The amino acid sequences of two large glycopeptides derived from the carbohydrate-carrying region of α1-acid glycoprotein

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE AMINO ACID SEQUENCES OF TWO LARGE GLYCOPEPTIDES DERIVED FROM THE CARBOHYDRATE-CARRYING REGION OF al-ACID

K. S&mid,

M. Ishiguro,

J. Emura,

Department University

of Biochemistry, Medical Center,

Satoko Motoyama

Boston Boston,

GLYCOPROTEIN

Isemura,

H. Kaufmann

and Toshiko

University School of Medicine, Mass. 02118, U.S.A.

Boston

December 10, 1970

Received

fragmentation with cyanogen bromide and subsequent reducSUMMARY: Specific tion and carboxymethylation of a,-acid glycoprotein, a normal human plasma globulin, permitted isolation of a large fragment which was shown to represent the amino-terminal half and to contain the total carbohydrate moiety of this protein. The amino acid sequences of two large glycopeptides derived from this fragment were established. One glycopeptide was composed of 22 amino acid residues and one carbohydrate unit, and the other consisted of 65 amino acid residues and carried four carbohydrate units.

INTRODUCTION a,-Acid extensively (2)

glycoprotein studied

(3)

has been done concerning

eral

chain

amino protein

of its the

which (1,5)

(4)-

elucidation

consists have

of two large account

been reported

paper segments for

we wish

probably

regard

moiety.

of approximately

studied

the

amino

linkages amino

acid

the most

to the

However,

of the sequence

little

of its

210 amino acid

of this residues

of this

work

single

poly-

acids.

sequences plasma

structure

Sev-

directly globulin.

The

conjugated

(1). to describe

of the polypeptide

40% of all

1) is

with

carbohydrate

and carboxyl-terminal

have also

see Ref.

glycoprotein

to the carbohydrate-protein

In this

which

(1,4)

investigators

adjacent

review

human plasma

and biosynthesis

peptide

(for

amino

acid

280

briefly backbone residues

the amino of al-acid and carry

acid

sequences

glycoprotein the five

(6)

. Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

carbohydrate units of this protein.

Specific

cleavage by cyanogen bromide

and subsequent reduction and carboxymethylation ted isolation

of this glycoprotein

of the amino-terminal half of the total

polypeptide

permit-

chain to

which these carbohydrate units are attached.

MATERIALSANDMETHODS al-Acid

glycoprotein

was isolated

from Cohn Fraction VI of pooled

normal humanplasma (7) and appeared homogeneousas judged by several criteria

of purity

including

amino- and carboxyl-terminal

amino acid analyses

(1,4). For the initial

fragmentation

of this protein

the highly specific

cyanogen bromide reaction was used in presence of 6 M guanidine hydrochloride (8).

After

removal of the reagents by gel filtration

the modified glycoprotein

through Sephadex 625,

was reduced with mercaptoethanol and carboxymethy-

3 SEPHADEX

3

2 EFFLUENT

F-igure 1:

G-100

4 VOLUME

(liters)

Separation of the CNRr-fragments of al-acid by gel filtration through Sephadex G-100.

281

5

glycoprotein

Vol. 42, No. 2, 1971

lated

(9).

BIOCHEMICAL

Subsequent

isolation

of

analysis tein

does not from

shown

to carry

three

appeared

and proved

derived

chromatography

the expected

chromatographed

possess

a free

pared

tryptic from

through tide

total

this

from each other

electrophoresis

fraction.

acid

was carried

man degradation

techniques

aminopeptidase-M. the

larger

peptides

S-,

In order were

acid

(11)

was passed a glycopepwere

separated of the

and CM-celluloses,

were

and high

voltage

prepared

in pure

G-25 and by high 1.8,

3.4 and 6.4

the peptides

and glyco-

and subtractive

(12)

carboxypeptidases

their

further

pre-

analyses.

with

to establish

were

into

pH-values:

the direct

hydrolyzed

digests

Sephadex

sequencesof

and hydrolysis

of frag-

combination

peptides

Dowex-50,X2,

acid

sequence

Sephadexes

at two of the following

out employing

acid

of each digest

The carbohydrate-free

of the amino

I was

separation

through

amino

glycopro-

Fragment

Each digest

on DEAE-,

on Dowex-1,X2,

acid

must have been

and peptic

a gross

filtration

amino

of the protein.

by an appropriate

and amino-terminal

Determination' peptides

to obtain

I when re-

a,-acid

(4).

glycoprotein.

purified

gel

electrophoresis

and by amino

fragment

of the amino

Chromatography

form by chromatography voltage

this

moiety

permitted

Fragment

Since

group,

column

by amino-terminal

The glycopeptides

and further

(10).

1).

groups.

chymotryptic

column

and BioRex-40,

G-100

of the glycoprotein

of or-acid

procedures:

Dowex-1,X2

a-amino

investigation

G-50

and a peptide

as judged

carbohydrate

portion

(Figure

of a-amino

(TPCK-trypsin),

a Sephadex

following

fragments

the amino-terminus

For the subsequent ment I,

on a Sephadex

homogeneous

to be free

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

complete

A and B and

sequences,

by chymotrypsin,

Ed-

some of

trypsin

or

thermolysin.

RESULTS AND DISCUSSION The obtained

'Further sequences

results

are

summarized

technical procedures necessary for are described in recent publications 282

in Figures

the

2 and 3.

elucidation (10,13,14).

Only

of amino

the

acid

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure

2

THE AMINO ACID SEQUENCE OF A GLYCOPEPTIDE OF al-ACID

GLYCOPROTEIN

WHICH CONSISTS OF 22 AMINO ACID RESIDUES AND ONE CARBOHYDRATE UNIT

Cys-Ala-Asn-Leu-Val-Pro-Val-Pro-Ile-Thr-Asn*---------------------

Ala-Thr-Leu-Asp-Arg-Ile-Thr-Gly-Lys-Trp-Phe -----_--------------c

The solid line indicates the amino acid peptide and the broken line that of the The attachment of the carbohydrate unit the number of the carbohydrate units is

sequences

of those

the amino

acid

report. acid

residues

were

consisting 3) were

carbohydrate found

carboxyl-terminal

These

five

acid

and glycopeptides presented

sequence

in the

unit

of 65 amino established. moiety

unequivocally

two figures

(Figure

acid

to asparagine

carbohydrate (6).

residues

in

possesses

with

this

22 amino

of a very

and carrying

glycoprotein

yielded

included

2) and that

five

of or-acid

are which

residues

These

to be attached

which

of a glycopeptide

and one carbohydrate

(Figure

the total units

sequences

The amino

glycopeptide units

peptides

sequence of the main peptic glycomajor chymotryptic glycopeptide. is indicated by the sign CdO, and referred to in the text.

large

4 carbohydrate units All

account carbohydrate

the following

sequences:

sequences

support

1.

-Asn-Ala-Thr-

2.

-Asn-Lys-Ser-

3.

-Asn-Lys-Thr-

4.

-Asn-Thr-Thr-

5.

-Asn-Gly-Thr-

the

theory

of Eylar

283

(15)

and Neuberger

and

for

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

284

Vol. 42, No. 2, 1971

Marshall

(16)

proteins

are

tripeptide: tances

according linked

between

hydrate the

two adjacent

backbone

of the of

halves

charges:

the amino-terminal

carboxyl-terminal

of this

half

residues, three

five

half

both

and the

of globular

are included is

units,

acid

carbohydrate

chain

possesses as inferred

CNBr-fragments

carry includes

a relatively from

high

from

were supported and the National Health Service

vary carbo-

can be seen

the total

poly-

inasmuch

free

of sugar.

of electrostatic

residues number

the composition

derived

is

number

16 sialyl

the dis-

between

asymmetric chain

a large

the general

that

It along

surprisingly

glyco-

as determined,

residues

units

of the polypeptide

half

ACKNOWLEDGMENT: These studies Science Foundation (GB-24813) Sciences, United States Public

is

in

the finding

as far

21 and 10 amino

the glycoprotein

However,

tein

that

interest

carbohydrate

to 16,

units

2 and 3, 3 and 4 and 4 and 5, respectively.

distribution

acid

residues

Of further

as the carboxyl-terminal

amino

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the carbohydrate

to asparaginyl

and amount

units

peptide

to which

-Asn-X-($!$)-.

considerably

that

BIOCHEMICAL

(1) of basic

of a,-acid

and the and acidic glycopro-

it. by grants Institute (GM-10374

from the National of General Medical and l-K3-GM-32,160).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Jeanloz,

R. W. in The Glycoproteins, A. Gottschalk, Ed., Elsevier, New York, 1966, p. 362. Wagh, P. V., Bornstein,.I., and Winzler, R. J., J. Biol. Chem., 244, 658 (1969). Schachter, H., Jabbal, I., Hudgin, R. L., and Pinteric, L., J. Biol. Chem., 245, 1090 (1970). Ikenaka,

T.,

Bammerlin, H., and Schmid, K., J. Biol. Chem., 241, 5560 (1966). Proc., 29, 600 (1970). Emura, J., and Sdhmid, K., Federation Satake, M., Okuyama, T., Ishihara, K., and Schmid, K., Biochem. J., 95, 749 (1965). Burgi, W., and Schmid, K., J. Biol. Chem., 236, 1066 (1961). Gross, E. and Witkop, B., J. Biol. Chem., 237, 1856 (1962). Ishiguro, M., Yamamoto, A., and Schmid, K., Biochim. Biophys. Acta, 194, 91 (1969). Hirs, C. H. W., Ed., Enzyme Structure, Methods in Enzymology, Vol. 11, Academic Press, New York, 1967. Edman, P., and Begg, G., Europ. J. Biochem., 1, 80 (1967). Elzinga, M., Lai, C. Y., and Hirs, C. H. W., Arch. Biochem. Biophys., 123, 353 (1968). Shinoda, T., Titani, K., and Putnam, F. W., J. Biol. Chem., 245, 4463, 4475, 4488 (1970). 285

Vol. 42, No. 2, 1971

14. 15. 16.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mitchel,

R. E. J., Chaiken, I. M., and Smith, E. L., 245, 3485 (1970). Biol., lo, 89 (1966). Eylar, E. H., J. Theor. R. D., in Carbohydrates Neuberger, A., and Marshall, Schultz, H. W., Cain, R. F., and Woolstad, Westport, Conn. 1969.

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Chem.,

and Their Roles, R. W., Eds., Avi,