Isolation of cDNA clones for human β-glucocerebrosidase using the λgtll expression system

Vol. 123, No. 2, 1984 September

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17, 1984

Pages

ISOLATION

OF cDNA CLONES FOR HUMAN B-GLUCOCEREBROSIDASE USING THE XgtII EXPRESSION SYSTEM

Edward I. Ginns, Prabhakara V. Choudary, Brian M. Martin, Winfield, Barbara Stubblefield, June Mayor, Denise Merkle-Lehman, Gary J. Murray, Lisa A. Bowers, and John A. Barranger

Suzanne

Developmental and Metabolic Neurology Institute of Neurological and Communicative National Institutes of Health Bethesda, Maryland 20205

National

Received

574-580

July

18,

Branch, Disorders

and Stroke,

1984

Two cDNA clones (XGC-1 and XGC-2) for human B-glucocerebrosidase [EC 3.2.1.451 have been isolated from a human hepatoma library in AgtII by immunological screening using monospecific polyclonal antibody for 8-glucocerebrosidase. Restriction endonuclease mapping indicates that these clones are probably identical in size, each with a 1900 bp insert. The 50 kDa size of the insert-encoded polypeptfde produced by these clones in fusion with 6-galactosidase of xgtII in E. coli BNN103 is consistent with the size of the nascent form of B-glucocerebrosidase. These fusion proteins are shown by Western blotting to react with antibody to B-glucocerebrosidase. Amino acid sequence deduced from the nucleotide sequence of the insert ir pGC-1 is identical to known amino acid sequence of B-glucocerebrosidase, and thus, confirms that the clones are specific for 8-glucocerebrosidase.

The deficiency

of the membrane-associated

B-glucocerebrosidase comnon Jewish

[EC 3.2.1.451

genetic

disorder

results

(1,2).

phenotypes:

Type 1 (chronic,

(acute

neuronopathic),

and Type 3 (chronic

species

are present

cross-reacting extracts

(6,7)

proteins as well

Abbreviations 0006-291X/84 Copyright AN righrs

by the

in electrophoretic material

arises

(CRM) demonstrable

(4,5).

from

abnormal

The absence

pattern

disease, occurs

non-neuronopathic), neuronopathic)

in fibroblast

Type 2 (3).

apparent of residual

These

molecular

Horseradish-Peroxidase.

(Ashkenazi)

$1.50 574

tissue

of 6-glucocerebrosidase

CRM in

between

HRP --

weight

and other

of complementation

predilection

distinct

B-glucocerebrosidase

processing

ethnic

the most

as three

post-translational

as the

0 1984 by Academic Press. Inc. of reproduction in ai?\ form reserved.

disorder

different

patterns

The characteristic

(4,5).

each phenotype mutant

distinguishable

glycoprotein

in Gaucher's

This

clinical

phenotypes

lysosomal

of only

of the phenotypes

Type 1

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 2, 1984

Gaucher's

disease

mutations

in the 8-glucocerebrosidase

the

phenotypic In order

provide

(2)

classes

for

we report

gene(s)

using

a cDNA library

cloned

by their

the

gene as part reactivity

strains,

are

involved

genetics

diagnosis,

in agtII

of this

we undertook

which

isolation

is

present

of cDNA clones (8-11).

of a B-galactosidase

multiple

allelic

in determining

disease the

and to

cloning

of

in low abundance. for

Recombinant fusion

to 6-glucocerebrosidase

cDNA library,

that

disease.

Materials Bacterial

hypothesis

locus

human 6-glucocerebrosidase paper

the

the molecular

tools

In this

the

support

of Gaucher's

to understand

more sensitive

mRNA for

further

protein

the

8-glucocerebrosidase phage were

that

expressed

identified

antibody.

and Methods

and reagents.

A human hepatoma cDNA library in xgtII and the E.coli strain BNNIO3 were generously provided by Drs. John 0 Briena and Jefnwetb (Neurosciencea and Biologyb Departments, University of California, San Diego) (10,ll). strains Y1088, Y1089, and Y109O were obtained from Drs. R. Young --E.coli and R. Davis (Biochemistry Department, Stanford University School of Medicine) (8,9). Restriction endonucleases and sequencing reagents were obtained from Bethesda Research Laboratories (Gaithersburg, Maryland) and New England Biolabs (Beverly, Massachusetts). Goat antirabbit HRP antibody and 4-Chloro-1-Naphthol were purchased from Biorad (Rockville Center, New York). Library

screening.

The preparation of monospecific rabbit antibody to human placental glucocerebrosidase has been described (4). This antibody was used to screen the library and in immunoblotting experiments. Recombinants that expressed cross-reactive material to 8-glucocerebrosidase were identified as described by Young and Davis (8,9), except that goat antirabbit HRP second antibody was used to visualize plaques on the BA85 nitrocellulose (Schleicher and Schuell) filters using 4-chloro-l-naphthol (10,ll). Positive recombinants were grown in NZYCM medium. The phage were purified on cesium chloride step gradients, and the DNA was isolated and purified using the SDS-EDTA protocol (12). Fusion

protein

identification.

Lysates were prepared from large scale, temperature induced, AGC-I/ BNN103 and AGC-2/BNN103 as described (10,ll) and electrophoresed on SOS These gels were either stained for protein or polyacrylamide gels (13). analyzed using imnunoblotting techniques (4,14). Subcloning The phage on alkaline and used

and plasmid

DNA preparations.

cDNA inserts were purified from the EcoRl digests of the recombinant NACS prepac (BRL) and ligated into the EcoRl site of the bacterial phosphatase-treated EcoRl digest of pBR322 (New England Biolabs) to transform C600. Large scale plasmid preparations of --E.coli 575

Vol. 123, No. 2, 1984

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

subclones pGC-1 and pGC-2 were performed using standard protocols (12). Plasmid DNA was isolated using the alkaline lysis method and further purified on cesium chloride equilibrium gradients (12). Nucleotide

sequence

analyses.

Plasmids, pGC-1 and pGC-2, were linearized (using Eco RV) and sequenced by the dideoxynucleotide chain termination procedure (15) as modified for double stranded template (16) using EcoRl and Hind111 universal pBR322 primers (New England Biolabs), or a 17-mer oligonucleotide mix derived from 8-glucocerebrosidase amino acid sequence. Protein

sequencing

and oligonucleotide

synthesis.

Amino acid sequences were determined for the N-terminus and a number of tryptic peptides from homogenous human placental B-glucocerebrosidase (17) using a Beckman 890M sequencer. Selected regions of the amino acid sequences were used as the basis for synthetic oligonucleotide sequences (57). These oligomers, synthesized by the triester method, were purchased from either OCS Labs (Denton, Texas) or New England Biolabs (Beverly, Massachusetts).

Results Approximately were

screened

(8,9),

2.4

by the --in situ

as modified

one hundred screening single

and forty

plaques

(Figure

by virtue

Preliminary

(Figure

1C).

primer

extension

(17).

A comparison

sequence 2) with

of the

inserts

(15,16)

using

of the

insert

clones

were

acid

inserts

approximately

(18).

into

17-mer

acid

a region

acid

sequence

to each other from

homogeneous

This

XGC-1 and XGC-2 as specific

established

chosen

for

clones

of

further proteins.

appeared

identical.

1900 bp in length pBR322 and sequenced

deduced

primer

the

nucleotide

match

(Figure

from

(17).

by

as the

of perfect

In addition, different

human placental identity

to B-glucocerebrosidase. 576

stage

and fusion

and to four

the

further

to the

oligonucleotide

sequence

of pGC-1 showed

derived sequence

of their

Out of

through

processed were

subcloned

amino

were

(11).

taken

maps of these

the

amino

were

Two clones

size

were

B-glucocerebrosidase

oligonucleotides

clones

cDNA library

of Young and Davis

and deWet --et al forty

endonuclease

pGC-1 and pGC-2 hybridized

amino

large

cDNA inserts These

procedure

isolates,

1A and 1B).

restriction

the human hepatoma

(10)

positive

of the

The EcoRl-excised

--et al

primary

putative

from

immunological

by O'Brien

and six

analysis

x 107 phage

and Discussion

synthetic

B-glucocerebrosidase of both

the

Vol.

123,

No.

2, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

6.6KbFlasmid

4.4Kb-

2.OKb-

Insert

1.4Kbl.lKb0.9Kb-

Figure

Inserts

in fusion fusion

1:

Isolation and characterization of cDNA clones. (A/B): Screening of human hepatoma cDNA/xgtll library with antibody to human 6-glucocerebrosidase. (A): Intermediate screening (third round) of putative positive reccmbinant phage (approximately 104). The dark spots on the nitrocellulose filter represent positive plaques. (B): Final screening of the purified positive phage (XGC-1). of EcoRl digests (C): Agarose gel (1.2%) electrophoresis pGC-1 and pGC-2. The cDNA inserts are approxiof subclones mately 1900 bp in size relative to the A/Hind III and 0X174/ Hae III standards.

of

both

with

of the

suggests

that

addition

to PEPTIDE

Figure

reacted of the

50 kDa size

and

B-galactosidase

protein

A comparison

xGC-1

with size

the clones those

coding

of Xgtll antibody

of the

nascent

form probably for

AGC-2 encoded, (Figure

in --E.coli 3).

BNN103, On Western

to B-glucocerebrosidase

inserts

of the clones

of B-glucocerebrosidase contain

untranslated

a 50 kDa protein blots,

(Figure

this 4).

pGC-1 and pGC-2 to the protein

(19)

sequences

in

+glucocerebrosidase.

GT33

2:

Schematic representation of amino acid sequence deduced from the nucleotide sequence of the pGC-1 insert. This sequence is identical to the amino acid sequence of the peptide GT33 from human placental p-glucocerebrosidase.

577

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No.

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ZookDa

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-

- 165kDa 114kDa 92.5kDa -

67kDa -

46kDa -

a Figure

3:

b

c

d

e

SDS-polyacrylamide gel (8%) of cell lysates stained with Coomassie-blue. Lane a, protein standards; lane b, E.coli BNN103; lane c, xgtll/BNN103; lane d, XGC-l/BNNlD3 and-e xGC-Z/BNN103. The 6-galactosidase (A tll) and B-galactosidaseB-glucocerebrosidase fusion proteins are 9 XGC-1 and xGC-2) seen at 114 kDa (lane c) and 165 kDa (lanes d and e), respectively.

e,

200kDa 165kDa 92.5kDa 68kDa-

ab Figure

4:

Western blot as described BNN103; lane

of in

c,

cd the the

fusion text.

proteins detected by 1251~Protein Lane a, E.coli BNN103; lane b, AGC-l/BNN103; and lane d, xGC-2/BNN103. 578

A igtll/

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 123, No. 2, i984

The cDNA clones more detailed

study

responsible

for

confirmation

of the

for

common to several

phenotypes

of Gaucher's

B-glucocerebrosidase

progress

processes

length

should

be possible

responsible

for

as well

as

locus

for

information that

using in our

lysosomal

mechanisms

structural

compartmentalization Thus,

a

cDNA and subsequently

may provide

hydrolases.

now facilitate

disease,

of the

of full

and cellular

will and molecular

localization

Analysis

lysosomal

further

mutations

chromosomal (20).

to enzyme synthesis

molecular

allelic

the diverse

clones

a model,

human B-glucocerebrosidase

of the

glucocerebrosidase genomic

for

pertaining

could

be

Gaucher's

disease

understanding

storage

as

of the

disorders.

Acknowledgement We thank

Dr.

and generosity fully

John

in sharing

acknowledge

the

Shelby

Berger,

David

Barbara

Wold,

Roscoe

Beverly

Smith

for

financial

O'Brien

support

help Lanar,

and Dr.

the

given Joseph

0. Brady,

helping

agtII

with

of the Gaucher's

deWet for

hepatoma

their

Richard

Young,

Tager,

Hisao

Fukushima,

Mook and Ad Konigs.

preparation Disease

suggestions

cDNA library.

by Drs.

George the

Jeff

We also Ronald

Davis,

Hiroto

Okayama,

We thank

of the manuscript. Research

grate-

Foundation

The is appreciated.

References

1. 2.

3.

4. 5. 6.

Brady, R. O., Kanfer, J. N. and Shapiro, D. (1965) Biochem. Biophys. Res. Commun. 18, 221-225. Kolodny, E. H., Ullman, M.D., Mankin, H. J., Raghavan, S. S., Topol, J. and Sullivan, J., (1982) in Gaucher Disease: A Century of Delineation and Research, Desnick, R. J., Gatt, S. and Grabowski, G. A. (eds.), Alan R. Liss, Inc., New York, pp. 33-65. Brady, R. 0. and Barranger, J. A. (1983) In: The Metabolic Basis of Inherited Disease. Stanbury, J. B., Wyngarden, J. B., Fredrickson, D. S., Goldstein, J. L. and Brown, M. S., eds. McGraw-Hill Book Co., Inc., New York, 5th Edition, pp. 842-856. Ginns, E. I ., Brady, R. O., Pirruccello, S., Moore, C., Sorrell, S., Furbish, F. S., Murray, G. J., Tager, J. M. and Barranger, J. A. (1982) Proc. Nat'l. Acad. Sci., USA 79, 5607-5612. Ginns, E. I., Tegelaers, F. P. W., Barneveld, R., Brady, R. O., Tager, J. M., Galjaard, H., Reuser, A. J. U. and Barranger, J. A., (1983) Clin. Chim. Acta. 131, 283-287. Wenger, D. A., Roth, S., Duloh, T., Grover, W. D., Tucker, S. H., Kaye, E. M. and Ullman, M. D., (1983) Ped. Res. -17: 344-348.

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10.

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Gravel, R. A. and Leung, A. (1983), Human Genetics 65, 112-116. Young, R. A. and Davis, R. W. (1983), Proc. Nat'l. Acad. Sci., USA 80. 1194-1198. Young, R. A. and Davis, R. W. (1983) Sci. 222, 778-782. O'Brien, J. S., deWet, J., Fukushima, H., Wilcox, E., Dewji, N., McGee, J., Warner, T., Yoshida, A., Fluharty, A., Hill, F. and Helinski, D., (1984), In: Molecular Basis of Lysosomal Storage Diseases, Academic Press, New York (in press). DeWet, J., Wilcox, E., Fukushima, H., Dewji, N., O'Brien, J. S. and Helinski, D. (submitted 1984) Nut. AC. Res. Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning: A Laboratory Manual (1982), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Laernnli, U. K. (1970), Nature (London) 227, 680-685. Towbin, H., Staehelin, T. and Gordon, J. (1979), Proc. Nat'1 Acad. Sci., U.S.A. 76, 4350-4354. Sanger, F., Nicklen, S. and Coulson, A. R. (1977), Proc. Nat'l. Acad. Sci., U.S.A. 74, 5463-5467. Wallace, R. B., Johnson, M. J., Suggo, S. V., Miyoshi, K., Bhatt, R. and Itakura, K. (1981) Gene 16, 21-26. Martin, B. M., Murray, G. J., Coligan, J. E., Raum, M., Brady, R. 0. and Barranger, J. A. (1984) Fed. Proc. 43, 2639. Ginns, E. I., Martin, 8. M., Choudary, P. V., Winfield, S., Stubblefield, B., Mayor, J., Murray, G. J., Bowers, L. A. and Barranger, J. A. (1984) Fed. Proc. 43, 1653. Erickson, A. H., Ginns, E. I., Barranger, J. A., Brady, R. 0. and Blobel, G. (1983) Fed. Proc. 42, 1912. Barneveld, R. A., Keijzer, W., Tegelaars, F. P. W., Ginns, E. I,, Geurts von Kessel, A., Brady, R. D., Bararnger, J. A., Tager, J. M., Galjaard, H. and Westerveld, A. and Reuser, A. J. J. (1983) Hum. Genet. 64, 227-231.

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