Identification of novel proteins synthesized in bone cells by antibody screening of a cDNA expression library

Vol. 151, No. 1, 1988 February 29, 1988

BIOCHEMICAL

IDENTIFICATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 382-387

OF NOVEL PROTEINS

SYNTHESIZED

IN BONE CELLS

BY ANTIBODY SCREENING OF A cDNA EXPRESSION Elka

Nutt,

Chris

Dunwiddie,

John

W. Jacobs

LIBRARY

and Ellen

Simpson

Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486 Received

January

18,

1988

SUMMARY: Novel proteins synthesize predominantly in bone have been identified by antibody screening of bone cell cDNA expression libraries. Two unique cDNAs were identified whose structures do not match any known nucleic acid or protein sequence in the NIH computer bank. The first cDNA clone, BP-I, encoded a mRNA of 2300 bases in size which was expressed at high levels in rat calvarial bone cells and placenta. A 17/2.8 rat osteosarcoma cells, second clone, BP-II, encoded a mRNA of 1500 bases which was expressed at high levels in 17/2.8 osteosarcoma cells and in salivary gland. Expression of both mRNAs in osteosarcoma cells was modulated by the calciotropic hormone, vitamin D. Southern blot analyses indicated that the two cDNAs represented distinct, single copy genes in the rat genome. These novel gene products may serve as B 1988 ’ potential new markers to study bone turnover in metabolic bone disease. Academic

Press,

The

Inc.

organic

component

collagen

with

the

proteins

(1).

These

proteins

found

in

isolation

and

achieved, and

bone (reviewed

noncollagenous of

them

which

these

still

exists

markers In

are

proteins

serve

in

identifying turnover

both

of

proteins

a

number osteocalcin, 1).

are

and have as markers

been

as

tools

the

of

these large to

as

(2).

The

have

been

proteins

osteopontin, the

functions

antibodies

examine

the

of to

interest

as potential

clinical

the

a

extent

Considerable

turnover.

study

I

as well

matrix

part,

proteins to

bone

although

utilized

Type

noncollagenous

osteonectin,

unknown,

of bone

insoluble of

proteins,

on

In

bone-specific

and

90% mixture

bone-specific

or

reference

new

a

concentrated

protein in

approximately

development

to

of

the

approach

to

phenotype. present

novel libraries

OC06-291X/88 Copyright All rights

available

bone

the

identify DNA

bone

are

for

osteoblast

which gla

is

representing

include

characterization

including

number

bone 10%

proteins

serum

sialoprotein

these

of

remaining

study,

mRNAs which were

we have are

utilized

expressed

screened

predominantly with

$1.50

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

a molecular

382

antibodies

biology in

bone. prepared

Recombinant against

BIOCHEMICAL

Vol. 151, No. 1, 1988

partially-purified clones

that

certain

bone reacted

the

antibodies.

identified

unidentified

heretofore, these

proteins

with

clones

cDNA

matrix

cloned

cDNAs encoded

can be regulated identified

bone

by the

may serve

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

to

DNA sequence

in

the

proteins.

screening

are

calciotropic

new protein

unique

level

D.

The proteins

for

bone

for,

indicated

at high

markers

that

coded

procedure

vitamin

cDNA

indicated

analyses

expressed

hormone,

identify

analyses

Hybridization

mRNAs which

as potential

utilized

in

that

bone

and

we have

turnover.

MATERIALS AND METHODS cDNA Librarv Screening Messenger RNA was prepared from a rat osteosarcoma cell line (3) and utilized to construct a cDNA expression library in the bacteriophage vector lambda GTll (4). The amplified library contained 7.8 x 105 independent clones with an average insert size of 1.05 kb. The amplified library was plated on E. coli 1090 strain and induction of protein synthesis was accomplished with isopropyl beta-D-thioglactopyranoside (ITPG). Expressed antigens were immobilized on nitrocellulose filters and screened with an antiserum raised to partially purified bone matrix proteins. 1251-goat-anti-rabbit ant ibody was used as the secondary antibody to visualize positive clones. Bacteriophage plaques showing a positive signal by autoradioagraphy were cored, replated and rescreened to plaque homogeneity. Recombinant cDNAs selected for further analysis were subcloned into M-13 vectors for dideoxy-sequencing (5). Northern Blot Analvsis - Intact RNA was isolated from a variety of rat tissues as well as two rat osteosarcoma cell lines, ROS 25/l and ROS 1.7/28, according Poly A RNA was prepared from total RNA from to the procedure of Chirgwin (3). 17/28 cells by oligo-(dT)-cellulose chromatography (6). Twenty micrograms of total RNA (as determined by optical density at 260 nM) or 3 ug of poly-A RNA were loaded per lane and then fractionated on 1.2% agarose12.2M formaldehyde gels containing 0.5 ug/ml ethidium bromide. The RNAs were then transferred to nitrocellulose according to the procedure of Thomas (7). The relative amounts of RNA were determined by densitometric analysis of hybrid images utilizing a Hoefer GS 300 transmittance scanning densitometer. As positive controls and for quantitation purposes, filters were subsequently rehybridized with a radiolabelled actin cDNA probe. Southern Blot Analysis - High molecular weight genomic DNA extracted from rat kidney was digested with various restriction endonucleases, fractionated on 0.7% agarose gels and transfersed to nitrocellulose according to the procedure Hybridizations were done at 43OC for 16-20 hours with the of Southern (8). addition of nick-translated 32P-labeled probe (2.5~10~ cpm/ml). The blots times in 2xSSC (2OxSSC is 3M NaC1/0.3M sodium were initially washed three citrate/O.l% sodium dodecyl sulfate/O.l% sodium pyrophosphate) at room temperature. The blots were then washed twice at 50°C in 0.4xSSC/O.l% sodium dodecyl sulfate/O.l% sodium pyrophosphate. The filters were then sealed in plastic bags and exposed to x-ray film. RESULTS AND DISCUSSION As

the

antisera

heterogeneous identified. two

cDNA

their

size,

(data

not

which mixture

Based clones

was

utilized

bone

matrix

on a preliminary were

appeared shown).

of

identified to

These

code two

in screen

that for,

these

proteins

utilizing

hybridize

heretofore,

cDNA clones 383

studies

a number

was

Northern with

mRNAs,

uncharacterized were

raised

of distinct

designated

analyses,

that,

based cell

proteins

a

were

blot bone

bone

to

clones

on

mRNAs I and

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I NUCLEOTIDE SEQUENCE AND DEDUCEDAMINO ACID SEQUENCEOF BP-I AND BP-II

cDNAs

10 Gly Ala Ala His Ser Leu Pro Thr Ser Val Pro Ala Ala Leu Leu Leu Leu Asp Tyr 5'-GGA GCA GCC CAC TCC CTT CCT ACC TCG GTT CCT GCA GCG CTA CTG CTC CTG GAT TAC 20 30 Pro Pro Asp Arg Ile Ser Leu Phe Leu His Asn Asn Glu Val Tyr His Glu Pro His CCG CCG GAC AGG ATC TCT CTT TTC CTT CAC AAC AAC GAG GTG TAC CAC GAG CCT CAC 40 50 Ile Ala Asp Ala Gly His Ser Pro Gly Pro Leu Leu Ser Cys Lys Ala Ser Gly Ala ATT GCA GAT GCT GGC CAC AGT CCA GGA CCA CTT CTC AGC TGT AAA GCT AGT GGG GCC 60 70 Arg Gly Gly Leu Ser Ser Gly Glu Ala Arg Asp Met Ala Met Asp Ser Cys Arg Gln AGA GGA GGC CTG AGC TCA GGG GAG GCC AGG GAC ATG GCC ATG GAT AGC TGT CGG CAG 80 Asn Pro Ser Val AAC CCG AGT GTG-3'

10 Gln Pro Ser Asn Val His Ser Gly Ile Phe Leu Pro Asp Gln Gln Gln Asp His Val 5'-CAG CCG AGC AAC GTT CAC TCC GGC ATT TTT CTT CCA GAC CAA CAG CAG GAT CAT GTC 20 30 Leu Ile Ala Phe Asp Phe Thr Ala Leu Ser Arg Arg Ile Thr Thr Asp Ala Asp Leu CTT ATC GCC TTT GAT TTC ACC GCT CTA TCG AGA CGG ATC ACT ACT GAC GCA GAT CTC 40 50 Gly Ile Leu Pro Asp Lys Ser Ser Gln Arg Phe Glu Ala Gly Phe His Ile His Leu GGC ATT TTA CCG GAC AAA TCG TCC CAG CGT TTC GAA GCG GGA TTT CAC ATA CAT CTG 60 70 Arg Leu Ser Ser Ala Ala Tyr Ser Ser Gly His Ser Val Arg Cys Val Asn Pro Gln AGG CTT TCC AGC GCT GCG TAT AGC TCG GGT CAT AGC GTG CGA TGC GTC AAT CCA CAG 80 Ser Leu Tyr Ile Glu His Asp TCG CTC TAC ATT GAA CAT GAT-3'

II

(BP-I,

sequence BP-II

BP-II)

is

sequence vector

stressed

complete

both

The in

Table

which

is

in-frame would

that

of

1.

The with

utilized the

structure

sequence of

their

and

amino the

be expressed

antibodies protein

strands

DNA sequence

shown

and which

bone-matrix be

and

analysis.

acid

the

shown BP-I

sequence

in

and II, 384

gene

of and

screening

Figure but

sequence shown

bacteriophage

library

subjected

protein

B-galactosidase by the

in

DNA were

deduced

does

1

does

dideoxy BP-I

is

that

the

lambda

not

GT-11 by the

It represent

the

and

protein

recognized

procedure.

represent

to of

structure

should the of

BIOCHEMICAL

Vol. 151, No. 1, 1988

AND BIOPHYSICAL

4

5

RESEARCH COMMUNICATIONS

6 23.1 9.4 6.7 4.3

Figure

the

1.

expressed

six of

2.3 2.0 Southern blot analysis of BP-II (Lanes l-3) and BP-II (Lanes 4-6) cDNA clones. High molecular weight rat DNA (15 ug) was digested with with a number of restriction enzymes and hybridized radiolabelled BP-I and BP-II cDNAs. Shown here are two such experiments: Lanes 1 and 5, double enzyme digestions with EcoRl and BamHl, Lanes 2 and 6, double enzyme digestions with EcoRl and HindIII; Lanes 3 and 4, phage lambda DNA molecular weight markers. Size, in base pairs of MW markers is listed on the right.

epitope

possible the

bank

reading

reading

frames

frames

shown

whether

blot in

1,

nick-translated likely, examine

the

distribution

with

osteosarcoma

cells rat

intense.

The

only

utilizing

the

total

indicated

of that

the

NIH

all none

computer

8,

a radiolabelled

10).

It actin

should in

each

lane

cDNA probe 385

to

hybridization that,

hybridization proteins,

and,

3) D3,

a

Northern

of rat

tissues.

2200

bases

although

showed be

restriction

genes

these

vitamin shows

amounts

As

hybridized

these

distinct

Hybridization

which

and minor loaded

mRNAs

tissue,

kidney.

different

from

genes,

most

genome.

encoded

a number

rat

and

mRNA/DNA

1,25(OH)2

bone tissue

rat

in

a mRNA of 9,

data

distinct

from

with

recognize

the

2

of

electrophoresis

these

from

&and.

RNA were

in

of

to

digested

cDNA probes

Figure

(Lane

was placenta, salivary

searches

in

DNA extracted

The

mRNAs which

calvarial other

with

cDNAs.

mRNAs by

intensely

from

and

products

utilized

RNA prepared

prepared probe

mRNAs were

gel

genes

then

of

these

cDNA hybridizes

BP-I

copy

biosynthesis.

experiment

lung

the

expression

of

protein

DNA sequences listed

DNA was

BP-II

were

size of

accumulation

of

and

single

cDNAs

1)

Computer

proteins

agarose

that

represent

two rat

by

BP-I

two

complete known

performed

15 ug of

indicate

The

bone

was

fractionated

experiments

the any

the

analysis

Figure

enzymes,

of

antibodies.

Program).

establish

Southern

by the

encoded

(Intelligenetics To

ug)

recognized

and

regulation regulator hybridization

also

of BP-I

osteoblast-like, be

signal

seen

in

RNA

was

not

as

to

the

hybridization was seen

that

of

Radiolabelled

the

equivalent control

equivalent

to

tissue

blot

can

subsequent

indicated

the

a known

the

of hybridization stressed

2)

the

in

significant

assays

in

brain,

amounts

(20

experiments quantities

of

Vol. 151, No. 1, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

WW

I

2

3

4

5

Figure

2.

Figure

3.

RNA were

7

9

in

experiment,

the

10

tissue distribution of messenger RNA blot to show to BP-I cDNA. Source of RNA is shown at the top of the figure. Amount of RNA loaded in each lane is as follows: Lanes 1-9, 20 ug RNA; Lane 10, 40 ug. RNA Northern blot to show tissue distribution of messenger Source of RNA is listed at top of the hybridizing to BP-II cDNA. figure. Amounts of total RNA loaded from left to right are: lung, 10 and 20 ug; liver, 10 ug; placenta, 10 and 20 ug; spleen, 10 and 20 ug; brain 10 and 20 ug; salivary, 10 and 20 ug; 17/2.8 bone cells, 10, 20 and 20 ug.

most

each

tissue

but

utilizing

significant

(17/2.8

osteosarcoma

prepared

from

1800

6

Northern hybridizing

analyzed

of

type Again,

6

(data

not

salivary,

BP-II

was to

with

placenta

Figure

radiolabelled

hybridization cells)

shown).

lesser

cDNA as

RNA prepared

amounts

and lung.

3 shows

of

the the

from

probe.

bone

hybridization

The predicted

size

same cells

to

of

this

RNA

mRNA is

bases. Further

levels known

studies

of

the

two

regulator

(17/2.8) encoding

grown

BP-I images

by vitamin

of

the

this

or

4A,

mRNA (Lanes by

BP-II

vitamin 3 and

mRNA as

RNA (“t”

lanes,

Figure

in a

state

fashion

in

finding

adds

levels bone further

related

of

both

cells

by

BP-I the

support to bone

cell

4B).

Figure

we have used summary, expressed in bone.

our

the

4B,

levels

of levels

of

densitometric in

the

relative

rehybridization

of RNA were analyzed initial and limited

that

the

enhancement the

with

36

of mRNA

analysis

regulated

in

hormone,

hypothesis

D for

fold

subsequent

mRNAs are

D, a cells

vitamin

reduction

Again,

and BP-II

vitamin

D downregulated

calcium-regulating

to

lo-‘M

a 2.5

two-fold

state

osteosarcoma

Densitometric

vitamin

shown

showing

of

indicated

In contrast,

images

Rat

D upregulated 4).

steady

by 1,25(0H)2

presence

autoradiography

mRNA.

hybrid

regulation

whether

biosynthesis.

absence

Figure

examine

probes showed similar quantities (data not shown). Thus, in these

steady

some role

in

to

hormonal protein

the

related

the

of

with actin experiment

In proteins

in

D of BP-I

analysis

undertaken

cell

visualized

mRNA encoding levels

bone

As can be seen

hybrid

then

mRNAs showed

of

were

hours.

were

in each studies, an

vitamin

BP-I

and

approach

to

inverse D.

BP-II

This

may have

function. a molecular By

antibody 386

biology

screening

of

a

identify cDNA

novel

expression

Vol. 151, No. 1, 1988 2 1

BIOCHEMICAL

3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4

cells

17/2.8

6

-18s

Figure

library

4.

of bone cells with 1,25(OH)2D3 and examination of Treatment steady state levels of BP-I and BP-II mRNAs.. Cells were grown in 36 hours before messenger RNA was prepared 10mgM Vitamin D3 for and subjected to Northern blot analysis. A) BP-I mRNA:Lanes 1 and lanes 3 and 4, 20 ug total 2, 20 ug total RNA from untreated cells; RNAfrom vitamin D-treated cells. B) BP-II mRNA: Lanes labelled ‘1-‘1 , 20 ug of total RNAfrom untreated cells; lanes labelled "+", 20 ug RNA from vitamin D-treated cells.

mRNA, unique gene products

prepared from bone cell

The expression exclusively,

of

these

in bone.

novel

products

The protein

structures

can now be used to prepare synthetic use of these proteins

is

peptides,

as markers of protein

seen predominantly predicted

but,

not

by these cDNA clones

and used to study the potential

turnover

and to study the development of the osteoblast

were identified.

in metabolic

bone disease

phenotype in cultured

cells.

ACKNOWLEDGEMENTS We thank Gabriel Eilon for the rat osteosarcoma cells 17/2.8 osteosarcoma cells.

and Sevgi Rodan for the

REFERENCES 1.

Termine, J.D., (1983) In: Bone and Mineral Research (W.A. Peck, Ed.), 1, pp 144-156.Excerpta Medica, Amsterdam.

2.

Stenner,D.D., Romberg, R.W., Tracey, R.P., Katzmann, J.A., Mann, K.G. (1984) Proc. Natl. Acad. Sci. 81,2868-2872.

3.

Chirgin, J.M., Przybyla, Biochemistry 18,5294-5299.

4.

Young, R.A. and Davis, R.W. (1983) PNAS80,1194-1198

5.

Sanger, F., Nicklen, USA 74,5463-5467.

6.

Aviv,

7.

Thomas, P. (1980) Proc. Natl.

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Southern, E.M. (1975) J. Mol. Biol.

S.,

A.E.,

MacDonald,

R-J.,

Rutter,

and Coulson, A.R. (1977) Proc.

H., Leder, P. (1972)

Proc. Natl.

Riggs,

B.L.,

W.J.

(1979)

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