Discovery

and

characterization

of

a

putrescine

oxidase

from

Rhodococcus

erythropolis

NCIMB

A

gene

encoding

a

putrescine

oxidase

-LRB-

PuORh

EC

-RRB-

was

identified

from

the

genome

of

Rhodococcus

erythropolis

NCIMB

The

gene

was

cloned

in

the

pBAD

vector

and

overexpressed

at

high

levels

in

Escherichia

coli

The

purified

enzyme

was

shown

to

be

a

soluble

dimeric

flavoprotein

consisting

of

subunits

of

kDa

and

contains

non-covalently

bound

flavin

adenine

dinucleotide

as

a

cofactor

From

all

substrates

the

highest

catalytic

efficiency

was

found

with

putrescine

-LRB-

KM

μM

kcat

s

-RRB-

PuORh

accepts

longer

polyamines

while

short

diamines

and

monoamines

strongly

inhibit

activity

PuORh

is

a

reasonably

thermostable

enzyme

with

at

°C

of

h.

Based

on

the

crystal

structure

of

human

monoamine

oxidase

B

we

constructed

a

model

structure

of

PuORh

which

hinted

to

a

crucial

role

of

for

substrate

binding

Mutation

of

this

residue

resulted

in

a

drastic

drop

-LRB-

five

orders

of

magnitude

-RRB-

in

catalytic

efficiency

Interestingly

the

mutant

enzyme

showed

activity

with

monoamines

which

are

not

accepted

by

wt-PuORh

Introduction

Flavoprotein

oxidases

catalyze

the

oxidation

of

a

wide

range

of

compounds

while

at

the

same

time

they

reduce

oxygen

to

hydrogen

peroxide

They

are

valuable

biocatalysts

for

the

oxidative

activation

of

biomolecules

as

they

usually

selectively

oxidize

their

substrate

at

a

specific

position

leaving

other

positions

unaffected

Due

to

their

ability

to

use

molecular

oxygen

as

electron

acceptor

no

expensive

coenzymes

like

NAD

-LRB-

P

-RRB-

H

are

needed

This

makes

oxidases

inexpensive

and

rather

straightforward

in

usage

compared

to

other

redox

enzymes

By

far

the

best

studied

oxidase

is

glucose

oxidase

from

Aspergillus

niger

This

enzyme

has

been

applied

for

decades

mostly

for

diagnostic

applications

-LRB-

Wilson

and

Turner

-RRB-

Another

example

is

pyranose

oxidase

-LRB-

-RRB-

from

Peniophora

gigantea

which

oxidizes

specifically

the

position

of

glucose

and

has

been

applied

for

the

chemoenzymatic

synthesis

of

rare

sugars

and

sugar-based

synthons

-LRB-

Giffhorn

et

al.

-RRB-

Human

monoamine

oxidases

-LRB-

MAO-A

and

MAO-B

-RRB-

are

important

for

the

oxidation

of

neurotransmitters

-LRB-

Abell

and

Kwan

-RRB-

An

engineered

mutant

of

the

homologous

A.

niger

enzyme

-LRB-

MAO-N

-RRB-

has

been

applied

in

an

elegant

biocatalytic

deracemization

process

to

obtain

enantiomerically

pure

chiral

amines

-LRB-

Carr

et

al.

Dunsmore

et

al.

-RRB-

So

far

the

most

described

oxidases

are

from

eukaryotic

origin

and

heterologous

expression

of

the

recombinant

protein

in

Escherichia

coli

for

large

scale

enzyme

production

can

be

problematic

In

a

search

for

novel

bacterial

oxidases

we

looked

in

sequenced

genomes

and

identified

genes

encoding

novel

oxidases

By

this

we

discovered

oxidases

primarily

acting

on

alditols

-LRB-

Heuts

et

al.

-RRB-

and

phenolic

compounds

-LRB-

Jin

et

al.

-RRB-

These

oxidases

both

from

actinomycetes

could

be

highly

overexpressed

in

E.

coli

Another

way

of

searching

for

novel

bacterial

oxidases

is

to

screen

unsequenced

bacterial

genome

libraries

directly

for

genes

encoding

enzyme

activity

As

actinomycetes

appear

to

be

rich

in

oxidases

we

have

chosen

to

explore

these

organisms

for

relevant

oxidases

Using

a

plate-based

screening

protocol

for

oxidase

activity

-LRB-

Alexeeva

et

al.

-RRB-

we

screened

a

genomic

DNA

library

of

the

actinomycete

Rhodococcus

erythropolis

NCIMB

to

find

novel

oxidases

acting

on

polyols

and/or

amines

In

this

paper

we

describe

the

discovery

and

characterization

of

a

novel

flavin-containing

oxidase

primarily

acting

on

putrescine

This

putrescine

oxidase

-LRB-

PuORh

-RRB-

shares

sequence

identity

with

the

enzyme

from

Micrococcus

rubens

-LRB-

PuOMr

Ishizuka

et

al.

-RRB-

We

show

that

PuORh

is

highly

overexpressed

in

E.

coli

and

can

be

easily

purified

Using

a

structural

model

we

were

able

to

identify

a

glutamate

residue

-LRB-

-RRB-

that

is

crucial

for

substrate

binding

and

we

could

alter

the

substrate

specificity

by

protein

engineering

Materials

and

methods

Chemicals

Restriction

enzymes

were

from

Roche

and

New

England

Biolabs

One-shot

electrocompetent

E.

coli

cells

and

the

TOPO

TA

Cloning

Kit

were

purchased

from

Invitrogen

Plasmid

isolation

was

performed

using

the

Qiagen

DNA

purification

kit

Oligonucleotides

were

purchased

from

Sigma

Nitrocellulose

filters

-LRB-

Protran

mm

μm

pore

size

-RRB-

were

from

Schleicher

and

Schuell

BioScience

Dassel

-LRB-

Germany

-RRB-

Deprenyl

and

rasagiline

were

a

kind

gift

from

Prof.

A.

Mattevi

-LRB-

University

of

Pavia

Italy

-RRB-

All

other

chemicals

were

of

analytical

grade

The

genomic

DNA

library

from

R.

erythropolis

NCIMB

was

provided

by

DSM

-LRB-

Geleen

The

Netherlands

-RRB-

Constructs

were

sequenced

at

GATC

Biotech

-LRB-

Kostanz

Germany

-RRB-

Plate-based

screening

method

for

oxidase

activity

A

gene

library

of

R.

erythropolis

NCIMB

in

was

screened

for

oxidases

using

the

plate-based

oxidase

activity

screening

method

adapted

from

the

group

of

Turner

-LRB-

Alexeeva

et

al.

-RRB-

This

gene

library

was

constructed

by

partial

digestion

of

genomic

DNA

from

R.

erythropolis

NCIMB

by

Fragments

of

kb

were

isolated

ligated

in

-LRB-

cut

with

BamHI

-RRB-

and

followed

by

transformation

of

E.

coli

Colonies

were

collected

from

plate

stored

as

glycerol

stock

and

plasmids

were

isolated

The

total

amount

of

plasmids

contained

a

mean

insert

size

of

kb

and

of

self-ligated

vector

molecules

Electrocompetent

E.

coli

cells

were

transformed

with

the

gene

library

and

the

transformed

cells

were

diluted

in

Luria

Bertani

-LRB-

LB

-RRB-

medium

to

obtain

single

colonies

on

plate

The

diluted

cell

suspensions

were

plated

on

nitrocellulose

filters

The

filters

were

placed

on

top

of

LB

agar

containing

mg/ml

kanamycin

and

incubated

for

h

at

°C

Subsequently

the

nitrocellulose

filters

were

transferred

to

empty

petri

dishes

and

stored

at

°C

to

partially

lyse

the

cells

Each

filter

was

submersed

with

ml

mM

sodium

phosphate

buffer

pH

containing

-LRB-

w/v

-RRB-

agarose

U/ml

Horseradish

peroxidase

-LRB-

HRP

-RRB-

and

a

mixture

of

mM

μM

cholesterol

mM

xylitol

mM

sarcosine

mM

l-alanine

mM

d-glucose

mM

d-galactose

mM

lactose

and

mM

d-glucosamine

Plates

were

incubated

at

room

temperature

and

regularly

checked

by

visual

inspection

for

color

formation

in

and

around

colonies

Positives

were

picked

and

cultivated

overnight

in

ml

liquid

LB

medium

and

subjected

to

another

screening

cycle

to

assure

that

color

formation

was

caused

by

single

clones

Sequence

analysis

To

identify

unique

positive

clones

plasmids

were

isolated

and

subjected

to

restriction

analysis

by

EcoRI

Inserts

were

sequenced

and

open

reading

frames

-LRB-

ORFs

-RRB-

were

identified

using

the

ORF

Finder

tool

of

the

National

Center

for

Biotechnology

Information

-LRB-

NCBI

-RRB-

-LRB-

-RRB-

A

BLAST

search

-LRB-

blastp

-RRB-

was

performed

with

the

protein

sequence

of

PuORh

using

the

BLAST

function

of

the

NCBI

website

-LRB-

-RRB-

Cloning

and

expression

of

the

gene

encoding

PuORh

To

overexpress

the

novel

putrescine

oxidase

the

corresponding

gene

-LRB-

puoRh

-RRB-

was

amplified

from

the

fragment

of

genomic

DNA

of

R.

erythropolis

using

the

following

primers

GCTCCATATGCCTACTCTCCAGAGAGATG

-LRB-

NdeI

site

shown

in

italics

-RRB-

and

GCTCAAGCTTTCAGGCCTTGCTGCGGGCG

-LRB-

HindIII

site

shown

in

italics

-RRB-

The

amplified

gene

was

purified

from

gel

and

ligated

between

the

NdeI

and

HindIII

restriction

sites

of

the

pBAD/Myc-His

vector

-LRB-

Invitrogen

-RRB-

The

plasmid

carrying

the

puoRh

gene

-LRB-

pBADpuoRh

-RRB-

was

transformed

to

E.

coli

cells

and

spread

on

LB

agar

plates

containing

μg/ml

of

ampicillin

E.

coli

cells

containing

pBADpuoRh

were

tested

for

overexpression

of

the

protein

at

and

°C

and

at

arabinose

concentrations

of

and

-LRB-

w/v

-RRB-

Cell

extracts

and

cell-free

extracts

of

E.

coli

containing

pBADpuoRh

were

analyzed

on

a

sodium

dodecyl

sulfate

polyacrylamide

gel

electrophoresis

-LRB-

SDS-PAGE

-RRB-

gel

to

find

the

best

conditions

for

overexpression

of

soluble

PuORh

Enzyme

purification

To

obtain

purified

protein

E.

coli

containing

pBADpuoRh

was

cultivated

for

h

at

°C

in

l

of

terrific

broth

medium

containing

μg/ml

ampicillin

and

-LRB-

w/v

-RRB-

arabinose

Cells

were

harvested

by

centrifugation

for

min

at

rpm

at

°C

The

supernatant

was

discarded

and

the

pellet

was

suspended

in

ml

of

mM

Tris

HCl

pH

and

sonicated

for

min

to

break

the

cells

The

cell

extract

was

centrifuged

for

min

at

rpm

at

°C

to

remove

the

broken

cells

and

obtain

cell-free

extract

From

this

cell-free

extract

PuORh

was

purified

using

a

Q-Sepharose

anion

exchange

column

Unbound

protein

fractions

were

washed

from

the

column

with

mM

Tris

HCl

buffer

pH

PuORh

was

eluted

from

the

column

with

a

mM

Tris

HCl

buffer

pH

by

increasing

the

KCl

concentration

Eluted

fractions

containing

PuORh

were

combined

concentrated

using

an

Amicon

filter

and

desalted

using

a

HiPrep

Desalting

Column

-LRB-

Amersham

Biosciences

-RRB-

Enzyme

activity

assay

and

determination

of

steady-state

kinetic

parameters

PuORh

activity

was

measured

at

°C

using

a

peroxidase-coupled

assay

containing

azino-bis

-LRB-

acid

-RRB-

-LRB-

ABTS

-RRB-

as

the

chromogenic

substrate

-LRB-

Childs

and

Bardsley

-RRB-

The

formed

by

PuORh

can

be

coupled

to

the

Horseradish-peroxidase-mediated

oxidation

of

ABTS

resulting

in

the

formation

of

a

green

product

that

can

be

measured

at

nm

-LRB-

mM

cm

-RRB-

The

standard

assay

mixture

with

ABTS

-LRB-

HRP-ABTS

assay

-RRB-

contained

μM

putrescine

mM

Tris

HCl

buffer

pH

μM

ABTS

U

of

Horseradish

peroxidase

μl

of

enzyme

dilution

-LRB-

appropriately

diluted

-RRB-

and

a

fixed

amount

of

substrate

The

reaction

was

started

with

adding

the

enzyme

solution

Alternatively

mM

-LRB-

AAP

-RRB-

and

mM

acid

-LRB-

DCHBS

-RRB-

were

used

as

chromogenic

substrates

for

HRP

-LRB-

Federico

et

al.

HRP-AAP/DCHBS

assay

-RRB-

The

oxidation

of

these

substrates

results

in

the

formation

of

a

purple

product

which

can

be

measured

at

nm

-LRB-

mM

cm

-RRB-

For

the

determination

of

kinetic

parameters

PuORh

activity

was

measured

at

different

substrate

concentrations

at

°C

using

the

HRP-ABTS

enzyme

assay

The

kinetic

parameters

of

the

mutant

enzymes

-LRB-

PuORh

and

PuORh

-RRB-

for

putrescine

were

determined

by

measuring

directly

the

production

at

nm

-LRB-

M

cm

-RRB-

Data

were

fitted

with

Origin

using

the

Michaelis

Menten

equation

for

enzyme

kinetics

Influence

of

temperature

and

pH

on

enzyme

activity

and

stability

To

determine

the

optimal

temperature

for

PuORh

activity

the

oxidase

activity

was

measured

at

temperatures

between

and

°C

Before

adding

the

enzyme

the

assay

mixture

was

equilibrated

for

min

to

assure

the

right

temperature

The

influence

of

temperature

on

the

enzyme

stability

was

determined

by

incubating

portions

of

the

enzyme

at

and

°C

Twenty-microliter

samples

were

taken

placed

on

ice

after

which

activity

was

measured

In

both

experiments

the

activity

was

determined

with

the

HRP-AAP/DCHBS

activity

assay

using

μM

putrescine

and

μM

PuORh

The

pH

optimum

for

putrescine

oxidase

was

determined

by

measuring

the

activity

at

different

pH

values

at

°C

The

following

buffers

were

used

mM

Pipes

buffer

-LRB-

pH

-RRB-

mM

Tris

HCl

-LRB-

pH

-RRB-

and

mM

Ches

buffer

-LRB-

pH

-RRB-

The

enzyme

activity

was

measured

using

the

HRP-AAP/DCHBS

assay

Inhibition

experiments

Rasagiline

deprenyl

and

cyclopropylamine

were

tested

to

probe

whether

they

were

able

to

inhibit

PuORh

by

the

formation

of

a

covalent

adduct

with

the

flavin

adenine

dinucleotide

-LRB-

FAD

-RRB-

cofactor

as

is

the

case

for

monoamine

oxidase

B

-LRB-

Binda

et

al.

-RRB-

For

μl

of

a

μM

PuORh

solution

an

absorbance

scan

was

recorded

from

nm

After

the

addition

of

μl

of

mM

inhibitor

-LRB-

final

inhibitor

concentration

μM

-RRB-

absorbance

spectra

were

recorded

regularly

in

time

to

see

if

addition

of

the

inhibitor

would

cause

an

alteration

of

the

typical

FAD

spectrum

of

PuORh

For

cyclopropylamine

also

a

final

inhibitor

concentration

of

mM

was

tested

as

described

above

Butylamine

aminoethanol

ethylenediamine

and

-LRB-

-RRB-

trimethylammonium

were

tested

as

competitive

inhibitors

of

PuORh

For

this

the

Michaelis

constant

-LRB-

KM

-RRB-

of

PuORh

for

putrescine

was

determined

for

three

different

concentrations

for

each

of

these

amino

compounds

with

the

HRP-ABTS

assay

Inhibition

constants

-LRB-

KI

-RRB-

for

each

compound

were

calculated

with

the

following

formula

Where

-LSB-

I

-RSB-

represents

the

inhibitor

concentration

KM

inhibitor

the

apparent

Michaelis

constant

in

presence

of

the

inhibitor

and

the

KI

the

inhibition

constant

Analytical

methods

All

absorbance

spectra

were

recorded

in

mM

Tris

HCl

pH

at

°C

on

a

Perkin

Elmer

Lambda

spectrophotometer

From

a

cuvet

containing

μM

PuORh

all

oxygen

was

removed

by

flushing

with

argon

and

an

absorbance

spectrum

was

recorded

from

to

nm

After

adding

μM

putrescine

another

spectrum

was

recorded

for

the

reduced

enzyme

Reoxidation

was

monitored

by

collecting

spectra

in

time

after

the

cuvet

was

exposed

to

air

A

spectrum

of

the

unfolded

enzyme

was

recorded

by

adding

SDS

and

heating

for

min

at

°C

Modeling

and

mutant

construction

Based

on

the

structure

of

MAO-B

in

complex

with

rasagiline

-LRB-

Binda

et

al.

-RRB-

a

model

of

PuORh

was

made

using

the

CHPmodels

Server

Mutants

were

constructed

by

Quick

Change

PCR

PuORh

was

made

by

a

substitution

of

the

codon

GAG

for

GCG

using

the

following

primers

CGAGGTAGTGCAGGCGGTGTACGACAACACC

and

GGTGTT

GTCGTACACCGCCTGCACTACCTCG

-LRB-

mutated

codon

underlined

-RRB-

For

construction

of

PuORh

the

codon

GAG

was

replaced

by

CTG

using

the

following

primers

CGAGGTAGTGCAGCTGGTGTACGACAACACC

and

GGTGTTGTCGTACACCAGCTGCACTACCTCG

-LRB-

mutated

codon

underlined

-RRB-

Both

mutants

were

expressed

in

E.

coli

TOP

and

cultivated

and

purified

in

the

same

way

as

wild-type

PuORh

Nucleotide

sequence

accession

number

The

nucleotide

sequence

of

the

PuORh

encoding

gene

-LRB-

puoRh

-RRB-

has

been

submitted

to

GenBank

under

accession

number

Results

Identification

of

a

novel

putrescine

oxidase

To

identify

novel

oxidases

acting

on

alcohols

and/or

amines

a

genomic

library

from

the

actinomycete

R.

erythropolis

NCIMB

was

screened

using

a

plate-based

oxidase

assay

Positive

clones

expressing

oxidase

activity

could

be

identified

by

their

purple

color

formation

From

transformants

were

observed

to

produce

a

purple

color

around

the

colony

Retransformation

to

E.

coli

cells

and

repeated

screening

on

plate

resulted

in

three

positive

clones

which

showed

oxidase

activity

Restriction

analysis

with

EcoRI

and

PvuI

showed

a

different

insert

for

each

clone

indicating

that

these

were

independent

clones

Sequencing

of

one

of

these

clones

revealed

an

inserted

fragment

of

genomic

DNA

of

bp

On

this

fragment

an

ORF

was

identified

which

shares

sequence

identity

-LRB-

at

the

amino

acid

level

-RRB-

with

putrescine

oxidase

-LRB-

EC

-RRB-

from

M.

rubens

a

FAD-containing

amine

oxidase

that

catalyzes

the

oxidative

deamination

of

putrescine

-LRB-

Ishizuka

et

al.

-RRB-

When

we

tested

the

cell

extract

of

the

clone

containing

the

above-mentioned

insert

of

bp

we

found

that

it

was

indeed

active

with

putrescine

The

other

two

positive

clones

probably

contained

the

same

gene

as

for

both

clones

by

using

the

primers

and

a

PCR

product

was

seen

on

gel

Gene

cloning

overexpression

and

protein

purification

For

overexpression

the

PuORh

encoding

gene

was

cloned

into

the

pBAD/Myc-His

A

vector

behind

the

araBAD

promotor

-LRB-

without

His-tag

-RRB-

and

the

plasmid

carrying

the

puo

gene

was

transformed

to

E.

coli

Several

temperatures

-LRB-

and

°C

-RRB-

and

inducing

conditions

-LRB-

L

-LRB-

-RRB-

arabinose

-RRB-

were

tested

to

find

the

optimal

conditions

for

overexpression

The

gene

was

well

expressed

at

arabinose

concentrations

of

and

as

judged

by

a

dominant

protein

band

of

kDa

on

SDS-PAGE

At

and

°C

the

protein

was

mainly

present

in

the

soluble

fractions

while

at

°C

the

unsolube

fractions

contained

most

of

the

overexpressed

protein

The

overexpressed

protein

did

not

show

any

fluorescence

under

UV-light

upon

SDS-PAGE

which

is

an

indication

that

this

protein

does

not

contain

a

covalently

histidyl-bound

flavin

cofactor

-LRB-

Fraaije

et

al.

-RRB-

PuORh

was

purified

from

a

culture

containing

arabinose

that

was

grown

for

h

at

°C

Due

to

the

high

level

of

overexpression

-LRB-

mg

-RRB-

PuORh

could

be

easily

purified

in

one-step

by

anion

exchange

chromotography

SDS-PAGE

analysis

of

the

purified

protein

revealed

that

besides

the

major

protein

band

present

at

around

kDa

there

is

a

minor

band

corresponding

to

a

protein

mass

which

is

approximately

kDa

larger

-LRB-

Fig.

lane

A

-RRB-

It

was

concluded

that

this

must

be

the

product

of

the

same

gene

with

a

His-tag

extension

as

this

minor

protein

band

could

be

removed

by

an

additional

purification

step

using

a

Ni-agarose

column

-LRB-

Fig.

lane

B

-RRB-

Although

in

the

plasmid

pBADpuoRh

the

stop

codon

-LRB-

TGA

-RRB-

was

properly

introduced

it

is

known

that

in

some

cases

this

stop

codon

can

be

translated

-LRB-

MacBeath

and

Kast

-RRB-

With

the

constructed

plasmid

this

would

indeed

result

in

a

kDa

larger

protein

Fig.

gel

of

purified

recombinant

PuORh

Lane

A

Purified

PuORh

Lane

B

Purified

PuORh

after

removal

of

His-tagged

PuORh

Lane

C

Low

molecular

weight

marker

Spectral

and

molecular

properties

of

PuORh

PuORh

shows

a

typical

flavoprotein

spectrum

with

absorbance

maxima

at

and

nm

-LRB-

Fig.

-RRB-

In

the

presence

of

trichloroacetic

acid

PuORh

precipitated

and

was

visible

as

a

white

pellet

after

centrifugation

The

supernatant

contained

a

yellow

color

and

its

absorbance

spectrum

with

maxima

at

and

nm

was

typical

for

that

of

free

FAD

This

shows

that

the

FAD

cofactor

in

PuORh

like

in

PuOMr

is

not

covalently

bound

At

room

temperature

the

addition

of

SDS

did

not

unfold

PuORh

indicating

this

flavoprotein

is

rather

robust

When

μM

putrescine

was

mixed

with

PuORh

under

anaerobic

conditions

the

FAD

cofactor

was

completely

reduced

-LRB-

Fig.

dotted

line

-RRB-

Heating

the

same

sample

for

min

at

°C

completely

unfolded

the

enzyme

as

can

be

seen

from

the

resulting

free

FAD

UV/VIS

spectrum

-LRB-

Fig.

dashed

line

-RRB-

The

reduced

enzyme

could

be

rapidly

and

fully

reoxidized

by

addition

of

oxygen

indicating

that

the

enzyme

is

a

true

oxidase

From

the

difference

in

absorbance

between

protein-bound

FAD

and

free

FAD

the

molar

extinction

coefficient

for

PuORh

-LRB-

-RRB-

was

calculated

mM

cm

Based

on

the

protein

concentration

as

determined

by

Wadell

's

method

-LRB-

Wolf

-RRB-

and

the

FAD

concentration

-LRB-

-RRB-

a

protein/FAD

ratio

of

was

calculated

This

is

consistent

with

what

has

been

described

earlier

for

PuOMr

-LRB-

Desa

-RRB-

Incubation

of

PuORh

with

additional

FAD

did

not

yield

an

increase

of

FAD

incorporation

or

oxidase

activity

Gel

filtration

experiments

revealed

that

PuORh

is

mainly

present

as

a

dimer

of

kDa

This

corresponds

well

with

the

theoretical

monomeric

mass

of

Da

Fig.

properties

of

PuORh

Absorbance

spectra

of

oxidized

native

-LRB-

solid

line

-RRB-

and

unfolded

-LRB-

dashed

line

-RRB-

PuORh

are

shown

After

addition

of

μM

putrescine

under

anaerobic

conditions

the

native

enzyme

is

fully

reduced

-LRB-

dotted

line

-RRB-

Temperature

and

pH

dependence

of

activity

and

stability

To

investigate

the

effect

of

temperature

on

enzyme

activity

and

stability

both

were

determined

at

different

temperatures

The

optimal

temperature

for

enzyme

activity

was

found

to

be

°C

Temperature

stability

experiments

revealed

that

PuORh

is

a

reasonable

thermostable

enzyme

At

°C

half

of

the

activity

was

lost

after

h

while

at

°C

such

a

degree

of

inactivation

was

reached

only

after

day

PuORh

has

a

rather

sharp

pH

optimum

for

activity

with

putrescine

with

a

peak

around

pH

Below

pH

no

significant

activity

can

be

detected

Substrate

specificity

Several

alcohols

amino

alcohols

and

amines

were

tested

as

substrate

and

if

they

turned

out

to

be

converted

by

PuORh

the

steady-state

kinetic

parameters

were

determined

at

pH

Enzyme

activity

was

found

with

several

aliphatic

diamines

amino

alcohols

and

polyamines

From

Table

it

is

clear

that

putrescine

is

by

far

the

best

substrate

With

aromatic

amines

and

amino

alcohols

no

activity

was

observed

An

increase

in

carbon

chain

length

of

the

diamine

from

to

C-atoms

resulted

in

a

more

than

drop

in

catalytic

efficiency

-LRB-

kcat/KM

-RRB-

For

diamines

containing

or

C-atoms

and

for

n-butylamine

no

activity

could

be

detected

These

amines

were

found

to

strongly

inhibit

the

enzyme

-LRB-

Table

-RRB-

Apparently

they

bind

efficiently

but

are

not

converted

Clearly

a

minimum

of

two

amino

groups

is

required

to

be

a

substrate

for

PuORh

and

these

amino

groups

should

be

preferably

C-atoms

apart

Polyamines

like

spermine

and

spermidine

were

also

converted

by

PuORh

but

are

poor

substrates

Compared

to

putrescine

is

also

a

poor

substrate

while

is

not

accepted

at

all

This

again

indicates

that

two

amino

groups

are

essential

for

efficient

catalysis

and

it

confirms

that

PuORh

is

a

true

amine

oxidase

Inhibition

studies

showed

that

aminoethanol

is

a

very

effective

competitive

inhibitor

of

PuORh

-LRB-

KI

μM

-RRB-

Apparently

this

amino

alcohol

binds

very

strongly

in

the

active

site

while

it

can

not

be

oxidized

When

we

tested

which

contains

an

aminoethanol

moiety

we

found

that

this

compound

is

readily

oxidized

l-Ornithine

was

found

to

be

a

very

poor

substrate

Apparently

the

presence

of

a

negatively

charged

and

relative

bulky

carboxylic

acid

group

at

the

position

prevents

efficient

amine

oxidation

Taken

together

it

can

be

concluded

that

PuORh

has

a

narrow

substrate

specificity

and

is

very

selective

for

putrescine

Table

kinetic

parameters

of

PuORh

at

pH

ND

Not

determinedaValues

of

putrescine

oxidase

from

Micrococcus

rubens

-LRB-

Okada

et

al.

-RRB-

Table

constants

of

competitive

inhibitors

of

PuORhaValues

of

putrescine

oxidase

from

Micrococcus

rubens

-LRB-

Swain

and

Desa

-RRB-

Model

structure

of

PuORh

reveals

a

key

role

for

in

substrate

binding

PuORh

shares

sequence

identity

with

human

monoamine

oxidase

B

-LRB-

MAO-B

-RRB-

which

is

a

kDa

FAD-containing

protein

involved

in

the

oxidation

of

neurotransmitters

and

other

arylalkylamines

like

benzylamine

and

phenylethylamine

-LRB-

Shih

et

al.

-RRB-

Based

on

the

structure

of

MAO-B

-LRB-

Binda

et

al.

-RRB-

it

was

possible

to

construct

a

model

for

PuORh

The

C-terminal

membrane

anchor

of

MAO-B

is

absent

in

the

resulting

model

structure

of

PuORh

This

could

explain

why

PuORh

is

well

expressed

in

the

cytosol

as

a

soluble

protein

in

contrast

to

MAO-B

which

is

membrane

associated

-LRB-

Binda

et

al.

-RRB-

In

the

model

the

active

site

of

the

PuORh

exhibits

a

similar

architecture

as

that

of

MAO-B

-LRB-

Fig.

-RRB-

The

catalytically

important

aromatic

cage

present

in

flavin-containing

amine

oxidases

-LRB-

Li

et

al.

-RRB-

is

formed

by

and

The

other

two

key

amino

acid

residues

and

involved

in

non-covalent

FAD

binding

-LRB-

Binda

et

al.

-RRB-

can

also

be

found

in

the

model

of

PuORh

However

there

are

also

some

striking

differences

In

MAO-B

FAD

is

covalently

attached

to

at

the

position

of

the

flavin

PuORh

has

an

alanine

residue

at

this

position

-LRB-

-RRB-

which

precludes

covalent

binding

of

FAD

Furthermore

the

active

site

of

PuORh

seems

to

be

narrower

than

that

of

MAO-B

due

to

the

presence

of

and

in

PuORh

-LRB-

and

in

MAO-B

-RRB-

The

model

of

PuORh

also

shows

a

glutamic

acid

residue

-LRB-

-RRB-

pointing

towards

the

of

the

FAD

while

MAO-B

contains

a

tyrosine

at

this

position

-LRB-

-RRB-

The

presence

of

such

a

negatively

charged

amino

acid

in

the

active

site

has

already

been

predicted

for

PuOMr

several

decades

ago

by

Swain

and

Desa

They

suggested

the

presence

of

a

negatively

charged

carboxyl

group

in

the

active

site

as

PuOMr

was

found

to

be

irreversibly

inactivated

by

carbodiimides

-LRB-

Swain

and

Desa

-RRB-

The

presence

and

position

of

can

well

explain

the

difference

in

substrate

specificity

between

MAO-B

and

PuORh

PuORh

prefers

short

aliphatic

diamines

while

MAO-B

is

active

with

a

range

of

aromatic

monoamines

Probably

is

involved

in

binding

the

protonated

amino

group

of

the

diamine

substrate

while

the

other

amino

group

is

positioned

in

the

aromatic

cage

near

the

of

the

flavin

Such

a

mode

of

binding

for

putrescine

is

consistent

with

our

PuORh

model

To

confirm

this

hypothesis

we

mutated

into

an

alanine

-LRB-

PuORh

-RRB-

and

a

leucine

residue

-LRB-

PuORh

-RRB-

and

found

that

the

catalytic

efficiency

for

putrescine

drops

respectively

and

-LRB-

Table

-RRB-

Fig.

site

residues

in

the

structure

of

MAO-B

and

in

the

model

of

PuORh

In

MAO-B

the

inhibitor

rasagiline

is

covalently

bound

to

the

flavin

In

the

model

of

PuORh

the

substrate

putrescine

is

modeled

in

the

active

siteTable

kinetic

parameters

for

putrescine

of

wild-type

PuORh

and

of

the

mutants

and

at

pH

Km

-LRB-

μM

-RRB-

kcat

-LRB-

s

-RRB-

kcat/Km

-LRB-

s

mM

-RRB-

PuORh

PuORh

PuORh

The

distance

between

and

the

of

the

isoalloxazine

ring

of

the

flavin

cofactor

-LRB-

Å

-RRB-

may

explain

the

specificity

for

the

specific

chain

length

of

the

substrate

To

be

a

substrate

a

minimal

carbon

chain

length

of

C-atoms

is

required

Shorter

diamines

can

bind

to

but

can

not

reach

the

flavin

and

therefore

are

strong

competitive

inhibitors

of

PuORh

-LRB-

Table

-RRB-

In

case

of

longer

diamines

or

polyamines

the

position

of

the

amino

group

that

is

oxidized

is

less

favorable

compared

to

the

case

of

putrescine

resulting

in

a

decrease

in

catalytic

efficiency

Aliphatic

monoamines

like

n-butylamine

will

bind

to

via

the

amine

moiety

but

can

not

be

oxidized

and

therefore

are

also

inhibitors

of

PuORh

Initial

substrate

screening

using

plates

for

the

mutants

PuORh

and

PuORh

revealed

that

both

showed

some

activity

with

aromatic

amines

like

and

with

aliphatic

monoamines

-LRB-

e.g.

kcat/KM

for

n-butylamine

s

mM

at

pH

for

PuORh

-RRB-

Unfortunately

accurate

determination

of

the

kinetic

parameters

of

the

mutant

enzymes

is

not

straightforward

due

to

their

low

substrate

affinity

which

requires

the

use

of

high

concentrations

of

amines

causing

interference

with

the

peroxidase-based

assay

Discussion

In

an

attempt

to

discover

novel

bacterial

oxidases

with

biocatalytic

potential

a

putrescine

oxidase

from

R.

erythropolis

NCIMB

was

identified

This

putrescine

oxidase

was

discovered

by

using

a

plate-based

screening

method

for

oxidase

activity

which

has

been

used

before

to

screen

for

improved

mutants

during

directed

evolution

experiments

-LRB-

Alexeeva

et

al.

-RRB-

Our

study

shows

that

this

method

is

also

valuable

for

the

discovery

of

novel

bacterial

oxidases

of

unsequenced

genomes

Although

the

physiological

substrate

of

the

discovered

putrescine

oxidase

was

not

present

during

screening

clones

expressing

this

oxidase

could

still

be

detected

based

on

their

oxidase

activity

As

polyamines

like

putrescine

spermine

and

spermidine

are

present

in

most

living

cells

including

E.

coli

-LRB-

Tabor

and

Tabor

-RRB-

this

resulted

in

the

formation

of

hydrogen

peroxide

and

detection

of

positive

clones

The

physiological

role

of

putrescine

oxidase

in

R.

erythropolis

is

probably

related

to

polyamine

degradation

-LRB-

Large

-RRB-

which

is

supported

by

the

presence

of

a

neighboring

gene

encoding

a

putative

aldehyde

dehydrogenase

on

the

sequenced

DNA

fragment

-LRB-

data

not

shown

-RRB-

PuORh

displays

sequence

identity

with

PuOMr

which

is

the

only

bacterial

putrescine

oxidase

that

has

been

characterized

so

far

A

BLAST

search

with

the

amino

acid

sequence

of

PuORh

resulted

in

a

high

number

of

homologous

-LRB-

putative

-RRB-

flavin-containing

amine

oxidases

which

are

widely

distributed

among

most

kingdoms

of

life

The

seven

closest

homologs

-LRB-

sequence

identity

-RRB-

can

all

be

found

in

actinomycetes

These

sequences

all

contain

the

active

site

glutamate

-LRB-

-RRB-

involved

in

substrate

binding

the

alanine

residue

-LRB-

-RRB-

which

excludes

covalent

FAD

binding

and

the

residues

which

limit

the

size

of

the

active

site

cavity

-LRB-

and

-RRB-

Most

likely

they

represent

a

clade

of

orthologous

putrescine

oxidases

which

are

not

active

with

monoamines

within

the

family

of

-LRB-

putative

-RRB-

flavin-containing

amine

oxidases

-LRB-

Supplementary

information

ESM

-RRB-

PuORh

shares

some

properties

with

PuOMr

Both

enzymes

are

soluble

dimeric

proteins

of

approximately

kDa

Interestingly

both

contain

only

mol

of

non-covalently

bound

FAD

per

mole

of

dimer

which

is

rather

unique

among

flavoproteins

The

substrate

specificity

of

PuORh

is

very

narrow

like

PuOMr

the

enzyme

is

very

specific

for

putrescine

Polyamines

are

also

accepted

but

aliphatic

monoamines

are

not

converted

at

all

In

general

PuORh

appears

to

have

a

higher

affinity

for

its

substrates

than

PuOMr

-LRB-

Km

for

putrescine

vs

μM

-RRB-

The

narrow

substrate

specificity

of

PuOMr

for

di

and

polyamines

was

already

rationalized

by

the

suggestion

of

the

presence

of

an

anionic

point

in

the

active

site

This

anionic

point

likely

due

to

a

carboxylate

function

binds

one

positively

charged

amino

group

of

the

substrate

while

another

amino

group

is

oxidized

-LRB-

Swain

and

Desa

Okada

et

al.

-RRB-

Based

on

sequence

alignment

between

PuORh

and

PuOMr

and

a

structural

model

of

PuORh

we

can

now

identify

as

the

anionic

point

in

PuOMr

The

presence

of

a

carboxylate

function

as

a

manner

to

bind

a

positively

charged

amino

group

is

not

restricted

to

putrescine

oxidase

In

polyamine

oxidase

-LRB-

PAO

-RRB-

a

negatively

charged

carboxylate

ring

can

be

found

at

one

side

of

its

substrate

tunnel

to

guide

polyamine

substrate

molecules

into

the

active

site

-LRB-

Binda

et

al.

-RRB-

In

addition

in

PAO

two

glutamate

residues

-LRB-

and

-RRB-

are

present

in

the

active

site

opposite

to

the

of

the

flavin

However

these

residues

have

been

suggested

to

be

protonated

-LRB-

Binda

et

al.

-RRB-

Moreover

in

PAO

and

are

in

close

proximity

to

a

substrate

secondary

amine

group

which

will

be

oxidized

while

in

PuORh

interacts

with

the

-LRB-

primary

-RRB-

amine

group

which

will

not

be

oxidized

The

amine

group

to

be

oxidized

in

PuORh

is

placed

between

and

near

the

of

the

isoalloxazine

ring

This

structural

feature

resembles

the

aromatic

cage

found

in

other

flavin-containing

amine

oxidases

like

MAO-A

-LRB-

De

Colibus

et

al.

-RRB-

MAO-B

-LRB-

Binda

et

al.

-RRB-

and

PAO

-LRB-

Binda

et

al.

-RRB-

For

MAO-B

this

aromatic

cage

has

been

shown

to

play

a

steric

role

in

substrate

binding

and

in

flavin

accessibility

and

helps

to

increase

the

substrate

amine

nucleophilicity

-LRB-

Li

et

al.

-RRB-

Polyamines

like

putrescine

cadaverine

spermine

and

spermidine

are

related

to

cell

growth

and

differentiation

processes

Increased

levels

in

body

fluids

and

tissue

occur

in

cancer

patients

and

therefore

polyamines

represent

important

cancer

markers

-LRB-

Casero

and

Marton

-RRB-

Polyamines

are

also

used

to

monitor

food

freshness

as

an

increased

level

can

be

found

in

spoiled

food

due

to

amino

acid

degradation

-LRB-

Bardócz

-RRB-

Due

to

efficient

heterologous

expression

and

its

catalytic

properties

PuORh

may

develop

as

a

valuable

diagnostic

enzyme

for

the

detection

of

low

amounts

of

putrescine

and

polyamines

Moreover

based

on

the

structural

model

enzyme

redesign

may

also

allow

creation

of

mutants

that

can

be

used

for

synthetic

purposes

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

ESM

Unrooted

phylogenetic

tree

representation

of

PuORh

homologs

which

exhibit

over

sequence

identity

-LRB-

at

the

amino

acid

level

-RRB-

throughout

the

whole

sequence

In

gray

the

clade

of

putrescine

oxidases

MAO-B

Human

monoamine

oxidase

B

PuORh

putrescine

oxidase

from

Rhodococcus

erythropolis

NCIMB

PuOMr

putrescine

oxidase

from

Micrococcus

rubens

-LRB-

DOC

kb

-RRB-

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

putrescine oxidase
flavin
amine
activity screening


Serologic and immunohistochemical prognostic biomarkers of cutaneous malignancies Biomarkers are important tools in clinical diagnosis and prognostic classification of various cutaneous malignancies . Besides clinical and histopathological aspects -LRB- e.g. anatomic site and type of the primary tumour , tumour size and invasion depth , ulceration , vascular invasion -RRB- , an increasing variety of molecular markers have been identified , providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities , up to even changing existing classification systems . Recently published gene expression or proteomic profiling data relate to new marker molecules involved in skin cancer pathogenesis , which may , after validation by suitable studies , represent future prognostic or predictive biomarkers in cutaneous malignancies . We , here , give an overview on currently known serologic and newer immunohistochemical biomarker molecules in the most common cutaneous malignancies , malignant melanoma , squamous cell carcinoma and cutaneous lymphoma , particularly emphasizing their prognostic and predictive significance . Introduction Biomarkers play an important role in the diagnosis and prognostic classification of various cancer entities and can moreover be useful in monitoring the patient 's clinical course of disease and response to therapy . In general , biomarkers are proteins , less often carbohydrates or lipids , and their expression profiles are associated with malignant disease . In the majority of cases , the marker molecules are expressed by the tumour cells themselves or by cells of the tumour microenvironment . Thus , most biomarkers can primarily be found in malignant tissues , but after active secretion or passive release at tumour cell destruction also become detectable in body fluids like blood , lymph or urine . Besides morphological and histopathological biomarkers -LRB- anatomic site and type of the primary tumour , tumour size and invasion depth , ulceration , vascular invasion -RRB- , an increasing variety of molecular markers have been identified , providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities , up to even changing existing classification systems . Recently published gene expression , as well as proteomic profiling data , indicates new candidate molecules involved in skin cancer pathogenesis , which may after further validation represent new markers superior to existing ones . This ongoing process of biomarker identification and validation would result in a rapidly changing molecular view and classification of skin cancers . Malignant melanoma Serologic markers of malignant melanoma Despite numerous therapeutic options , the prognosis of malignant melanoma , once metastasized , is still poor . Thus , the search for reliable methods to detect metastases as early as possible and to identify patients with high risk of disease progression is of major importance . The serological parameters most widely used for the early detection of a tumour relapse or metastasis in the follow-up of melanoma patients are the melanocyte lineage/differentiation antigens S100-beta and melanoma inhibitory activity -LRB- MIA -RRB- -LRB- see Table 1 and Fig. 1 -RRB- . Both proteins are with high , but not exclusive , specificity expressed by melanoma cells and thus correlate with the patient 's tumour load . Table 1Serologic markers of malignant melanomaSerologic markerSelected literatureMelanocyte lineage/differentiation antigensS100-beta -LSB- 30 , 71 , 32 , 33 , 18 , 43 , 28 -RSB- MIA -LRB- melanoma inhibitory activity -RRB- -LSB- 9 , 8 , 10 , 74 , 28 -RSB- Tyrosinase -LSB- 2 -RSB- 5-S-Cysteinyldopa -LSB- 91 , 34 -RSB- L-Dopa/L-tyrosine -LSB- 75 -RSB- Proangiogenic factorsVEGF -LRB- vascular endothelial growth factor -RRB- -LSB- 83 , 56 , 12 -RSB- BFGF -LRB- basic fibroblast growth factor -RRB- -LSB- 83 , 12 -RSB- IL-8 -LRB- Interleukin-8 -RRB- -LSB- 68 , 83 , 12 -RSB- Molecules involved in cell adhesion and motilitysICAM-1 -LRB- soluble intracellular adhesion molecule 1 -RRB- -LSB- 34 , 87 , 94 -RSB- sVCAM -LRB- soluble vascular cell adhesion molecule 1 -RRB- -LSB- 26 , 87 -RSB- Matrix metalloproteinases -LRB- MMP -RRB- -1 and 9 -LSB- 63 , 53 -RSB- Cytokines and cytokine receptorsIL-6 -LRB- Interleukin-6 -RRB- -LSB- 50 , 73 -RSB- IL-10 -LRB- Interleukin-10 -RRB- -LSB- 21 , 51 -RSB- sIL-2R -LRB- soluble interleukin-2-receptor -RRB- -LSB- 11 , 58 -RSB- HLA moleculessHLA-DR -LRB- soluble HLA-DR -RRB- -LSB- 62 -RSB- sHLA-class-I -LRB- soluble HLA-class I -RRB- -LSB- 89 -RSB- OthersLDH -LRB- lactate dehydrogenase -RRB- -LSB- 72 , 18 , 6 -RSB- CRP -LRB- C-reactive protein -RRB- -LSB- 19 -RSB- Albumin -LSB- 72 -RSB- TuM2-PK -LRB- Tumour pyruvate kinase type M2 -RRB- -LSB- 81 -RSB- sFas/CD95 -LSB- 84 -RSB- YKL-40 -LSB- 69 , 70 -RSB- CYT-MAA -LRB- cytoplasmic melanoma-associated antigen -RRB- -LSB- 85 -RSB- HMW-MAA -LRB- high-molecular-weight melanoma-associated antigen -RRB- -LSB- 85 -RSB- Fig. 1Schematic presentation of the cellular localization and function of the melanoma biomarkers S100-beta , tyrosinase and melanoma inhibitory activity -LRB- MIA -RRB- The S100 protein is a 21-kDa thermo-labile acidic dimeric protein , which was originally isolated from the central nervous system -LRB- CNS -RRB- -LSB- 49 -RSB- . It consists of two subunits , alpha and beta , in the combinations alpha/alpha , alpha/beta and beta/beta . S100 affects the assembly and disassembly of microtubules and also interacts in a calcium-dependent manner with the p53 tumour suppressor gene . The beta subunit is expressed in cells of the central nervous system as well as in cells of the melanocytic lineage . Therefore , S100-beta measured in the cerebrospinal fluid was known as a marker of CNS damage -LSB- 59 -RSB- , years before S100-beta was shown to be a useful serum marker in melanoma -LSB- 30 -RSB- . MIA was originally detected in melanoma cell culture supernatant -LSB- 9 -RSB- and was shown to exert an important role in cell -- matrix interaction and metastasis -LSB- 8 -RSB- . Serum S100-beta has been shown to be superior compared to MIA , as an early indicator of tumour progression , relapse or metastasis -LSB- 18 , 43 -RSB- , and its distribution as a serum biomarker of melanoma , therefore , is the broadest -LSB- 36 -RSB- . Both markers have been shown to be useful prognostic markers in melanoma patients with distant metastases -LRB- stage IV , classification system of the American Joint Committee on Cancer , AJCC , see Fig. 2 -RRB- -LSB- 71 , 33 -RSB- , but fail to provide prognostic significance in early stages of melanoma , especially in patients who are tumour-free after surgical procedures -LSB- 74 -RSB- . Because of the strong correlation of their serum concentrations with the patients ' tumour load , S100-beta and MIA are useful markers in the monitoring of therapy response in advanced metastatic melanoma patients -LRB- AJCC stage IV -RRB- -LSB- 32 -RSB- . Again , none of both markers are suitable indicators of therapy response in tumour-free early-stage melanoma patients -LRB- AJCC stage II or III -RRB- . Moreover , S100-beta has been shown to fail to identify patients with lymph node micrometastases detected by sentinel node procedure -LSB- 1 -RSB- . Fig. 2Kaplan -- Meier survival curves of 65 patients with advanced metastatic melanoma -LRB- AJCC stage IV -RRB- . Patients with elevated serum levels of S100-beta show a significantly reduced survival probability compared to patients with normal S100-beta serum concentrations -LRB- P = 0.003 by log rank test ; detection system LIAISON Sangtec 100 -RRB- The strongest prognostic serum biomarker in advanced metastatic melanoma is lactate dehydrogenase -LRB- LDH -RRB- , an unspecific marker indicating high tumour load in a variety of tumour entities , including melanoma . Studies comparing LDH , S100-beta and MIA using multivariate data analysis showed LDH to be the strongest independent prognostic factor in stage IV melanoma patients -LSB- 18 -RSB- . Due to its high prognostic significance , coupled with its easy , cost-efficient and widely distributed detection methodology , serum LDH is the only molecular marker that has been included in the current melanoma staging and classification system of the AJCC -LSB- 6 -RSB- . It , moreover , serves as a stratification parameter in most randomized therapy trials performed in stage-IV melanoma and may also be used to monitor therapy response in these patients . A variety of other molecules of peripheral blood have been described as markers of tumour load and disease progression in melanoma . These biomarkers are derived from different fields like melanocytic differentiation -LRB- e.g. tyrosinase ; see Fig. 1 -RRB- , tumour angiogenesis -LRB- e.g. VEGF , bFGF , IL-8 -RRB- , cell adhesion and motility -LRB- e.g. ICAM-1 , MMPs -RRB- , cytokines and their receptors -LRB- e.g. IL-6 , IL-10 -RRB- , antigen presentation -LRB- e.g. HLA molecules -RRB- , tumour cell metabolism -LRB- e.g. TuM2-PK -RRB- , apoptosis -LRB- e.g. Fas/CD95 -RRB- and many others -LRB- see Table 1 -RRB- . However , neither one of these markers could be confirmed to be superior to S100-beta or LDH in reflecting the prognosis of patients in advanced disease stages , nor could any marker be shown to be of strong prognostic relevance in early stage tumour-free patients . The serum proteomic profiling is an innovative approach to identify new , potentially better serological biomarkers in melanoma . This methodology offers the possibility of screening the whole serum proteome for markers , which match different criteria like prognostic significance , prediction of therapy response , etc. . Using this technology , marker proteins from thematic fields , different from the above-mentioned ones , might be found and thereafter validated for their clinical use . The first promising results have been obtained and are currently tested in large sets of serum samples -LSB- 46 -RSB- . Immunohistochemical markers of malignant melanoma Cutaneous malignant melanoma regularly develops from the radial to the vertical growth phase and thereafter to metastatic disease . The variability of this clinical course is only partially explained by morphological and histopathological parameters like primary tumour localization , patient gender and age , mitotic rate , tumour thickness and ulceration . There is a need to identify molecular variables , which help to assign patients to specific risk groups . The number of modalities for diagnosing and subclassifying malignant melanomas is rapidly increasing and includes immunohistochemistry of tissue sections and microarrays , gene expression profiling , comparative genomic hybridization and mutational analysis . These methodologies promise to improve our prognostic classification systems , as well as our diagnostic and therapeutic potential . For diagnostic purposes , a small panel of melanocytic lineage markers , e.g. S100 , MART-1 / MelanA and gp100/HMB45 , is sufficient to distinguish melanoma from non-melanocytic cancers . For the differentiation between benign and malignant melanocytic lesions , a review of immunohistochemical markers is given in ref . -LSB- 44 -RSB- . The present review focuses on newer markers with potential prognostic impact for the disease . For this purpose , the situation is more complex . The transformation from benign melanocytes to metastatic melanoma is the result of a compilation of genetic alterations crucial to cell division , differentiation , anti-apoptosis , invasion , angiogenesis and sustenance in a microenvironment distant from the point of origin of the cell . Several marker molecules involved in these genetic alterations have been identified , and their expression in primary melanoma has been studied and correlated with the prognosis . Table 2 gives a current overview on already identified biomarkers , whose abnormal expression is associated with the patient 's prognosis . It may be expected that the most detailed prognostic classification will result not from one , but rather from a panel of multiple biomarkers from this list . Table 2Immunohistochemical markers of malignant melanoma associated with prognosisAssociation with poor prognosisSelected literatureMelanocyte lineage/Differentiation antigens gp100/HMB45Increased expression -LSB- 52 -RSB- Tumour suppressors/oncogenes/signal transducers p16 INK4ADecreased expression -LSB- 47 , 3 -RSB- PTENDecreased expression -LSB- 48 -RSB- pRb -LRB- retinoblastoma protein -RRB- Inactivation due to protein phosphorylation -LSB- 65 -RSB- EGFR -LRB- epidermal growth factor receptor -RRB- Increased expression -LSB- 80 -RSB- p-Akt -LRB- activated serine-threonine protein kinase B -RRB- Increased expression -LSB- 17 -RSB- c-KitExpression -LSB- 35 , 82 -RSB- c-mycIncreased expression -LSB- 42 -RSB- AP-2 -LRB- activator protein-2alpha -RRB- transcription factorLoss of nuclear AP-2 expression -LSB- 7 -RSB- HDM2 -LRB- human homologue of murine mdm2 -RRB- Increased expression -LSB- 61 -RSB- bcl-6Expression -LSB- 3 -RSB- Cell cycle associated proteins Ki67 -LRB- detected by Mib1 -RRB- Increased expression -LSB- 29 , 3 , 57 -RSB- Cyclin A , B , D , EIncreased expression -LSB- 24 , 25 -RSB- p21CIP1Decreased expression -LSB- 3 -RSB- GemininIncreased expression -LSB- 92 -RSB- PCNA -LRB- proliferating cell nuclear antigen -RRB- Increased expression -LSB- 92 -RSB- Regulators of apoptosis bcl-2Increased expression -LSB- 78 -RSB- bax Decreased expression -LSB- 23 -RSB- BakDecreased expression -LSB- 23 -RSB- APAF-1 -LRB- Apoptotic protease activating factor-1 -RRB- Decreased expression -LSB- 27 -RSB- SurvivingIncreased expression -LSB- 78 -RSB- Molecules involved in angiogenesis LYVE-1 -LRB- lymphatic vascular endothelial hyaluronan receptor-1 -RRB- Increased expression -LSB- 16 -RSB- PTN -LRB- pleiotrophin -RRB- Increased expression -LSB- 93 -RSB- Molecules involved in cell adhesion and motility P-CadherinStrong cytoplasmic expression -LSB- 5 -RSB- E-CadherinDecreased expression -LSB- 4 -RSB- Beta-cateninLoss of nuclear staining -LSB- 5 -RSB- Integrins beta1 and beta3 Increased expression -LSB- 66 -RSB- MMPs -LRB- matrix metalloproteinases -RRB- Increased expression -LSB- 63 -RSB- DysadherinIncreased expression -LSB- 54 -RSB- CEACAM1 -LRB- carcinoembryonic-antigen-related cell-adhesion molecule 1 -RRB- Increased expression -LSB- 79 -RSB- Osteonectin -LRB- also termed BM40 or SPARC -LRB- secreted protein , acidic and rich in cysteine -RRB- -RRB- Increased expression -LSB- 45 -RSB- Others TA -LRB- telomerase activity -RRB- Increased expression -LSB- 13 -RSB- MelastatinDecreased expression -LSB- 22 -RSB- ALCAM/CD166 -LRB- Activated leukocyte cell adhesion molecule -RRB- Increased expression -LSB- 77 -RSB- CXCR4 receptorIncreased expression -LSB- 67 -RSB- MetallothioneinIncreased expression -LSB- 88 -RSB- In a recent retrospective study , frozen tissue samples from primary melanomas with long-term clinical follow-up were analysed using a pangenomic oligonucleotide microarray -LSB- 92 -RSB- . The authors describe a signature of 174 genes , identifying patients at risk to develop distant metastasis . From these 174 genes , 141 were under-expressed and 33 were over-expressed in primary melanomas of patients who remained free of metastasis for 4 years . About 30 of these 174 genes have already been studied in melanoma and include genes involved in cell cycle -LRB- CKS2 , CDC2 , CCNB1 , CENPF and DHFR -RRB- , mitosis -LRB- HCAP-G and STK6 -RRB- , mitotic spindle checkpoint -LRB- BUB1 -RRB- , inhibition -LRB- BIRC5 -RRB- or stimulation -LRB- GPR105 -RRB- of apoptosis , DNA replication -LRB- TOP2A , RRM2 , TYMS , PCNA , MCM4 and MCM6 -RRB- , stress response -LRB- GLRX2 , DNAJA1 , HSPA4 , HSPA5 , HSPD1 and TXNIP -RRB- , ubiquitin cycle -LRB- SIP -RRB- , actin and calmodulin binding -LRB- CNN3 -RRB- , intracellular signalling -LRB- STMN2 -RRB- , negative regulation of the Wnt signaling pathway -LRB- CTNNBIP1 -RRB- , inhibition of MITF expression -LRB- EMX2 -RRB- , regulation of proteolysis -LRB- TNA -RRB- , testis cancer -LRB- CML66 -RRB- and metastasis suppression -LRB- NME1 -RRB- . Winnepenninckx et al. -LSB- 92 -RSB- recommend the use of a battery of antibodies that may improve the future determination of prognosis and treatment stratification . The determination of karyopherin-alpha2 , MCMs -LRB- minichromosome maintenance proteins -RRB- , geminin and PCNA over-expression could be used for screening in melanoma patients with a poor clinical outcome . Squamous cell carcinoma of the skin While primary cutaneous squamous cell carcinomas -LRB- SCC -RRB- are usually easily treatable , they have the potential to recur locally and even metastasize , then leading to a significant morbidity and mortality . Therefore , it is important to identify those tumours that are more aggressive and require closer follow-up and additional treatments , such as lymph adenectomy or radiation therapy . Established prognostic factors include anatomic site of primary , tumour size , depth of invasion , rapid growth , grade of differentiation , perineural invasion , history of previous treatment , host immunosuppression , and etiologic factors such as burn scars , radiation and chronic ulceration . The histological subtypes of SCC have also been considered as a factor in determining the prognosis -LSB- 60 -RSB- . Only few molecular markers are known to be associated with progression or prognosis of cutaneous SCC . In the following , we give an overview on some recently described proteins whose abnormal expression contributes to a malignant phenotype in this cancer entity . STAT3 , a member of the signal transducer and activator of transcription -LRB- STAT -RRB- family of transcription factors is a known regulator of cell motility . The expression of phosphorylated STAT3 -LRB- p-STAT3 -RRB- was described to be stronger in poorly differentiated than in well-differentiated SCCs . Moreover , the percentage of tumour cells expressing p-STAT3 correlated with the depth of tumour invasion and with metastasis formation -LSB- 76 -RSB- . E-Cadherin is a Ca -LRB- 2 + -RRB- - dependent , intercellular adhesion molecule that is specifically expressed in epithelial cells and tissues and functions by maintaining intercellular connections . In some types of carcinomas , E-cadherin expression of tumour cells is decreased in association with metastasis . In cutaneous SCC , a decreased expression of E-cadherin in the primary lesion is correlated with the development of regional lymph node metastasis -LSB- 41 -RSB- . Additionally , a decreased expression is more often associated with well-differentiated than with poorly differentiated SCC . Therefore , E-cadherin might be useful as a marker for the metastatic potential of well-differentiated SCC -LSB- 41 -RSB- . The simultaneous detection of p-STAT3 and E-cadherin may contribute to the prediction of prognosis of cutaneous SCC patients -LSB- 76 -RSB- . Another marker to distinguish between well-differentiated and poorly differentiated SCC is Ets-1 -LSB- 37 -RSB- . Ets-1 is a transcription factor regulating the expression of various genes including matrix metalloproteinases -LRB- MMPs -RRB- . Therefore , Ets-1 might be important in the pathogenesis of invasive SCC . MMP-12 was found to be expressed by tumour cells in squamous cell carcinoma of the vulva . Its expression correlates with invasiveness , while that of macrophages predict a better clinical outcome -LSB- 38 -RSB- . A cell surface marker , CD44 , is a glycoprotein widely distributed in the extracellular matrix . CD44 isoforms , which arise from alternative mRNA splicing , were found to be implicated in the formation of tumour metastasis . In a study by Rodriguez-Rodriguez et al. , it was shown that lymph node metastases of cutaneous SCC of the vulva were immunoreactive for CD44 -- 9v -LSB- 64 -RSB- . Also , CD44 -- 10v expression was present in 78 % of tumours compared to only 56 % of normal epithelium . CD44 -- 10v membrane expression , but not cytoplasmic expression , correlates with disease recurrence -LSB- 64 -RSB- . In ocular squamous cell carcinomas , over-expression of CD44 -- 6v is correlated with tumour progression and metastasis -LSB- 55 -RSB- . Cutaneous T-cell lymphomas Cutaneous T-cell lymphomas -LRB- CTCL -RRB- are a heterogeneous group of cutaneous non-Hodgkin 's lymphomas . In this cutaneous malignancy , the tumour cells home to and persist in the skin , producing a broad spectrum of clinical entities . The prognosis of CTCL depends on histologic and molecular aspects . The new WHO/EORTC classification for cutaneous lymphomas comprises mature T-cell and natural killer -LRB- NK -RRB- - cell lymphomas , mature B-cell lymphomas and immature haematopoietic malignancies . Marker proteins for the diagnosis of CTCL include , for example , CD2 , CD3 , CD4 , CD5 , CD7 , CD8 , CD14 , CD16/56 , CD19 , CD25 , CD45 , CD45RA and CD45R0 -LSB- 90 -RSB- . The probability of survival in CTCL can be accurately predicted by a formula based on the clinical CTCL-Severity-Index -LRB- CTCL-SI -RRB- -LSB- 20 -RSB- , which evaluates the involvement of the skin , lymph nodes , blood and visceral organs -LSB- 40 -RSB- . Besides clinical and morphological parameters , several molecules have been investigated in CTCL that are involved in general cellular signalling processes , regulation of cellular proliferation and apoptosis , like Jun , Myc , c-myb , p53 , STATs , bcl-2 , Fas/CD95 and SOCS-3 , or contribute to the putative immunopathology of the disease such as expression of inhibitory MHC receptors -LRB- ILT2/CD85j -RRB- , NK receptors -LRB- p140/KIR3DL2 -RRB- and dendritic cell defects -LRB- CD40 -RRB- . The abnormal expression of these molecules could be relevant for the prognosis of CTCL , as it has been shown for other tumour entities -LSB- 39 -RSB- . With regard to serological biomarkers in CTCL , it has been shown that the serum concentrations of the soluble alpha-chain of the interleukin-2 receptor -LRB- sIL-2R -RRB- as well as lactate dehydrogenase -LRB- LDH -RRB- strongly correlate with lymph node size , but only sIL-2R significantly correlates with the severity of skin manifestations in erythrodermic patients -LSB- 86 -RSB- . Moreover , sIL-2R was demonstrated to be produced at a relatively low rate by tissue-based lymphoma cells , whereas large-cell transformation in CTCL results in a marked increase in the sIL-2R production in some patients -LSB- 86 -RSB- . In addition to sIL-2R , neopterin and beta2-microglobulin have been shown to be significantly elevated in the serum from patients with Sezary syndrome . Thus , sIL-2R seems to be the most sensitive marker , which is typically increased in Sezary syndrome . Concerning the outcome of the disease , in terms of disease progression versus non-progression , only neopterin showed a significant prognostic value in non-leukemic CTCL patients -LSB- 31 -RSB- . Conclusion and future directions Taken together , molecular markers provide additional and much more detailed information for the prognostic classification of cutaneous malignancies . Currently , this is particularly true for malignant melanoma , but will certainly also affect other entities in due time . In addition to the serological and immunhistochemical biomarkers discussed here , genetic abnormalities have recently been recognized to influence the prognosis of cancer patients in higher extents than previously assumed . With regard to malignant melanoma , a new classification system was proposed combining genetic aberrations with histomorphological changes , resulting in new insights into the pathogenesis of this malignancy -LSB- 14 , 15 -RSB- . It may be expected that the rapidly increasing knowledge of molecular mechanisms would lead to mainly biomarker-based , rather than morphology-based , classification systems that might facilitate an individualized , molecular-driven cancer therapy .

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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 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 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 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 B 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 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 B 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 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 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 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 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 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 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

skin cancer
biomarker
prognosis
serum
immunohistochemistry