Amyloid fibrils formed from a segment of the pancreatic islet amyloid protein

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 608-614

Vol. 155, No. 2, 1988 September 15, 1988

AMYLOID FIBRILS FORMED FROM A SEGMENT OF THE P A N C R E A T I C ISLET A M Y L O I D P R O T E I N George

G.

Glenner, M.D. E. D a v i d Eanes, and C l a y t o n A. Wiley, M.D.

University

of C a l i f o r n i a , San D i e g o La Jolla, CA 92093

Ph.D.*

(M-012)

* N a t i o n a l I n s t i t u t e s of D e n t a l R e s e a r c h , N a t i o n a l I n s t i t u t e s of Health, B e t h e s d a , MD 20892

Received July 18, 1988

SUMMARY: A s y n t h e t i c p e p t i d e f o r m e d from r e s i d u e s 20-29 of the p a n c r e a t i c islet a m y l o i d p r o t e i n has the c o n f i r m a t i o n of a t w i s t e d g-pleated sheet protein s u g g e s t i n g it is a p o t e n t i a l c o n t r i b u t o r toward amyloid fibril formation in the islets of L a n g e r h a n s in Type 2 d i a b e t e s m e l l i t u s . ©1988AcademicPress,lnc.

Amyloid arranged

in

a

conformation fibrils is

fibrils

chain

usually

of

prealbumin

varies

variants

inflammatory

are

disease

(6,7)

in

the

amyloid

sheet

or

twisted

nature with

of the p r o t e i n the

is

fibrils

protein

dialysis

seen

in

where

the

fibril

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in "any form reserved.

the

in

fibrils

protein the

608

light

which

myeloma

SAA

of

the

deposits

in

(3),

in of

plaques

in

of

the

~2

deposits

thyroid

Recently

(2),

protein

cerebral composed

it

polypeptide

Localized as

sheet amyloid

polyneuropathy

are

(8).

the

with

multiple

(5).

carcinoma

comprising

of

sites

g-pleated

condition

amyloidosis;

such

(or p o l y p e p t i d e s )

composing

amyloidotic

amyloidosis

medullary the

clinical

deposits

in f a m i l i a l

(reactive)

Alzheimer's

precalcitonin

proteins

systemic

in

fibers

and

of

immunoglobulin

microglobulin

amyloid

The

e.g.

an

composed

g-pleated

(i).

associated

are

g

protein where

a

protein

of

insulinomas

and

in

Vol. 155, No. 2, 1 1 9 8 8

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

I

CGRP

A

C

D

T

A

T

C

V

T

I0 H R

L

A

G

L

L

S

R

S

lAP

K

C

N

T

A

T

C

A

T

Q

R

L

A

N

F

L

V

H

S

20

CGRP

G

G

V

V

K

N

N

F

V

P

30 T N

V

G

S

K

A

F

lAP

S

N

N

F

G

A

I

L

S

S

T

V

G

S

N

T

Y

Amino acid (underlined)

the

of

islets

mellitus

reveals

it

It

to

is not

has This

have

pancreas

isolated

analysis 46%

of

(i0).

evident

that

the

to

specific by

the

for

method

cysteine

solvents

of

Attempts

were

from

to

20

A

to

29

in

study

was

with

protein

a C-

carrier the

of

of

obtain

(IP20)

IP20

further

to

(ii)

a

lAP,

calcitonin

order

Merrifield

solubilize

unsuccessful.

the

In

sequence

protein;

residues

coupling to

with

2 diabetes

acid

decapeptide

and

for

type

amyloid

CGRP.

of M a r g l i n

of

amino

i)

this

incorporated (12).

islet

sequence

lAP

59%

its

(Table

that

Residues

in

and

the

homology

CGRP

immunization

coupling

the

been

homologous

synthesized terminal

of

peptide,

is

antibodies

for

Langerhans

(9).

gene-related

IAP

sequence of lAP and CGRP. from 20-29 show no homology.

patients

determined

N

usual

IP20

was

performed

by

undertaken.

Material Congo

red

previously

staining described

microscope.

beta

(i).

of

lAP

strand

examination The

methods

studies

of

and

(6)

microscopy

utilized

staining (6).

CGRP turn

this

material were

those

beta

of

recorded

609

on

Orthoplan

the

hydropathy,

as

film

polarizing

employed

evaluate

X-ray

performed

was

were

to

(13).

was

an

methods

Indices

were

and

photographs

Methods

polarization

Negative

ultrastructural structure

and

and

secondary

alpha

helix,

crystallographic

previously using

for

either

described a

57.3mm

Vol. 155, No. 2, 1988

(4589,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4589R2)

powder

camera

or and

a

l14.6mm

CuK~

(4589R3)

diameter

Debye-Scherrer

radiation.

Result~ The smeared this

synthesized

on

a

material

amyloid

sharp

4.75

figure

~

broad much

X-ray

diffraction

fairly

a

slide

revealed

fibrils.

dominant

of

glass

line

at

than a

the

features

characteristic

Figure

with

I.

in

A.

of

sheet the A

line of

microscopic an

average

the

is

a k-pattern structure

8.6

color

(14).

features Principal A.

These

twisted

k-pleated

of

amyloid

fibrils

examination of

of

indicative

of

is

is

more

Besides in

the

among two

that a

less

the

strong, than

the

suggestive some

very

pattern these

sheet

are

structure,

(I).

~ with

filamentous narrowings

X-ray diffraction pattern demonstrating major bands at 4.64 ~ ("back-bone" spacing) (arrow) and 8.3 A ("sidechain" spacing) (arrowhead) indicative of a twisted gpleated sheet structure.

610

are

latter

d-spacings

revealed 50-80

i)

IP20

somewhat and

water,

staining

(Fig.

for

a

width

red

revealed

value

other

distilled

Congo

pattern

line.

at

in

polarization

This

characteristic

Electron

dried.

crystallography

4.64

weaker

dispersed

air

green

scatter,

is

structures

a

4.64

k-pleated

background

defining

and

feature

features

the

was

characteristic

twisted

weaker

IP20

Vol. 155, No. 2, 1988

Figure 2.

consisting revealed the beta

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Electron mierograph Bar = 0.i #m.

of

twists

distinct

various

(Fig,

2).

differences

indices

examined

of

70-80

A w i d e fibrils

Only between

e. g.

the IAP

alpha

of

hydropathic and

CGRP

helix,

beta

(Fig.

IP20.

index 3)

strand

of and

turn. Discussion The

results

comprising

of

residues

the

20-29

examination of

the

lAP

of

the

molecule

synthetic

peptide

reveal

to

it

have

2.2

1.3

mx 0,4 -.0.5

~. -1.4 .,~

-2.3 -3.2

:-CGRP

~'-.,-~ IA P

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

Amino A c i d s Figure 3.

Hydropathy Index shows dramatic hydrophobicity b e t w e e n C G R P a n d I A P 29. 611

distinction from residues

in

20-

Vol. 155, No. 2, 1988 the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tinctorial,

characteristics twisted this

of

of

sheet

lAP

of t h e s e

to

contributor

to

lAP

the

other

protein.

toto

of

into

by

intrinsic fibrils

can be free

energy Many

N-pleated known

influenced

adherence

(19).

therefore,

Chou

and

activity

the

of

of

20-29

sheet

been

a

N2

of

to

the

vast

that

segment

glucagon sheet

the

destined

of

the

have

into

by

the

Nover

a

form

high

amyloid

intrinsic

and p o l y - l - l y s i n e

fibrils

in

amyloid

have

an

N

amyloid

created

(18)

to

of

intact

intrinsic

majority

(20)

of

form

created

devoid

segments

being

microglobulin

(15)

in

of

significant

proteins

lAP are

fibrils

18-28

found

the

conformation

amyloid

residues

On

of

synthetic

capable

although

as

to

to

the have

antigen,

the

N

(21)

introduction

region

that,

these

when

reside

612

and

the

of

cell

ability

the

on

the

light

binding

to r e g u l a t o r

(inherent to

of

N-structure

selective

applied

suggested

"in its

Based

immunoglobulin that

tight

molecules

subsequently

of

seemed

case

significance

proposed.

~-structure

of

glucagon may

been

it

in this

suggested

(22)

functional

variable

and

binding

Fasman

that

(6) has

have

(20),

the

proteins,

influenced

residues

reveals

CGRP.

N-configuration

Of those

in

of

chains

hormonal

of

of

system.

polypeptide

We,

the

and

conformation

interaction

shown

proteins

(17)

suggestions sheet

that

the N - p l e a t e d

have

to N - p l e a t e d

to the

that

decapeptide

insulin

converted

from

index

mellitus.

However,

both

hydropathic

2 diabetes

proteolysis

structure,

conformation

in Type

N-structure.

(19).

the

as

fibrils

N-structure

i.e.

ultrastructural

deposition

disease

Prealbumin

and

its

Usually

amyloid

intrinsic

fibrils

to

synthetic

(13,16).

it

conclude

can h a v e

Alzheimer's

structure. 50%

and

proteins

The

fibrils

we

investigations

amyloid

protein

distinguish

of L a n g e r h a n s

Recent

(15) The

in c o n f e r r i n g

molecule

islets

fibrils

fibrils.

findings

a major the

crystallographic

amyloid

N-pleated

region

basis

x-ray

or

induced)

receptors

that

the

to a s s u m e

or

(20).

biologic a folded

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

conformation

upon

the

of

adoption

Recently

the

interaction a

having

protein,

suggested of

soluble, noted

associated a wide

a

small,

such

that

of

as

variety

(23)

retinol

these

the

it

is

possible

just

the

the

amino

understanding

and

the

of

structure

this

basic

molecules

of

are

labile

has

a

influenced

either both. seem the

It

to

have

potential

was an for

interaction,

capacity.

Since

structure

methodologies

architecture

the

sparingly

three-dimensional

newer

protein

binding

diverse

retinol-binding

protein-protein

in a r e c e p t o r

i.e.

binding.

three

or

all

~-strueture

sequence,

receptor

structure

proteins

predict

receptors"

e.g.

that

A),

including

native

upon

K-structure,

the

to

acid

specific

structure

mammalian

of f u n c t i o n s carrier

its

(Vitamin

Thus,

a molecular

from

that

conjugated

as

yet

of

significant

receptor.

not

compact

commonality

proteins

binding

more

with

are

more

making

feasible

(24).

Acknowledgment We w i s h of

Patrick

Olshefski grant

Burrola

for

#DK34609

and K i d n e y

to a c k n o w l e d g e

the from

Disease,

in

peptide

the

excellent

the u l t r a s t r u c t u r a l synthesis.

the N a t i o n a l

This

Institute

technical studies

study

was

assistance and

Dennis

supported

of D i a b e t e s ,

by

Digestive

NIH. References

1

.

2. 3. 4.

5 .

6. 7.

Eanes, E.D. and Glenner, G.G. (1968) J. Histoehem. C y t o e h e m . 16. 673-677. Glenner, G.G., Terry, W., Harada, M., Isersky, C., and Page, D. (1971) S c i e n c e 172. 1 1 5 0 - 1 1 5 1 . Dwulet, F.E. and Benson, M.D. (1984) Prec. Natl. Aead. Sci. U S A 81. 694-698. G o r e v i e , P.D., Munoz, P.C., Casey, T.T., D i R a i m o n d o , C.R., Stone, W.J., Prelli, F.C., R o d r i g u e s , M.M., Poulik, M.D., and Frangione, B. (1986) Prec. Natl. Acad. Sci. U S A 83. 7908-7912. Rosenthal, C.J., Franklin, E.C., Frangione, B., and G r e e n s p a n , J. (1976) J. Immunol. 116. 1 4 1 5 - 1 4 1 8 . G l e n n e r , G.G. and W o n g C.W. (1984) Biochem. B i o p h y s . Res. Commun. 120. 885-890. G l e n n e r , G.G. and W o n g C.W. (1984) Biochem. B i o p h y s . Res. Commun. 122. 1 1 3 1 - 1 1 3 5 .

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8 .

9.

I0. ii. 12. 13. 14. 15. 16.

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