The biochemical and functional heterogeneity of circulating human plasma fibronectin

Vol. 119, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 30, 1984

Pages 1174-1180

THE BIOCHEMICAL AND FUNCTIONAL HETEROGENEITY OF CIRCULATING HUMAN PLASMA FIBRONECTIN B. J. Dept.

Boughton

and A. W. Simpson

of Haematology, Birmingham

University B15 2TI-I

of Birmingham U.K.

Received February 15, 1984 Human plasma fibronectin purified by affinity chromatography, consisted of homogeneous 215 kD protein subunits when assessed by On isoelectric focusing SDS polyacrylamide gel electrophoresis. however, 5 separate fractions were present, with isoelectric points ranging from 5.6 to 6.1. Isoelectric focusing and similar but not immunofixation of native plasma produced identical appearances. Only 15% of the total plasma fibronectin cerevisiae opsonically stimulated the ingestion of Saccharomyces and this opsonic fibronectin by human peripheral blood monocytes, was confined to the fraction with an isoelectric point of 6.1.

When

purified

by

fibronectin

appears

examined

to

(1).

proteins

doing

and in

locomotion

(2).

fibrinogen

and for coagulation

bacteria

(3,4).

and are phagocytosed

opsonic

however,

This

presence

same protein

(3).

isoelectric

function

heterogeneity

not

to

0

1984

focusing

demonstrate

previously

by Academic Press, Inc.. in any form reserved.

of reproduction

affinity

to

in

sites

wound

debris

in

functional

and an assay

this

on macrophages

heterogeneity

following

and

lipotechoic

opsonized

a biochemical

1174

roles

bacterial

and

collagen,

of cell

functional

described.

for

to

of different

0006-291X/84 $1.50 Copyright All rights

adhesion

receptor

In the

ultra-

morphology,

opsonization

wide

two-

cytoskeletal

and micro-organisms

(7).

(PAGE),

cellular

its

binds

when

analytical

points

fibronectin

by the

utilized

cell

fibronectin

specific

been explained

with

micro-organisms

A (5,6)

to

the

interacts

plasma

protein,

electrophoresis or

and in the

human

homogeneous

gel

so modifies

Thus,

and Protein

way adhere

It

In addition

healing,

have

be a single

immunoelectrophoresis

centrifugation

within

chromatography,

by SDS polyacrylamide

dimensional

acid

affinity

has

domains

experiments of

the

protein's

and functional

we

BIOCHEMICAL

Vol. 119, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Human Plaa anticoagulated

was obtained by centrifugation of with l/10 vol. trisodium citrate.

fresh

whole

blood

Fibronectin was purified in the presence of 5mM benzamidine under non-denaturing conditions using gelatin-Sepharose and arginineSepharose as previously described (1). For some experiments fibronectin was purified using a polyclonal antifibronectin antibody linked to Sepharose (8). Fibronectin concentrations were measurer! phoresis as previously described (9).

by

immunoelectro-

SDS polyacryla,mide gel electrophoresis (PAGE) was performed in 6.5% gels using 1 mg/ml protein reduced with 10% B mercaptoethanol -1% SDS. MW markers were obtained from Pharmacia and the protein bands were stained with PAGE Blue 90. Isoelectric focusing of purified protein was performed in 5% polyacrylamide gels containing 2.4 ol (w/v) Ampholine (LKB Instruments) giving a pH range 3.5 to ‘b.5. The gels were prefocused for 20 mins. and 10 ul protein (400 mg l-') was applied 1 cm from the cathode. This was then focused for 1% hrs. at 10 'C using 1.5 kv and 1 watt/cm gel. The gels were then fixed with 10% trichloracetic acid/5% sulphosalicylic acid and stained with PAGE Blue 83. Human and bovine carbonic anhydrase B and R lactoglobulin A were used as markers - pH 6.6, 5.8 and 5.2 respectively. Isoelectric focusing of plasma 1% agarose containing 6.3% Pharmalyte (Pharmacia Ltd.) 12% sorbitol and 3% polyethylene was prefocused for 20 mins., and 20 ~1 samples of plasma glycol, diluted 1:lO in distilled water were applied 1 cm from the cathode and focused for 1% hour at 10 'C using 1.5 Kv and 0.5 watts/cm gel. Gels were immunifixed with an undiluted polyclonal sheep antihuman fibronectin antibody (Immunodiagnostics Research Laboratory, Birmingham University) for 16 hours at 22 'C washed for 24 hours in 0.9 NaCl, dried and stained with Page Blue 83. Opsonic activity of plasma or purified fibronectin was assayed a previously described (10). 0.5 ~1 were used to opsonize 5 x 10 8 Saccharomyces cerevisiae for 2 hr. at 37 OC. The opsonized yeast were washed twgice in Hank's Balanced Salt Solution (HBSS) and added to 5 x 10 human peripheral blood monocytes in 1:l (v/v) RPMI-1640/HBSS. After f hr. at 37 'C. phagocytosed yeast were counted using an electronic particle size analyser. RESULTS Figure

(1)

purified

freshly

t

homogeneous as assessed

300 and 400 mgl-'

ingestion

(Mean

the

fibronectin

containing the

shows

215 kD protein by SDS-PAGE.

fibronectin

In

comparison,

purified

fibronectin

identical opsonized

Native

respectively

of 0.35 + 0.07 and 0.52 +_ 0.04 yeasts

1 SEM).

subunits

plasma

stimulated per monocyte

concentrations 0.32

of

+ 0.12

and 0.50

of 2

Vol. 119, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

"=:L*

18

36 60 67

Figure

(1) SDS PAGE of two samples of 1 Gl-1 plasma fibronectin, The left gelatin-Sepharose and arginine-Sepharose. shows markers with molecular weights in kD.

0.02 yeasts

per

different

to

significant

loss

purification

of

of

was not

examine

the

shows purified Saccharomyces opsonic

with

protein's

during opsonic

opsonization, activity

fibronectin

activity

a

the

curing

wide

number of yeast 85% of

with

and after 1176

12-

only

opsonization of adsorbed protein

binding

sites

protein

were

conducted

to

protein.

Table

(1)

4 successive

aliquots

of

The concentration before

that

the

experiments

adsorbed

its

of

range

no

following

percentage

of unadsorbed

cerevisiae.

indicating

activity

was aosorhed

Since

factor.

significantly

(2) shows however

over that

not

plasma,

The relative

fixed

indicating

are

opsonic

Figure

cerevisiae.

a limiting

unadsorbed

the

plasma.

remained

concentrations

obtained

plasma fibronectin

Saccharomyces

protein

its

those

from

15% of native

These results

monocyte.

purified on hand track

each

of

fibronectin

adsorption

was

and stage,

BIOCHEMICAL

Vol. 119, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-'

0

30

60 MINUTES

Figure

(2)

that

unable

In did

plasma

function

after

b .nd

to

inert.

of

180

150

INCUBATION

Human plasma containing 60-450 mgl-l fibronectln opsonize Saccharomyces cerevisiae (see text). Only fibronectin was adsorbed by yeast during 0-180 min. 37 ‘C regardless of the concentration of protein point is the mean of 2 determinations.

indicate

stages

120

90

45OmglL

to

yeast,

and

contrast,

the

stimulate

ingestion

fibronectin as

measured

83% of

2 adsorptions, that

protein of

this

therefore

is the

The

major

to

have

first

two

proportion no

opsonic

(1) 1

2

3

4

330 330

330 330

FIBRONE-E TIN mgl

BEFORE AFTER

400 350

350 330

OPSONIC ACTIVITY %

BEFORE AFTER

100 48

48 6

6 2

2 1

the opsonic activity of purified consecutive, 0.5 hr. adsorptions The initial opsonic activity (0.5 denoted as 100%. Fibronectin at of yeasts retalned 100% opsonic

1177

is

opsonically

system.

Table

Gbronectin concentration and fibronectin before and after 4 with Saccharomyces cerevisiae. yeasts ingested per monocyte) is 37 ‘C for 2 hrs. in the absence activity.

protein

during

yeasts.

assay

initial

protein

adsorbed

appears by

this

the

was used to 12-15X of the incubation at used. Each

Vol. 119, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5.2

d

c b

1

03

2

3

Figure

Figure

a

0

6.6

4

4

1

Plasma fibronectin purified on gelatin Sepharose and arginine was isoelectrically focused on polyacrylamide. Sepharose, (1) shows that the purified protein contained 5 major bands with isoelectric points 5.66to 6.1. (2) yfd (3) show 0.5 ml protein adsorbed with 5 x 10 and 5 x 10 Saccharomyces cervisiae respectively. The band at pH 6.1 is selectively adsorbed. The position of marker proteins is indicated by the pH value of each.

(3)

(4)

Isoelectric

focusing

of

native

plasma

on

15

agarose.

fibronectin free plasma (2) and (3) plasma from The position of marker proteins is shown individuals. 8.15 (b) 7.35 (c) 6.05 (d) 6.55 and (e) 5.85.

Figure after

(3)

isoelectric

purified very

shows

appearances

focusing

similar

points

from

5.6 to 6.1.

by Saccharomyces adsorbed

this

preferentially. of

fibronectin

Five

procedure, Figure

cerevisiae,

freshly

at

purified

(a)

gel.

separate with

Proteins

(3) also the Figure

can also 1178

of

shows that

fraction (4)

gave

fractions

a range

shows

are

isoelectric when adsorbed

with

a p1 of that

be distinguished

pH

protein

and antifibronectin-Sepharose,

appearances. by

of

on polyacrylapl3e

on gelatin-Sepharose

distinguished

fractions

the

(1)

2 normal

6.1 is

separate in

native

BIOCHEMICAL

Vol. 119, No. 3, 1984

plasma. than

The pH of

those

of

the

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

isoelectric

purified

points

protein.

showed no immuno-reactivity

in this

were

however

Fibronectin

free

higher plasma

preparation.

DISCUSSION Cell

membrane

their

solubility

towards

is

molecular

purification

of

proteolysis

(13).

presence

of proteolytic

fragments

have not

correspond

to

detected

in

for

molecular

the

different

this

study

charge

isoelectric

unknown

and

the

purified

protein

using

of circulating

protein

weight,

raises

a number

of

produced

by a wide

in

possible

that

different

tissues.

may have VIII

different

specific

and Clq

plasma

Secondly,

and may thus

variety

fibronectins

with have 1179

capable

and do not

The reasons

separate actin,

separate

for

the

presently

plasma

and

separate

distinction of

of

proteolytic

are

of

or

subfractions

the

forms

between

similar

questions.

these

interactions

also

fractions

fractions

important

molecular

plasma

native

the

stimulate

weight

and the

and non-opsonic

plasma

responsible

these

fibronectin

low

Such

The finding

unclear.

in-vitro

in

focusing.

between

opsonic

fibronectin

(15).

differences

differences

plasma

and are

isoelectric

of

chemical

when

fibronectin

molecular

migration

are also

seen

in circulating

homogeneous

by to

Various

division

been detected

the

(1).

(8,14)

cell

and the

chromatography

plasma

phagocytosis

fibroblast

not

affinity

of

protein,

attributed is

inhibitors

fragments

monocyte

stimulating

by

fibronectin

obtained

been

picture

purified

proteolytic

inhibit

has

This

is

as a homogeneous

by

reactivity

Plasma

fibronectins

plasma

fibronectin

and by their

(11,12).

regarded weight

can be distinguished

weights

antibodies

generally

of

weight

fibronectins

and molecular

monoclonal

however range

and plasma

molecular

First,

since

of cell

types,

it

are

derived

plasma

functions.

from

fibronectins

fibrinogen,

is

factor Thirdly,

Vol.

119,

No.

there

is

3, 1984

good

BIOCHEMICAL

evidence

fixed

macrophage

clear

circulating

vascular

the

humans

however,

fibronectin syndrome

there

levels (18).

proportion

of

fibronectins

(16).

and Since

the

total

may now

pathophysiology

of

a poor

the opsonic

consumptive

direct

correlation of

a more

patients

effects

on

loss

and

(17).

In

new

total

with

comprises

detailed

to

lung

hctween

protein,

failure

fluid as the

fibronectin

plasma permit

such

survival

produces

subsequent

possible

organs

COMMUNICATIONS

depletion

transvascular

vital

is

The

or

increases of

RESEARCH

f'ibronectin

microemboli,

function

BIOPHYSICAL

plasma

dysfunction

endothelium

impairs

that

AND

assays

plasma

shock only of

examination

lung

a minor separate of

the

opsoninopathy.

ACKNOWLEDGEMENT B. J.

Boughton

is

a Leukaemia

Research

Fund

Senior

Lecturer.

REFERENCES

(1) Vuento, Yamada, Mosesson, 13’; (41 Kuusela, i5j (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18)

M., Vaheri, A. (1979) Bioch. J. 183 331-337. X. M., Olden, K. (1979) Nature 275, 179-184. M. W., Amrani, D. L. (1980) Blood 56(2) 145-158. P. (1978) Nature 276, 718-720. Courtney, H. S., Simpson, W. A., Beachey, E. H. (1982) J. Bact. 153(2) 763-770. Doran, J. E., Raynor, R. H. (1981) Infect. Imm. 33(3) 683689. Bevilacqua, M. P., Amrani, D., Mosesson, M.W., Bianco, L. (1981) J. Exp. Med. 153, 42-60. Czop, J. Ii., Austen, K. F. (1982) J. Imm. 129(6) 2678-2681. Boughton, B. J., Simpson, A. W., Chandler, S. (1983) Lancet (1) 121-122. Simpson, A. W., Boughton, B. J. (1983) Imm. Methods 63, 321-327. Proctor, R. A., Mosher, D. F., Olbrantz, P. J. (1982) J. Biol. Chem. 257(24) 14788-14794. Atherton, B. T., Hynes, R. 0. (1981) Cell 25(l) 133-141. Mosesson, M. W. (1978) Ann. N.Y. Acad. Sci. 312, 11-30. Ehrlich, M, I., Krushell, J. S., Blumenstock, F. A., Kaplan, J. (1981) J. Lab. Clin. Med. 98(2), 263-271. Humphries, M. J., Sobhy, R. A. (1983) Nature 305; 811-813. Saba, T. M.Blumenstock, F. A., Weber, P., Kaplan, J. E. (1978) Ann. N.Y. Acad. Sci. 312, 43-55. Niehaus, G. D., Schumaker, P. T., Saba T. M.,(1980) J. Appl. Physiol. 49(4) 693-699. Rubli, E., Bussard, S., Frei, E. Lundgerard Hansen P., Pappova, E. (1983) Ann. Surg. 197, 310-317.

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