Separation and characterization of two bovine alpha1-fetoprotein molecular variants by concanavalin A-Sepharose chromatography

BIOCHEMICAL

Vol. 82, No. 2, 1978 hioy

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

30,1978

SEPARATION AND CHARACTERIZATION

OF TWO BOVINE ALPHAi-FETOPROTEIN

492-497

MOLECULAR

VARIANTS BY CONCANAVALIN A-SEPHAROSE CHROMATOGRAPHY P.C.W.

Lai

and F.L.

Lorscheider

Division of Medical Physiology, Faculty of Medicine, and The Alberta Children's Hospital Research Centre, Received

March

The University of Calgary Calgary, Alberta, Canada

20,1978

SUMMARY. Two molecular variants of bovine alphal-fetoprotein were separated by affinity chromatography of fetal calf serum on a concanavalin A-Sepharose column. Radialimmunodiffusion assay of bovine alphaI-fetoprotein revealed that 29% of the alphaI-fetoprotein in fetal serum lacked concanavalin A-binding activity whilst 71% of the alphal-fetoprotein was capable of binding to the lectin. These two bovine alpha]-fetoprotein variants show antigenic identity suggesting that the polypeptide chain rather than the carbohydrate moiety of the alphal-fetoprotein molecule is the antigenic determinant. INTRODUCTION Alphal-fetoprotein in the diagnosis sinus (3,

tumors

There

AFP.

of certain (1,

Recent

4).

(AFP)",

is

rat

investigations evidence (5)

shown

(10-14).

chromatography

concanavalin capable

a-D-glucopyranosyl objectives variants characterize

of fetal

activities molecular

molecules

10,

possessing

were

related

to separate

OOOS-29ur/78/0822-0492$01.00/0 492

9) and

different

parameters

(7).

separated

by

on insolubilized Concanavalin

residues

A is

or (16,

and identify

A-Sepharose

by immunochemical

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

to possess

a-D-mannopyranosyl

AFP by concanavalin variants

13, 14).

of

Furthermore,

in man (8,

AFP variants,

or sterically

study

7).

variants

fluid

fetus

AFPs possess

(6,

demonstrated

of rat

(7,

of the

and function

mammalian

serum or amniotic

reported

and endodermal

on the structure

certain

significance

monitoring

15) and estrogen-binding

characterization

of the present

these

that

as several

end-groups

of bovine

focused

of AFP have been

A, has been

of binding

gastrointestinal

and estrogen-binding

(9,

clinical

in the prenatal

to suggest

potencies

Immunochemical

established

of liver,

have

to exist

Variants

immunosuppressive

column

types

2) and as a marker

immunosuppressive AFP has been

has a well

chromatography

methods.

17).

molecular and to

The

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

Rabbit anti-bovine

AFP. AFP (35 vg) purified from the concanavalin A-reactive fraction of fetal calf serum as previously described (18) was dissolved in 0.4 ml of 0.12 M Tris-HCl, pH 8.1, emulsified with an equal volume of complete Freund's adjuvant (Calbiochem, La Jolla, CA, USA) and injected subcutaneously into a rabbit. Injection was repeated twice at lo-day intervals and the antiserum was obtained 10 days after the final injection. ImmnologicaZ techniques.

Quantitation of AFP was carried out using radialimmunodiffusion assay (19) as we have previously employed (18). standardized fetal calf serum used in the assay was calibrated against purified bovine AFP. Immunological comparison of antigens was carried 1% (w/v) agarose gel by Ouchterlony double immunodiffusion method (20) Tandem crossed immunoelectrophoresis (21).

The our out in and by

Affinity

chromatography. Concanavalin A-Sepharose (Pharmacia, Uppsala, Sweden) was packed in a 0.9 x 12 cm column and equilibrated with the starting buffer (0.1 M sodium acetate, 1 m&f Ca2+, 1 mM Mg2+, 1 mM Mn2+, 0.02% NsN3, pH 6.0) at 21°C. Fetal calf serum, (0.5 ml; 55-60 days gestation; GIBCO, Grand Island, NY, USA) estimated to contain 3 mg AFP/ml and 31 mg total protein/ml, was mixed with 1 ml of the starting buffer and chromatographed on the column. After washing with 12 ml of the starting buffer, the column was eluted with 0.1 M a-methyl-D-glucoside (Sigma Chemical Co., St. Louis, MO, USA) dissolved in the starting buffer. The flow rate was maintained at 12 ml/h throughout the chromatographic procedures and 1.1 ml - fractions were collected. Fractions containing proteins not bound to the column were pooled and subsequently rechromatographed on a fresh concanavalin A-Sepharose column under identical conditions to confirm that this protein fraction Fractions from both chromatographic lacked concanavalin A-binding activity. runs were monitored for protein by absorbance at 280 nm or by the method of AFP concentration in each fraction was estimated by Lowry et al (22). radialimmunodiffusion assay as described above and the presence of AFP in each fraction was also identified by analytical disc polyacrylamide gel electrophoresis (23).

RESULTS AND DISCUSSION The elution

profile

concanavalin

A-Sepharose

protein

consisted

the which

peak

column were

glucoside.

bound

could disc

column

is

serum

do not of total

The presence

chromatographed

illustrated

in Fig.

A-nonreactive

represented

variants

proteins

eluted

with

separated

electrophoresis

differ protein

493

first

proteins

that

of AFP. the

column

protein

two

The

mobility. the

to

a-methyl-D-

assay

in electrophoretic

of AFP in the

not bound

by affinity

revealed

and AFP from

The first

A-reactive

by radialimmunodiffusion gel

on a

1 (left).

concanavalin

and subsequently

AFP molecular

polyacrylamide

recoveries

90% respectively.

peak

be detected

variants

calf

of concanavalin

to the

Two distinct

AFP molecular analytical

column

and the second

chromatography analytical

of fetal

were

91% and

peak was not

due

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Alpha, -, +

Alpha, fetoprotein*

fetoprotem

Albumin

AlbumIn+

+

FRACTION

NUMBER

FRACTION

NUMBER

(Left) Elution profile of the affinity chromatography of fetal Fig. 1. calf serum on a concanavalin A-Sepharose column. Closed circles: absorbance at 280 nm; open circles: AFP concentration. Abbreviations: a, sample application; b, elution with starting buffer; c, elution with 0.1 M a-methyl-D-glucoside added to starting buffer. Analytical disc polyacrylamide gel electrophoresis: I, fetal calf serum; II, concanavalin A-nonreactive fraction; III, concanavalin A-reactive fraction. Horizontal bar represents fractions pooled. (Right) Elution profile of the rechromatography of pooled concanavalin A-nonreactive fractions. Disc polyacrylamide gel electrophoresis of the fractions are inserted. Abbreviations a-c, same as above.

to overloading

of the

A-nonreactive that (Fig.

this

column

fraction protein

fraction

because

on a fresh indeed

rechromatography

concanavalin lacked

of the

A-Sepharose

concanavalin

concanavalin

column

A-binding

confirmed activity

1, right). Table

1 shows

the percentage

distribution

494

of total

serum protein

and

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1. Relative distribution of total fetal calf serum proteins and AFP respectively in concanavalin A-reactive and concanavalin A-nonreactive fractions eluted from the affinity chromatography of the serum on a concanavalin A-Sepharose column. Protein concentration was determined by the method of Lowry et al (22).

% eluted in concanavalin A-nonreactive fraction total

serum proteins

AFP

AFP in

the concanavalin

Fig.

2 demonstrates

AFPs show antigenic finding

suggests

chain

rather

than

the bovine

that

precipitating

identity

between

concanavalin fraction 32

29

71

concanavalin

by Ouchterlony

that

in contrast

to bovine

its

carbohydrate

moiety

The antiserum antibodies

in

68

and nonreactive

the

identity

AFP molecule.

contain

crossed

A-reactive

% eluted A-reactive

fractions. A-reactive

double

diffusion

fetuin

(24)

is

method.

was specific

variants

This

the polypeptide

the antigenic

to non-pregnant

the two AFP molecular

and nonreactive

for

determinant

of

AFP and did

not

cow serum.

The reaction

was confirmed

by Tandem

of

immunoelectrophoresis.

The ability

of AFP to bind

species.

Human APP

contrast,

rat

(10,

existence

AFP consists

13,

of only

concanavalin

Vallette AFP molecule was based

14).

A-weakly-reactive In the present

two forms

of bovine

et al

(7)

suggested

may have a role on the

finding

is

that

study,

not that

among mammalian

A-reactive

forms,

(7).

In

namely

concanavalin

and concanavalin

A-reactive

we have

AFP, i.e.

demonstrated

concanavalin

The species

variants.

A interaction

A varies

concanavalin

of as many as three

A-nonreactive

AFP-concanavalin

entirely

almost

concanavalin

A-nonreactive, variants

is

to concanavalin

the

A-reactive

difference

with

and

respect

known. the carbohydrate

in estrogen-AFP the concanavalin

495

interaction. A-reactive

moiety

of the rat

Their or "high

hypothesis

to

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

double immunodiffusion gel plate. Antigens: Fig. 2. Ouchterlony pooled concanavalin A-nonreactive fractions; B, pooled concanavalin A and B obtained from affinity chromatography A-reactive fractions; C, non-pregnant cow serum. Antiserum: D, rabbit anti-bovine text);

carbohydrate"

form has low

A-nonreactive

or "low

hormone.

Their

The present species

inasmuch

shows

calf

consists

of substantial

carbohydrate"

serum

(25)

chromatography that

is not

a significant

this

steroid

of the that

lack

of

human AFP A-reactive

form.

applicable

to the bovine

estrogen-binding

protein

demonstrated

of a concsaavalin

that

carcinoembryonic

reported

bovine

on a concanavalin

from human hepatic also

for

or concanavalin

and yet we have amounts

the concanavalin

affinity

finding

(see AFP.

that

this

A-nonreactive

glycoprotein or "1~

form.

We have demonstrated

note

the

such a hypothesis AFP is not

while

by the correlation

carbohydrate"

that

as bovine

in fetal

form has high

of human AFP with

of the "high

study

la-estradiol

was supported

activity

mostly

for

carbohydrate"

hypothesis

estrogen-binding consists

affinity

A,

to contain

heterogeneous

A-Sepharose

antigen,

metastases

AFP is

another

of rectal

concanavalin

column. carcino-fetal

and pancreatic A-reactive

496

It

by affinity is

interesting

protein

isolated

adenocarcinoma

and nonreactive

to

was

variants

BIOCHEMICAL

Vol. 82, No. 2, 1978

(26).

At present,

heterogeneity

the significance

of AFP in

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

and the

nature

the development

chemical

of bovine

fetuses

of the remain

to be

elucidated. ACKNOWLEDGEMENTS This investigation was supported Canada grant no. MA 5292 and a grant Research Foundation.

by the Medical from The Alberta

Research Council of Children's Hospital

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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