Domain

analysis

of

lipoprotein

LppQ

in

Mycoplasma

mycoides

subsp

mycoides

SC

The

lipoprotein

LppQ

is

the

most

prominent

antigen

of

Mycoplasma

mycoides

subsp

mycoides

small

colony

type

-LRB-

SC

-RRB-

during

infection

of

cattle

This

pathogen

causes

contagious

bovine

pleuropneumonia

-LRB-

CBPP

-RRB-

a

devastating

disease

of

considerable

socio-economic

importance

in

many

countries

worldwide

The

dominant

antigenicity

and

high

specificity

for

M.

mycoides

subsp

mycoides

SC

of

lipoprotein

LppQ

have

been

exploited

for

serological

diagnosis

and

for

epidemiological

investigations

of

CBPP

Scanning

electron

microscopy

and

immunogold

labelling

were

used

to

provide

ultrastructural

evidence

that

LppQ

is

located

to

the

cell

membrane

at

the

outer

surface

of

M.

mycoides

subsp

mycoides

SC

The

selectivity

and

specificity

of

this

method

were

demonstrated

through

discriminating

localization

of

extracellular

-LRB-

i.e.

in

the

zone

of

contact

with

host

cells

-RRB-

vs.

integral

membrane

domains

of

LppQ

Thus

our

findings

support

the

suggestion

that

the

accessible

N-terminal

domain

of

LppQ

is

surface

exposed

and

such

surface

localization

may

be

implicated

in

the

pathogenesis

of

CBPP

Introduction

Mycoplasma

mycoides

subsp

mycoides

small

colony

type

-LRB-

SC

-RRB-

is

an

extra-cellular

pathogen

living

in

close

association

with

host

cells

It

causes

contagious

bovine

pleuropneumonia

-LRB-

CBPP

-RRB-

an

acute

subacute

or

chronic

infection

of

the

respiratory

system

in

cattle

with

a

mortality

rate

of

up

to

causing

severe

losses

in

livestock

production

in

particular

on

the

African

continent

-LRB-

Provost

et

al.

Food

and

Agriculture

Organization

of

the

United

Nations

-RRB-

Lipoproteins

are

usually

strongly

antigenic

membrane

proteins

known

to

play

a

central

role

in

interactions

between

bacteria

and

eukaryotic

cells

particularly

with

respect

to

adhesion

and

to

stimulate

the

release

of

pro-inflammatory

cytokines

-LRB-

Mühlradt

and

Frisch

Herbelin

et

al.

Brenner

et

al.

Marie

et

al.

Calcutt

et

al.

Belloy

et

al.

-RRB-

Lipoproteins

have

been

put

forward

as

possible

virulence

factors

of

pathogenic

mycoplasmas

-LRB-

Dyson

and

Smith

Vilei

et

al.

Pilo

et

al.

-RRB-

Membrane

lipoprotein

LppQ

is

the

predominant

antigen

of

M.

mycoides

subsp

mycoides

SC

and

shows

the

strongest

signal

on

immunoblots

containing

total

antigen

from

this

pathogen

reacted

with

serum

from

cattle

that

have

suffered

from

CBPP

It

induces

a

specific

early

and

persistent

immune

response

in

infected

animals

-LRB-

Abdo

et

al.

-RRB-

LppQ

is

encoded

as

a

precursor

-LRB-

of

amino

acids

-RRB-

with

a

consensus

sequence

for

prokaryotic

signal

peptidase

II

and

a

lipid

attachment

site

-LRB-

Figure

-RRB-

The

leader

sequence

of

LppQ

shows

a

typical

transmembrane

structure

with

a

significant

helix

formation

capacity

-LRB-

Abdo

et

al.

-RRB-

LppQ

was

shown

to

be

a

membrane

protein

by

Triton

phase

partitioning

and

lipidation

was

demonstrated

by

palmitic

acid

radiolabelling

-LRB-

Abdo

et

al.

-RRB-

The

C-terminal

part

of

LppQ

was

found

to

possess

repeated

integral

membrane

structures

rich

in

hydrophobic

and

aromatic

amino

acids

which

have

pore

formation

potential

and

immunoblot

analysis

showed

that

the

C-terminal

domain

possesses

no

particular

immunogenicity

as

serum

derived

from

cattle

naturally

infected

with

M.

mycoides

subsp

mycoides

SC

did

not

react

against

it

-LRB-

Abdo

et

al.

-RRB-

In

contrast

the

N-terminal

domain

of

LppQ

has

three

strongly

hydrophilic

domains

and

was

shown

to

be

the

origin

of

the

strong

antigenic

response

against

LppQ

in

naturally

infected

cattle

From

these

data

Abdo

and

collaborators

deduced

that

the

N-terminal

part

of

LppQ

is

exposed

at

the

outer

surface

of

M.

mycoides

subsp

mycoides

SC

-LRB-

Abdo

et

al.

-RRB-

Fig.

of

lipoprotein

LppQ

-LRB-

Panel

A

-RRB-

LppQ

protein

sequence

Repeats

are

indicated

by

dashed

arrows

The

symbol

corresponds

to

the

signal

peptidase

II

cleavage

site

which

allows

for

the

obtainment

of

mature

LppQ

protein

-LRB-

Panel

B

-RRB-

The

C-terminal

repeats

rich

in

hydrophobic

and

aromatic

residues

as

identified

with

the

MEME

software

which

build

the

transmembrane

region

of

LppQ

Black

background

indicates

conserved

residues

The

consensus

sequence

is

shown

-LRB-

Panel

C

-RRB-

Model

for

protein

topology

of

LppQ

in

the

membrane

lipid-bilayer

of

M.

mycoides

subsp

mycoides

SC

Repeat

numbers

and

amino

acid

positions

for

the

beginning

of

mature

LppQ

-LRB-

-RRB-

the

beginning

of

C-terminal

portion

-LRB-

-RRB-

and

the

protein

end

-LRB-

-RRB-

are

indicated

The

N-terminal

part

is

supposed

to

be

anchored

in

the

lipid-bilayer

by

the

lipid

anchor

of

the

residue

The

three

hydrophilic

domains

of

the

N-terminal

part

which

were

found

with

significant

scores

for

coiled-coil

tertiary

structure

-LRB-

Abdo

et

al.

-RRB-

are

depicted

as

globular

items

The

C-terminal

part

consists

of

a

transmembrane

region

built

up

of

the

repeated

integral

membrane

structures

whose

consensus

sequence

is

shown

at

the

bottom

Due

to

the

strong

antigenicity

of

the

extracellular

N-terminal

part

of

LppQ

and

its

specificity

for

M.

mycoides

subsp

mycoides

SC

a

recombinant

peptide

comprising

amino

acids

of

the

LppQ

precursor

protein

was

used

to

develop

a

specific

and

sensitive

ELISA

assay

for

the

serological

detection

of

CBPP

in

cattle

-LRB-

Bruderer

et

al.

-RRB-

Furthermore

LppQ

seems

to

be

involved

in

the

inflammatory

processes

of

M.

mycoides

subsp

mycoides

SC

as

cattle

immunized

with

the

recombinant

peptide

showed

an

increased

susceptibility

to

infection

with

M.

mycoides

subsp

mycoides

SC

and

exhibited

more

severe

symptoms

of

CBPP

than

unvaccinated

animals

-LRB-

Nicholas

et

al.

-RRB-

As

LppQ

is

apparently

involved

in

the

molecular

mechanisms

of

pathogenicity

of

M.

mycoides

subsp

mycoides

SC

-LRB-

Nicholas

et

al.

Pilo

et

al.

-RRB-

we

have

investigated

the

topology

of

LppQ

in

M.

mycoides

subsp

mycoides

SC

by

means

of

high

resolution

field

emission

scanning

electron

microscopy

-LRB-

SEM

-RRB-

with

particular

respect

to

the

predicted

localization

of

LppQ

on

the

mycoplasmal

cell

surface

This

technique

allows

proteins

to

be

localized

on

mycoplasmas

unequivocally

and

with

high

accuracy

and

resolution

-LRB-

Pilo

et

al.

-RRB-

Here

we

provide

evidence

that

the

N-terminal

part

of

LppQ

is

located

on

the

surface

of

the

mycoplasmal

cell

membrane

and

is

accessible

from

the

extracellular

side

while

the

C-terminal

part

of

LppQ

is

not

exposed

Materials

and

methods

Bioinformatic

analysis

Prediction

of

membrane

topology

was

carried

out

by

using

the

softwares

TMpred

-LRB-

Hofmann

and

Stoffel

-RRB-

TopPred

-LRB-

von

Heijne

-RRB-

and

Phobius

-LRB-

Käll

et

al.

-RRB-

Protein

secondary

structure

was

predicted

with

the

MINNOU

software

available

on

the

web

page

-LRB-

Cao

et

al.

-RRB-

Determination

of

the

C-terminal

repeats

of

lipoprotein

LppQ

was

assessed

by

the

MEME

software

version

at

-LRB-

Bailey

and

Elkan

-RRB-

M.

mycoides

subsp

mycoides

SC

strain

and

sample

preparation

M.

mycoides

subsp

mycoides

SC

strain

Afadé

was

used

in

the

present

study

This

strain

was

isolated

in

at

Farcha

Laboratory

N'Djamena

Chad

from

a

cow

from

Afadé

Northern

Cameroon

which

suffered

from

acute

CBPP

The

strain

Afadé

is

known

to

be

highly

virulent

-LRB-

Vilei

and

Frey

-RRB-

and

was

used

previously

for

experimental

infection

studies

-LRB-

Abdo

et

al.

-RRB-

It

was

grown

in

standard

mycoplasma

medium

-LRB-

Axcell

Biotechnologies

St.

Genis

l'Argentière

France

-RRB-

by

inoculation

of

μl

of

a

frozen

stock

culture

into

ml

of

medium

and

incubation

for

days

at

°C

Growth

and

handling

of

live

M.

mycoides

subsp

mycoides

SC

were

performed

in

a

biological

safety

laboratory

fulfilling

the

containment

safety

standards

Crude

lysates

were

prepared

by

resuspending

harvested

mycoplasmas

in

TES

buffer

-LRB-

mM

Tris

HCl

mM

EDTA

NaCl

pH

-RRB-

at

a

concentration

of

approximately

cells/ml

-LRB-

Abdo

et

al.

-RRB-

Production

of

antibodies

against

recombinant

LppQ

peptides

The

polyhistidine-tagged

recombinant

peptides

LppQ-N

-LRB-

amino

acids

to

-RRB-

and

LppQ-C

-LRB-

amino

acids

to

-RRB-

from

the

N

and

C-terminal

portions

of

LppQ

respectively

were

produced

in

Escherichia

coli

strain

-LRB-

-RRB-

harbouring

the

respective

plasmids

as

reported

previously

-LRB-

Abdo

et

al.

Bruderer

et

al.

-RRB-

Recombinant

peptides

were

polyhistidine-tagged

at

both

their

N

and

C-termini

Purification

of

the

peptides

was

performed

by

chelation

chromatography

and

the

purity

of

the

peptides

was

analyzed

by

sodium

dodecyl

sulfate-polyacrylamide

gel

electrophoresis

-LRB-

SDS-PAGE

-RRB-

Polyclonal

monospecific

sera

directed

against

LppQ-N

and

LppQ-C

were

obtained

by

subcutaneous

immunization

of

rabbits

with

μg

of

purified

recombinant

polyhistidine-tagged

peptides

in

μl

of

mM

PBS

buffer

-LRB-

mM

mM

NaCl

pH

-RRB-

mixed

with

μl

of

Adjuvant

-LRB-

Gerbu

Biotechnik

GmbH

Gaiberg

Germany

-RRB-

Booster

immunizations

with

and

μg

of

protein

were

performed

and

later

respectively

The

rabbits

were

bled

days

after

the

last

booster

immunization

and

antisera

were

stored

at

°C

Immunoglobulin

G

-LRB-

IgG

-RRB-

fractions

were

purified

from

the

two

rabbit

antisera

with

the

HiTrap

Protein

G

kit

-LRB-

Amersham

Pharmacia

Biotech

Uppsala

Sweden

-RRB-

according

to

the

manufacturer

's

instructions

Immunoglobulins

from

pre-immune

sera

were

also

purified

The

purity

of

all

IgGs

was

analyzed

by

SDS-PAGE

Protein

concentrations

were

determined

by

using

the

method

of

Bradford

-LRB-

-RRB-

Immunoblot

analysis

Whole

cell

antigens

of

strain

Afadé

were

separated

by

gradient

SDS-PAGE

-LRB-

Ausubel

et

al.

-RRB-

and

blotted

onto

a

nitrocellulose

membrane

with

a

pore

size

of

μm

-LRB-

Bio-Rad

Laboratories

AG

Reinach

Switzerland

-RRB-

The

membrane

was

blocked

with

skim

milk

powder

in

TBS

buffer

-LRB-

mM

Tris

HCl

mM

NaCl

pH

-RRB-

for

min

at

room

temperature

Strips

were

incubated

with

corresponding

IgGs

at

a

concentration

of

μg/ml

-LRB-

diluted

-RRB-

overnight

at

°C

and

washed

three

times

with

TBS

buffer

for

min

Serum

obtained

from

a

cow

that

was

infected

experimentally

with

M.

mycoides

subsp

mycoides

SC

strain

Afadé

-LRB-

Abdo

et

al.

diluted

-RRB-

or

previously

prepared

-LRB-

Abdo

et

al.

-RRB-

rabbit

hyperimmune

sera

directed

against

LppQ-N

and

LppQ-C

-LRB-

diluted

-RRB-

were

used

as

controls

The

strips

were

incubated

for

min

at

room

temperature

with

alkaline

phosphatase-labelled

goat

anti-rabbit

IgG

-LRB-

H

L

-RRB-

-LRB-

Kirkegaard

Perry

Laboratories

Gaithersburg

MD

USA

-RRB-

diluted

or

monoclonal

antibody

anti-bovine

IgG

-LRB-

Sigma

Buchs

Switzerland

-RRB-

diluted

in

skim

milk

buffer

The

strips

were

washed

three

times

with

TBS

buffer

The

colour

reaction

was

initiated

with

mg/ml

nitroblue

tetrazolium

-LRB-

NBT

-RRB-

-LRB-

Roche

Diagnostics

Rotkreuz

Switzerland

-RRB-

and

mg/ml

phosphate

-LRB-

BCIP

-RRB-

-LRB-

Roche

Diagnostics

-RRB-

in

alkaline

buffer

-LRB-

mM

mM

mM

pH

-RRB-

and

stopped

with

distilled

water

Preparation

of

M.

mycoides

subsp

mycoides

SC

for

immunogold

labelling

Coverslips

that

had

been

coated

with

gold

or

platinum

and

treated

with

poly-l-lysine

-LRB-

Sigma

-RRB-

to

promote

cell

adhesion

were

immersed

into

ml

of

fresh

standard

medium

in

multiwell

plates

Thereafter

μl

of

mycoplasma

culture

were

added

and

the

cultures

were

incubated

for

to

days

at

°C

until

stationary

phase

Coverslips

with

adhering

mycoplasmas

were

washed

three

times

with

PBS

buffer

-LRB-

mM

mM

mM

NaCl

pH

-RRB-

at

°C

and

fixed

with

paraformaldehyde

in

PBS

for

min

at

room

temperature

After

fixation

samples

were

washed

with

PBS

buffer

and

blocked

with

BSA

and

M

glycine

in

PBS

for

min

at

room

temperature

Thereafter

mycoplasma

cells

were

incubated

with

anti-LppQ-N

or

anti-LppQ-C

IgGs

diluted

-LRB-

μg/ml

-RRB-

in

PBS

containing

BSA

overnight

at

°C

After

another

washing

step

with

PBS

buffer

for

min

mycoplasmas

were

labelled

with

nm

colloidal

gold-conjugated

goat

anti-rabbit

antibody

-LRB-

British

Biocell

International

Cardiff

UK

-RRB-

diluted

in

PBS

for

min

at

room

temperature

Coverslips

were

then

washed

with

M

cacodylate

buffer

-LRB-

pH

-RRB-

and

processed

for

SEM

Scanning

electron

microscopy

Coverslips

were

incubated

in

osmium

tetraoxide

with

ruthenium

red

in

M

cacodylate

buffer

-LRB-

pH

-RRB-

for

min

washed

with

M

cacodylate

buffer

dehydrated

through

an

ascending

ethanol

series

and

dried

by

evaporation

of

hexamethyldisilazane

-LRB-

Sigma

-RRB-

as

described

previously

-LRB-

Stoffel

et

al.

Stoffel

et

al.

Stoffel

and

Friess

-RRB-

Finally

they

were

mounted

on

metal

stubs

with

a

conductive

adhesive

-LRB-

Provac

AG

Balzers

Liechtenstein

-RRB-

Samples

were

examined

without

further

metal

coating

Secondary

electron

micrographs

and

corresponding

backscattered

images

were

obtained

with

a

fully

digital

field

emission

scanning

electron

microscope

DSM

Gemini

-LRB-

Zeiss

Oberkochen

Germany

-RRB-

at

an

accelerating

voltage

of

kV

a

working

distance

of

mm

and

a

magnification

from

to

Control

experiments

included

omission

of

primary

antibody

as

well

as

the

use

of

a

rabbit

anti-calcitonin

antibody

-LRB-

Anawa

Zurich

Switzerland

-RRB-

and

of

IgGs

from

rabbit

pre-immune

sera

as

irrelevant

substitutes

for

the

anti-LppQ

primary

antibodies

Results

Structure

of

the

C-terminal

domain

of

lipoprotein

LppQ

Conventional

predictors

of

membrane

topology

available

online

did

not

envisage

the

C-terminal

domain

of

LppQ

to

be

membrane-associated

However

determination

of

repeats

in

the

LppQ

protein

sequence

revealed

that

its

C-terminal

domain

represented

by

the

last

amino

acids

consists

of

a

region

built

up

of

repeated

units

rich

in

hydrophobic

and

aromatic

residues

-LRB-

Figure

-RRB-

These

repeat

units

of

amino

acids

were

all

found

to

adopt

a

particular

secondary

structure

consisting

of

helices

of

approximately

amino

acids

flanked

upstream

and

downstream

by

amino

acids

forming

random

coils

-LRB-

not

shown

-RRB-

Monospecificity

of

IgG

preparations

On

immunoblots

containing

total

antigen

of

M.

mycoides

subsp

mycoides

SC

strain

Afadé

purified

IgG

preparations

directed

against

the

N-terminal

and

against

the

C-terminal

halves

of

LppQ

each

identified

a

single

protein

band

with

an

apparent

molecular

mass

of

kDa

which

corresponds

to

the

calculated

size

of

kDa

of

LppQ

-LRB-

Figure

-RRB-

Reference

rabbit

hyperimmune

sera

directed

against

LppQ-N

and

LppQ-C

collected

almost

a

decade

ago

gave

identical

results

Control

experiments

with

IgGs

purified

from

pre-immune

sera

did

not

detect

any

corresponding

antigen

-LRB-

Figure

-RRB-

Fig.

analysis

of

total

antigen

of

M.

mycoides

subsp

mycoides

SC

with

anti-LppQ-N

and

anti-LppQ-C

Approximately

μg

of

total

antigen

of

strain

Afadé

per

lane

was

separated

by

gradient

SDS-PAGE

transferred

onto

a

nitrocellulose

membrane

and

probed

with

the

serum

from

a

cow

infected

experimentally

with

strain

Afadé

-LRB-

Abdo

et

al.

-RRB-

or

with

IgGs

from

rabbit

pre-immune

sera

or

from

antisera

against

LppQ-N

and

LppQ-C

respectively

Reference

rabbit

hyperimmune

sera

directed

against

LppQ-N

and

LppQ-C

obtained

almost

a

decade

ago

-LRB-

Abdo

et

al.

-RRB-

were

also

used

The

arrowhead

indicates

the

position

of

LppQ

on

the

immunoblots

Morphology

of

the

mycoplasmas

Electron

microscopy

showed

cultivated

M.

mycoides

subsp

mycoides

SC

to

be

basically

spherical

in

shape

They

usually

occurred

as

single

cells

but

pairs

or

small

groups

of

up

to

five

organisms

were

also

noted

Cell

diameter

varied

between

and

nm

and

the

cell

surface

was

slightly

grainy

and

irregular

-LRB-

Fig.

C

E

-RRB-

Between

fully

grown

microorganisms

small

granular

debris

were

observed

Fig.

electron

micrographs

of

M.

mycoides

subsp

mycoides

SC

with

anti-LppQ-N

and

anti-LppQ-C

antibodies

Secondary

electron

microscope

images

-LRB-

A

C

E

-RRB-

reveal

the

spherical

shape

and

rough

cell

surface

of

the

mycoplasmas

Corresponding

backscattered

electron

micrographs

-LRB-

B

D

F

-RRB-

show

indirect

immunolabelling

with

nm

colloidal

gold

antibodies

directed

against

the

N-terminal

extracellular

domain

of

LppQ

-LRB-

B

-RRB-

In

contrast

LppQ

is

not

detected

with

anti-LppQ-C

antibody

-LRB-

D

-RRB-

Labelling

is

abolished

when

an

anti-calcitonin

antibody

is

used

as

an

irrelevant

primary

antibody

-LRB-

F

-RRB-

Immunogold

labelling

Labelling

of

mycoplasmas

on

coverslips

with

anti-LppQ-N

antibodies

yielded

a

SEM

signal

of

moderate

to

high

density

at

the

cell

surface

-LRB-

Fig.

-RRB-

Specific

labelling

of

granular

debris

was

also

present

In

contrast

labelling

with

anti-LppQ-C

antibodies

was

absent

in

most

of

the

cases

-LRB-

Fig.

-RRB-

and

appeared

to

be

insignificant

and

coincidental

in

few

cases

-LRB-

Table

-RRB-

as

also

observed

in

the

three

control

experiments

whereby

labelling

was

investigated

in

the

absence

of

primary

antibodies

and

when

pre-immune

serum

or

anti-calcitonin

antibodies

were

used

-LRB-

Fig.

-RRB-

Overall

there

were

significant

differences

between

the

number

of

particles

labelled

per

cell

by

using

anti-LppQ-N

antibodies

and

those

labelled

in

the

other

four

experiments

-LRB-

P

Table

-RRB-

Table

analysisStatistical

dataTreatmentAnti-LppQ-N

Anti-LppQ-C

Anti-calcitoninPre-immune

serumNo

primary

antibodySingle

cells

per

a

Standard

deviationbCalculated

by

the

analysis

of

variance

-LRB-

anova

-RRB-

method

single

factor

Differences

between

the

number

of

particles

labelled

per

cell

by

using

anti-LppQ-N

antibodies

and

those

labelled

in

the

other

four

experiments

were

analysed

Discussion

The

goal

of

the

present

study

was

to

discriminate

the

distribution

of

extracellular

vs.

integral

membrane

domains

of

lipoprotein

LppQ

in

M.

mycoides

subsp

mycoides

SC

at

the

ultrastructural

level

Amino

acid

sequence

analysis

for

hydrophobicity

by

the

method

of

Hopp

and

Woods

-LRB-

-RRB-

revealed

the

N-terminal

portion

of

LppQ

to

be

particularly

hydrophilic

-LRB-

Abdo

et

al.

-RRB-

Thus

the

lipidated

N-terminal

domain

was

predicted

to

be

exposed

at

the

extracellular

surface

of

the

plasma

membrane

-LRB-

Abdo

et

al.

-RRB-

On

the

other

hand

the

C-terminal

portion

consists

of

a

region

built

up

of

hydrophobic

repeated

units

and

not

nine

as

reported

previously

-LRB-

Abdo

et

al.

-RRB-

The

repeat

units

were

found

to

consist

each

of

approximately

amino

acids

forming

helices

delimited

on

both

sides

with

random

coils

i.e.

conformations

without

a

regular

secondary

structure

and

were

not

predicted

to

be

membrane-associated

by

bioinformatic

analysis

Since

transmembrane

helices

are

mostly

composed

of

hydrophobic

amino

acid

and

the

degree

of

hydrophobicity

of

the

C-terminal

domain

of

LppQ

is

clearly

significant

the

C-terminal

portion

was

expected

to

be

an

integral

membrane

domain

-LRB-

Fig.

-RRB-

It

has

to

be

noted

at

this

point

that

lipoproteins

generally

do

not

possess

additional

membrane-spanning

domains

-LRB-

MSD

-RRB-

beyond

their

N-terminal

lipid

anchors

However

there

are

relevant

examples

from

Gram-positive

bacteria

which

contradict

this

general

statement

about

lipoproteins

and

substantiate

instead

the

occurrence

of

such

additional

MSDs

These

examples

include

lipoproteins

CtaC

and

QoxA

of

Bacillus

subtilis

-LRB-

Bengtsson

et

al.

Antelmann

et

al.

-RRB-

as

well

as

the

proven

lipoprotein

of

Mycoplasma

pneumoniae

ATPase

-LRB-

Pyrowolakis

et

al.

-RRB-

High

resolution

scanning

electron

microscopy

in

combination

with

immunogold

labelling

revealed

extracellular

epitopes

with

antibodies

against

LppQ-N

but

no

signal

above

the

background

of

the

three

negative

controls

was

detected

with

the

antibody

against

LppQ-C

These

results

substantiate

the

predicted

localization

of

the

two

domains

as

they

unambiguously

corroborate

the

accessibility

of

the

N-terminal

domain

at

the

extracellular

side

of

the

plasma

membrane

The

failure

to

immunolabel

the

C-terminal

epitopes

is

an

indication

that

they

may

be

imbedded

in

the

membrane

However

one

can

not

rule

out

the

possibility

that

such

epitopes

-LRB-

i

-RRB-

are

buried

in

the

centre

of

a

globular

protein

-LRB-

ii

-RRB-

are

masked

by

other

surface

components

or

-LRB-

iii

-RRB-

are

not

recognized

in

their

native

forms

by

rabbit

antibodies

produced

by

inoculation

of

the

recombinant

peptide

Beyond

providing

ultrastructural

evidence

for

the

location

of

the

hydrophilic

and

hydrophobic

domains

of

LppQ

these

results

also

demonstrate

that

immunogold

labelling

in

scanning

electron

microscopy

allows

for

subtle

topographical

discrimination

and

that

selective

visualization

of

peptides

being

exposed

at

the

outer

surface

of

microorganisms

can

be

achieved

with

high

accuracy

and

reliability

The

domain

analysis

of

LppQ

which

demonstrates

the

outstanding

surface

localization

of

the

strongly

hydrophilic

N-terminal

part

of

the

lipoprotein

may

explain

the

strong

humoural

response

elicited

by

this

lipoprotein

during

infection

of

cattle

by

M.

mycoides

subsp

mycoides

SC

-LRB-

Abdo

et

al.

-RRB-

It

has

to

be

demonstrated

yet

whether

LppQ

is

also

able

to

induce

any

cell-mediated

immune

response

as

it

is

more

often

the

case

for

cytoplasmic

proteins

Absence

of

a

cell-mediated

immune

response

would

explain

the

lack

of

protection

in

animals

immunized

with

purified

recombinant

LppQ

In

this

respect

it

should

be

noted

that

immunization

of

cattle

with

the

purified

recombinant

N-terminal

domain

of

LppQ

elicited

inflammatory

processes

of

M.

mycoides

subsp

mycoides

SC

and

exacerbated

the

clinical

signs

of

CBPP

after

experimental

infection

compared

to

non-immunized

animals

-LRB-

Nicholas

et

al.

-RRB-

Conclusions

Direct

visualization

of

the

N-terminal

part

of

LppQ

at

the

outer

surface

of

M.

mycoides

subsp

mycoides

SC

as

demonstrated

herein

provides

evidence

for

the

contention

that

LppQ

is

located

to

the

extracellular

side

of

the

plasma

membrane

of

mycoplasmas

and

that

it

is

easily

accessible

by

the

humoural

immune

system

of

infected

animals

upon

contact

of

host

cells

with

M.

mycoides

subsp

mycoides

SC

B I O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

contagious bovine pleuropneumonia (cbpp)


RGD-avidin

biotin

pretargeting

to

αvβ3

integrin

enhances

the

proapoptotic

activity

of

TNFα

related

apoptosis

inducing

ligand

-LRB-

TRAIL

-RRB-

Recombinant

TNF-related

apoptosis-inducing

ligand

-LRB-

TRAIL

-RRB-

is

considered

a

powerful

and

selective

inducer

of

tumor

cell

death

We

hypothesize

that

TRAIL

's

potential

as

anticancer

agent

can

be

enhanced

further

by

promoting

its

accumulation

in

tumor

tissue

For

this

purpose

we

developed

TRAIL

complexes

that

bind

to

angiogenic

endothelial

cells

We

employed

an

avidin

biotin

pretargeting

approach

in

which

biotinylated

TRAIL

interacted

with

RGD-equipped

avidin

The

assembled

complexes

killed

tumor

cells

-LRB-

Jurkat

T

cells

-RRB-

via

apoptosis

induction

Furthermore

we

demonstrated

that

the

association

of

the

RGD-avidin-TRAIL

complex

onto

endothelial

cells

enhanced

the

tumor

cell

killing

activity

Endothelial

cells

were

not

killed

by

TRAIL

nor

its

derived

complexes

Our

approach

can

facilitate

the

enrichment

of

TRAIL

onto

angiogenic

blood

vessels

which

may

enhance

intratumoral

accumulation

Furthermore

it

offers

a

versatile

technology

for

the

complexation

of

targeting

ligands

with

therapeutic

recombinant

proteins

and

by

this

a

novel

way

to

enhance

their

specificity

and

activity

Introduction

TNF

ligands

have

strong

potential

as

anti-tumor

agents

acting

in

different

ways

on

either

tumor

cells

or

tumor

vasculature

or

by

promoting

the

induction

of

tumor-directed

immune

responses

-LSB-

-RSB-

The

broad

clinical

use

of

TNF

ligands

is

prohibited

however

due

to

lack

of

tumor

selectivity

causing

various

side

effects

A

recently

identified

TNF

family

member

is

TNF

related

apoptosis-inducing

ligand

-LRB-

TRAIL

-RRB-

-LSB-

-RSB-

Many

tumor

types

are

sensitive

to

TRAIL

or

become

sensitive

after

treatment

with

chemotherapeutic

drugs

-LSB-

-RSB-

Soluble

TRAIL

variants

showed

no

cytotoxic

effects

on

most

primary

human

cells

and

were

well

tolerated

in

mice

and

chimpanzees

-LSB-

-RSB-

However

the

selectivity

of

TRAIL

for

tumor

cells

over

normal

cells

is

lost

when

TRAIL

forms

multimeric

complexes

For

example

it

has

been

observed

that

cross-linked

TRAIL

induces

apoptosis

more

efficiently

than

non-aggregated

trimeric

TRAIL

-LSB-

-RSB-

but

such

cross-linked

TRAIL

also

caused

cell

death

in

normal

hepatic

cells

-LSB-

-RSB-

It

seems

therefore

attractive

to

design

clustered

TRAIL

variants

with

enhanced

specificity

for

tumor

tissue

The

increased

tissue

retention

of

cellular

targeted

TRAIL

variants

may

counterbalance

or

prevent

side

effects

elsewhere

in

the

body

In

the

present

paper

we

investigated

the

development

of

TRAIL

complexes

equipped

with

targeting

ligands

directed

to

the

tumor

vasculature

Enhanced

retention

of

TRAIL

complexes

at

the

tumor

vasculature

will

enhance

their

accumulation

in

the

tumor

tissue

thereby

favoring

the

desired

therapeutic

activity

and

reducing

potential

toxicity

Several

recent

papers

described

the

development

of

recombinant

TNF

family

members

with

enhanced

target

specificity

One

approach

is

to

modify

the

intrinsic

specificity

of

TRAIL

for

its

target

Death-receptor

specific

by

substituting

amino-acids

located

in

the

receptor

binding

interface

of

TRAIL

-LSB-

-RSB-

Development

of

chimaeras

is

another

successful

approach

Linkage

of

TRAIL

to

scFv

antibody

fragments

yielded

constructs

which

bind

to

additional

surface-exposed

receptors

on

tumor

cell

types

-LSB-

-RSB-

Similarly

other

TNF

family

members

like

Fas-ligand

and

TNFα

-LRB-

have

been

conjugated

to

tumor-cell

directed

targeting

ligands

yielding

chimeric

proteins

with

full

or

even

enhanced

therapeutic

activity

-LSB-

-RSB-

Although

the

recombinant

approach

has

been

applied

successfully

for

the

production

of

chimeric

proteins

low

production

yields

may

hamper

the

feasibility

of

such

a

strategy

Especially

when

multiple

targeting

ligands

are

introduced

into

a

protein

subunit

which

is

preferred

for

high

avidity

binding

aberrant

folding

of

chimeric

proteins

can

occur

In

the

current

study

we

therefore

pursued

a

chemical

approach

in

which

TRAIL

was

biotinylated

and

complexed

to

avidin

equipped

with

multiple

targeting

ligands

Avidin

biotin

complexation

is

a

widely

accepted

approach

in

diagnostic

applications

that

employ

immunodetection

steps

and

is

also

considered

an

attractive

strategy

for

the

preparation

of

tumor

targeted

complexes

especially

in

so-called

pretargeting

approaches

in

which

the

complexes

are

assembled

in

vivo

at

the

cell

surface

of

tumor

cells

Commonly

such

strategies

employ

biotinylated

tumor-specific

antibodies

and

a

therapeutic

or

diagnostic

cargo

coupled

to

avidin

-LSB-

-RSB-

The

approach

presented

in

this

paper

differs

to

the

above-discussed

strategies

in

several

aspects

First

we

now

equipped

TRAIL

with

targeting

ligands

directed

to

angiogenic

endothelium

rather

than

to

tumor

cells

allowing

an

enhanced

binding

to

the

tumor

blood

vessels

The

vascular

wall

is

in

direct

contact

with

the

systemic

circulation

which

makes

it

an

easily

attainable

target

for

therapeutic

proteins

Furthermore

intratumoral

pressure

which

hampers

the

penetration

of

macromolecules

into

tumor

tissue

will

not

affect

the

homing

to

of

RGD-equipped

complexes

to

the

angiogenic

endothelium

Second

we

have

prepared

a

different

type

of

targeted

avidin

that

can

interact

directly

via

covalently

attached

RGD-groups

with

tumor

endothelial

cells

Thus

the

developed

avidin

biotin-TRAIL

complexes

will

home

to

angiogenic

endothelium

without

the

need

of

complexation

with

a

biotinylated

antibody

thereby

leaving

its

biotin-binding

sites

available

for

complexation

with

the

biotinylated

TRAIL

The

functionalization

of

avidin

with

RGD-homing

ligands

was

carried

out

using

a

cyclic

RGD-peptide

with

high

affinity

and

specificity

for

αvβ3

integrin

on

angiogenic

endothelium

-LSB-

-RSB-

We

furthermore

applied

a

polyethylenglycol

-LRB-

PEG

-RRB-

linker

for

coupling

of

the

RGD-ligand

which

brings

several

advantages

such

as

a

reduced

non-specific

binding

of

the

avidin

and

reduced

immunogenicity

-LSB-

-RSB-

The

now

developed

RGDPEG-avidin

conjugates

contain

the

RGD

homing

ligand

attached

to

the

distal

end

of

the

PEG

groups

In

order

to

minimize

loss

of

functional

TRAIL

activity

we

have

compared

two

different

biotinylation

strategies

that

react

at

different

functional

groups

in

the

protein

We

used

a

conventional

Biotin-NHS

reagent

that

binds

to

primary

amino

groups

in

the

protein

and

we

employed

the

Biotin-ULS

-LRB-

ULS

universal

linkage

system

-RRB-

reagent

which

reacts

at

methionine

residues

Figure

shows

a

schematic

representation

of

the

developed

complexes

and

their

interaction

with

target

cells

The

products

were

evaluated

for

their

association

with

αvβ3

integrin

expressing

endothelial

cells

and

for

their

antitumor

activity

in

both

endothelium

and

cultured

tumor

cells

Our

results

demonstrate

that

recombinant

TRAIL

can

be

complexed

with

endothelial

specific

targeting

ligands

in

such

a

way

that

it

maintains

its

extensive

cell

killing

activity

either

in

solution

or

when

displayed

on

the

surface

of

endothelial

cells

Fig.

representation

of

RGD-avidin

TRAIL

complexes

Soluble

recombinant

human

TRAIL

was

modified

with

biotin

groups

via

two

different

reagents

that

either

react

at

lysine

or

methionine

residues

Biotinylated

TRAIL

was

complexed

to

an

avidin-based

targeting

device

equipped

with

approximately

RGD-peptide

ligands

per

avidin

molecule

A

hydrophilic

PEG-linker

was

used

to

append

the

RGD-peptides

to

avidin

The

assembled

complexes

can

bind

to

tumor

blood

vessels

via

RGD

interactions

while

apoptosis

can

be

induced

via

TRAIL/DR

ligation

Materials

and

methods

Preparation

and

characterization

of

RGDPEG-avidin

Avidin

was

modified

with

heterobifunctional

PEG

groups

that

can

react

with

primary

amino

groups

in

avidin

after

which

RGD-peptide

groups

can

be

conjugated

to

their

distal

ends

The

hydrophilic

PEG

groups

of

kDa

will

furthermore

reduce

non-specific

protein

interactions

Briefly

avidin

-LRB-

mg

nmol

Sigma

-RRB-

was

dissolved

in

PBS

at

mg/ml

and

treated

with

a

molar

excess

of

the

heterobifunctional

vinylsulfone-polyethylenglycol-N-hydroxysuccinimide

ester

-LRB-

mg

nmol

VNS-PEG-NHS

Nektar

Alabama

USA

-RRB-

which

had

been

dissolved

in

water

at

a

concentration

of

mg/ml

The

PEG

linker

was

added

drop

wise

The

reaction

mixture

was

protected

from

light

with

tin

foil

and

incubated

for

h

at

room

temperature

on

a

spiramix

rollerbank

Meanwhile

the

RGD

peptide

c

-LRB-

RGDf

-LRB-

ε-S-acetylthioacetyl

-RRB-

K

-RRB-

-LRB-

Ansynth

Service

Roosendaal

The

Netherlands

-RRB-

was

dissolved

at

mg/ml

in

a

acetonitrile/water

mixture

The

peptide

was

added

drop

wise

to

the

reaction

mixture

at

a

molar

ratio

of

after

which

freshly

prepared

hydroxylamine

was

added

to

a

final

concentration

of

mM

The

reaction

was

carried

out

overnight

at

room

temperature

while

protected

from

light

Remaining

VNS

groups

were

quenched

by

addition

of

cysteine

-LRB-

molar

excess

over

the

amount

of

avidin

-RRB-

after

which

the

product

was

purified

by

size

exclusion

chromatography

on

a

Highload

superdex

column

-LRB-

Amersham

Biosciences

-RRB-

pre-equilibrated

in

PBS

The

final

product

RGDPEG-avidin

was

stored

in

PBS

at

°C

RGDPEG-avidin

conjugates

were

characterized

for

protein

content

by

BCA

protein

assay

-LRB-

Pierce

-RRB-

Coupling

of

RGDPEG

groups

to

avidin

was

demonstrated

by

SDS-PAGE

performed

on

a

mini-Protean

II

system

-LRB-

Bio-Rad

-RRB-

using

Ready

Gels

-LRB-

Bio-Rad

The

Netherlands

-RRB-

Duplicate

gels

were

either

stained

for

protein

-LRB-

Coomassie

Brilliant

Blue

staining

-RRB-

or

blotted

on

a

PVDF

membrane

-LRB-

Roche

Mannheim

Germany

-RRB-

The

membrane

was

blocked

with

BSA

and

subsequently

incubated

with

an

in-house

raised

anti-RGD

polyclonal

rabbit

antiserum

specific

for

the

conjugated

RGD-peptide

-LSB-

-RSB-

Blots

were

further

developed

with

goat-anti-rabbit

peroxidase

-LRB-

GARPO

-RRB-

DAB

detection

The

grade

of

RGD-pegylation

was

furthermore

determined

by

MALDI-TOF

analysis

as

described

previously

-LSB-

-RSB-

Expression

and

purification

of

recombinant

human

His-tagged

TRAIL

A

cDNA

corresponding

to

human

soluble

TRAIL

-LRB-

amino

acids

-RRB-

was

cloned

downstream

of

the

hexahistidine

-LRB-

His

-RRB-

tag

sequence

in

-LRB-

Novagen

-RRB-

using

NdeI

and

BamHI

restriction

sites

Escherichia

coli

-LRB-

-RRB-

-LRB-

Invitrogen

-RRB-

was

subsequently

transformed

with

the

His-TRAIL

construct

The

transformed

host

was

grown

in

shake

flasks

at

four

liter

scale

as

described

previously

-LSB-

-RSB-

The

culture

was

centrifuged

at

rpm

and

the

supernatant

was

discarded

The

isolated

pellet

was

resuspended

in

three

volumes

of

extraction

buffer

-LRB-

mM

phosphate

buffer

pH

containing

mM

NaCl

-LRB-

v/v

-RRB-

glycerol

and

mM

β-mercaptoethanol

-RRB-

Cells

were

disrupted

using

sonication

and

extracts

were

clarified

by

centrifugation

at

g.

Subsequently

the

supernatant

was

loaded

on

a

cobalt-charged

IMAC

TALON

column

-LRB-

Clontech

-RRB-

and

His-TRAIL

was

eluted

with

a

step

gradient

of

extraction

buffer

containing

M

imidazole

Trimeric

His-TRAIL

was

purified

by

size-exclusion

chromatography

on

a

Hiload

Superdex

column

-LRB-

Amersham

Biosciences

-RRB-

to

remove

impurities

The

final

product

was

flash

frozen

and

stored

at

°C

in

storage

buffer

-LRB-

phosphate

buffer

pH

containing

mM

NaCl

mM

DTT

and

μM

-RRB-

Purified

protein

was

pure

as

determined

by

colloidal

Coomassie

Brilliant

Blue-stained

SDS

PAGE

gel

Preparation

and

characterization

of

bio-NHS-TRAIL

and

bio-ULS-TRAIL

We

studied

two

different

linkage

strategies

to

introduce

biotin

into

His-TRAIL

by

applying

either

a

conventional

biotinylation

reagent

that

reacts

with

primary

amino

groups

in

the

protein

-LRB-

biotin-NHS

-RRB-

or

one

that

reacts

to

methionine

residues

-LRB-

biotin-ULS

ULS

universal

linkage

system

-RRB-

Such

an

alternative

strategy

may

prove

less

harmful

to

the

therapeutic

activity

of

TRAIL

depending

on

the

extent

to

which

side

chain

residues

in

the

active

domains

are

modified

Biotin-ULS

was

provided

by

Kreatech

Biotechnology

-LRB-

Amsterdam

The

Netherlands

-RRB-

All

syntheses

were

performed

in

a

molar

ratio

of

biotinylation

reagent

over

TRAIL

His-TRAIL

in

storage

buffer

was

extensively

dialyzed

against

PBS

containing

glycerol

at

°C

using

Slide-A-Lyzer

dialysis

cassettes

-LRB-

MWCO

Pierce

-RRB-

to

remove

DTT

and

other

components

of

the

storage

buffer

that

can

interfere

in

the

reactions

Typically

the

purified

His-TRAIL

-LRB-

ug

nmol

-RRB-

at

mg/ml

was

mixed

either

with

Sulfo-NHS

biotin

-LRB-

Pierce

-RRB-

-LRB-

mg/ml

in

DMF

-RRB-

or

with

biotin-ULS

-LRB-

mg/ml

in

mM

NaCl

-RRB-

after

which

the

mixtures

were

incubated

for

h

at

°C

Unreacted

biotinylation

reagent

was

removed

by

dialysis

against

PBS

at

°C

The

products

were

sterilized

by

filtration

via

disposable

μm

filters

and

stored

at

°C

Biotin-TRAIL

conjugates

were

characterized

for

protein

content

using

BCA

protein

assay

-LRB-

Pierce

-RRB-

The

relative

number

of

coupled

biotin

molecules

was

determined

by

anti-biotin

ELISA

Wells

were

coated

with

serial

dilutions

of

the

biotinylated

proteins

for

h

at

room

temperature

washed

with

PBS

containing

and

incubated

for

h

at

room

temperature

with

streptavidin-peroxidase

complex

-LRB-

Dako

in

PBS

-RRB-

followed

by

a

standard

incubation

with

OPD

The

protein

concentration

at

which

of

the

maximum

absorbance

was

measured

-LRB-

-RRB-

was

calculated

by

nonlinear

regression

-LRB-

Graphpad

Prism

-RRB-

and

used

to

calculate

relative

biotin

protein

ratios

in

each

conjugate

The

grade

of

biotinylation

was

furthermore

assayed

by

MALDI-TOF

analysis

as

described

above

for

RGDPEG-avidin

Cells

Jurkat

T

cells

were

cultured

in

RPMI

culture

medium

-LRB-

containing

mM

HEPES

and

l-glutamine

-RRB-

supplemented

with

heat-inactivated

FCS

-LRB-

Bodinco

Alkmaar

The

Netherlands

-RRB-

mM

sodium

pyruvate

mM

l-glutamine

mM

and

mg/ml

gentamycin

sulfate

obtained

from

BioWhittaker

and

μg/ml

fungizone

-LRB-

Bristol-Meyers

Woerden

The

Netherlands

-RRB-

HUVEC

were

isolated

by

the

UMCG

Endothelial

Cell

Facility

by

previously

described

methods

-LSB-

-RSB-

Primary

isolates

were

cultured

in

gelatin-coated

tissue

culture

flasks

-LRB-

Corning

Costar

The

Netherlands

-RRB-

at

°C

under

air

The

culture

medium

hereafter

referred

to

as

EC

medium

consisted

of

RPMI

-LRB-

BioWhittaker

Verviers

Belgium

-RRB-

supplemented

with

heat

inactivated

fetal

calf

serum

-LRB-

Integro

Zaandam

The

Netherlands

-RRB-

mM

l-glutamine

-LRB-

Invitrogen

Breda

The

Netherlands

-RRB-

U/ml

heparin

-LRB-

Leo

Pharmaceutical

Products

Weesp

The

Netherlands

-RRB-

U/ml

penicillin

-LRB-

Yamanouchi

Pharma

Leiderdorp

The

Netherlands

μg/ml

streptomycin

-LRB-

Radiumfarma-Fisiopharma

Italy

-RRB-

and

μg/ml

endothelial

cell

growth

factor

supplement

extracted

from

bovine

brain

After

attaining

confluence

cells

were

detached

from

the

surface

by

trypsin

EDTA

-LRB-

mg/ml

in

PBS

-RRB-

treatment

and

split

in

ratio

Cells

were

used

up

to

passage

four

Binding

of

RGDPEG-avidin

to

HUVEC

Binding

affinity

of

RGDPEG-avidin

to

expressed

on

the

surface

of

HUVEC

was

determined

by

competitive

displacement

of

Echistatin

which

is

a

known

ligand

for

αvβ3

-LSB-

-RSB-

Confluent

monolayers

of

HUVEC

in

plates

-LRB-

Costar

-RRB-

were

incubated

with

cpm

either

in

the

presence

of

excess

of

RGDPEG-avidin

free

RGD-peptide

or

avidin

as

competitors

-LRB-

competitors

all

in

a

concentration

of

μM

-RRB-

or

a

serial

dilution

of

RGDPEG-avidin

and

free

RGD-peptide

Competition

experiments

were

carried

out

with

divalent

cation

containing

binding

buffer

-LRB-

mM

Tris-HCl

pH

mM

NaCl

mM

mM

mM

and

BSA

-RRB-

at

°C

for

h.

Cells

were

washed

three

times

with

binding

buffer

and

lysed

with

M

NaOH

Radioactivity

was

counted

in

a

Packard

RIASTAR

multiwell

gamma

counter

-LRB-

GMI

Minnesota

USA

-RRB-

Data

were

analyzed

by

non-linear

regression

using

the

GraphPad

Prism

program

Complexation

of

biotinylated

TRAIL

with

RGDPEG-avidin

We

studied

the

complexation

of

biotinylated

TRAIL

with

RGDPEG-avidin

in

an

ELISA-like

experimental

setup

Wells

were

coated

with

ng

of

avidin

or

RGDPEG-avidin

overnight

at

room

temperature

blocked

with

BSA

-LRB-

in

PBS

-RRB-

and

incubated

with

ng

of

biotinylated

TRAIL

for

h

at

room

temperature

After

the

wells

were

washed

with

the

amount

of

TRAIL

associated

with

the

avidin

was

detected

by

anti-TRAIL

immunodetection

-LRB-

anti-TRAIL

antibody

Alexis

Breda

The

Netherlands

-RRB-

for

h

followed

by

standard

detection

with

GARPO/OPD

Apoptotic

activity

of

biotinylated

TRAIL

The

apoptosis

inducing

activity

of

the

biotinylated

products

was

evaluated

on

Jurkat

T

cells

and

on

endothelial

cells

using

a

cell

viability

assay

and

a

caspase

activity

assay

Experiments

were

conducted

with

the

single

cell

types

Jurkat

tumor

cells

or

HUVEC

or

with

a

combination

of

tumor

and

endothelial

cells

Assessment

of

TRAIL

activity

by

MTS

cell

viability

assay

Briefly

cells

were

seeded

in

flat-bottom

plates

at

a

density

of

-LRB-

Jurkat

-RRB-

or

-LRB-

HUVEC

-RRB-

in

μl

medium

supplemented

with

the

indicated

compounds

Concentrations

of

TRAIL

bio-NHS-TRAIL

and

Bio-ULS-TRAIL

ranging

from

μg/ml

to

ng/ml

were

tested

on

Jurkat

Only

one

concentration

-LRB-

ng/ml

-RRB-

was

tested

on

HUVEC

The

MTS

cell

viability

assay

-LRB-

Promega

-RRB-

was

performed

after

h

of

incubation

according

to

the

manufacturer

's

recommendations

Experiments

were

performed

in

triplicate

Assessment

of

TRAIL

activity

by

caspase

assay

We

investigated

the

induction

of

apoptosis

in

Jurkat

T

cells

using

a

caspase

activity

assay

-LRB-

Promega

-RRB-

employing

luminescence

detection

Different

experimental

settings

were

evaluated

either

reflecting

direct

incubation

of

the

products

with

tumor

cells

or

settings

in

which

biotinylated

TRAIL

was

complexed

to

RGDPEG-avidin

and/or

anchored

onto

endothelial

cells

First

we

determined

pro-apoptotic

effects

of

non-complexed

TRAIL

About

Jurkat

cells

were

incubated

with

biotinylated

TRAIL

at

a

concentration

of

ng/ml

Experiments

were

carried

in

plates

in

μl

medium

supplemented

with

the

indicated

compounds

for

h

after

which

μl

of

caspase

substrate

was

added

to

the

cells

followed

by

luminescence

detection

with

a

microplate

luminometer

according

to

the

provider

's

instructions

Next

we

determined

proapoptotic

effects

of

RGDPEG-avidin

TRAIL

complexes

in

Jurkat

cells

These

experiments

were

performed

in

the

presence

of

endothelial

cells

to

allow

adherence

of

the

RGD-equipped

complexes

to

αvβ3

integrin

expressing

cell

layers

HUVEC

were

preincubated

with

μg/ml

RGDPEG-avidin

for

h

at

room

temperature

after

which

the

excess

of

non-cell

associated

RGDPEG-avidin

was

removed

by

washing

with

PBS

Control

experiments

included

RGDPEG-avidin

treated

HUVEC

that

were

not

washed

and

cells

to

which

no

RGDPEG-avidin

was

added

All

wells

were

supplemented

with

biotinylated

TRAIL

-LRB-

ng/ml

in

culture

medium

-RRB-

after

which

Jurkat

cells

-LRB-

cells

in

μl

of

medium

-RRB-

were

added

Cells

were

incubated

for

h

at

°C

after

which

caspase

activity

was

assayed

as

described

above

Experiments

were

conducted

in

triplicate

Statistical

analysis

Statistical

analysis

was

performed

using

Student

's

two-tailed

t-test

assuming

equal

variances

Difference

were

considered

to

be

significant

when

P

unless

otherwise

stated

Results

Preparation

and

characterization

of

RGDPEG-avidin

We

modified

avidin

with

PEG-linked

RGD-peptides

to

obtain

an

avidin

molecule

which

could

bind

to

angiogenic

endothelial

cells

Successful

synthesis

of

RGDPEG-avidin

was

proven

by

several

analytical

techniques

SDS-PAGE

analysis

-LRB-

Fig.

-RRB-

showed

a

single

band

for

avidin

of

approximately

kDa

corresponding

to

the

weight

of

one

of

its

four

subunits

Different

protein

bands

were

observed

for

RGDPEG-avidin

with

molecular

weights

corresponding

to

the

unmodified

subunit

and

to

subunits

modified

with

one

or

multiple

RGDPEG

moieties

Second

Western

blot

of

the

product

showed

positive

staining

of

these

bands

by

an

anti-RGD-peptide

antiserum

Due

to

the

size

heterogeneity

of

the

PEG

polymer

the

RGD-band

stained

in

a

broad

streak

rather

than

a

discrete

band

-LRB-

Fig.

-RRB-

Of

note

the

tetrameric

band

of

avidin

was

not

observed

due

to

the

denaturating

conditions

of

SDS-PAGE

Third

we

analyzed

the

product

by

MALDI-TOF

mass

spectrometry

-LRB-

Fig.

-RRB-

which

showed

peaks

corresponding

to

avidin

subunits

modified

with

RGDPEG

to

a

different

degree

From

the

average

molecular

weights

-LRB-

Mn

-RRB-

we

calculated

a

relative

derivatization

ratio

of

RGDPEG

groups

per

avidin

subunit

or

a

total

of

RGDPEG

groups

per

tetrameric

avidin

Fig.

of

RGDPEG-avidin

Panel

A

and

B

SDS-PAGE

separation

followed

by

CBB

protein

staining

-LRB-

a

-RRB-

or

Western

blotting

and

anti-RGD

detection

-LRB-

b

-RRB-

lane

MW

marker

lane

avidin

lane

RGDPEG-avidin

Unmodified

avidin

was

separated

into

its

kDa

subunits

while

RGDPEG-avidin

was

disassembled

into

different

bands

corresponding

to

subunits

with

or

attached

RGDPEG

groups

The

intrinsic

heterogeneity

of

PEG

polymers

can

be

observed

in

the

broadly

stained

band

of

the

RGDPEG-avidin

Western

blot

Panel

C

MALDI-TOF

analysis

of

RGDPEG-avidin

Upper

line

RGDPEG-avidin

bottom

line

avidin

During

ionization

the

tetrameric

proteins

have

disassembled

into

subunits

yielding

a

single

peak

for

avidin

and

multiple

peaks

for

RGDPEG-avidin

corresponding

to

modification

degree

Next

we

investigated

the

interaction

of

RGDPEG-avidin

with

HUVEC

which

express

when

being

at

a

subconfluent

state

To

determine

binding

affinity

of

RGDPEG-avidin

for

we

studied

its

capability

to

compete

with

the

ligand

While

RGDPEG-avidin

completely

displaced

from

the

cells

similar

to

the

free

RGD-peptide

avidin

did

not

affect

binding

of

the

radiolabeled

peptide

-LRB-

Fig.

-RRB-

The

sigmoid

displacement

curve

with

RGDPEG-avidin

-LRB-

Fig.

-RRB-

demonstrated

a

increase

in

affinity

-LRB-

nM

-RRB-

as

compared

to

the

single

RGD-peptide

-LRB-

nM

-RRB-

Fig.

of

RGDPEG-avidin

with

endothelial

cells

Subconfluent

HUVEC

were

incubated

with

radiolabeled

echistatin

which

is

a

known

ligand

for

αvβ3

integrin

and

RGDPEG-avidin

free

RGDpeptide

or

unmodified

avidin

Panel

A

Binding

specificity

Echistatin

was

displaced

by

excess

of

RGDPEG-avidin

or

free

RGD-peptide

-LRB-

both

at

μM

-RRB-

but

not

by

excess

of

avidin

P

Panel

B

Affinity

of

binding

Echistatin

binding

was

displaced

by

different

concentrations

of

the

added

competing

ligands

showing

the

increased

affinity

of

RGPEG-avidin

over

the

single

RGDpeptide

ligand

Preparation

and

characterization

of

biotinylated

TRAIL

Since

the

modification

of

side-chain

residues

may

diminish

the

activity

of

a

therapeutic

protein

we

studied

two

different

biotinylation

strategies

that

target

chemical

modifications

at

different

amino

acid

residues

i.e.

at

lysine

groups

-LRB-

biotin-NHS

-RRB-

or

methionine

residues

-LRB-

biotin-ULS

-RRB-

Such

different

labeling

positions

at

the

surface

of

the

protein

may

correlate

to

differences

in

pharmacological

activity

of

the

biotinylated

proteins

Anti-biotin

ELISA

-LRB-

Fig.

-RRB-

demonstrated

a

higher

biotin/TRAIL

modification

of

biotin-NHS

over

biotin-ULS

which

is

in

line

with

the

lower

abundance

of

methionine

residues

as

compared

to

the

number

of

lysine

residues

We

tried

to

confirm

these

data

by

analysis

of

the

biotinylation

extent

by

MALDI-TOF

mass

spectrometry

which

provided

reliable

data

on

the

average

number

of

incorporated

RGDPEG

units

in

avidin

Although

this

approach

succeeded

for

biotin-NHS-TRAIL

-LRB-

Fig.

-RRB-

demonstrating

the

incorporation

of

about

biotin

group

per

subunit

of

TRAIL

MALDI-TOF

analysis

failed

to

detect

biotin-ULS

modification

of

TRAIL

Most

likely

the

mass

ionization

step

induced

the

dissociation

of

biotin-ULS

from

the

protein

From

the

collective

results

we

concluded

that

both

strategies

had

yielded

biotinylated

TRAIL

with

approximately

biotin

groups

per

protein

-LRB-

summarized

in

Table

-RRB-

Fig.

of

biotinylated

TRAILs

Panel

A

Anti-biotin

ELISA

with

streptavidin-HRP

Signals

were

corrected

for

background

and

expressed

relative

to

the

maximum

detected

intensity

Panel

B

MALDI-TOF

analysis

of

TRAIL

and

biotin-NHS-TRAILTable

of

biotinylated

TRAIL

variantsRelative

biotin

content

-LRB-

Biotin/TRAIL

-RRB-

aActivity

in

cell

viability

assay

-LRB-

in

ng/ml

-RRB-

bActivity

in

caspase

assayc

-LRB-

-RRB-

TRAIL

aExpressed

as

mole

biotin

bound

per

mole

of

trimeric

TRAIL

Relative

biotin

content

was

determined

by

MALDI-TOF

analysis

-LRB-

bio-NHS-TRAIL

-RRB-

or

ELISA

-LRB-

BIO-ULS-TRAIL

-RRB-

bAs

determined

after

h

of

incubation

with

Jurkat

cellscAs

determined

after

h

of

incubation

at

ng/ml

with

Jurkat

cells

We

studied

complexation

of

biotinylated

TRAIL

with

RGDPEG-avidin

that

had

been

coated

in

plates

Highest

binding

was

observed

for

Bio-NHS-TRAIL

probably

due

to

its

higher

biotin

content

while

bio-ULS-TRAIL

showed

approximately

less

binding

-LRB-

Fig.

-RRB-

Unmodified

His-TRAIL

showed

non-specific

binding

to

RGDPEG-avidin

as

well

despite

the

absence

of

biotin

groups

in

this

product

Non-specific

binding

of

TRAIL

to

RGDPEG-avidin

was

however

far

lower

than

binding

to

unmodified

avidin

most

likely

due

to

the

PEGylation

-LRB-

data

not

shown

-RRB-

Fig.

of

biotinylated

TRAIL

with

RGDPEG-avidin

plates

were

coated

overnight

with

ng

RGDPEG-avidin

and

incubated

with

indicated

compounds

for

h.

TRAIL

binding

was

assessed

by

anti-TRAIL

immunodetection

and

expressed

relative

to

the

highest

signal

obtained

with

biotin-NHS-TRAIL

Experiments

were

performed

in

triplicate

Cytotoxic

and

proapoptotic

activity

of

TRAIL

complexes

To

test

whether

our

products

were

functionally

active

i.e.

capable

of

inducing

cell

death

and

apoptosis

we

assessed

their

activity

on

Jurkat

T

leukemic

cells

and

HUVEC

endothelial

cells

Figure

shows

the

dose-dependent

cytotoxic

effects

at

Jurkat

cells

after

treatment

for

h.

Both

His-TRAIL

Bio-NHS-TRAIL

and

Bio-ULS-TRAIL

effectively

killed

Jurkat

cells

with

values

between

ng/ml

and

ng/ml

-LRB-

Table

-RRB-

While

the

Bio-NHS

modification

resulted

in

a

considerable

reduction

in

activity

compared

to

the

unmodified

protein

Bio-ULS-TRAIL

displayed

almost

similar

activity

as

unmodified

TRAIL

The

pro-apoptotic

activity

of

His-TRAIL

and

biotinylated

TRAIL

derivatives

in

Jurkat

cells

was

furthermore

determined

by

analyzing

caspase

activity

as

shown

in

Fig.

These

experiments

confirmed

the

relative

higher

activity

of

bio-ULS-TRAIL

versus

bio-NHS-TRAIL

and

the

absence

of

caspase

induction

by

RGDPEG-avidin

-LRB-

data

not

shown

-RRB-

In

contrast

to

Jurkat

cells

no

cytotoxic

effects

were

observed

when

HUVEC

were

incubated

with

the

products

-LRB-

Fig.

-RRB-

Fig.

activity

assays

Panel

A

Cell

viability

of

Jurkat

T

cells

after

h

of

incubation

with

indicated

compounds

Cell

viability

was

assessed

by

MTS

assay

Experiments

were

performed

in

triplicate

Panel

B

Caspase

activity

in

Jurkat

T

cells

after

h

of

incubation

with

ng/ml

of

the

indicated

TRAIL

derivatives

RLU

relative

light

units

Panel

C

Cell

viability

-LRB-

MTS

assay

-RRB-

of

HUVEC

after

h

of

incubation

with

ng/ml

of

indicated

compounds

Finally

we

investigated

the

proapoptotic

effects

of

RGDPEG-avidin

TRAIL

complexes

in

Jurkat

cells

These

experiments

were

performed

in

the

presence

of

endothelial

cells

to

allow

adherence

of

the

RGD-equipped

complexes

to

αvβ3

integrin

expressing

cell

layers

First

we

determined

the

effects

of

avidin

TRAIL

complexes

formed

in

the

presence

of

an

excess

of

RGDPEG-avidin

As

can

be

observed

in

Fig.

such

complexes

with

both

types

of

biotinylated

TRAIL

showed

similar

activity

to

the

non-complexed

biotinylated

TRAIL

derivatives

-LRB-

striped

bars

versus

white

bars

respectively

-RRB-

Furthermore

in

agreement

with

Fig.

Bio-NHS-TRAIL

showed

a

reduced

activity

as

compared

to

unmodified

TRAIL

while

Bio-ULS-TRAIL

retained

its

activity

Fig.

activity

of

RGDPEG-avidin

TRAIL

complexes

Assays

were

performed

in

plates

seeded

with

HUVEC

that

were

subsequently

incubated

at

room

temperature

with

RGDPEG-avidin

-LRB-

μg/ml

-RRB-

and

biotinylated

TRAIL

-LRB-

ng/ml

-RRB-

to

allow

assembly

of

the

complexes

Next

Jurkat

T

cells

were

added

followed

by

h

of

incubation

at

°C

after

which

the

activation

of

caspase

was

determined

by

a

luminescence

based

assay

Experiments

were

carried

out

in

the

absence

of

RGDPEG-avidin

-LRB-

control

experiment

white

bars

-RRB-

in

the

presence

of

excess

of

RGDPEG-avidin

-LRB-

striped

bars

molar

excess

over

biotinylated

TRAIL

-RRB-

or

in

wells

incubated

with

RGDPEG-avidin

that

had

been

washed

before

addition

of

biotin-TRAIL

and

Jurkat

cells

-LRB-

black

bars

-RRB-

P

versus

TRAIL

P

versus

biotin-TRAIL

in

the

absence

of

RGDPEG-avidin

A

striking

result

was

observed

when

the

excess

of

RGDPEG-avidin

was

removed

before

addition

of

TRAIL

and

Jurkat

cells

leaving

only

endothelial-associated

RGDPEG-avidin

available

for

complexation

with

biotinylated

TRAIL

This

procedure

significantly

potentiated

the

proapoptotic

effect

for

both

Bio-NHS-TRAIL

and

Bio-ULS-TRAIL

the

latter

even

beyond

the

activity

of

unmodified

TRAIL

Discussion

In

the

present

study

we

describe

the

development

and

functional

evaluation

of

RGD-avidin

TRAIL

complexes

These

targeted

complexes

of

TRAIL

showed

enhanced

binding

to

endothelial

cells

and

maintained

their

pro-apoptotic

activity

As

a

result

of

the

binding

to

angiogenic

endothelial

cells

of

these

complexes

an

improved

tumor

accumulation

and

enhanced

residence

within

tumor

tissue

can

be

expected

Such

an

improvement

in

the

pharmacokinetic

profile

of

TRAIL

may

further

enhance

its

therapeutic

efficacy

and

enable

a

reduction

in

dosing

frequency

αvβ3

integrin

is

a

validated

target

for

drug

delivery

and

imaging

approaches

directed

to

angiogenic

endothelium

One

of

the

most

popular

ligands

for

αvβ3

integrin

is

the

cyclic

RGD-peptide

cyclo

-LRB-

RGDfK

-RRB-

Due

to

the

special

constrained

conformation

of

the

cyclic

RGD-peptide

and

the

d-phenylalanine

amino

acid

its

binding

motif

adopts

a

high

affinity

conformation

for

αv-integrins

-LSB-

-RSB-

The

cyclo

-LRB-

RGDfK

-RRB-

peptide

has

been

used

successfully

for

the

delivery

of

a

wide

range

of

drug

or

gene

carrying

systems

-LRB-

e.g.

liposomes

polymers

adenoviruses

-RRB-

-LSB-

-RSB-

Since

the

cyclopeptide

is

an

unnatural

peptide

it

can

not

be

encoded

by

a

DNA

sequence

and

requires

chemical

synthesis

and

hence

chemical

coupling

techniques

to

prepare

multivalent

targeting

devices

The

present

paper

describes

a

versatile

approach

in

which

multivalent

RGD-avidin

is

complexed

with

biotinylated

TRAIL

The

RGD-peptides

are

linked

to

avidin

via

bifunctional

PEG

linkers

thereby

surrounding

the

avidin

by

a

hydrophilic

PEG

shell

tethered

with

targeting

ligands

PEGylation

is

a

widely

accepted

methodology

for

improvement

of

therapeutic

proteins

and

affects

both

the

immunological

properties

as

well

as

the

renal

filtration

of

proteins

-LSB-

-RSB-

The

functionalization

of

avidin

with

RGD-PEG

groups

thus

will

combine

several

advantageous

properties

RGDPEG-avidin

displayed

nanomolar

affinity

for

αvβ3

integrin

expressing

endothelial

cells

which

is

in

good

agreement

with

the

data

obtained

with

other

RGD-equipped

proteins

prepared

in

our

group

-LSB-

-RSB-

We

have

compared

two

different

biotinylation

procedures

which

react

at

different

amino

acid

side

chain

residues

While

biotin-NHS

couples

at

lysine

residues

biotin-ULS

binds

at

methionines

residues

in

TRAIL

The

methionines

in

TRAIL

are

not

solvent

exposed

as

can

be

derived

from

the

crystal

structure

of

TRAIL

-LSB-

-RSB-

Therefore

the

most

likely

binding

site

for

biotin-ULS

is

the

methionine

in

the

HIS-tag

of

the

recombinant

protein

In

contrast

most

lysine

residues

are

readily

accessible

including

ones

at

positions

that

will

interface

with

the

Death

receptor

This

could

explain

why

Biotin-ULS-TRAIL

better

maintained

its

proapoptotic

activity

than

biotin-NHS

derivatized

TRAIL

Whether

the

greater

loss

in

activity

of

biotin-NHS-TRAIL

is

due

to

the

relative

higher

extent

of

derivatization

or

to

modifications

of

lysine

residues

in

the

activity

domains

of

protein

can

not

be

concluded

from

our

data

Upon

complexation

of

biotinylated

TRAIL

with

RGDPEG-avidin

a

bulky

structure

has

been

introduced

onto

the

surface

of

the

therapeutic

protein

The

assembled

complexes

however

fully

maintained

their

TRAIL

activity

indicating

that

steric

hindrance

did

not

interfere

in

the

TRAIL-death

receptor

ligation

Figure

showed

similar

proapoptotic

activities

for

either

biotinylated

TRAIL

or

the

complexes

formed

in

the

presence

of

excess

of

RGDPEG-avidin

Remarkably

we

observed

an

enhancement

of

caspase

activity

when

the

excess

of

RGDPEG-avidin

was

removed

leaving

only

surface-exposed

RGDPEG-avidin

at

the

endothelial

cell

layer

One

of

the

explanations

that

may

account

for

this

is

the

assembly

of

multiple

TRAIL

molecules

onto

a

single

avidin

thereby

affording

a

mega'TRAIL

complex

with

enhanced

death-inducing

activity

as

was

also

observed

for

multimeric

forms

of

Fas-ligand

-LSB-

-RSB-

Such

types

of

complexes

would

facilitate

the

close

assembly

of

multiple

death

receptors

thereby

potentiating

the

induction

of

death

signaling

The

presence

of

an

excess

of

RGDPEG-avidin

would

avoid

the

formation

of

such

complexes

as

both

components

were

added

in

a

molar

ratio

of

avidin

TRAIL

Gently

washing

of

the

endothelial

cells

after

RGDPEG-avidin

incubation

removed

the

excess

of

unbound

RGDPEG-avidin

leaving

only

cell-associated

avidin

after

incubation

at

room

termperature

These

conditions

therefore

provided

an

excess

of

biotinylated

TRAIL

over

avidin

now

favoring

the

formation

of

multimeric

TRAIL

complexes

Alternatively

the

exposure

of

TRAIL

via

RGD-PEG-avidin

onto

the

surface

of

endothelial

cells

may

explain

its

increased

activity

as

has

been

observed

for

chimeric

TRAIL

variants

directed

to

tumor

receptors

-LSB-

-RSB-

HUVEC

were

not

killed

by

TRAIL

or

its

derivatives

as

has

been

described

by

others

-LSB-

-RSB-

On

the

other

hand

although

TRAIL

does

not

induce

apoptosis

in

endothelial

cells

it

activates

other

processes

such

as

production

of

NO

and

reactive

oxygen

species

which

may

alter

the

tumor

responsiveness

to

TRAIL

-LSB-

-RSB-

This

can

not

explain

the

results

shown

in

Fig.

since

endothelial

cells

were

preincubated

with

RGDPEG-avidin

only

and

preincubations

were

performed

at

room

temperature

rather

than

at

°C

Such

effects

of

RGD-equipped

TRAIL

may

occur

however

in

vivo

In

addition

tumor

derived

growth

factors

might

sensitize

endothelial

cells

to

TRAIL

's

apoptosis

inducing

capacity

similar

to

the

findings

reported

for

TNF-sensitivity

-LSB-

-RSB-

The

complexation

of

biotinylated

TRAIL

with

RGDPEG-avidin

and

its

association

with

angiogenic

endothelial

cells

may

afford

an

increased

accumulation

into

the

tumor

blood

vessels

similar

to

the

enhanced

tumor

accumulation

of

other

RGD-modified

proteins

-LSB-

-RSB-

An

unresolved

issue

is

whether

RGD-avidin/TRAIL

complexes

bound

to

the

tumor

vasculature

can

interact

with

tumor

cells

behind

the

endothelial

lining

in

vivo

To

interact

with

tumor

cells

and

induce

tumor

apoptosis

TRAIL

complexes

bound

to

the

tumor

vasculature

should

dissociate

and

penetrate

into

the

tumor

tissue

Several

considerations

may

be

important

in

this

respect

such

as

the

expression

of

αvβ3

integrin

on

both

luminal

and

abluminal

side

of

the

endothelium

and

the

fact

that

many

tumor

cells

express

αvβ3

integrin

-LSB-

-RSB-

Both

of

these

will

favor

the

interaction

of

RGD-avidin

complexed

TRAIL

with

tumor

cells

Furthermore

RGD-avidin

binding

to

endothelial

cells

is

less

tight

than

the

typical

antibody

binding

This

may

favor

redistribution

of

the

complexes

upon

their

initial

binding

to

αvβ3

integrin

Homing

to

the

tumor

vasculature

will

provide

elevated

concentrations

within

the

tumor

microenvironment

which

may

subsequently

provide

elevated

intratumoral

concentrations

Together

with

the

observed

enhancement

of

TRAIL

activity

this

may

favor

a

more

efficient

induction

of

apoptosis

in

tumor

cells

To

summarize

we

have

prepared

a

novel

type

of

RGD-targeted

TRAIL

with

binding

specificity

for

angiogenic

endothelium

and

maintenance

of

strong

cytotoxic

activity

Whether

in

vivo

application

of

this

product

will

lead

to

an

enhanced

tumor

accumulation

still

needs

to

be

investigated

We

now

succeeded

in

coupling

the

well

known

homing

device

RGD

to

the

TRAIL

molecule

without

losing

the

apoptosis-inducing

activity

of

TRAIL

or

the

receptor-binding

properties

of

RDG

peptides

An

enhanced

activity

could

even

be

demonstrated

in

vitro

This

may

in

combination

with

an

improved

pharmacokinetic

profile

induced

by

the

PEG

and

RGD

moieties

lead

to

improved

effects

of

TRAIL

in

vivo

Lastly

the

followed

synthetic

approach

offers

opportunities

for

the

complexation

with

alternative

targeting

ligands

that

bind

directly

to

tumor

cells

such

as

tumor-specific

antibodies

or

peptide

ligands

O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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