Modification of human erythrocyte ghosts with transglutaminase

Vol. 67, No. 3, 1975

BIOCHEMICAL

MODIFICATION

OF HUMAN

WITH L. Lorand,

October

RESEARCH COMMUNICATIONS

ERYTHROCYTE

GHOSTS

TRANSGLUTAMINASE

R. Shishido,

K. N. Parameswaran

Department of Biochemistry University, Evanston, Illinois and Medicine, University Received

AND BIOPHYSICAL

and T. L. Steck

and Molecular Biology, Northwestern 60201 and Departments of Biochemistry of Chicago, Chicago, Illinois 60637

1.3,1975

Summary: Guinea pig liver transglutaminase was shown to catalyze the incorporation of dansylcadaverine and putrescine into two major protein fractions of human erythrocyte ghosts. As judged by sodium dodecylsulfate gel electrophoresis under reducing conditions, one of these is a high molecular weight polymer which may contain spectrin. The other corresponds to band 3, an 88, 000 dalton polypeptide. Amine substrates of transglutaminase were synthesized with specific properties to further explore this useful enzymatic technique of covalently labelling proteins in erythrocyte ghosts and in other biological membranes.

There

is a family

displacement

of transamidating

reactions

residues

in proteins.

enzymes

are already

cross-linking

specifically From

Cross -linking

copulation

plug in seminal

sources

as diverse

(12), sea urchin

for the selective intermolecular

blood coagulation

fluid (2) are outstanding

eggs (13).

of promoting

Though

properties

(9), hair follicle considerable

are discernable

the covalent

incorporation 1158

Copyright D 19 75 by Academic Press, Inc. AN rights of reproduction in ar1.y form reserved.

of these

post-translational

(1) or the formation

examples.

(3), platelets

several

y-glutamyl-c-lysine

transferases,

as blood plasma

(8, 9), red blood cells

and catalytic

the ability

during

nucleophilic

of some glutamine

point of view,

as endo-y-glutamine:c-lysine

(6, 7), muscle

ficities

of fibrin

by producing

which catalyze

the y-amides

the physiological

proteins

bridges.

from

involving

known to be responsible

of native

best designated

enzymes

Such enzymes, have been isolated

(4), placenta (10, ll),

differences

(5), liver

coagulating

gland

of substrate

speci-

within the group, of a variety

of

they all share

of synthetic

primary

Vol. 67, No. 3, 1975

amine

derivatives

i.e.,

PCONH,

BIOCHEMICAL

AND BIOPHYSICAL

at neutrality

into a number

(H,NR)

+ H,NR __j

The enzymes of the synthetic

PCONHR

display

amine

by dansylcadaverine

to c-lysine

residues

In general,

the presence

transamidases

human erythrocyte

perhaps

the least specific

cadaverine

hyaline

might

paper describes

reticulum

as synthetic

embryos

substrates

phosphate

proteins results

14C-Putrescine

for labelling.

with

and dansyl-

Previously,

of proteins

erythrocyte

membrane

of the

ghosts (18), rabbit

(19).

AND METHODS

red blood cell ghosts were prepared

blood in 5 mM sodium

the forma-

transglutaminase,

modification

(13), mouse

(18) and fibroblast MATERIALS

Human

inhibits

our preliminary

of this group of enzymes.

of sea urchin

sarcoplasmic

amines

also be useful for modifying

has been used for the covalent

layer

in which cross-linking

ghosts using guinea pig liver

were selected

this technique

side chains analogous

systems

of synthetic

among these --

(17).

The present

isolated

the R group

-- comprise

In biological

bridges

in cell membranes.

towards

the best substrates

(14-16)

in proteins.

tion of protein-to-protein

(PCONH,)

+ NH,.

and, interestingly,

occurs,

of proteins

a high degree of specificity

exemplified

by such enzymes

RESEARCH COMMUNICATIONS

buffer,

from freshly

pH 8, by the procedure

outdated

bank

of Fairbanks

et al. (20) and used promptly. Transglutaminase as a frozen buffer

solution

of pH 7.5,

containing containing

14C-Putrescine dansylcadaverine

was isolated

from

guinea pig livers

25 mg protein

per ml of 50 mM Tris

chloride

1 mM of EDTA.

(60 mCi per mmole)

was purchased

(14) was a gift from AB Kabi,

as well as dithiothreitol

(7) and was stored

(Pierce

Chemical

Stockholm.

Co. ), calcium

1159

from Amersham-Searle; These compounds chloride

and EDTA

Vol. 67, No. 3, 1975

were dissolved

in 50 mM Tris

Reaction comprised

BIOCHEMICAL

mixtures

in cellulose

nitrate

were allowed phosphate

to proceed

buffer

chloride.

dithiothreitol

(pH 8) was admixed

The sediments of 5% sodium

in buffer

and centrifuged

were dissolved dodecylsulfate;

of Fairbanks

which incorporated handlamp

(17).

experiments

with 14C-putrescine

a Packard aliquots

liquid

of the detergent

dansylcadaverine doubly labelled

Aliquots

chloride

(pH 8),

of 50 ti were taken for

visualized

blue R (20).

peroxide

under a TJV

The gels pertaining

Model

blue,

to the

photographed

24), sliced

trans-

for the assay of radioactivity

counter

(Model

of the ghosts treated

prior

Bands

with ink and the gels

with Coomassie

Spectrophotometer

scintillation

were mixed

unreacted

by Steck and Yu (21).

were marked

were stained

solutions

The sedimented

50 mM Tris

could be readily

with hydrogen

Tri-Carb

1.5 ml cold 5 mM sodium

gels (100 mA per gel; 100 min) according

by Coomassie

and scanned at 540 rnv (Beckman and treated

(20).

of fluorescence

for proteins

The reactions

by adding 150 ul of water and 50 rJ1 of a

et al. (20) as modified

were stained

versely

chloride.

in order to remove

50% sucrose;

dansylcadaverine Positions

mM

50 111of 1 mM EDTA

and the ghosts sedimented.

on 5% polyacrylamide

to the protocol

In controls,

and calcium

5 mM EDTA and 200 mM of dithiothreitol electrophoresis

10 cll of 0 -5.6

10 ul of 0.2 M dithiothreitol

at 3’7’ for 3 hr at which time

ghosts were resuspended amines.

tubes (Spinco No. 303369) typically

20 ~1 of transglutaminase,

20 cll of 40 mM calcium

was added --in lieu of enzyme,

RESEARCH COMMUNICATIONS

buffer of pH 7.5.

or of 0 - 5 mM dansylcadaverine,

and, finally,

mixture

chloride

100 111of packed ghosts,

14C-putrescine

AND BIOPHYSICAL

to electrophoresis

3385).

(22) in

Occasionally,

with 14C-putrescine

and

so as to be able to obtain

gel bands. RESULTS

Guinea pig liver

AND DISCUSSION

transglutaminase

is a calcium 1160

dependent

enzyme

(6).

As

Vol. 67, No. 3, 1975

-

BIOCHEMICAL

I- ‘-I-

iI 2

0

AND BIOPHYSICAL

IO

RESEARCH COMMUNICATIONS

I I I

3 I4.2 4.5 4.1

5 6

7

20

40

50

DISTANCE

30

60

MIGRATION,

OF

70

mm

Fig. 1. Electrophoretic profiles of human erythrocyte ghosts after exposure to transglutaminase and 14C-putrescine in the absence of calcium ions. Open bars denote radioactivity found in gel slices. The photographic insert shows the protein pattern after staining with Coomassie blue; numbering of bands corresponds to that given in the literature (23).

such, red cell ghosts treated

with transglutaminase

of EDTA showed an electrophoretic of untreated

ghosts (20).

dansylcadaverine

incorporation

Thus,non-enzymatic base formation)

Similarly,

or 14C-putrescine

and no appreciable

uptake

pattern

(Fig.

treatment produced

no visible

ions,

substrates

in the gels

bands (Fig.

(for example,

1).

by Schiff-

extent with red cell membranes.

transglutaminase

1161

from that

fluorescence

of 14C into any of the protein

of these amine

of calcium

1) indistinguishable

of an excess

in EDTA with this enzyme plus

does not occur to any appreciable

In the presence

in the presence

brought

about the in-

Vol. 67, No. 3, 1975

BIOCHEMICAL

18

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

I6 -

l4-

I2 -

IO -

8-

6-

4-

2-

O-

1

I

I

0

IO

I 20

30

40

50

60

70

profiles of human erythrocyte ghosts after reaction Fig. 2. Electrophoretic withansglutaminase and 14C-putrescine in the presence of calcium ions. Presentation as in Fig. 1. 1162

Vol. 67, No. 3, 1975

corporation tions.

BIOCHEMICAL

of dansylcadaverine

Intense

labelling

This situation linked

glutaminase

of synthetic

Fig.

l),

amines

may contain

since these are missing

peptides

of about 240,000

dense reticulum

which catalyzes

susceptible the formation

25).

Evidently,

proteins. cross-

on top of gels after trans-

(designated

to covalent

as bands 1 and 2 in profile.

These poly-

are thought to be associated membrane

cross-linking

of v-glutamyl-c-lysine

crossbridges.

cross-linked

this process

in a

(23) and

by a transamidase

by a variety

of dansylcadaverine

to inhibit

Apparently

is enzymatically

surface of the plasma

the concentrations

ployed were not sufficient

remaining

daltons

be noted that bands 1 and 2 are readily

2).

of certain

from the electrophoretic

and 215,000

frac-

(17).

spectrin

at the cytoplasmic

may be particularly

and labelling

weight material

treatment

(Fig.

to what happens when fibrin

protein

at the top of the gel

(not shown) and radioactivity

is analogous

The high molecular

into two major

remaining

caused both cross-linking

in the presence

RESEARCH COMMUNICATIONS

and “C-putrescine

was found in material

as seen by its fluorescence transglutaminase

AND BIOPHYSICAL

It should of reagents

or 14C-putrescine

appreciably

(23-

em-

at the monomeric

state. The second,

less intensely

labelled

region

on the gels corresponds

band 3, an 88,000

dalton polypeptide,

believed

diffusion

(26,27).

While

can be selectively

to form

covalent

dimers

dimeric

form was seen either

Singer

with mouse erythrocyte The selective

in situ should provide -addition cadaverine

this protein

both in the membrane

modification

in this system

of membrane

containing

(29)] or an isotope

in facilitated

disulfide

and in solution (Fig.

cross-linked (24,25,28),

no

2) or in that of Dutton and

ghosts (18).

a useful tool for labeling

to the compounds

to be involved

to

label

proteins

and cross-linking

a fluorescent

reporter

(e. g. 14C-putrescine), 1163

with transglutaminase studies.

In

group [e. g. dansylwe tested a number

Vol. 67, No. 3, 1975

of synthetic

BIOCHEMICAL

amines

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as transglutaminase

substrates

the following

seem to be of special

spin-labelled

N-(5’-Aminopentyl)-2,2,5,5-tetramethyl-3-pyrrolin-l-oxyl-3-

carboxamide;

interest

and, for obvious

(b) di- and tri-iodinated

in membrane

compounds,

diiodo-L-tyrosine-N-(5’-aminopentyl)amide cadaverine); amide

(c) amines

which antibodies

aniline

(or 2,4-dinitrophenylcadaverine);

N-(5-Aminopentyl)

are readily

available:

. Details

et al.,

data) will be the subject

to transglutaminase transglutaminase,

is some evidence

that the spectrin

for the anchoring

and thus may actually An enzyme

system

catalyzing

have some physiological

lysine

bridges

of synthesis

and evaluation

of a separate

polypeptides

as to whether its function

in ghosts

stiffens

the clot structure

reticulum

just as trans(30,3I).

on the cytoplasmic

interactions

Since formation

the possibility

role is worth considering. 1164

proteins

of the cell (cf. 23).

relevance.

by amide

There

side provides

of transmembrane

cross-linking

molecules

may somehow

skeleton,

the reversible

between spectrin

(P. Stenberg

paper.

of the spectrin

the outside

change in free energy,

such a dynamic

(or 2,4-dinitrobenzene-

and positioning

influence

might

with little

N-(5-Aminopentyl)-2,4-dinitro-

state of the spectrin

molecules

group

Since red cells are known to contain a

arises

the physical

between fibrin

the framework

attention.

the question

to regulating

amidation

of methods

susceptibility

merits

haptenic

NE-Aminocaproyl-p-nitroanilide;

sulfonylcadaverine)

the extreme

(or 2,3,5-

with a nitrobenzene

-2,4dinitrobenzenesulfonamide

unpublished

-

N-(5’-Aminopentyl)-3-acetamido-2,4,6-

against

pertain

such as $-Acetyl-3,5

and N-(5’-Aminopentyl)-2,3,5-triiodobenzamide

triiodobenzoylcadaverine);

(a) nitroxide

-5 -acetamido-2,4,6-triiodo-N-methylisophthalam

(or Iothalamoylcadaverine);

Finally,

research:

(or #-acetyl-3,5-diiodotyrosyl-

N-(5 ‘-Aminopentyl)

triiodobenzamide

reasons,

of spectrin

molecules

of y-glutamyl-c

exchange

could proceed

of transglutaminase

playing

Vol. 67, No. 3,1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This work was supported at Northwestern University by USPHS Research Career Award to L. Lorand and by grants from the National Heart and Lung Institute, National Institutes of Health (HL-02212 and HL-16346) and at the University of Chicago by a grant (BC-95C) from the American Cancer Society.

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