Amino acid sequence of galactosamine-containing glycopeptides in the hinge region of a human immunoglobulin D

Vol. 105, No. 3, 1982 April 14, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1066-1071

ACID SEQUENCE OF GALACTOSAMINE-CONTAINING GLYCOPEPTIDES REGION OF A HUMAN IMMUNOGLOBULIN D

AMINO

Tatsunori

Takayasu, Satoru Tomotaka Shinoda,

Suzuki, Tsuneo

Fuyuki Kametani, Nobuhiro Takahashi, Okuyama and Eisuke Munekata

Department of Chemistry, Tokyo Metropolitan University, 158 and Institute of Applied Biochemistry, University Ibarakiken, 305, Japan Received

February

IN THE HINGE

Setagaya-ku, of Tsukuba,

Tokyo

15, 1982

Amino acid sequence and the location of seven galactosamine oligosaccharide moieties of the hinge region of the 6 chain of human IgD NIG-65 have been determined. These oligosaccharide moieties are distributed in two distinct fashions: 1) three clusters each consisting of five amino acid residues with two consecutive attachment sites either Ala-X-Ala-Ser-Ser or Ala-X-Ala-Thr-Thr, where X can be any amino acid including proline, 2) one triplet sequence Val-Pro-Thr with one attachment site. We propose two rules with regard to the acceptor sequence for galactosamine oligosaccharides, the quintet sequence rule and triplet sequence rule. Of two different chain

types

of human IgD,

virtually

N-acetylglucosamine

in the Fc region(l)

established location

has recently

in part

to our result the number

kinds,

heavy chains views roles

the

as well

N-acetylgalactosamine

of five

class,

on the relationship

its

WAH(5),

NIG-65.

which

Since

of oligosaccharide are

of the

are now avaiable

apparently

immunoglobulin

is contrasting

needed classes

for

additional

data

to have general and their

biological

and functions. This

communication

describes

galactosamine-containing

the

glycopeptides,

isolation and amino and the tentative

galactosamine

oligosaccharide

moieties

In connection

to our

a new hypothesis

galactosamine-oligosaccharide

results,

will

MATERIALS

in the hinge

be also

region

acid sequences of location of seven of a human IgD.

on the acceptor

sequences

AND METHODS

0006-291X/82/071066-06$01.00/0 0 1982 by Academic Press, Inc. in an-v form reserved.

of reproduction

of

described.

Human IgD NIG-65 was isolated from the plasma of a patient with myeloma by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and Bio gel A-5m gel filtration. Although it existed

Copynghi .A// righa

heavy

tentative

much data

human immunoglobulins,

chain,

between

protein

protein

principal

6

6

As to the other

oligosaccharide,

for

for

and the location

of four

remaining

been reported

in the

as by other(4).

type

obtained

identified

oligosaccharide has been found type numbers and the location have been

and their

by the authors(2,3)

carbohydrate,

for

of oligosaccharide

1066

multiple in

BIOCHEMICAL

Vol. 105, No. 3, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

it was shown to be essentially homomultiple bands on isoelectrofocusing, SDS-polyacrylamide gel electrophoresis, and geneous by immunoelectrophoresis, The isolation, purification and characterization N- and C-terminal analyses. procedures have been described by Shinoda, et al(2). Fd(t) fragment was prepared from the Fab(t) fragment after the complete reduction and aminoethylation according to the procedure previously described(6,7) and was shown to be homogenous in SDS-polyacrylamide gel electrophoresis and N-terminal analysis. Purified aminoethyl(AE-)Fd(t) fragment(l20 mg in O.lM NH HC03) was t! ,3) and the digested with 2.6 mg of TPCK-trypsin in the way as described( digest was chromatographed on a column of DEAE-Sephadex A-25(1.5 x 42 cm). The column was eluted at room temperature first with 360 ml of 0.Ol.M NH4HC0310% 1-propanol and then by a linear gradient of an increasing NH HC03 concentration(O.Ol-0.6M) containing 10% 1-propanol. Flow rate was 1 %.4 ml/h, and fractions of 4.4 ml were collected. Peptides were further purified by gel filtration with Bio gel F-6 colum(l.5 x 98 cm) in O.lM NH4HC0 at room temperature. Another portion of the AE-Fd(t) fragment(100 mg in ;I2 ml O.lM NH4HC03) was also digested with 3.4 mg of V8 protease at 37°C for 20 h and the The V8 peptides were isolated and purified in the digest was lyophilyzed. same manners as described above. HF treatment of the GalN-containing peptides was carried out in the similar way as reported(8) and the deglycosilated peptides were purified either by gel filtration with Sephadex G-25 or ion-exchange column chromatography with DEAE-Sephasose CL-6B according to the methods described(2). Amino acid sequence analyses and identification of PTH-amino acids by High Performance Liquid Chromatography were carried out as essentially described(6,7).

RESULTS AND DISCUSSION Following trypsin

the chromatography

digest

an elution (GalN

of the completely

pattern

shown

T) was eluted

further

purified

composition residues, component

which

cause

in table

of steric

and aminoethylated

indicated

had six

cleavage hinderance

with

did

the

at lysyl-alanine

of the fragment,

in the

peptide

figure.

not contain

sequence

It

amino

was

acid

any glucosamine

Upon sequence analysis about 10% of an additional

to contain

by massive

Fd(t)

of P-6 and its

1. The peptide

more residues

A-25

A GalN-containing

as Frl

on a column

observation(2,3).

was shown

of DEAE-Sephadex

was obtained.

filtration

the previous the peptide

at incomplete

reduced

in Fig.l(A)

at the position by gel

was given

, confirming

Lys-

on a column

up to 11th minor

Trp-Pro-Glu-Ser-Pro-

bond with

trypsin,

GalN oligosaccharide

probably

moieties

be-

adjacent

to it. To facilitate

sequence

determination

HF to remove GalN oligosaccharide chromatography HF). shown

The peptide

of the peptide

in Table

ses of the were Fd(t)

fragment

from

was first

and was purified , giving

acid

residues

by manual

of the sequence GalN-containing

of the completely

the procedures

similar 1067

exchange

peptide,

GalN-T(

and its

GalN content The first

Edman degradation GalN-Sl reduced

to those

with

amount.

was determined peptides

treated

by ion

a single

10% of the original

was determined

the V8 digest

through

moieties filtration

of 32 amino to about

2. The rest

two different

isolated

by gel

consisted

to have decreased

sequence rized

followed

the peptide

was 23

as summa-

by sequence

analy-

and GalN-S2,

which

and aminoethylated

applied

to the tryptic

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3.

d 0

50

15~1

100

A

Fr.1 fraction

200 number(L.4

ml)

NH4 HC03

0

B

Fig.

1.

peptides.

F-r. 1

An elution

S2 were

respectively P-6,

whose

GalN-Sl by manual and 11th

steps

were

for

consisted

because tially

in Fig.l(B).

in pure were

200 number(7.8

ml)

HF-treated

1) composition

I except for the sequence

directly

throughout 8th,

12th with tryptic

but

were

data

peptide

GalN-T(BF),

obtained

the results The se-

The PTH-derivaidentified,

for

but were

the sequence

moiety,

HF-treated(par-

and 2) all

the composition

8th

V8 glycopeptide

residues.

directly

throughout

to N-acetylgalactosamine

the sequence

4 threonine residues found in as shown in table 2. Altogether

with

the 7th,

from

The other

not

each linking

1068

deduced

Edman degradation.

were

and GalN-

filtration

from

5 threonine

by manual

with

1.

was determined

peptide,GalN-T(HF). including

GalN-Sl

the gel

in Table

The PTH-derivatives

and 13th

residue

following

determined

tryptic

chromatography

and Fr2,

The sequence

2).

of 18 residues

to be threonine deglycosylated)

forms

column

Frl

summerized

of 17 residues. not

the 7th,

From peaks

obtained

was determined from

ion exchange

compositions

consisted

,GalN-S2

deduced

of the

Edman degradation(Table

obtained

tives

profile

was shown

Bio

gel

I50 fraction

Separation of trypsin(A) and S.aureus V8 protease peptides from aminoethyl-Fd(t) fragment. Separations were carried out with DEAE-Sephadex A-25 column(l.5 x 42 cm) at room temperature. Details are described in the text.

V8 peptides

quence

tboFr.2

the amino

were of the

recovered 35-residue

acids in

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1

AMINO ACID COMPOSITIONOF ~INEWAINIK IN THE HIKE REGIONOF A HUMANIgD NIG65. GalN and GlcN were determined after hydrolysis with 6N x1 for 24 h at 11O'C. Values of less than 0.2 were anitted except GalN and GlcN.

-IIDES

GalN-T

GalN-T (HF)

GalN-Sl

GalN-S2

1.0

0.8

0.9

0.9

1.8

1.0 5.7 4.0 5.2 4.3 2.0 8.9 0.9

2.0 1.0 6.2 3.3 3.9 3.3 2.0 8.5 1.1

1.2

1.2

2.6 0.0

0.2 0.0

LYS

His Ar& Asp Thr Ser Glu PI-0 GlY Ala Val Met Ile Leu

2.0 0.9 5.0 0.9

1.1 2.6 4.4 3.2

1.3 1.8 3.8

3.6 0.9

1.0

TY~ Phe GalN GlcN

peptide

which

covered

determined(Table

2).

The tentative difference

location

or serine

and by the

at a given assignment

,24th,

25th,

quences

GalN-oligosaccharide

step

of peptides

failure

and 30th

the

containing

each has two consective

region

sequence

was

was assigned

and after

determination.

by

the HF treatment

the PTH-derivative

to each of the

threonine

shown inTable2,

before

to detect

during

of one GalN

29th

The sequence

1.7 0.0

of seven GalN-oligosaccharides

in the GalN content

of the peptides allowed

the entire

0.5 0.0

of threonine This

operation

7th and 8th serine,

one to 11th

residues.

these are GalN-binding

three characteristic sites either Ser-Ser

quintet seor Thr-Thr,

but neither Ser-Thr nor Thr-Ser sequence: they are 4-Ala-Gln-Ala-Ser-Ser8, 21-Ala-Lys-Ala-Thr-Thr-25 and 26-Ala-Pro-Ala-Thr-Thr-30, respectively. basic

sequence

any amino acid "quintet rule"

can be drawn including

as the acceptor

hinge

region

other

GalN-oligosaccharide-containing

oligosaccharide

as Ala-X-Ala-Ser-Ser(or

proline.

of human identified

6 chain

The finding sequence

for

and that

it

Thr-Thr), allowed

6 hinge 1069

X can be

us to propose

a

the GalN-oligosaccharide may also

glycoproteins. in the

where

exist

in the

be applicable

for

Six of seven in this

form.

The

GalN-

The

the

Vol. 105, No. 3, 1982

BIOCHEMICAL

Table 2.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

M.IINO ACID SIQJFNCC OF ~SAMINF-CONTAINIMS GLYCOPIlPTIDES IN TKE IIINGE REGION OF A HUMAN IgD r:IG-65 12

GalN-T

(major)

3

4

5

6

7

8

9

11

Ala-Gin-Ala(Serfier)Val-Pro(Thr)Ala-Gln-ProCHO CHO CHO

i (minor)(Trp-Pro-G111-Ser-Pro(Lys)Ala-Gln-Ala(S~r~~r))

trio ccl0

Ala-Gln-Ala-Ser-Ser-Val-Pro-Thr-Ala-Gln~~~~~~~~777 Pro-Gln-Ala-Clu-Gly(Ser)Leu-Ala(Lys)Ala---J 7 --I / 7 --7 --7 -7 --7 -7 Thr-Thr-Ala77-7 ~ __ __ Ser-Pro-Lys-Ala-Gin-Ala(SerEer)Val-Pro( I ---T ---? ---T ---? 1 CHO CHO 1 ---J --7 --7 Thr)Ala-Gin-Pro-Gln-Ala-Glu CHO---7-777‘-7 Gly-Ser-Leu-Ala-Lya -Ala(TQrmQr)Ala-PrO-1 -1 1-1 CKO CHO -7 --7 --7 Ala(Thr)@hr)ArS-Asn-Thr-Gly-ArS 7 CA0 CA0 1 1 1 -7 1 --7 --7

GalN-T(HF)

-

10

-

GalN-Sl

Gall<-S2

TOTAL SEQUENCE qlo NH*-Ser-Pro-Lys-ALA-Gin-ALA-SER PI Pro-Gin-Ala-Glu-Gly-Scr-Lclj-ALA-L,

cyo cyo -SER-Val-PRO-THR-Ala-Glncijo ci'o ~,s-ALA-T~IR-TI11~-AI,A-proII

t

cyo cyo ALA-THR-THH-Arg-Asn-Thr-Gly-Arp,-COOH 4

remaining

is

sequence

linked

contrasting

oligosaccharide the doublet appeared

to the threonine

to the case of the

sequence

in the different

We would

ancies

with

rides between an individual

reported

of Pro-Ser

have a galactosamine rule.

are

moieties

o(1 hinge

in the hinge

protein, that

in the 9-Val-Pro-Thr-11

to be linked

in which

region(g).

the attachment

and location

in

sequence

reported

is possible appear some

of galactosamine

but our present result and ether(5), sample variation rather than technical 1070

residue to for

the

of the galactosamine-oligo-

the threonine residue in the triplet residue has a cis-configuration. There to the number

the GalN-

more generality

to

regard

all

The triplet is also

suggesting

triplet

to the serine

dl-microglobulin,

oligosaccharide(lO),

suggest

saccharide moiety when the proline

residue

this

only discrep-

oligosaccha-

may be attributed

problems.

to

Vol. 105, No. 3, 1982 The presence human

6

chain

GalN,

which

the GalN-rich

is

rather segment

proteolytic

caused

by massive

possible

of seven

AND BIOPHYSICAL

GalN-oligosaccharides

is characteristic

various

the restricted

BIOCHEMICAL

uncommon of the enzymes.

of

the high

in immunoglobulins.

This

might

of

the hinge

region

local

concentration

of the of

As in the case of the dl to be very

be due to the steric moieties

Such characteristic significance

in

& h' in g e was shown

GalN-oligosaccharide

region.

biological

because

RESEARCH COMMUNICATIONS

which

may have

resistant

to

hinderance

are distributed some correlations

along to

IgD.

ACKNOWLEDGEMENTS Department of Medicine, Osaka University We thank Dr. Akira Shimizu, School of Medicine for providing myeloma plasma NIG-65, and Dr. A. Yamada for general discussion. This work was supported in part by a grant-in-aid from the Ministryof Education, Science and Culture of Japan, and by research funds from the Ito Science Foundation.

REFFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Prah1,J.W. and Grey,H.M. (1970) Biochemistry 9, Spiegelberg,H.L., 2115-2122 Shinoda,T., Takahashi,N., Takayasu,T., Okuyama,T. and Shimizu,A. (1981) Proc. Natl. Acad. Sci. USA 78, 785-789 Takayasu,T., Takahashi,N. and Shinoda,T. (1980) Biochem. Biophys. Res. Commun. 97, 635-641 Lin,L.-C. and Putnam,F.W. (1981) Proc. Natl. Acad. Sci. USA 78, 504-508 Putnam,F.W., Takahashi,N., Tetaert,D., Debuire,B. and Lin,L.-C. (1981) Acad. Sci. USA 78, 6168-6172 Proc. Natl. Takahashi,N., Takayasu,T., Isobe,T., Shinoda,T., Okuyama,T. and Shimizu, A. (1979) J. Biochem. 86, 1523-1535 Takayasu,T., Takahashi,N., Shinoda,T., Okuyama,T. and Tomioka,H. (1981) J. Biochem. 89, 421-436 Mort,A.J. and Lamport,D.T.A. (1977) Anal. Biochem. 82, 289-309 Liu,Y.-S.V., Low,T.L.K., Infante,A. and Putnam,F.W. (1976) Science 193 I 1017-1020 Takagi,T., Takagi,K. and Kawai,T. (1981) Biochem. Biophys. Res. Commun. in press

1071