

defined.

heterogeneity

of adenosine

One hypothesis

in activity

severe

species

severe

case,

and 10.2

peak was approximately molecular

by

of adenosine

of adenosine

in both

form of adenosine were

peaks

with

of the gradient combined

up to

subjects,

7.1 to 7.4;

the patient

corresponding

further

of the molecular

If this

in this

for

control

three

of 3.2;

one molecular

the

three

in

CPM above

or on enzyme profile from

from

(5,600

at -700C

exhibited

S20w values

The presence

with

(S20w,

calculations.

and 0.03

Storage

analysis,

for

the patient

in the

human hemoglobin

for

7.8222.59

homogenates

activity

bottom

4.3),

on enzyme activity

1B) displayed

each molecular

locus.

were respectively.

splenic

at the

migrated

and a patient

1A) with

observed

on the

subjects

tissue

effect

The total

preparation.

was reproducible

gene

(Fig.

calculated

a Gilson

of 5 control

splenic

The splenic

the

were

with

deaminase

protein,

(Fig.

collected

a

of adenosine

to zone-sedimentation

activity

using

activity

immunodeficiency

analysis.

and then

The specific

combined

sedimentation

(Szow,

of crude

SW41 rotor

were

(Szow)

(200~1)

as standards

with

had no apparent

gradients

were prepared

were used

from

36 months

on the

of 255 pl

serum albumin

prepared

pmoles/hr/mg

Samples

of the S20w to the distance

homogenates

background)

(8).

coefficients

Bovine

(S20w,

gradients

at 4’C in a Spinco

Fractions

RESULTS AND DISCUSSION. ____-

severe

layered

30 hours

relationship

gradient

sucrose

previously

Sedimentation

of a linear

isokinetic

were

ultracentrifuge.

microfractionator.

sucrose

as described

and standards

at 40,000

Beckman LS-50

basis

10% to 28.2%

pH 7.4,

homogenate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

proposed

deaminase

a mutation of all

592

by Edwards

was the product

involving forms

deaminase

one of these of the

enzyme.

in normal --et al

(5)

of a specific loci

should

Our finding

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

i:\l! 10

Fig. 1 - Sucrose density in splenic homogenate. combined immunodeficiency.

that

two molecular with

present,

is markedly

therefore,

inconsistent

Nishihara are

severe

--et al

(11)

interconvertible. deaminase

molecular

heterogeneity

provide

further

The substantial homogenate be

attributed

from

25 NUMBER

of adenosine

combined

35

are

immunodeficiency,

reduced

40

TOP

in activity hypothesis.

suggest

that

the molecular

for

this

contamination

genetic

(10)

and

deaminase

that

all

locus

events.

is,

forms

and that

Our observa-

interpretation.

adenosine with

is

of

is

tissue,

of Ressler

results

of a single

tissue

form which

of adenosine

from post-translational

evidence residual

forms

in splenic

to normal

The studies

of their

the control

results

the

when compared

this

are under

absent

while

with

the patient to

30

deaminase

One interpretation

adenosine

tions

20 FRACTION

gradient ultracentrifugation of adenosine deaminase A. Control Subject. B. Patient with severe Note different ordinates.

forms

a patient

15

deaminase

combined occuring 593

activity

immunological after

the

in the deficiency patient's

splenic cannot death

since

of the

Vol. 57, No. 3, 1974

examination

BIOCHEMICAL

of another

enzyme in the

phoribosyltransferase,

E.C.2.4.2.8)

enzyme was present. deaminase

present

enzyme.

These

activity

in splenic

tissue

from

form

The initial

suggestion

severe

combined

affecting that

both

the

Recent

(12) * this

is not

whereas this

for

the

the

using

case since

locus

type

protein

deaminase

would

or of a markedly

than

a low

level

Although reflection

studies

deaminase do not

deaminase

transport

adenosine

the

In either

cogent

a general

related

possibility disorder

or processing

HL-A

that

is on chromosome

the possibility

20

that

adenosine

deaminase

could

not

be

evidence

the gene coding

for

of no enzyme

case an absence,

deaminase

does not

is causally

for

rather

be expected.

adenosine it

loci

indicate

to the production

would

deletion,

to the

we do provide

involving

molecule.

deletion

on observations

to the HL-A loci

to lead

deficiency

genetic

the gene for

deletion

that

deaminase

in culture

study

this

competence.

was based

However,

for

immunodeficiency

of catalytic

deaminase

6 (13).

activity,

deficiency

exclude

may reflect

recognition,

aberrant

genetic

cells

In the present

a large

we can now suggest

combined

to be linked

of the

deaminase

be due to a large

involving

be expected

of catalytic

of a large

adenosine

for

gene coding

of adenosine

gene unrelated

of evidence. since

severe

form

the adenosine

adenosine

a low level

hybrid

excluded

adenosine

with

mouse-human the

above,

and immune function,

are on chromosome

hypothesis

residual

appeared

immune response

this

the

phos-

the mammalian

to another

deaminase

and an important

against

cited

could

was due to a deletion

by this

reasons

the association

deaminase

HL-A loci

association

that

immunodeficiency

studies

only

enzyme with

that

adenosine

that

the patient

of the

adenosine

locus

the

(hypoxanthine-guanine

be attributed

suggest

is due to a mutant

with

for

cannot

observations

same tissue revealed

In addition,

activity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of antigens

necessarily

is not

follow

the deficiency function

which

or mediators

a

that

to immune dysfunction.

that of cell

deficiency

Our

of adenosine also

affects

of the

immune

the

response. REFERENCES 1.

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E.R., Anderson, Lancet 2, 1076.

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B.,

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