Understanding

Marine

Mussel

Adhesion

In

addition

to

identifying

the

proteins

that

have

a

role

in

underwater

adhesion

by

marine

mussels

research

efforts

have

focused

on

identifying

the

genes

responsible

for

the

adhesive

proteins

environmental

factors

that

may

influence

protein

production

and

strategies

for

producing

natural

adhesives

similar

to

the

native

mussel

adhesive

proteins

The

production-scale

availability

of

recombinant

mussel

adhesive

proteins

will

enable

researchers

to

formulate

adhesives

that

are

water-impervious

and

ecologically

safe

and

can

bind

materials

ranging

from

glass

plastics

metals

and

wood

to

materials

such

as

bone

or

teeth

biological

organisms

and

other

chemicals

or

molecules

Unfortunately

as

of

yet

scientists

have

been

unable

to

duplicate

the

processes

that

marine

mussels

use

to

create

adhesive

structures

This

study

provides

a

background

on

adhesive

proteins

identified

in

the

blue

mussel

Mytilus

edulis

and

introduces

our

research

interests

and

discusses

the

future

for

continued

research

related

to

mussel

adhesion

Introduction

to

Biological

Adhesion

Biomimetics

Biomimetic

materials

are

designed

to

mimic

compounds

with

a

biological

origin

The

biomaterial

development

field

is

actively

pursuing

the

use

of

various

synthetic

and

natural

protein

and

carbohydrate-containing

compounds

The

production

of

ecologically

friendly

materials

also

is

of

importance

as

a

result

of

a

rapid

increase

in

the

world

's

population

and

the

subsequent

environmental

problems

associated

with

product

wastes

-LRB-

Arora

and

Arora

-RRB-

The

key

interest

in

biomimetic

adhesives

is

the

interaction

between

the

material

and

a

surface

whether

that

surface

is

of

biological

or

nonbiological

origin

Scientific

studies

in

the

interdisciplinary

field

of

biomaterial

research

include

areas

such

as

surface

characterization

protein

adsorption

and

adhesion

behavior

-LRB-

Kirkpatrick

et

al.

-RRB-

Table

briefly

describes

biomolecules

with

adhesive

properties

used

for

biomimetic

research

Many

of

the

proteins

are

scleroproteins

which

contribute

mechanical

strength

to

supporting

structures

in

animals

Waite

-LRB-

b

-RRB-

has

commented

on

the

durability

of

these

proteins

in

terms

of

relative

insolubility

resistance

to

proteolytic

hydrolysis

or

other

chemical

dissolution

Repetitive

amino

acid

motifs

and

intermolecular

and

intramolecular

cross-linking

are

common

DOPA-containing

scleroproteins

produced

through

posttranslational

modification

of

tyrosine

by

various

organisms

leads

to

further

stabilization

or

curing

of

scleroproteins

involved

in

adhesion

and/or

protection

of

more

flexible

structural

proteins

Table

Research

targets

for

biomaterial

developmentBiological

TargetDescriptionKeratinA

hard

durable

insoluble

structural

protein

that

is

the

primary

component

of

horns

hoofs

feathers

skin

hair

and

nails

a

scleroproteinElastinAn

insoluble

protein

found

in

connective

tissue

and

known

for

its

elasticity

and

similarity

to

collagen

a

scleroproteinCollagenA

tough

insoluble

inelastic

protein

with

high

tensile

strength

that

serves

as

the

support

structure

in

skin

tendons

and

bone

a

scleroproteinSilksHigh

tensile

strength

protein

fibers

that

contain

various

proteins

-LRB-

fibroin

spidroin

-RRB-

most

commonly

from

spiders

and

silkwormsFibrin

-LRB-

and

other

coagulation

system

proteins

-RRB-

A

sticky

insoluble

clot-forming

protein

formed

by

constituents

in

the

blood

a

scleroproteinChitinA

specialized

carbohydrate

containing

nitrogen

-LRB-

nitrogenous

polysaccharide

-RRB-

found

in

the

cell

walls

of

certain

fungi

and

in

the

exoskeletons

of

arthropodsCelluloseAn

insoluble

complex

carbohydrate

-LRB-

polysaccharide

composed

of

linked

glucose

units

-RRB-

main

constituent

of

the

cell

walls

of

plantsMucinA

nitrogenous

conjugated

protein

-LRB-

protein

linked

to

a

sugar

-RRB-

found

in

mucous

secretions

acts

as

a

lubricant

and

protects

body

surfaces

The

production

of

an

underwater

adhesive

that

mimics

the

properties

of

marine

mussels

is

a

challenge

that

has

received

considerable

attention

Many

of

the

mussel

adhesive

proteins

identified

to

date

are

polyphenolic

proteins

Polyphenolic

proteins

are

nontoxic

biodegradable

and

have

low

immunogenic

qualities

that

make

them

highly

attractive

for

environmental

medical

and

industrial

purposes

Biological

Adhesion

Naturally

produced

adhesives

are

common

in

many

biological

systems

and

are

known

for

their

superior

strength

and

durability

compared

with

man-made

materials

Examples

of

specialized

biological

systems

that

generate

a

vast

amount

of

adhesives

research

include

bacteria

spiders

marine

tubeworms

sea

cucumbers

barnacles

and

mussels

Many

bacteria

synthesize

exopolysaccharides

extracellular

protective

adhesive

matrixes

Bacteria

can

form

layers

or

biofilms

on

a

variety

of

surfaces

by

embedding

cells

in

exopolysaccharides

The

polysaccharide-based

holdfast

of

the

aquatic

bacterium

Caulobacter

crescentus

has

been

shown

recently

to

demonstrate

the

strongest

adhesive

force

measured

in

bacteria

-LRB-

Tsang

et

al.

-RRB-

Spiders

also

express

adhesive

proteins

by

synthesizing

different

types

of

silk

all

of

which

have

high

tensile

strength

extensibility

and

an

energy-dissipative

viscoelastic

response

that

is

not

matched

by

synthetic

polymers

-LRB-

Zhou

et

al.

Vollrath

-RRB-

Current

research

with

spider

silks

involves

the

production

of

spider

silk

in

other

organisms

a

technique

known

as

recombinant

protein

expression

-LRB-

Piruzian

et

al.

-RRB-

Many

marine

organisms

have

developed

adhesive

strategies

to

deal

with

the

dynamic

ocean

environment

particularly

at

the

tidal

interface

Marine

invertebrates

attach

permanently

or

temporarily

to

inanimate

and

sometimes

living

surfaces

Therefore

adhesive

mechanisms

have

evolved

to

promote

attachment

The

marine

polychaete

Phragmatopoma

californica

is

a

tubeworm

that

builds

protective

tubes

with

secreted

proteinaceous

cement

mixed

with

shells

and

sand

particles

from

the

sea

floor

The

cement

adheres

rapidly

to

a

variety

of

materials

in

seawater

-LRB-

Stewart

et

al.

Zhao

et

al.

-RRB-

The

sea

cucumber

-LRB-

Holothuria

forskali

-RRB-

reacts

defensively

through

the

discharge

of

Cuvierian

tubules

that

ensnare

their

threat

Biochemical

analysis

of

the

tubules

indicates

a

protein

to

carbohydrate

ratio

with

a

high

proportion

of

highly

insoluble

protein

The

soluble

protein

component

appears

to

contain

up

to

ten

glycine

and

acidic

amino

acid-rich

proteins

ranging

in

size

from

to

kilodaltons

-LRB-

DeMoor

et

al.

-RRB-

Barnacles

adhere

directly

and

permanently

to

surfaces

such

as

underwater

substrata

-LRB-

ship

hulls

oil

platforms

and

pipelines

-RRB-

Initial

attachment

of

barnacle

cyprid

larvae

is

via

o-quinone

cross-linking

that

resembles

the

dihydroxyphenylalanine

-LRB-

DOPA

-RRB-

containing

adhesive

proteins

of

Mytilus

spp.

-LRB-

Wiegemann

-RRB-

but

the

adult

barnacle

cement

is

substantially

different

comprised

of

three

groups

of

proteins

that

contain

high

levels

of

the

amino

acids

serine

threonine

glycine

and

alanine

-LRB-

Kamino

et

al.

Wiegemann

-RRB-

A

pattern

of

short

alternating

regions

of

hydrophobic

and

hydrophilic

residues

throughout

the

largest

of

the

three

groups

of

proteins

has

been

noted

and

suggested

that

these

alternating

motifs

may

have

a

role

in

assembly

in

seawater

-LRB-

Kamino

et

al.

-RRB-

Marine

and

freshwater

mussel

adhesive

proteins

differ

from

barnacle

cement

proteins

because

of

the

presence

of

repetitive

amino

acid

motifs

-LRB-

characterized

by

a

high

polyphenolic

content

-RRB-

high

levels

of

the

modified

amino

acid

and

chemical

modifications

-LRB-

hydroxylations

-RRB-

to

specific

amino

acids

-LRB-

Burzio

et

al.

-RRB-

At

least

ten

different

adhesive-related

proteins

from

the

marine

mussel

M.

edulis

have

been

identified

reflecting

the

greater

ease

of

working

with

the

adhesive

structures

of

this

organism

and

also

its

popularity

as

a

target

for

biomimetic

research

Marine

mussels

such

as

the

blue

mussel

M.

edulis

attach

to

a

variety

of

surfaces

in

an

aqueous

environment

by

using

a

natural

adhesive

that

is

incredibly

strong

and

durable

There

are

no

synthetic

glues

that

can

be

similarly

applied

in

an

aqueous

environment

and

are

impervious

to

water

and

turbulent

forces

Previous

research

has

shown

that

one

of

the

proteins

in

the

adhesive

Mytilus

edulis

foot

protein

-LRB-

-RRB-

bonds

to

glass

plastic

wood

concrete

and

Teflon

Nine

other

adhesive-related

proteins

from

M.

edulis

have

been

identified

to

date

A

tenth

is

implicated

but

has

not

been

isolated

The

precise

mechanism

for

assembly

of

the

ten

collagens

such

as

precollagen-D

P

-LRB-

variant

and

-RRB-

and

NG

proximal

matrix

thread

protein

-LRB-

and

a

-RRB-

and

a

polyphenol

oxidase

is

not

understood

There

also

may

be

additional

proteins

involved

in

the

formation

of

the

adhesive

Figure

illustrates

adhesion

of

M.

edulis

to

seaweed

other

mussels

and

a

stainless

steel

surface

Figure

edulis

attachment

to

-LRB-

a

-RRB-

seaweed

-LRB-

b

-RRB-

other

mussels

and

-LRB-

c

-RRB-

a

stainless

steel

surface

Adhesion

Mechanisms

in

Mussels

The

mollusk

byssus

evolved

to

anchor

postlarval

organisms

during

metamorphosis

Some

species

of

mussels

have

retained

the

byssus

in

the

adult

animal

permitting

a

transition

from

a

bottom-dwelling

existence

to

an

epibenthic

lifestyle

Within

the

Mytilidae

examples

of

mussels

across

this

range

of

marine

lifestyles

can

be

observed

from

the

burrowing

Geukensia

demissa

to

Mytilus

edulis

and

Bathymodiolus

childressi

which

live

entirely

exposed

attached

to

hard

surfaces

and

intermediate

species

such

as

Modiulus

modiolus

which

can

survive

under

mixed

conditions

-LRB-

Brazee

and

Carrington

-RRB-

Common

features

of

the

byssus

in

the

Mytilidae

include

a

root

attached

to

the

byssal

retractor

muscle

a

stem

extending

from

the

root

and

individual

byssal

threads

which

are

attached

to

overlapping

cuffs

of

the

root

The

strength

of

mussel

adhesive

scleroproteins

is

attributed

to

the

introduction

of

cross-links

between

polymer

chains

of

individual

adhesive

proteins

-LRB-

a

process

called

curing

-RRB-

Curing

or

hardening

of

the

polyphenolic

mussel

proteins

is

believed

to

require

a

catecholic

precursor

-LRB-

e.g.

DOPA

-RRB-

and

the

presence

of

a

catechol

oxidase

-LRB-

Waite

-RRB-

Individual

adhesive

proteins

from

mussels

are

produced

by

the

foot

organ

of

the

animal

The

proteins

are

stockpiled

in

the

foot

then

secreted

or

released

into

the

byssal

groove

which

creates

a

template

for

thread

and

plaque

formation

to

form

strong

attachments

underwater

The

reactive

oxidized

form

of

DOPA

quinone

is

thought

to

provide

the

moisture-resistance

characteristic

of

mussel

underwater

adhesion

-LRB-

Yu

and

Deming

Yu

et

al.

-RRB-

DOPA

is

formed

from

the

hydroxylation

of

tyrosine

residues

by

a

polyphenoloxidase

-LRB-

tyrosinase

-RRB-

DOPA

can

complex

with

metal

ions

and

oxides

-LRB-

-RRB-

and

semimetals

such

as

silicon

thus

explaining

its

ability

to

adhere

to

rocks

and

glass

-LRB-

Sever

et

al.

-RRB-

Sun

and

Waite

-LRB-

-RRB-

have

reported

an

incremental

posttranslational

modification

of

tyrosine

to

DOPA

from

the

proximal

end

to

distal

tips

of

threads

coupled

with

a

selective

localization

of

metals

-LRB-

aluminum

calcium

iron

and

silicon

-RRB-

and

an

associated

iron

gradient

The

sequestering

of

iron

occurs

during

feeding

rather

than

after

threads

have

been

formed

Thus

iron

is

incorporated

in

a

chemical

gradient

pattern

similar

to

that

of

DOPA

during

secretion

of

byssal

threads

and

plaques

The

identification

of

variants

of

the

DOPA-rich

adhesive

foot

protein

from

Mytilus

californianus

-LRB-

-RRB-

further

supports

the

intricate

role

of

DOPA

in

marine

mussel

adhesion

-LRB-

Zhao

et

al.

-RRB-

The

catecholic

content

of

mussel

adhesive

proteins

has

been

linked

to

hydrogen-bonding

and

metal-liganding

-LRB-

strong

chelating

-RRB-

capabilities

-LRB-

Monahan

and

Wilker

Deming

-RRB-

Examples

of

proteins

undergoing

the

o-quinone

intermediate

process

-LRB-

quinone

tanning

-RRB-

include

collagen

-LRB-

mussel

byssal

threads

-RRB-

cellulose

chitin

and

mineral

deposits

-LRB-

mussel

shells

-RRB-

Figure

illustrates

the

chemical

steps

for

hydroxylation

of

tyrosine

and

DOPA

residues

in

M.

edulis

polyphenolic

proteins

Figure

of

tyrosine

residues

in

M.

edulis

polyphenolic

proteins

Other

constituents

of

mussel

adhesive

proteins

include

lysine

and

glycine

Lysine

may

contribute

to

adhesion

via

ionic

bonding

to

negatively

charged

surfaces

such

as

collagen

and

acidic

polysaccharides

-LRB-

Suci

and

Geesey

Olivieri

et

al.

-RRB-

and

intermolecular

cross-linking

with

o-quinones

Glycine

may

contribute

to

adhesion

through

the

open

extended

conformation

it

imparts

on

protein

structures

Histidine

an

amino

acid

exhibiting

a

gradient

pattern

and

present

in

high

concentrations

in

the

distal

region

of

byssal

threads

has

been

correlated

to

transition

metal

content

-LRB-

zinc

or

copper

-RRB-

and

adhesion

in

marine

mussels

-LRB-

Waite

et

al.

-RRB-

The

exceptional

strength

of

mussel

adhesive

proteins

is

undoubtedly

the

result

of

the

repetitive

nature

of

many

of

the

individual

proteins

-LRB-

decapeptide

and

hexapeptide

repeats

in

and

hydroxyproline

repeats

in

collagens

-RRB-

the

modification

of

individual

amino

acids

-LRB-

e.g.

hydroxylation

of

proline

and

tyrosine

-RRB-

and

the

gradient

nature

of

byssal

attachment

devices

This

gradient

nature

was

first

noted

by

Brown

in

-LRB-

referenced

by

Brazee

and

Carrington

-RRB-

who

observed

corrugated

proximal

-LRB-

to

the

organism

-RRB-

and

smooth

distal

regions

The

development

of

our

understanding

of

the

transition

of

mechanical

properties

along

the

thread

length

from

the

more

flexible

proximal

region

close

to

the

soft

tissues

of

the

mussel

through

the

collagenous

main

body

of

the

thread

to

the

stiffer

less

extensible

distal

region

and

the

exquisite

fine

tuning

of

protein

components

to

impart

these

properties

has

been

concisely

reviewed

recently

-LRB-

Brazee

and

Carrington

-RRB-

Total

mussel

adhesive

strength

is

a

function

of

the

individual

adhesive

proteins

secreted

by

the

animal

their

association

with

other

proteins

their

distribution

along

the

thread

and

their

proximity

to

materials

of

differing

moduli

Details

of

the

individual

adhesive

protein

components

are

described

in

the

next

section

Components

of

Mussel

Adhesion

Attachment

in

Mussels

The

Byssus

The

byssus

an

exogenous

attachment

structure

was

first

described

in

-LRB-

Brown

-RRB-

High

concentrations

of

polyphenolic

proteins

-LRB-

e.g.

DOPA

-RRB-

the

presence

of

collagen

and

the

presence

of

a

catechol

oxidase

were

among

the

first

biochemical

observations

of

byssal

attachments

Environmental

factors

such

as

salinity

temperature

pH

season

and

substratum

choice

as

well

as

biological

factors

such

as

age

and

metabolic

state

of

the

animal

affect

the

efficiency

and

strength

of

byssal

attachment

to

surfaces

-LRB-

Van

Winkle

Crisp

et

al.

Carrington

-RRB-

The

stem

is

rooted

in

the

byssal

retractor

muscles

at

the

base

of

the

foot

organ

The

byssal

threads

flexible

structures

of

variable

dimensions

-LRB-

mm

diameter

cm

length

-RRB-

and

strength

originate

from

the

stem

A

byssal

thread

consists

of

a

flexible

collagenous

inner

core

surrounded

by

a

hardened

cured

polyphenolic

protein

Numerous

researchers

photographed

the

collagen

core

in

the

-LRB-

Brown

-RRB-

well

before

three

unique

collagenous

proteins

were

identified

and

characterized

by

Qin

and

Waite

-LRB-

-RRB-

The

outer

polyphenolic

protein

believed

to

undergo

a

quinone

tanning-type

reaction

with

a

specialized

catechol

oxidase

enzyme

is

Designation

of

this

byssal

thread

polyphenolic

adhesive

protein

as

well

as

subsequent

adhesive

proteins

identified

in

Mytilus

edulis

is

preceded

by

the

genus

and

species

The

byssal

structure

culminates

in

a

polyphasic

plaque

of

varying

size

dependent

on

both

the

size

of

the

animal

and

the

age

of

the

byssus

-LRB-

Crisp

et

al.

-RRB-

Plaques

are

commonly

only

mm

in

diameter

where

they

meet

the

thread

and

to

diameter

at

the

substrate

interface

Plaque

formation

occurs

from

the

deposition

of

proteins

that

originate

in

the

foot

organ

To

date

five

specialized

adhesive

proteins

have

been

identified

in

byssal

plaques

from

M.

edulis

and

Figure

illustrates

the

anatomy

of

M.

edulis

and

the

associated

byssus

structures

Figure

illustrates

the

location

of

the

adhesive-related

proteins

identified

in

the

byssus

of

M.

edulis

Figure

of

M.

edulis

mussel

and

byssus

structures.Figure

of

adhesive-related

proteins

identified

in

the

byssus

of

M.

edulis

Mechanical

Properties

of

Mussels

The

most

extensive

research

about

the

adhesive

mechanical

properties

of

mussels

has

been

with

M.

edulis

The

breaking

energy

of

intact

byssal

threads

is

reported

to

be

Jm

versus

tendon

-LRB-

Jm

to

Jm

-RRB-

and

silk

-LRB-

Jm

to

Jm

-RRB-

-LRB-

Denny

-RRB-

Bond

strengths

are

between

to

Nm

depending

on

the

surface

for

adhesion

-LRB-

Waite

-RRB-

Byssal

thread

strength

at

the

distal

portion

of

threads

is

as

strong

as

a

vertebrate

tendon

but

three

to

five

times

more

extensible

-LRB-

Qin

and

Waite

-RRB-

Byssal

thread

strength

at

the

proximal

portion

of

threads

is

weaker

but

to

times

more

extensible

Strain

energy

density

of

threads

approaches

that

of

silk

approximately

six

times

tougher

than

a

tendon

-LRB-

Smeathers

and

Vincent

Coyne

et

al.

-RRB-

Byssal

threads

can

recover

initial

length

and

stiffness

if

given

sufficient

relaxation

time

-LRB-

Bell

and

Gosline

-RRB-

the

most

studied

mussel

adhesive

protein

has

adhesive

properties

comparable

to

synthetic

cyanoacrylate

and

epoxy

resins

These

resin

adhesives

are

popular

because

of

their

high

bond

strengths

quick

polymerization

and

ability

to

bond

to

a

number

of

substrates

-LRB-

metals

glass

ceramic

-RRB-

-LRB-

Savla

Coover

and

McIntire

-RRB-

Research

related

to

the

mechanical

properties

of

individual

M.

edulis

adhesive

proteins

is

limited

because

of

the

difficulty

in

obtaining

large

quantities

of

isolated

proteins

for

adhesive

testing

Byssal

Thread

Proteins

Byssal

Thread

Polyphenolic

Protein

was

the

first

polyphenolic

protein

to

be

identified

in

the

mussel

byssus

-LRB-

Waite

and

Tanzer

Waite

-RRB-

The

primary

location

of

is

in

the

byssal

threads

cross-linked

via

a

polyphenol

oxidase

to

form

a

hardened

sheath

around

the

flexible

collagenous

inner

core

Byssal

plaques

contain

of

as

well

is

a

large

-LRB-

amino

acids

-RRB-

basic

hydrophilic

protein

with

very

little

secondary

structure

and

a

molecular

mass

of

kDa

based

on

mass

spectroscopy

-LRB-

Filpula

et

al.

-RRB-

Decapeptide

and

hexapeptide

repeats

containing

numerous

posttranslational

modifications

-LRB-

of

the

amino

acid

residues

are

hydroxylated

-RRB-

provided

the

first

indication

of

an

adhesive-related

protein

unlike

any

others

identified

in

nature

The

hexapeptide

repeat

is

made

up

of

the

amino

acids

AKPTYK

The

major

decapeptide

consensus

repeat

consisting

of

the

amino

acids

AKPSYP

P

TYK

-LRB-

in

which

P

represents

proline

Y

represents

DOPA

P

represents

and

P

represents

-RRB-

occurs

times

in

DOPA

residues

constitute

of

the

protein

-LRB-

Waite

-RRB-

The

open

conformation

of

the

protein

is

believed

to

allow

functional

groups

full

accessibility

for

cross-linking

interactions

with

other

proteins

and

a

variety

of

surfaces

including

glass

Teflon

and

metals

has

been

characterized

as

possessing

random

coil

conformations

with

helix-like

decapeptide

segments

under

physiologic

conditions

-LRB-

Haemers

et

al.

-RRB-

Our

laboratory

recently

has

deduced

complementary

DNA

-LRB-

cDNA

-RRB-

sequences

for

-LRB-

GenBank

Accession

number

and

-RRB-

Other

Mytilus

mussel

species

contain

a

protein

analogous

to

with

differences

in

the

decapeptide

repeat

frequency

residue

composition

and

nonrepetitive

regions

these

species

include

M.

galloprovincialis

-LRB-

-RRB-

-LRB-

Inoue

and

Odo

-RRB-

M.

coruscus

-LRB-

-RRB-

-LRB-

Inoue

et

al.

-RRB-

M.

trossulus

-LRB-

Inoue

et

al.

-RRB-

M.

californianus

Conrad

California

mussel

-LRB-

Waite

-RRB-

M.

chilensis

-LRB-

Pardo

et

al.

-RRB-

and

M.

sp.

-LRB-

Wang

et

al.

-RRB-

like

adhesive

proteins

from

mussels

outside

of

the

Mytilus

genus

also

have

been

identified

Dreissena

polymorpha

zebra

mussel

-LRB-

-RRB-

-LRB-

Rzepecki

and

Waite

Anderson

and

Waite

-RRB-

Dreissena

bugensis

quagga

mussel

-LRB-

-RRB-

-LRB-

Anderson

and

Waite

-RRB-

Perna

viridis

green

mussel

-LRB-

-RRB-

-LRB-

Ohkawa

et

al.

-RRB-

Perna

canaliculus

green

shell

mussel

-LRB-

-RRB-

-LRB-

Zhao

and

Waite

-RRB-

Guekensia

demissa

ribbed

mussel

-LRB-

Waite

et

al.

-RRB-

Limnoperna

fortunei

Asian

freshwater

mussel

-LRB-

-RRB-

-LRB-

Ohkawa

et

al.

-RRB-

Aulacomya

ater

-LRB-

Saez

et

al.

-RRB-

and

Choromytilus

chorus

-LRB-

Pardo

et

al.

-RRB-

Polyphenolic

proteins

from

other

marine

mussels

also

have

been

compared

with

-LRB-

Rzepecki

et

al.

-RRB-

requires

oxidization

of

tyrosine

residues

by

catechol

oxidase

tyrosinase

or

sodium

periodate

for

conversion

to

the

reactive

DOPA

residues

required

for

strong

adhesion

The

enzyme

catalyzing

oxidation

may

serve

as

an

oxidative

agent

and

as

a

copolymer

Molecular

oxygen

can

be

used

to

further

oxidize

DOPA

to

a

quinone

Possible

cross-linking

agents

are

oxygen

polyvalent

metal

ions

-LRB-

and

-RRB-

aldehydes

and

many

types

of

bi/polyfunctional

cross-linkers

The

hydroxylation

of

DOPA

from

incubation

with

tyrosinase

has

been

shown

to

form

-LRB-

TOPA

-RRB-

in

a

decapeptide

related

to

-LRB-

Burzio

and

Waite

-RRB-

The

prospective

functions

of

TOPA

isomers

have

included

metal

binding

wound

healing

and

adhesion

has

been

previously

commercialized

as

a

source

for

mussel

adhesive

protein

Companies

supplying

have

obtained

adhesive

protein

from

the

byssal

structures

by

using

protein

extraction

techniques

-LRB-

Sigma-Aldrich

Adhesive

Protein

Swedish

BioScience

Laboratory

MAP

BD

Biosiences

Clontech

Cell-Tak

-RRB-

and

recombinant

protein

techniques

using

synthetic

gene

constructs

-LRB-

Genex

Corp.

AdheraCell

-RRB-

However

currently

there

are

no

commercial

sources

for

pure

because

of

the

high

cost

of

extraction

methods

and

inconsistencies

in

quality

of

protein

from

recombinant

protein

techniques

-LRB-

Cell-Tak

Cell

and

Tissue

Adhesive

is

a

formulation

of

multiple

polyphenolic

proteins

from

Mytilus

edulis

-RRB-

The

laboratory-prepared

products

have

not

demonstrated

comparable

strength

to

the

natural

protein

Our

laboratory

is

currently

conducting

research

on

the

large-scale

production

of

recombinant

protein

-LRB-

Silverman

and

Roberto

-RRB-

Byssal

Thread

Polyphenol

Oxidase

The

Enzyme

Commission

of

the

International

Union

of

Biochemistry

classifies

each

enzyme

into

six

major

groups

according

to

the

reactions

catalyzed

Enzymes

are

given

specific

nomenclature

with

the

letters

EC

prefacing

the

specific

subclass

with

its

major

grouping

-LRB-

-RRB-

Polyphenol

oxidases

fall

under

the

broad

enzyme

grouping

of

oxidoreductase

They

are

oxygen

transferring

copper

metalloproteins

having

both

catecholase

-LRB-

act

on

diphenols

-RRB-

and

cresolase

-LRB-

act

on

monophenols

-RRB-

activity

Polyphenol

oxidases

catalyze

the

orthohydroxylation

of

phenols

to

catechols

and

the

dehydrogenation

of

catechols

to

orthoquinones

A

monophenol

monooxygenase

-LRB-

EC

-RRB-

more

traditionally

called

tyrosinase

can

oxidize

tyrosine

to

L-DOPA

and

L-DOPA

to

o-quinone

-LRB-

Worthington

-RRB-

A

catechol

oxidase

-LRB-

EC

-RRB-

can

oxidize

L-DOPA

to

o-quinone

The

enzymatic

formation

of

o-quinone

is

a

browning/tanning/curing

reaction

that

occurs

in

plants

bacteria

and

animals

-LRB-

Waite

-RRB-

The

adhesive

properties

of

marine

mussels

have

been

demonstrated

by

metal

complex

formation

of

DOPA

and

the

intermolecular

cross-linking

of

o-quinone

with

lysine

residues

Very

little

definitive

information

exists

about

the

polyphenol

oxidase

-LRB-

s

-RRB-

present

in

M.

edulis

that

is

responsible

for

tyrosine

and

DOPA

conversions

in

byssus

formation

A

cytochemical

study

of

the

enzyme

gland

in

the

foot

organ

of

M.

edulis

demonstrated

phenol

oxidase

activity

in

the

Golgi

complex

but

not

the

secretory

granules

-LRB-

Zuccarello

-RRB-

Waite

-LRB-

-RRB-

measured

catechol

oxidase

activity

from

enzyme

gland

extractions

and

whole

byssus

structures

The

native

enzyme

was

shown

to

prefer

diphenols

over

monophenols

as

substrates

A

single

purified

protein

was

not

obtained

and

the

enzyme

was

estimated

to

have

subunits

and

a

molecular

weight

of

kDa

Burzio

-LRB-

-RRB-

extracted

two

catechol

oxidases

from

M.

edulis

byssal

threads

plaques

and

feet

Both

the

byssal

catechol

oxidase-high

molecular

weight

-LRB-

BCO-H

kDa

-RRB-

and

byssal

catechol

oxidase-low

molecular

weight

-LRB-

BCO-L

kDa

-RRB-

isozymes

only

oxidized

catechols

to

o-quinones

and

their

amino

acid

composition

was

similar

to

other

catechol

oxidases

found

in

nature

Again

homogenous

purification

was

not

possible

Recently

Hellio

et

al.

-LRB-

-RRB-

purified

a

kDa

phenol

oxidase

monomer

from

mussel

feet

The

substrate

specificity

of

the

enzyme

was

greatest

toward

dihydroxyphenols

-LRB-

L-DOPA

and

catechol

-RRB-

followed

by

trihydroxyphenols

suggesting

that

there

may

be

at

least

two

forms

of

phenol

oxidase

involved

in

mussel

adhesion

Extremely

low

relative

activity

was

observed

with

monohydroxyphenols

Thus

the

specific

identification

of

the

polyphenol

oxidase

enzyme

-LRB-

s

-RRB-

involved

in

byssus

formation

continues

to

elude

researchers

Various

compounds

have

been

found

to

inhibit

the

activity

of

the

M.

edulis

phenol

oxidases

identified

above

oxygen

competitors

-LRB-

cyanide

and

nitrogen

-RRB-

metal

chelators

-LRB-

diethyldithiocarbamate

-LRB-

DETC

-RRB-

and

ethylenediaminetetraacetic

acid

-LRB-

EDTA

-RRB-

-RRB-

a

reducing

agent

-LRB-

L-ascorbic

acid

-RRB-

and

the

competitive

inhibitors

benzoic

acid

and

L-DOPA

with

respect

to

and

L-DOPA

-LRB-

Waite

Burzio

Hellio

et

al.

-RRB-

There

is

currently

extensive

research

in

the

area

of

anti-fouling

methods

to

prevent

attachment

of

marine

and/or

freshwater

mussels

to

surfaces

These

studies

rely

on

understanding

the

biochemical

properties

of

mussel

adhesive

proteins

including

polyphenol

oxidase

and

the

surface

chemistry

of

paints

varnishes

and

other

materials

Byssal

Thread

Collagens

All

collagens

in

nature

contain

a

triple-helical

domain

with

the

sequence

repeat

-LRB-

Gly-X-Y

-RRB-

n

in

which

Gly

is

glycine

X

often

is

proline

and

Y

is

usually

hydroxyproline

This

triple-helical

configuration

provides

mechanical

strength

but

on

its

own

offers

little

flexibility

and

elasticity

The

collagens

present

in

M.

edulis

byssal

threads

contain

additional

sequence

domains

that

make

the

byssal

thread

approximately

six

times

tougher

than

the

human

Achilles

tendon

-LRB-

Smeathers

and

Vincent

Coyne

et

al.

-RRB-

Two

M.

edulis

collagens

appear

in

a

gradient

fashion

in

byssal

threads

and

a

third

is

distributed

evenly

throughout

the

thread

Analogous

byssal

collagens

have

been

identified

in

M.

galloprovincialis

-LRB-

Lucas

et

al.

Hassenkam

et

al.

-RRB-

Proximal

Collagen

-LRB-

Col-P

-RRB-

The

proximal

region

of

a

byssal

thread

-LRB-

the

region

closest

to

the

animal

-RRB-

consists

of

a

unique

natural

co-polymer

termed

proximal

collagen

-LRB-

Col-P

-RRB-

-LRB-

Coyne

et

al.

Qin

and

Waite

-RRB-

This

protein

is

an

incredible

shock

absorber

with

extensibility

The

precursor

collagen

-LRB-

PreCol-P

-RRB-

has

a

molecular

mass

of

kDa

and

consists

of

seven

domains

representative

amino

and

carboxyl

termini

a

large

central

collagenous

domain

-LRB-

kDa

-RRB-

flanked

by

elastin-like

domains

-LRB-

kDa

each

-RRB-

and

small

histidine-rich

domains

-LRB-

kDa

-RRB-

that

flank

the

elastin-like

domains

A

small

acidic

patch

is

present

between

the

collagen

and

elastin-like

domains

at

the

carboxy

end

of

the

protein

The

collagen

domain

coupled

with

the

presence

of

the

two

elastin-like

domains

provides

for

an

incredibly

tough

and

extensible

proximal

region

in

byssal

threads

Histidine-rich

domains

are

believed

to

function

in

metal

binding

The

transition

metal

zinc

has

been

detected

in

M.

edulis

byssi

supporting

theories

of

mediated

cross-linking

in

the

terminal

domains

of

Col-P

Two

variants

of

PreCol-P

have

been

implicated

in

molecular

dovetailing

between

the

proximal

byssal

thread

and

the

byssal

stem

-LRB-

and

-RRB-

-LRB-

Coyne

and

Waite

-RRB-

The

presence

of

these

variants

suggests

a

mechanism

in

which

mussels

can

mitigate

stresses

by

overlapping

different

proteins

a

phenomenon

that

occurs

between

tendon

and

bone

-LRB-

Fukuta

et

al.

-RRB-

and

in

spiders

from

frame

silk

-LRB-

web

frame

and

radial

supports

-RRB-

to

viscid

-LRB-

glue-covered

-RRB-

silks

-LRB-

Guerette

et

al.

-RRB-

Distal

Collagen

-LRB-

Col-D

-RRB-

The

distal

region

of

a

byssal

thread

contains

another

natural

co-polymer

distal

collagen

-LRB-

Col-D

-RRB-

The

Col-D

is

stiffer

than

the

Col-P

The

precursor

collagen

-LRB-

PreCol-D

-RRB-

-LRB-

molecular

mass

kDa

-RRB-

also

contains

seven

separate

domains

representative

amino

and

carboxyl

termini

a

central

collagenous

domain

larger

than

PreCol-P

-LRB-

kDa

-RRB-

silk

fibroin-like

domains

flanking

the

collagen

domain

-LRB-

and

kDa

-RRB-

and

small

histidine-rich

domains

-LRB-

and

kDa

-RRB-

that

flank

the

silk-fibroin

domains

A

large

collagen

domain

-LRB-

repeats

of

Gly-X-Y

-RRB-

a

small

acidic

patch

and

the

histidine-rich

domains

are

similar

to

the

PreCol-P

structure

The

presence

of

silk-fibroin

domains

allow

for

extensibility

in

PreCol-D

as

is

the

case

for

elastin

in

PreCol-P

However

the

distal

region

of

byssal

threads

is

localized

in

straight

bundles

-LRB-

strong

and

stiff

-RRB-

whereas

the

collagen

fibers

are

coiled

in

the

proximal

region

-LRB-

pliable

and

elastic

-RRB-

-LRB-

Waite

et

al.

-RRB-

Before

the

characterization

of

PreCol-D

silk

fibroin

domains

had

been

previously

reported

only

in

arthropods

Pepsin-resistant

Nongradient

Collagen

-LRB-

Col-NG

-RRB-

The

entire

length

of

a

byssal

thread

contains

a

third

block

copolymer-like

protein

pepsin-resistant

nongradient

collagen

-LRB-

Col-NG

-RRB-

-LRB-

Qin

and

Waite

-RRB-

The

precursor

pepsin-resistant

nongradient

collagen

-LRB-

PreCol-NG

-RRB-

-LRB-

molecular

mass

kDa

-RRB-

is

believed

to

function

as

a

mediator

between

PreCol-D

and

PreCol-P

It

consists

of

a

central

collagenous

domain

-LRB-

kDa

-RRB-

an

acidic

patch

and

histidine-rich

termini

similar

to

PreCol-D

and

PreCol-P

The

regions

flanking

the

collagen

domain

contain

-LRB-

X-Glyn

-RRB-

m

repeats

-LRB-

which

are

similar

motifs

found

in

plant

cell

walls

-RRB-

in

addition

to

the

familiar

silk

fibroin-like

domains

that

are

present

in

PreCol-D

There

is

a

larger

distribution

of

tyrosine

in

the

histidine

domain

of

the

N

and

C

terminal

sequences

of

PreCol-NG

compared

to

the

other

two

collagens

The

elastic

domains

of

PreCol-P

the

silk

fibroin-like

domains

of

PreCol-D

and

the

plant

cell

wall-like

domains

of

PreCol-NG

characterize

the

unique

collagenous

block

copolymers

found

in

the

byssal

threads

of

M.

edulis

Proximal

Thread

Matrix

Protein

-LRB-

PTMP

-RRB-

The

proximal

portion

of

each

byssal

thread

contains

a

water-soluble

noncollagenous

protein

designated

proximal

thread

matrix

protein

-LRB-

PTMP

Sun

et

al.

-RRB-

This

kDa

matrix

protein

has

a

capacity

for

collagen

binding

and

resembles

the

von

Willebrand

factor

in

amino

acid

sequence

antigenicity

and

its

stiffening

effect

Two

variants

have

been

identified

in

M.

edulis

-LRB-

and

-RRB-

A

similar

cDNA

has

been

found

in

M.

galloprovincialis

Byssal

Plaque

Polyphenolic

Proteins

is

found

exclusively

in

byssal

plaques

constituting

from

to

of

the

total

plaque

proteins

Unlike

is

a

smaller

adhesive

protein

-LRB-

molecular

mass

kDa

-RRB-

with

only

to

mol

DOPA

and

no

hydroxylation

of

proline

to

or

The

DOPA

residues

occur

primarily

in

the

N

and

C

terminal

regions

of

the

protein

contains

considerable

secondary

structure

and

is

relatively

resistant

to

a

variety

of

proteases

-LRB-

compared

with

-RRB-

an

important

characteristic

for

integrity

of

the

byssal

plaque

The

high

cysteine

content

-LRB-

mol

-RRB-

coupled

with

tandem

repetitive

motifs

similar

to

epidermal

growth

factor

suggests

that

is

an

adhesive

protein

with

a

stabilization

role

in

the

byssus

-LRB-

Inoue

et

al.

-RRB-

A

multigene

family

may

exist

based

on

evidence

that

at

least

three

different

repetitive

motifs

have

been

identified

in

the

primary

protein

sequence

-LRB-

Rzepecki

et

al.

-RRB-

A

published

full-length

gene

sequence

for

is

not

available

However

an

investigation

of

cDNA

sequences

identified

by

our

laboratory

supports

the

multigene

family

premise

-LRB-

GenBank

Submission

numbers

and

-RRB-

Our

laboratory

is

currently

conducting

research

on

the

large-scale

production

of

recombinant

protein

-LRB-

Silverman

and

Roberto

-RRB-

As

is

the

case

with

other

mussel

species

contain

proteins

analogous

to

M.

galloprovincialis

-LRB-

-RRB-

-LRB-

Inoue

et

al.

-RRB-

M.

coruscus

-LRB-

-RRB-

-LRB-

Inoue

et

al.

-RRB-

and

D.

polymorpha

-LRB-

-RRB-

-LRB-

Rzepecki

and

Waite

-RRB-

Stewart

et

al.

-LRB-

-RRB-

reported

that

the

tube

cement

of

Phragmatopoma

californica

a

marine

polychaete

forms

solid

foam

-LRB-

similar

to

-RRB-

via

cross-linking

with

DOPA

is

the

smallest

byssal

adhesive

protein

identified

to

date

with

a

molecular

mass

of

to

kDa

-LRB-

Papov

et

al.

Warner

and

Waite

-RRB-

contains

no

repeats

to

mol

DOPA

and

a

prevalence

of

and

tryptophan

residues

Warner

and

Waite

-LRB-

-RRB-

identified

gene

variants

-LRB-

kB

-RRB-

of

in

the

foot

organ

however

only

four

or

five

proteins

have

actually

been

detected

in

plaques

deposited

on

glass

or

plastic

The

presence

of

a

gene

family

for

supports

the

primer-like

function

proposed

for

the

protein

in

adhering

to

substrata

One

hypothesis

has

been

that

deposition

of

a

specific

variant

is

dependent

on

the

surface

used

for

attachment

However

Floriolli

et

al.

-LRB-

-RRB-

reported

no

correlation

between

expression

and

surface

type

-LRB-

stainless

steel

glass

polyethylene

-RRB-

in

an

individual

mussel

Differences

in

cDNA

transcripts

were

found

between

animals

Zhao

et

al.

-LRB-

-RRB-

reported

different

variants

from

a

mussel

population

broken

into

fast

or

slow

electrophoretic

characteristics

upon

exposure

to

glass

coverslips

The

molecular

mechanism

-LRB-

s

-RRB-

for

the

numerous

and

variants

is

not

known

Variables

such

as

exposure

time

to

surfaces

water

temperature

and

the

age

of

the

animals

could

influence

expression

levels

As

is

the

case

with

and

other

mussel

species

contain

proteins

analogous

to

variants

M.

galloprovincialis

-LRB-

and

-RRB-

-LRB-

Inoue

et

al.

-RRB-

and

M.

californianus

-LRB-

variants

mentioned

above

-RRB-

-LRB-

Zhao

et

al.

-RRB-

is

another

protein

identified

in

byssal

plaques

with

a

molecular

mass

of

kDa

-LRB-

Weaver

Vreeland

et

al.

Warner

and

Waite

-RRB-

contains

elevated

levels

of

glycine

arginine

and

histidine

as

well

as

mol

DOPA

A

unique

tyrosine-rich

octapeptide

is

present

with

variations

in

residue

substitutions

giving

rise

to

a

family

of

proteins

This

very

large

protein

most

likely

serves

as

a

coupling

agent

in

the

thread-plaque

junction

designated

by

the

precollagens

and

the

byssal

plaque

protein

A

gene

sequence

for

has

not

been

identified

however

two-foot

protein

variants

from

M.

californianus

have

recently

been

identified

-LRB-

Zhao

and

Waite

-RRB-

is

the

most

recent

adhesive-related

byssal

plaque

protein

identified

from

Mytilus

edulis

-LRB-

Waite

and

Qin

-RRB-

is

a

relatively

small

protein

with

a

molecular

mass

of

kDa

a

mol

DOPA

content

and

the

presence

of

phosphoserine

Phosphoserine

is

known

to

occur

in

acidic

mineral-binding

motifs

of

proteins

that

bind

calcareous

materials

-LRB-

e.g.

osteopontin

-RRB-

therefore

its

presence

in

byssal

plaques

may

aid

in

adhesion

of

one

animal

to

a

neighboring

mussel

's

shell

was

formerly

associated

with

the

family

of

variants

and

similarly

plays

an

interfacial

role

as

a

primer

for

substrate

adhesion

An

adhesive

protein

analogous

to

has

recently

been

reported

to

be

present

in

M.

galloprovincialis

-LRB-

-RRB-

-LRB-

Hwang

et

al.

-RRB-

and

M.

californianus

-LRB-

variants

-RRB-

-LRB-

Zhao

and

Waite

-RRB-

Discovery

of

Additional

Foot

Proteins

was

identified

along

with

by

Zhao

and

Waite

-LRB-

variants

-RRB-

This

small

protein

-LRB-

kDa

-RRB-

contains

a

relatively

large

amount

of

tyrosine

and

a

small

amount

of

DOPA

Its

suggested

role

in

adhesion

may

be

to

provide

a

link

between

the

DOPA-rich

proteins

and

the

plaque

proteins

present

in

byssal

attachment

plaques

Adhesive

Testing

of

Mussel

Proteins

A

wide

variety

of

adhesive

tests

have

been

applied

to

intact

byssal

threads

plaque

portions

of

threads

or

materials

bonded

or

coated

with

individual

adhesive

proteins

Gross

comparative

tests

on

byssal

threads

were

described

earlier

in

this

review

Mussels

seem

to

prefer

surfaces

with

higher

critical

surface

energy

a

phenomenon

also

observed

in

barnacles

-LRB-

Crisp

et

al.

Waite

-RRB-

Plaques

were

shown

to

attach

more

strongly

to

slate

and

glass

than

plastic

acetal

-LRB-

acetate

-RRB-

paraffin

wax

and

polytetrafluoroethylene

-LRB-

PTFE

-RRB-

The

high-energy

hydrophilic

surfaces

-LRB-

glass

slate

-RRB-

had

smaller

plaque

surfaces

attached

to

substrates

and

smaller

contact

angles

compared

with

the

low-energy

hydrophobic

surfaces

-LRB-

wax

PTFE

-RRB-

Attachment

Failure

Byssal

attachments

can

fail

in

a

number

of

ways

A

byssal

plaque

can

cleanly

peel

away

from

a

surface

the

byssal

thread

itself

can

tear

or

fail

or

the

root

can

tear

away

from

the

animal

Failure

of

the

thread

is

the

most

common

cause

of

failure

of

byssal

attachment

If

byssal

threads

break

or

tear

away

from

the

animal

the

true

adhesive

force

is

underestimated

Therefore

when

analyzing

adhesive

forces

it

is

important

to

consider

the

type

of

failure

The

surface

for

attachment

source

of

foot

protein

age

of

animal

temperature

and

season

are

important

in

evaluating

the

strength

of

byssal

attachments

-LRB-

Crisp

et

al.

-RRB-

Adhesive

Techniques

Numerous

surfaces

and

techniques

are

used

to

evaluate

the

mechanical

properties

of

mussel

adhesive

proteins

Atomic

force

microscopy

-LRB-

AFM

for

surface

topography

-RRB-

attenuated

total

reflection

fourier

transform

infrared

spectroscopy

-LRB-

ATR/FT-IR

for

information

on

molecular

composition

bonding

conformation

and

orientation

with

respect

to

interfaces

-RRB-

surface

plasmon

resonance

-LRB-

SPR

to

study

interactions

of

samples

and

surfaces

-RRB-

and

tensometer

methods

-LRB-

for

tensile

strength

and

elastic

properties

-RRB-

are

common

techniques

used

in

adhesives

research

Table

provides

examples

of

studies

in

which

surfaces

have

been

tested

with

mussel

adhesive

proteins

or

synthetic

analogs

containing

repetitive

motifs

from

mussel

proteins

Table

Examples

of

materials

testing

with

mussel

adhesive

proteins

or

synthetic

analogs

containing

repetitive

motifs

from

mussel

proteinsSurfaces

TestedMussel

Protein

or

Synthetic

AnalogReferencesSlateSynthetic

recombinant

et

al.

-LRB-

glass

-RRB-

Plastic

acetal

-LRB-

acetate

-RRB-

Paraffin

waxPolytetrafluoroethylene

-LRB-

PTFE

-RRB-

PolystyreneRecombinant

et

al.

cobbleD

bugensis

and

D.

polymorphaAckerman

et

al.

steelStainless

steelMarine

concreteAckerman

et

al.

plywoodPolyvinyl

chloridePolymethylmethacrylate

-LRB-

Plexiglas

-RRB-

Ackerman

et

al.

D.

bugensis

and

D.

polymorphaDormon

et

al.

steelPolyvinyl

chlorideStainless

steelSiliconeSynthetic

recombinant

et

al.

terephthalate

-LRB-

PET

-RRB-

Teflon

AluminumSynthetic

polypeptide

mimics

of

marine

adhesivesYu

and

Deming

apatite

homo

and

copolypeptides

of

marine

and

related

adhesive

proteins

-LRB-

M.

edulis

M.

californianus

A.

ater

G.

demissa

liver

fluke

pearl

oyster

-RRB-

Yamamoto

et

al.

-LRB-

PTFE

-RRB-

Teflon

NylonIronSoda

glassMethyl

and

oligo

-LRB-

ethylene

oxide

-RRB-

terminated

self-assembled

-LRB-

Cell-Tak

-RRB-

and

fibrinogenHarder

et

al.

-LRB-

oxide

-RRB-

and

polylysineSuci

and

Geesey

octadecyl

methacrylateSuci

and

Geesey

and

Belfort

skinM

edulis

mussel

feet

extractNinan

et

al.

and

-LRB-

Cell-Tak

-RRB-

Hwang

et

al.

-LRB-

Plexiglas

-RRB-

PolystyreneAluminum

Brazee

and

Carrington

-LRB-

-RRB-

recently

provided

an

elegant

and

detailed

study

of

whole

byssal

thread

material

properties

comparing

threads

from

a

range

of

Mytilidae

This

study

also

compared

their

results

to

those

of

previous

studies

along

with

statistics

to

quantify

the

variation

in

the

results

Quasi-static

tensile

strength

tests

and

dynamic

testing

of

elastic

properties

-LRB-

extensibility

modulus

resilience

and

recovery

-RRB-

were

performed

and

correlated

with

morphometric

measurements

including

shell

weight

and

dimensions

and

thread

morphometry

Several

interesting

findings

were

reported

including

the

elliptical

cross-section

-LRB-

rather

than

a

regular

round

section

-RRB-

of

the

byssal

thread

novel

behavior

of

Modiolus

modiolus

threads

-LRB-

double

yield

under

strain

-RRB-

the

surprising

strength

of

Dreissena

polymorpha

threads

-LRB-

although

they

lack

a

collagen

core

-RRB-

and

the

overall

greater

strength

of

Mytilis

californianus

threads

-LRB-

suggested

to

be

a

factor

in

its

success

in

living

in

the

strong

tidal

environment

of

the

Pacific

coast

-RRB-

The

application

of

atomic

force

microscopy

-LRB-

AFM

-RRB-

is

revolutionizing

our

understanding

of

the

interactions

of

mussel

adhesive

proteins

with

surfaces

and

with

other

adhesive

proteins

that

make

up

the

byssal

thread

It

is

now

possible

to

measure

adhesion

energy

and

shear

forces

of

individual

adhesive

proteins

such

as

has

been

done

in

comparing

M.

edulis

foot

proteins

and

-LRB-

Lin

et

al.

-RRB-

Even

more

remarkably

the

cantilevers

of

an

AFM

can

be

coated

with

DOPA

residues

to

perform

single

molecule

measurements

and

quantify

the

differences

in

bond

strength

that

might

be

expected

between

unmodified

and

DOPA-modified

adhesives

-LRB-

Lee

et

al.

-RRB-

As

our

ability

to

perform

sensitive

measurements

with

small

amounts

of

protein

improves

along

with

the

increased

resolution

of

the

techniques

used

we

can

anticipate

that

our

understanding

of

the

interactions

of

adhesive

proteins

necessary

to

achieve

robust

adhesion

will

increase

Future

Supply

of

Adhesive

Proteins

by

Recombinant

Approaches

Background

An

impediment

to

further

understanding

the

unique

adhesion

system

of

M.

edulis

is

the

lack

of

availability

of

the

individual

protein

components

Large

quantities

of

M.

edulis

-LRB-

or

other

mussel

-RRB-

adhesive

proteins

are

needed

to

perform

research

and

development

for

commercial

adhesives

Current

methods

for

obtaining

the

adhesive

proteins

of

the

mussel

byssus

rely

largely

on

excision

of

the

byssus

followed

by

extraction

of

the

proteins

under

acidic

conditions

However

the

chemical

extraction

process

does

not

always

yield

pure

or

individual

adhesive

proteins

Approximately

M.

edulis

mussels

are

needed

to

produce

g

of

adhesive

from

byssal

structures

-LRB-

Strausberg

and

Link

Hwang

et

al.

-RRB-

At

least

mg

of

material

is

needed

for

performing

small

conventional

adhesive

tests

such

as

tensile

strength

or

wall-jet

analyses

The

sacrifice

of

such

a

large

number

of

animals

is

neither

environmentally

friendly

nor

economically

practical

Therefore

an

alternate

approach

to

obtain

large

amounts

of

adhesive

protein

components

is

necessary

for

further

conventional

adhesive

testing

formats

and

adhesive

formulation

development

Molecular

and

microbiology

techniques

can

be

used

for

the

directed

production

of

large

quantities

of

many

different

proteins

Prokaryotes

and

eukaryotes

are

used

as

hosts

in

recombinant

protein

expression

systems

Common

prokaryote

hosts

include

bacteria

-LRB-

Escherichia

coli

-RRB-

Saccharomyces

cerevisiae

-LRB-

baker

's

yeast

-RRB-

Pichia

pastoris

-LRB-

a

methylotrophic

yeast

-RRB-

or

Kluyveromyces

lactis

-LRB-

a

lactic-acid

producing

yeast

-RRB-

Eukaryote

hosts

include

plants

-LRB-

tobacco

potatoes

-RRB-

trees

-LRB-

loblolly-pine

-RRB-

and

mammals

-LRB-

rabbit

mouse

goat

-RRB-

Cell

culture

techniques

use

insect

plant

and

mammalian

cells

or

tissue

cultures

rather

than

whole

organisms

Variables

important

for

successful

recombinant

protein

expression

systems

include

a

codon-usage

compatible

host

promoter

transcriptional

and

translational

regulators

fine-tuned

cultivation

methods

targeted

recombinant

protein

purification

methodologies

posttranslational

modification

ability

and

three-dimensional

configuration

of

the

recombinant

protein

Codon

usage

refers

to

the

predominant

DNA

bases

that

code

for

specific

amino

acids

Codon

analysis

can

be

performed

for

the

source

organism

across

all

its

known

proteins

or

only

for

the

specific

protein

or

protein

class

of

interest

Possible

choices

for

mussel

adhesive

recombinant

protein

expression

include

a

eukaryotic

host

a

special

bacterial

line

-LRB-

E.

coli

strains

-RRB-

or

a

plant

source

such

as

tobacco

-LRB-

because

plant

cell

wall

proteins

are

repetitive

proteins

with

hydroxylations

-RRB-

Promoter

and

transcriptional

regulators

are

DNA

sequences

that

direct

gene

expression

They

can

be

native

to

the

host

artificially

added

to

the

host

DNA

or

incorporated

in

vector

systems

Translational

stop

signals

can

be

incorporated

into

the

DNA

genes

or

are

present

on

an

expression

vector

Prokaryotes

and

eukaryotes

use

different

regulators

for

gene

expression

Cultivation

factors

such

as

energy

sources

aeration

-LRB-

oxygen

-RRB-

temperature

and

induction

protocols

are

specific

to

the

host

organism

or

cell

type

and

are

dependent

on

the

quantity

used

for

production

Small-scale

cultivation

generally

involves

flasks

or

petri

dishes

and

volumes

less

than

L.

Larger-scale

cultivation

in

the

research

laboratory

setting

can

use

bioreactors

with

volumes

as

large

as

L.

Automated

monitoring

and

control

of

cultivation

variables

is

used

in

large-scale

recombinant

protein

production

Heterologous

recombinant

protein

production

occurs

simultaneously

with

the

host

's

production

of

its

own

proteins

Thus

harvesting

of

a

recombinant

protein

involves

its

identification

and

separation

from

the

host

's

native

proteins

Special

tags

that

can

be

incorporated

with

a

recombinant

protein

include

specific

amino

acid

repeats

-LRB-

histidine

-RRB-

fluorescent

molecules

-LRB-

green

fluorescent

protein

-RRB-

biotinylation

or

antibody

recognition

sequences

-LRB-

epitopes

-RRB-

The

specific

chemical

properties

of

proteins

also

can

be

used

for

isolation

and

purification

For

M.

edulis

adhesive

proteins

and

acidic

conditions

in

purification

steps

will

exclude

many

of

the

host

's

native

proteins

while

maintaining

the

integrity

of

the

mussel

proteins

One

caveat

in

heterologous

protein

production

however

is

that

correctly

folded

or

modified

proteins

are

not

always

produced

Prokaryotes

and

eukaryotes

can

modify

their

proteins

following

translation

with

specific

disulfide

bond

formation

for

correct

protein

folding

cleavage

of

precursor

protein

forms

to

yield

functional

proteins

glycosylation

of

amino

acid

residues

for

protein

stability

and

other

modifications

to

amino

acids

such

as

phosphorylation

acetylation

or

hydroxylation

-LRB-

as

in

the

case

with

many

M.

edulis

adhesive

proteins

-RRB-

Posttranslational

modifications

do

not

always

occur

naturally

in

heterologous

systems

therefore

in

vitro

methods

often

are

required

to

obtain

properly

modified

proteins

For

example

heterologously

produced

unhydroxylated

requires

treatment

with

mushroom

or

bacterial

tyrosinase

to

produce

hydroxylated

tyrosine

-LRB-

DOPA

-RRB-

at

positions

two

and

nine

of

the

decapeptide

repeat

-LRB-

Strausberg

and

Link

Filpula

et

al.

Kitamura

et

al.

-RRB-

Other

considerations

for

biologically

functional

recombinant

proteins

include

hyperglycosylation

inclusion

bodies

and

loss

of

expression

plasmids

with

a

scale-up

in

cultivation

volumes

The

Recombinant

Protein

Approach

The

recombinant

expression

of

cDNAs

or

synthetic

adhesive-related

proteins

modeled

from

organisms

other

than

mussels

has

been

reported

human

blood

coagulation

protein

Factor

XIIIa

in

the

yeast

S.

cerevisiae

-LRB-

Broker

et

al.

-RRB-

and

collagen

and

spider

silk

proteins

in

bacteria

yeast

insect

and

mammalian

cells

and

plants

-LRB-

Fahnestock

et

al.

Kieliszewski

and

Lamport

Scheller

et

al.

-RRB-

Researchers

have

to

some

extent

successfully

produced

Mytilus

mussel

adhesive

proteins

in

bacteria

yeast

mammalian

cells

and

plants

Filpula

et

al.

-LRB-

-RRB-

used

a

hybrid

combination

of

promoters

to

direct

the

production

of

tandem

repeats

in

S.

cerevisiae

A

single

copy

of

the

genomic

cDNA

clone

for

encoded

the

carboxy

terminus

including

decapeptide

and

one

hexapeptide

repeat

sequences

Synthetic

gene

constructs

carrying

between

one

and

four

copies

of

a

portion

of

the

genomic

sequence

for

were

shown

to

express

proteins

between

to

kDa

Amino

acid

analyses

of

the

four

different

recombinant

proteins

expressed

in

the

yeast

system

were

similar

to

unhydroxylated

natural

The

recombinant

protein

constituted

to

of

the

total

cell

protein

and

adhesive

tests

showed

water-resistant

bonding

after

in

vitro

modification

of

tyrosine

residues

to

DOPA

and

then

quinone

Salerno

and

Goldberg

-LRB-

-RRB-

expressed

a

synthetic

mussel

adhesive

protein

analog

in

E.

coli

using

a

promoter

system

and

a

repetitive

gene

cassette

that

encoded

the

consensus

decapeptide

repeat

of

The

synthetic

polydecapeptide

was

produced

from

a

base

pair

gene

-LRB-

decapeptide

repeats

-RRB-

and

constituted

up

to

of

total

cell

protein

Kitamura

et

al.

-LRB-

-RRB-

also

expressed

a

synthetic

mussel

adhesive

protein

analog

in

E.

coli

with

a

promoter

system

and

a

repetitive

gene

cassette

that

encoded

six

repeats

of

the

consensus

decapeptide

of

The

researchers

were

able

to

obtain

mg

of

the

model

peptide

per

liter

of

growth

medium

Surface

strength

and

contact-angle

were

measured

and

used

to

calculate

the

value

of

work

of

adhesion

-LRB-

WA

-RRB-

for

both

the

unmodified

and

mushroom-tyrosinase

treated

peptide

Adhesion

to

silicone

glass

polyethylene

terephthalate

and

Teflon

substrates

was

found

to

be

dependent

on

sodium

chloride

concentration

-LRB-

e.g.

sea

water

-RRB-

Our

laboratory

has

patented

a

technique

for

cloning

and

expressing

recombinant

and

in

S.

cerevisiae

from

cDNA

clones

-LRB-

Silverman

and

Roberto

b

-RRB-

We

are

currently

producing

and

in

fermentation

batches

-LRB-

unpublished

data

-RRB-

Purification

of

each

individual

recombinant

protein

is

in

progress

The

purified

products

will

be

analyzed

by

mass

spectrometry

-LRB-

MS

-RRB-

for

total

intact

mass

followed

by

MS

analysis

-LRB-

post-protease

digestion

-RRB-

Adhesive

studies

will

be

performed

in-house

and/or

through

interested

parties

after

the

confirmation

of

and

Recombinant

mussel

adhesive

proteins

from

M.

galloprovincialis

also

have

been

produced

Takeuchi

et

al.

-LRB-

-RRB-

cultured

mussel

foot

cells

from

M.

galloprovincialis

in

Petri

dishes

Gene-specific

probes

were

used

to

identify

the

stage

of

development

for

different

adhesive

proteins

Probes

for

the

genes

encoding

and

were

used

to

demonstrate

that

the

expression

of

the

byssal

protein

genes

in

M.

galloprovincialis

is

morphologically

programmed

-LRB-

Takeuchi

et

al.

-RRB-

The

findings

suggested

that

a

cell

culture

system

for

in

vitro

expression

of

byssal

adhesive

proteins

might

be

a

plausible

alternative

to

yeast

or

bacterial-based

expression

systems

Hwang

et

al.

-LRB-

-RRB-

produced

a

soluble

recombinant

fused

with

a

histidine

tag

in

E.

coli

A

purification

yield

of

was

obtained

from

a

L

working

volume

because

of

difficulties

in

removing

the

recombinant

protein

from

the

chromatographic

resin

used

to

bind

histidine

proteins

The

adhesive

properties

of

recombinant

were

compared

to

Cell-Tak

by

using

atomic

force

microscopy

-LRB-

AFM

-RRB-

material

surface

coating

-LRB-

on

glass

polymethylmethacrylate

polystyrene

a

silicone-based

antifouling

agent-coated

slide

and

aluminum

-RRB-

and

quartz

crystal

microbalance

-LRB-

QCM

-RRB-

techniques

Recombinant

was

shown

to

have

adhesive

abilities

comparable

to

and

sometimes

exceeding

those

of

Cell-Tak

The

adhesion

force

of

tyrosinase-treated

-LRB-

nN

-RRB-

was

higher

than

both

tyrosinase-treated

Cell-Tak

-LRB-

nN

-RRB-

and

untreated

Cell-Tak

-LRB-

nN

-RRB-

The

adhesion

ability

of

recombinant

to

a

glass

slide

and

a

poly

-LRB-

methyl

methacrylate

-RRB-

plate

was

comparable

to

Cell-Tak

but

better

than

Cell-Tak

on

a

silicone-based

antifouling

agent

coated

slide

And

adsorption

of

to

a

gold

surface

was

comparable

to

Cell-Tak

Hwang

et

al.

-LRB-

-RRB-

also

produced

recombinant

-LRB-

variant

A

-RRB-

with

a

histidine

tag

in

E.

coli

The

purification

yield

and

solubility

exceeded

that

of

the

above-mentioned

recombinant

The

adsorption

and

adhesion

force

were

comparable

to

Cell-Tak

but

less

than

recombinant

-LRB-

e.g.

nN

for

nN

for

Cell-Tak

and

nN

for

-RRB-

Hwang

and

colleagues

noted

the

low

production

yields

low

purification

yields

and

high

levels

of

insolubility

of

and

and

sought

to

improve

the

technique

for

recombinant

protein

expression

by

designing

novel

fusion

proteins

-LRB-

Hwang

et

al.

b

-RRB-

The

first

hybrid

MAP

mussel

bioadhesive

was

produced

from

a

fusion

protein

containing

six

decapeptide

repeats

at

both

the

N

and

C-termini

of

-LRB-

Hwang

et

al.

-RRB-

The

second

hybrid

MAP

mussel

bioadhesive

was

a

fusion

of

the

GRGDSP

residues

found

in

fibronectin

-LRB-

designated

RGD

-RRB-

to

the

C-terminus

of

-LRB-

Hwang

et

al.

-RRB-

The

authors

reported

greater

production

yields

easier

purification

and

improved

solubility

for

both

novel

fusion

proteins

and

In

addition

the

cell

adhesion

and

spreading

abilities

of

were

superior

to

Cell-Tak

poly-L-lysine

-LRB-

PLL

-RRB-

a

noncoated

surface

and

regardless

of

mammalian

cell

type

tested

-LRB-

human

or

hamster

-RRB-

The

first

non-Mytilus

recombinant

mussel

adhesive

protein

the

like

adhesive

protein

from

the

zebra

mussel

D.

polymorpha

was

expressed

by

Anderson

and

Waite

-LRB-

-RRB-

Recombinant

was

produced

as

a

maltose-fusion

protein

in

E.

coli

The

recombinant

protein

was

then

used

as

an

antigen

for

polyclonal

antibody

production

Immunologic

studies

definitively

revealed

the

presence

of

in

foot

organs

and

byssal

threads

of

D.

polymorpha

Whole

plants

have

been

used

to

synthesize

foreign/recombinant

proteins

such

as

antibodies

vaccines

and

industrial

enzymes

-LRB-

Doran

-RRB-

An

alternative

to

the

use

of

whole

plants

is

the

use

of

plant

tissue

cultures

Tissue

cultures

provide

a

technology

that

may

gain

momentum

because

of

the

ease

in

manipulation

of

culture

conditions

for

greater

foreign

protein

levels

There

is

considerable

expense

in

cell

culture

techniques

indicating

that

this

scheme

would

not

be

optimal

for

industrial

production

Tobacco

and

rice

are

the

most

commonly

used

plant

cell

cultures

used

to

date

Correct

posttranslational

modification

is

an

issue

when

using

plants

as

hosts

similar

to

bacterial

or

yeast

expression

systems

However

plants

may

be

very

good

hosts

for

production

of

the

repetitive

mussel

adhesive

proteins

Plant

cell

walls

contain

large

repetitive

proteins

similar

to

foot

analogs

and

the

diverse

collagens

found

in

byssal

threads

Dr.

Simon

McQueen-Mason

-LRB-

Department

of

Biology

University

of

York

United

Kingdom

-RRB-

has

investigated

the

ability

of

transgenic

tobacco

plants

to

produce

recombinant

spider

silks

and

because

of

their

similarity

to

the

repetitive

plant

cell

wall

proteins

expansin

and

extensin

-LRB-

-RRB-

His

group

continues

to

research

expansins

and

pectins

from

various

plant

species

-LRB-

Jones

et

al.

-RRB-

Recently

Patel

et

al.

-LRB-

-RRB-

reported

the

use

of

elastin-like

polypeptide

fusions

-LRB-

an

ELP

tag

-RRB-

to

enhance

the

accumulation

of

a

range

of

different

recombinant

proteins

-LRB-

human

murine

and

the

native

major

ampullate

spidroin

protein

gene

from

the

spider

Nephilla

clavipes

-RRB-

in

the

leaves

of

a

transgenic

tobacco

plant

The

fusion

protein

concentrations

in

the

plant

leaves

were

significantly

higher

than

the

target

recombinant

proteins

alone

in

all

instances

suggesting

that

this

type

of

fusion

tag

may

be

beneficial

for

producing

large

repetitive

recombinant

proteins

in

plant

hosts

Current

and

Future

Research

Areas

Related

to

Mussel

Adhesion

and

Adhesives

Commercialization

of

Mussel

Adhesive

Proteins

Numerous

economic

factors

are

important

in

the

production

and

synthesis

of

foreign

proteins

whether

using

microbial

cell

culture

animal

cell

culture

plant

tissue

culture

transgenic

plants

or

transgenic

animals

Production

costs

-LRB-

yield

for

cost

comparisons

-RRB-

safety

issues

-LRB-

for

therapeutic

use

-RRB-

and

stability

of

the

product

-LRB-

the

potential

for

the

protein

to

degrade

or

lose

function

during

extraction/purification

procedures

-RRB-

are

a

few

issues

that

require

careful

analysis

before

the

method

is

chosen

Regulatory

issues

relevant

to

Good

Manufacturing

Practice

-LRB-

GMP

-RRB-

for

production

of

therapeutic

proteins

must

be

followed

Any

product

containing

recombinant

mussel

adhesive

protein

will

require

extensive

testing

and

validation

from

health

environmental

and

adhesives

industries

before

commercialization

The

underwater

adhesion

of

the

marine

mussel

M.

edulis

has

intrigued

scientists

for

decades

Extensive

progress

has

been

made

in

elucidating

the

mechanisms

responsible

for

adhesion

in

understanding

adhesion

to

a

variety

of

substrates

and

in

producing

recombinant

proteins

and

materials

that

mimic

the

natural

mussel

adhesive

A

selection

of

current

and

future

research

areas

related

to

mussel

adhesives

is

discussed

below

Biofouling

In

mussel

biofouling

cost

the

Navy

more

than

in

the

areas

of

hull

scraping

and

excess

fuel

consumption

from

unnecessary

drag

on

large

vessels

-LRB-

Morgan

-RRB-

Understandably

the

Office

of

Naval

Research

and

other

organizations

have

invested

considerably

in

the

areas

of

antifouling

and

foul-release

Approaches

to

antifouling

and

bivalve

control

can

generally

be

classified

as

chemical

or

physical

-LRB-

hull

scraping

-RRB-

methodologies

Chemical

methods

have

received

the

most

attention

Until

recently

bis

-LRB-

tributyltin

-RRB-

oxide

was

a

popular

antifouling

component

of

boat

paints

However

heavy

metal-based

and

organotin

coatings

are

now

restricted

because

they

are

toxic

to

marine

and

freshwater

organisms

Synthetic

and

natural

biodegradable

compounds

are

currently

being

investigated

for

their

efficacy

as

antifouling

agents

Imides

-LRB-

special

types

of

amino

acid-based

compounds

-RRB-

terpenoids

-LRB-

multicyclic

structures

with

basic

carbon

skeletons

-RRB-

from

brown

alga

species

and

a

marine

sponge

proteases

from

a

marine

bacterium

phloroglucinol

compounds

-LRB-

white

crystalline

phenols

-RRB-

from

Eucalyptus

and

a

variety

of

chemicals

have

all

been

shown

to

inhibit

mussel

attachment

to

surfaces

-LRB-

Zentz

et

al.

Hellio

et

al.

Sera

et

al.

Venkateswaran

and

Dohmoto

Cope

et

al.

-RRB-

Experimentation

with

low-adhesion

surfaces

has

included

modified

glass

-LRB-

Yamamoto

et

al.

-RRB-

polyethylene

glycol

-LRB-

PEG

-RRB-

-LRB-

Dalsin

et

al.

-RRB-

and

various

other

polymeric

surfaces

-LRB-

Frank

and

Belfort

-RRB-

Antifouling

and

detachment

research

related

to

mussels

and

other

sessile

organisms

is

vitally

important

in

addressing

the

economical

problems

associated

with

biofouling

-LRB-

Taylor

Bellas

Bellas

et

al.

-RRB-

Mussels

from

Extreme

Environments

Hydrothermal

vents

and

cold

seeps

from

the

Gulf

of

Mexico

and

the

Japan

Trench

are

home

to

unique

combinations

of

sea

life

New

classes

of

animals

plants

and

bacteria

are

rapidly

being

identified

with

the

aid

of

special

underwater

submersibles

-LRB-

ALVIN

the

Johnson

Sea

Link

-RRB-

and

advances

in

molecular

biology

and

microbiology

Bathymodiolinae

is

a

subfamily

of

mussels

that

contains

species

harboring

methanotrophic

chemoautotrophic

and/or

both

types

of

bacterial

endosymbionts

within

the

mussel

's

gill

tissue

Geological

forces

have

caused

cracks

in

the

continental

slope

allowing

oil

natural

gases

such

as

methane

and

hydrogen

sulfide

and

related

substances

to

seep

into

the

ocean

environments

Bathymodiolinae

mussels

and

other

organisms

from

the

Gulf

of

Mexico

have

been

able

to

survive

under

anoxic

high-pressure

and

high-saline

-LRB-

brine

-RRB-

environments

for

centuries

Figure

illustrates

the

large

shells

and

byssal

plaques

and

threads

from

Bathymodiolus

childressi

mussels

obtained

by

Dr.

Charles

Fisher

from

Brine

Pool

in

the

Gulf

of

Mexico

-LRB-

MacDonald

and

Fisher

-RRB-

compared

with

M.

edulis

-LRB-

Figure

and

c

-RRB-

The

foot

organs

of

M.

edulis

and

B.

childressi

and

M.

edulis

-LRB-

Figure

and

d

-RRB-

also

are

shown

for

comparison

The

morphologic

differences

in

the

foot

organ

shell

and

gill

characteristics

between

the

two

marine

mussel

species

as

well

as

the

vast

differences

between

the

environmental

conditions

in

which

the

two

reside

may

be

important

variables

affecting

byssus

characteristics

-LRB-

material

properties

adhesion

mechanisms

individual

proteins

etc.

Brazee

and

Carrington

-RRB-

The

identification

of

adhesive

proteins

from

extreme

mussels

is

an

interesting

task

for

prospective

research

Figure

appearance

and

internal

organs

of

B.

childressi

and

M.

edulis

mussels

-LRB-

a

-RRB-

B.

childressi

shell

thread

and

plaques

-LRB-

b

-RRB-

B.

childressi

foot

organ

-LRB-

c

-RRB-

M.

edulis

shell

thread

and

plaques

-LRB-

d

-RRB-

M.

edulis

foot

organ

Novel

Applications

for

Mussel

Adhesive

Collagens

and

Polyphenol

Oxidase

Immobilization

of

mussel

adhesive

proteins

-LRB-

Mefp

class

and

collagens

-RRB-

on

solid

supports

may

be

exploited

for

uses

in

the

design

of

biosensors

immunosensors

or

artificial

tissue

scaffoldings

and

constructs

with

glucose

-LRB-

Saby

and

Luong

Newman

and

Setford

-RRB-

with

human

chorionic

gonadotrophin

-LRB-

Burzio

et

al.

-RRB-

collagens

as

supports

-LRB-

BD

Three-Dimensional

Collagen

Composite

Scaffold

BD

Biosciences

San

Jose

CA

-RRB-

The

identification

and

subsequent

use

of

the

polyphenol

oxidase

derived

from

M.

edulis

byssal

structures

also

could

be

exploited

as

a

thickening

agent

for

numerous

industries/applications

-LRB-

Yamada

et

al.

-RRB-

Medical

and

Dental

Adhesives

Current

adhesives

approved

for

medical

use

in

the

United

States

include

fibrin

sealants

albumin-based

compounds

glutaraldehyde

glues

cyanoacrylates

hydrogels

and

collagen-based

compounds

Fibrin

sealants

are

the

most

prevalent

They

consist

primarily

of

thrombin

and

fibrinogen

clotting

factors

in

blood

Fibrin

sealants

have

a

wide

range

of

uses

and

are

bioabsorbed

by

the

body

However

risks

of

products

isolated

from

blood

albeit

minimal

are

a

concern

with

this

class

of

tissue

adhesives

One

albumin-based

compound

approved

for

use

in

the

United

States

is

BioGlue

Surgical

Adhesive

-LRB-

CryoLife

Inc.

Kennesaw

GA

-RRB-

It

is

glutaraldehyde-based

or

gelatin-resorcinol-formaldehyde-glutaraldehyde-like

glue

-LRB-

without

the

formaldehyde

-RRB-

that

has

limited

U.S.

Food

and

Drug

Administration

approval

because

of

known

immunoreactivity

problems

The

cyanoacrylates

approved

for

use

in

the

United

States

are

stronger

than

the

fibrin

sealants

but

not

bioabsorbable

Their

use

is

restricted

to

external

or

temporary

applications

because

of

associations

with

carcinogenicity

inflammation

and

infection

Hydrogels

are

water-soluble

polyethylene

glycol

polymers

that

are

activated

by

light

Both

protein-based

and

DOPA-based

hydrogel

formulations

are

being

researched

-LRB-

Nowak

et

al.

Mo

et

al.

Liu

and

Li

-RRB-

Currently

their

use

is

limited

because

of

the

time

required

for

application

setting

and

bioabsorption

Collagen-based

adhesives

are

the

newest

type

of

tissue

adhesive

available

-LRB-

Koob

and

Hernandez

-RRB-

So

far

they

work

well

and

are

inexpensive

thus

adding

an

alternative

to

the

use

of

fibrin

sealants

Dr.

Robin

L.

Garrell

-LRB-

Organic

Chemistry

Department

University

of

California

Los

Angeles

-LSB-

UCLA

-RSB-

-RRB-

has

worked

on

elucidating

the

chemical

mechanisms

for

adhesion

and

cohesion

in

and

other

marine

biopolymers

to

develop

artificial

tissues

and

bioreactors

-LRB-

Ooka

and

Garrell

-RRB-

Future

dental

and

medical

adhesives

may

contain

mussel

adhesive

proteins

and/or

domains

from

mussel

adhesives

-LRB-

Tay

and

Pashley

Ninan

et

al.

Fulkerson

et

al.

Robin

et

al.

Schmidt

et

al.

-RRB-

Conclusions

Scientists

and

laymen

alike

have

been

fascinated

for

years

with

the

ability

of

mussels

to

cling

to

surfaces

under

water

The

ability

to

adhere

in

an

aqueous

environment

withstand

numerous

environmental

forces

and

resist

conventional

approaches

to

detachment

are

factors

that

continue

to

intrigue

researchers

today

During

the

last

two

decades

considerable

time

and

effort

has

been

spent

in

identifying

the

proteins

that

contribute

to

underwater

adhesion

by

marine

mussels

The

production-scale

availability

of

recombinant

mussel

adhesive

proteins

will

enable

researchers

to

develop

formulations

for

adhesives

in

which

there

exist

endless

applications

for

the

commercialization

of

water-impervious

ecologically

safe

adhesives

derived

from

mussels

adhesion
biomimetics
recombinant protein