Enzymatic transfer of oligosaccharide from oligosaccharide-lipids to an Asn-Ala-Thr containing heptapeptide

Vol. 81, No. 3, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

April 14, 1978

ENZYMATIC TRANSFER OF OLIGOSACCHARIDE FROM OLICKISACCHARIDE-LIPIDS ASN-ALA-THR

C. Ronin,

772-778

TO AN

CONTAINING HEPTAPEPTIDE

C. Granier*,

and S. Bouchilloux

J. Van Rietschotenl

Laboratoire de Biochimie Medicale, Faculte de Medecine& Bd J. Moulin F-13385 MARSEILLE Cedex 4, et Laboratoire de Biochimie , Faculte de MgdecineNord, Bd P. Dramard, F-13326 MARSEILLE Cedex 3, FRANCE.

Received

February

9,1978

SUMMARY. Thyroid rough microsomes cataiyzed the transfer of oligosaccharide from previously labeled thyroid [ManC] oligosaccharide-lipids to a synthetic dinitrophenylated heptapeptide containing a sequence Asn-Ala-Thr. The resulting product revealed an N-glycosidic attachment, probably to the asparaginyl residue in this sequence. The reaction which was time-dependent up to I h and exhibited an apparent Xm of 94 uM for the DNP-heptapeptide was favoured by dimethylsulfoxide and inhibited by EDTA. Several

lines

phosphosugar of proteins pathway

that

shown,

lipid-released

ted several suggested

Two papers of membrane

defined

proteins

globulin

light

enzymes (5) (6).

glycoproteins

acceptor

Abbreviation

that

for were

It

(7).

Its

: DNP-peptide,

way were

system structure

to newly GDP-[14C]Man,

moieties

(3,4).

exhibi-

The identities difficult

could

act

in

the glycosylation

of exogenous

a carbohydrate-free

kappa-type

proteins

containing

from structural

is

a necessary

but

we wish

of to

used as an efficient thyroid,

peptide.

the prewellimmuno-

at least examination

not

to report,

on a sequence

772

with

nevertheless

from pig

OQO6-291X/78/0813-0772$01,00/~

and mannose.

such a possibilitY,also

is based

dinitrophenylated

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

as donors

tissues.

In the work

has been

microsomal

lipids.

this

sulfitolyzed tripeptide

to this

substantiating

has been anticipated this

According

N-acetylglucosamine

oligosaccharide-lipids

or several

heptapeptide

in a rough

oligosaccharide

as in other

N-glycosylation

dinitrophenylated

in

as donors

that

function

the case of thyroid

acceptors

chain

I).

polyprenol

to the glycosylation

microsomes incubated I 4CJ oligosaccharide

a result

in

as well

: these

for

rough

glycosylated

Asn-X- z:

requirement

(2),

have reported

sence

one vacant

would containing

thyroid

approaches

proteins

in thyroid

numerous

with

that

membranes

in refer

and protein-releasedban-

from other

ascertain,

(reviewed

units

corm-non properties

the endogenous

to the realization

oligosaccharides

of pre-assembled

recently

led

in eukaryotic

sugars

pyrophospho

made proteins

have

contribute

from nucleotide

lipid

We have

of evidence

derivatives

of

sufficient a synthetic'

oligosaccharide

supplemented that

with[Manwas determined

14C]

Vol. 81, No. 3, 1978

for

a tryptic

glycopeptide

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from human thyroglobulin

(8)

:

DNP-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH2 EXPERIMENTAL : GDP-[u-'~C~ mannose (179 Ci/mol) and I-fluoro-2,4-dinitro-13,5-% Xaterials benzene (30 Ci/mmol) were from the Radiochem. Centre. Bio-Gels P-2 and P-4 were from Bio-Rad Sephadex G-15 from Pharmacia and carboxymethyl-cellulose (W-52) from Watman. The resin and the protected amino acids used for the solid phase synthesis of the peptide were from Beckman. Synthesis of the heptapeptide H-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH2 : This sequence of amino acids has been assembledx0.8 g of the benzhydrylamine derivative of 1 X cross-linked polystyrene (0.5 mequiv. NH2/g) by the solid phase method (IO) as described for the synthesis of apamin (11). After HP cleavage, the synthetic peptide was purified on a Sephadex G-15 column developed with 0.1 M ammonium acetate. Ion exchange chromatography on a carboxymethyl-cellulose column, equilibrated in 0.012 M ammonium acetate pH 6.0, revealed a nice well-retarded peak. After lyophilization the purity of this material was assessed by amino acid analysis after acid hydrolysis (Asn1.0, ThrlsO, Glu,.,, Ala2.0, LeulsO, Ang1.0) and total enzymatic digestion (Asn and Thr co-elute, Glu1 o, Ala2 o, leulmO, Are, ,). The yield of purified peptide over starting NH2 has been 53 X: Preparation of the DNP-, and 13H- DNP-heptapeptide(s) : 3 umoles of the heptapeptide were dissolved in 120 ul of 1 % triethylamine in water and reacted with 30 umolesoffluorodinitrobenzene dissolved in 240 ul of ethanol, for 90 min under vigorous stirring. The DNP-peptide was purified on a Bio-Gel P2 column developed with 0.1 M acetic acid. Amino acid analysis after acid hydrolysis showed the disappearance of one alanine. A similar dinitrophenylation was carried out with 0.8 nmol ofhepqeptide, but in the presence of fluorodinitro -13H benzene (250 nCi). The specific activity of the resulting purified '3H‘DNP-heptapeptide was 8 nCi/nmol. Preparation of IMan-14Cl oligosaccharide-lipids : They were labeled by incubating thyroid rough microsomes (40 mg of protein) with 14 nM GDP-114C; Man (10 uCi) in a final volume of 4 ml for 10 min at 37*C, as previously described (2). The delipidation procedure was slightly modified according to Lucas et al. (9) in order to minimize the contamination by dolichol-P-Man in the final CHC13/CH30H/H20 extract : in these conditions a DEAE-cellulose chromatography revealed 5 I of contamination and the radioactiyity pttained was 200 - 250 000 cpm, Standard assay for transfer of .ManC; oligosaccharide from -Man- 14 CI, oligosaccharide-lipids to the DNP-peptide (or to the peptide) : incubation mixtures contained thyroid rough microsonms (240 ng of protein), :Han-14C , saccharide-lipids (1 to 10 x IO3 cpm, supplied in 15 ~1 of 25 X dimethylsulfoxide), 530 ptl DNPheptapeptide, 20 mM Tris-HCl pH 7.4, 25 mM KCl, 5 mM MgCl 12 mM MnC12, 1.25 mM dithiothreitol and 0.25 M sucrose, in a final volume of 7 !? nl. Reactions were started with microsomes and stopped after 60 min at 37°C by adding 125 pl of ethanol. After staying for 16h at -17°C the supernatants were spotted on Nhatman 3 MM mper and submitted to electrophoresis in 1.5 N formic acid at 34 V/cm for 135 min. Radioactivity was counted in 1 cm wide strips of paper after solubilisation in 1 ml of water. RESULTS AND DISCDSSION Assay of the DNP-heptapeptide as a potential from lipid derivatives I Exogenous ,Man-'4Cjoligosaccharide-lipids

acceptor were

of ,Man-14C; incubated

with

oligosaccharide thyroid

rough

Vol. 81, No. 3, 1978

8lOCHEMlCAL

AND BIOPHYSICAL

Man I

10 :

DNP-pept.

0

RESEARCH COMMUNICATIONS

A

I 5 f

0 0

cm

microsomes

in

(Fig.

1).

compound yellow

of ethanol

the origin differs

color,

from

of oligosaccharides,

both

assays

with

of

known

530 pM DNP-peptide on a paper

As shown

strip

94 pI has been

deduced

from

and further

The supernatant incubation

from

in this

characterization

tected

from its

absorbance

at 360 nm).

lipids

in thyroid

: we have previously rough

for

cleaved

microsomes

incubation

represented

30 X of

an entire

compound

60 min.

is

migrating

The effect

shown in Fig.

reaction at 8 cm

of increasing

3. An apparent

Km of

would

reported by mild

(2),

from a preparative

on Bio-Gel

species This

DNP-peptide

issued

and chromatographed

moiety

in

The intermediate

by spotting

of the glycosylated

as a much larger

saccharide

present

migration).

precipitation

behaved

[Man-'4C1

were

data.

material latter

from its and from

which

donor.

of the labeled

labeled

form of the

of this

In the standard

(checked

DNP-peptide

of an ethanol

was concentrated

the labeled

at about

The mobility

peak has repeatedly

a plateau

these

(Z),

a glycopeptide.

2 the formation

of exogenous

efectrophoresis

15 cm, localized

at the origin

:

and

was observed

assay.

(approx.

electrophoretic

reaching

material

the control

incubation

without

on time,

the concentration Isolation

the label

in Fig.

was dependent

to stay

suggests

the total

by paper

of the [3H]DNP-heptapeptide)

some cleavage

in

a labeled in

DNP-heptapeptide,

examined

of the DNP-peptide

the new compound

the radioactivity mixture

was absent

the radioactivity

indicating

of unlabeled

were

of DNP-peptide,

that

and from

the absence

precipitation

which

that

mobility

or in

In the presence

8 cm from

cm

comparison of ethanol/water-soluble labels issued from assay of rough microsomes with [Man-14C] oligosaccharide(A) or absence (B) of exogenous DNP-heptapeptide. Markers and [14C) mannose. Arrow = origin.

the presence

the supernatants

m

B

a

Fig. I Electrophoretic a standard incubation lipid in the presence [3H] DNP-heptapeptide

B

DNP-pepl.

QD

acid

and our

than

P-4

(Fig.

4).

the DNP-heptapeptide

be expected on an aver

for

DNP-heptapeptide

The major (de-

a glycosylated

Mr of

from endogenous

scale

1500 for

the

saccharidehas a Mr of 940.

Vol. 81, No. 3, 1978

EROCHEMKAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

10 n b

x E n 0

5

0

I

10 0

I

I

20 30 MINUTES

I

I

I

40

50

60

0

DNP-

peptide

(mM)

Fig. 2 Time-course of labeling of the material migrating at 8 cm by paper trophoresis in formic acid. Conditions of a standard assay.

elec-

Fig. 3 Effect of varying the concentration of DNP-heptapeptide on the labeling of the material migrating at 8 cm by paper electrophoresis in formic acid. The inset shows the Lineweaver-Burk plot of the data.

4b

6-0

0’0

FRACTION

lb0 NUMBER

Fig. 4 Gel filtration on Bio-Gel P-4 of the [14C] labeled ethanol/water supernatant issued from a 2 ml incubation in standard conditions (except for a 20 X final concentration in dimethylsulfoxide). Running buffer : pyridine-acetate, pH 5.1 (1 N in pyridine). Column size : 0.8 x 75 cm. Fractions of 0.6 ml. Varkers : Blue Dextran , c3H] DNP-heptapeptide and L'4C] mannose.

Pooled

radioactive

was submitted distance

to paper

were

to assess

and the peptide,

reductive 0-glycosidic

alkaline

lyophilized.

electrophoresis

of 8 cm from origin

In order moiety

fractions

in not

the nature aliquots

treatments

or N-glycosidic

(data

yellow

acid

and found

formic

colored

material

to migrate

at a

shown).

of the of this

linkage lyophilized

generally linkages,

The pale

used

for

as previously

775

between

the

material selective reported

['"C: were

mannosylated submitted

cleavage (2).

to

of either It

was found

BIOCHEMICAL

Vol. 81, No. 3, 1978

AND 8lOPHYSlCAL

FRACTION

Fig. ved lated ride and

RESEARCH COMMUNICATIONS

NUMBER

5 Gel filtration on Bio-Gel P-4 of the [?lan-14C] oligosaccharid moiety cleaby strong reductive alkaline hydrolysis from the purified IMan- 12C) glycosyDNP-peptide, and comparatively (dotted line) of the ;Man-'4C1 oligosacchamoiety released by mild acid from the saccharide-lipid donor. Column size experimental conditions as in Fig. 4.

by gel

filtration

no detectable

on Bio-Gel change

P-4

(data

not

NaOH, 2 M Na BH4, at 80°C for moiety

was observed

at a position

(Fig.

close

that

a treatment

B-elimination

On the contrary

shown).

16h, an essentially

determined

after

peak

resulted

a treatment

complete

5) : the radioactive

to that

for

cleavage

now emerged

in a parallel

run

in

by 2 N

of the labeled

from

the column

Man-l4 CI* oligosac-

for

charide donor.

moieties that were cleaved by mild acid from the endogenously labeled Since deacylation is known to occur in strong reductive alkaline conditions ?I4 7 the material released from the . C , glycosylated DNP-peptide was treated for N-acetylation

of its

putative

glucosamine

electrophoresis

: it

behaved

as a neutral

origin,

the acid-released

as does

lipids

moiety

residues

(2),

compound

and stayed

issued

from

and examined

by paper

entirely

at the

.Man- 14C‘ saccharide-

(2). Altogether

these

results

provide

membranes

an enzyme is present

exogenous

oligosaccharide-lipids

reasonable

which

evidence

transfers

that

within

an oligosaccharide

to the asparaginyl

residue

the rough

unit

from

of the synthetic

DNP-heptapeptide. Other

properties Increasing

sing

agent,

extent

of the system the final

of dimethylsulfoxide,

from 5 to 25 X was found

of transfer.

When, instead,

of 0.02

Z the results

totally

inhibited

dependent

concentration

were

not

to have a slightly

we used Triton

satisfactorily

integrity

of the membrane 776

stimulatory

X-100

at a final

reproducible.

by 10 mM EDTA. These observations

on a relative

used as a disper-

suggest

effect

The reaction that

and on a metal

on the

concentration was

the enzyme is ion.

BIOCHEMICAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i 0

7.5

;;

s.0

I 025 0 TO t

cm 'cprofile of the ethanol/water soluble labels issued from an Fig. 6 Electrophorefi incubation of [ManC ] oligosaccharide-lipids with [%I] DNP+ep*eptide. The incubation was as described in Experimental except for being 700 uM in DNP-peptide. Double isotope counting using adequately quenched standards.

When the synthetic DNP-derivative deduced

it

from

heptapeptide

exhibited

about

the proportion migrating

new species, the DNP-group

beyond

terminating

acceptors

in

We have nevertheless reaction

occurs

with

to penetrate

a search

for

noticed

that

our system.

This

It

hydrophobic

probably

maining

of this

by paper

important

at origin),

migrating

there

has been evaluated

glycosylated

mannose

(12).

of the dinitrophecompetes

with

(of

the

it

(resulting

was a definite

the

initially

gosaccharide conformational Ser).

transferase.

Other

requirements Another

was observed

f3 ~ H,1 label

an ethanol

(Fig.

6) that,

associat

associated of

peak re-

ed with with

the material

this

double-labeled

DNP-heptapeptide

have been

present).

synthetic

analogs

are necessary

important

from

incuba-

in a 13Hl radioactivity,

3H radioactivity I 1 that 0.25 nmoles

49 nmoles

in a microsomal

In conclusion our data demonstrate the M-glycosylation defined peptide catalyzed by a microsomal oligosaccharide-lipid

(or

a similar various

and the supernatant

electrophdresis,

cleavage

at 8 cm. From the it

was used as acceptor

oligosaccharide-lipids,

analyzed

in spite

Thr

that

environment

activity

cleavage

as in a

group

transferase

cleavage

latter,

is probable

when assaying

an extensive

of its

electrophoresis

the lipophilic

of this

u-mannose

instead

of the

DNP-peptide.

by Adamany and Spiro

t3H]bNP-heptapeptide

[Man-14C)

precipitation

peak,

in

activity

by paper

the glycosylated

assay

of glycosylation.

When the tion

localized

the usefulness

has been reported

moiety

the acceptor

the peptide

As regards

observation

nylated

half

of label

facilitates

of the enzyme.

was used in a standard

problem

777

might

in addition will

of a synthetic : peptide help

to find

to the basic

be to assessthe

out

welloliif

sequence

specificity

of

some Asn-Xthis

BIOCHEMICAL AND EIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 3, 1978

transferase in lipid

as regards donors.

an exogenous comitant different

Finally,

peptide

protein approach

the length the ability

indicates

synthesis, (13)

attached

peptides

of the rough

of thyroid this

rough

transfer the proposal

occuring might

of the oligosaccharide

concluded

; alternatively

glycosylation

level

that

as recently

a lipid-mediated nascent

and composition

microsomes

might

correspond

to glycosylate

not be dependent

by Vargas

to the process

on a con-

and Caminatti

made by Kiely

on the hydrophobic

moiety

extremities occuring

et al.

from

a

(14)

of

of ribosome --in vivo

at the

E.R.

Acknowledgements : The authors sions. This work was supported fique et Technique (Convention and I.N.S.E.R.M. (U. 38).

wish to thank Dr. H. ROCHAT for fruitful1 discusby the DdlEgation G&&-ale 1 la Recherche Scienti75.7.0797), the C.N.R.S. (L.A. I78 and E.R.A. 617)

REFERENCES 1. 2. 3; 4.

Waechter, C.J. and Lennarz, W.J. (1976) Ann. Rev. Biochem., 45, 95-112 Bonin, C. and Bouchilloux, S. (1978) Biochim. Biophys. Acta, 539, in the press Ronin, C. and Bouchilloux, S. (1976) Biochim. Biophys. Acta, 428, 445-455 Spiro, M.J., Spiro, R.G. and Bhoyroo, V.D. (1976) J. Biol. Chem, 251, 6400-6408 5. Eagen, P.K., Hsu, A.F. and Heath, E.C. (1975) Fed. Proc., 34, 678 6. Pless, D.D. and Lennarz, W.J. (1977) Proc. Nat. Acad. Sci. USA, 74, 134-138 7. Marshall, R.D. (1974) Biochem. Sot. Symp.,40, 17-26 8. Rawitch, A.B., Liao, T.H. and Pierce, J.G. (1968) Biochim. Biophys. Acta, 160, 360-367 9. Lucas, J.J., Waechter, C.J. and Lennarz, W.J. (1975) J. Biol. Chem., 250, 1992-2002 lO.Erickson, B.W. and Merrifield, R.B. (1976) in:"The Proteins" vol.2 (H. Neurath and R.L. Hill eds., Academic Press, New Yom pp. 255-562 ll.Van Rietschoten, J., Granier, C., Rochat, H., Lissitzky, S. and Miranda, F. (1975) Eur. J. Biochem., 56, 35-40 12.Adamany, A.M. and Spiro, R.G. (1975) J. Biol. Chem., 250, 2830-2841 13.Vargas, V.I. and Carminatti, H. (1977) Mol. Cell. Biochem., 16, 171-176 14.Kiely, M.L.,McKnight ,G.S. and Schimke, T. (1976) J. Biol. Chem., 251, 5490-5495.

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