The Role of Glycosidically-Bound Mannose in the Cellular

12 The Role of Glycosidically-Bound Mannose in the

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Cellular Assimilation of β-D-Galactosidase JACK DISTLER, VIRGINIA HIEBER, ROY SCHMICKEL, and GEORGE W. JOURDIAN The Rackham Arthritis Research Unit and The Departments of Biological Chemistry and Pediatrics, The University of Michigan, Ann Arbor, MI 48109

While glycose-protein complexes have been recognized since the early 1800's (1,2), the role that carbohydrate residues play in the function of these biopolymers has until recently remained obscure. In the last 15 years evidence has accumulated that supports the participation of the carbohydrate portion of these complexes in cell-cell recognition (3), c e l l adhesion (4) and c e l l transformation processes (5). In addition, an increasing body of evidence now suggests that an initial step in the assimilation of glycoproteins involves the binding of specific carbohydrate residues of glycoproteins, called recognition markers, to specific cell surface receptors. The elegant pioneering experiments of Ashwell, Morell and co-workers (6) demonstrated that glycoproteins terminating in β-galactosyl residues are rapidly and selectively removed from the circulation by mammalian hepatocytes and that specific receptors for β-galactosyl residues occur on the plasma membranes (7). Other carbohydrate residues also serve as recognition markers for the c e l l u l a r assimilation of specific glycoproteins. Lunney and Ashwell (8) have shown that avian hepatocytes s p e c i f i c a l l y assimi late circulating glycoproteins that contain oligosaccharide chains terminating in β-N-acetylglucosaminyl residues. Mannosyl residues have been implicated as putative recognition markers for binding of gonadotropin to rat t e s t i s c e l l s (9), and the clearance of ribonuclease b (10), agalacto-orosomucoid (11), and β-glucuronidase (12) from the circulation by rat liver. In Neufeld obtained *This

t h e l a t e

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and colleagues from

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A r t h r i t i s ,

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Health,

and from

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Unless otherwise noted, all sugars are of the D-configiiration.

0-8412-0466-7/79/47-088-163$05.75/0 © 1979 American Chemical Society

Goldstein; Carbohydrate-Protein Interaction ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CARBOHYDRATE-PROTEIN

164 the in

abnormal

accumulation

f i b r o b l a s t s

(13).

The

enzymes

under

ries

to

lysosomal ical

studies

stimulated l u l a r

the

in

part

large to

the

surface

man,

Shapiro,

enzyme of

have

and

Neufeld

by

found

of


has

r i e s .

Bovine

highly

p u r i f i e d

coprotein was

suggested

a

found

of

to

of c l i n

have lysosomal

the

i n t r a c e l

that

under

mechanism

treatment

to

s p e c i f i c Hick

^ - a c e t y l -

that

e f f i c i e n t

for

involve

to

of

dTd

not

a s s i m i l a t i o n

A d d i t i o n a l l y ,

of

s p e c i

hypothesis

conditions

the

of


markers

t h i s

Kresse

of

and

von

N^acetyl-a-hexosaminidase


of

t h i s

carbohydrate generalized

by

c e l l

obtained

enzyme

by

with San-

mannose

mannosyl

by

the

well

the

cell

surface

parameters

or i t

proceeded

experiments

methyl was

receptors designed

found of

from

comprised This

mannose of

and

did

binding (Figure

g a n g l i o s i d o s i s

gly

and

10

explore

the

of

or

that of 1). the

a-

3-

whereas

(21).

By to


mannosyl

residues

above

k i n e t i c


was or

glycoproteins

the

The

enzyme

contributed

methyl

not

was

from

results

3-galactosidase serum

the

was

a The

observation

glycosides

with

be

mannose

These

binding

to

to

3-galactosidase

removed

enzyme.

the

laborato

preparation

(21_).

hypothesized

by

associated

generalized

When

were


the

enzyme,

( 1 8 J 9 , 2 0 ) .

mannosidase

that

in

3 - g a l a c t o s i 3 â s e - d e f i c i e n t

e i t h e r

the

established

receptors,

prompted

of

observation

i n h i b i t e d

the

residues

residues

and

residues

a s s i m i l a b i l i t y

marker

the

by

authors'

lysosomal

molecule

f i b r o b l a s t s .

p u r i f i e d

of

18

per

residues


the a

chromatography

assimilated

loss

in

3-galactosidase,

approximately

monosaccharides to

been

a f f i n i t y

s p e c i f i c

by

recognition

by

recently

3-galactosidase

ase

the

p a r t i a l l y

that

mannosides

tural

they

hypothesized

of

n i g e r ,

strengthened

l i v e r

such

which

skin

p a r t i c i p a t i o n

recognition

analogy

laborato

of

underlying

was

support

treatment

t e s t i c u l a r

concomitant

other

of


The

prevented


with

the

the

3-galactosidase

rapidly

Aspergillus

to

In

N-acetylglucosamine

generalized

with

that

containing of

treated

way

systems


reduced

for


enzyme

by

as

mutant


Evidence

residues

t h e i r

enzymes

periodate

Sandhoff

3-galactosidase Β

occur

correction

r e s u l t s

mechanism

study.

(16)

a c t i v i t y

f i l i p p o

the

disappointing,

the

model

(15).

with

enzymatic (17)

the

number

for

the

studied

as

under

3-hexosaminidase

Figura

a

therapy

been

lysosomal

receptors

enzyme

i d e n t i f i e d in

defective

c e l l s , f i n d

using

affect this

that

or

prompted

carbohydrate-containing

c e l l

deposits

mucopolysaccharidoses

subsequently

While

elucidate

enzymes

in

of

to

laboratories

translocation

binding

(14).

with

m a t e r i a l s .

lysosomal

d e f i c i e n t

findings

replacement

externally

storage

were

absent

diseases

have

Several f i c

These

attempts

enzymes, added

were

enzyme

storage

glycosaminoglycan patients

factors

that

study.

attempt

of

from

corrective

lysosomal c e l l s

derived

INTERACTION

of

of to

hypothesis

and

s t r u c -

3 - g a l a c t o s i d -



12. DisTLER E T A L .

Glycosidically Bound Mannose

I AÎSI SN-GLCNAC-(GLCNAC,

I

β-Galactosldase (and Inhibitors)

MAN)-*-KAN

ι « 4 I

165

SPECIFIC CELL-SURFACE RECEPTOR

^^^^ SURFACE-BOUND β-GALACTOSIDASE endocytosis LYSOSOMAL β-GALACTOSIDASE

Figure 1. Hypothetical mechanism for the selective assimilation of bovine tes ticular β-galactosidase by generalized gangliosidosis fibroblasts


166


C h a r a c t e r i s t i c s t i o n

System.

tion

of

hours

the

at

enzyme

which

associated b l a s t s ed

to

in

2).

the That

by

of

in

require c e l l s been

passive enzyme

used

to

tion

the

the

(Figure

4).

The

rapid

dence tion

for of

the

should

at

in

Figures

3

at

about

450

s e l e c t i v e presence surface

s e l e c t i v e

l i m i t i n g 4)

times

the

rate

In

for

for

of

to

the

and

glycoproteins

related

a b l e , t e i n

of

highly

a t t e n t i o n , f r a c t i o n

" i n h i b i t o r

obtained The

tion

with

c e l l

surface.

as

was a

for

i n h i b i t o r

the

have

been

yet of

C a l c u l a -

in

from

an the

f r a c t i o n , a

presented This

due

to

the

while

been an

c e l l

i s o l a t e d

i n i t i a l

of

3-Galacto-

of

the

3-galactosidase s u f f i c i e n t

not

quanti-

i n i t i a l l y

abundant

preparation

potent

of

s i t e s

c a l l e d

i n h i b i t o r of

i t s

of competi-

contained

f r a c t i o n

a v a i l -

glycopro-

subsequently

because

receptor

glycoprotein

and a s s i m i l a t e d

structure

Since on

by e v i -

s a t i s f i e d .

were

presumably the

be

Structures

obtained

was

was

Thus,

not

chemical

func-

medium

peroxidase.

have

(25).

l i n e a r

r e s u l t s

may

the

has gener-

additional

enzyme.

focused

f r a c t i o n , 1 '

3-galactosidase

a

receptors.

(from

of

by

3-galactosidase

the

by-product

a s s i m i l a t i o n , The

of

studied.

glycoprotein

glycoprotein

3-galactosidase

were

not

3-galactosidase

provides

3-galactosidase

therefore,

3-galactosidase.

the

does

growth


glycopeptides

p u r i f i e d

be

the of

c e l l

endocytosis

to

in

Carbohydrate

e l u c i d a t e

marker,

found

horseradish

the

that

of were

peroxidase

3-galactosidase for

were

number

3-galactosidase

endocytosis

recognition t i e s

of

of

c r i t e r i a

f l u i d

surface

3-galactosidase

during

order

that

receptors

Characterization s i d a s ê T

c e l l

concentrations

revealed

s p e c i f i c

step

was

of 50


peroxidase

a s s i m i l a t i o n of

the

Horseradish

enzyme

i n t e r n a l i z a t i o n

of

saturation



i d e n t i f i e d ,

the

i n c r e a s -

rate

approximating

process

passive


When the

receptors,

horseradish

receptors

absorptive

a

(Figure

demonstrat-

3-galactosidase

of

of

(23).

l i m i t e d

of

i n t e r n a l i z a t i o n of

occur.

a

rate

and

and

of

surface

not

the

to

f i b r o -

and

was



percent

peroxidase

enzyme

i f

the

demonstrat-

s u l f a t e - c o n t a i n -

medium,

Saturation

endocytosis,


the

3).

12

was

s t a i n i n g

of

levels

expected

c e l l

concentration

generalized

at

a s s i m i l a t i o n of


of

be the

f l u i d

measure

expected,

a l i z e d

If


with

of

culture

of

was

metabolism

f i b r o b l a s t s to

to

functional

accumulation

constant

(Figure

enzyme

was

up

enzyme

exposure

histochemical

c a t a b o l i c

added

would

(lj[,24).

by

d e f i c i e n t

became

adsorption

on

a s s i m i l a -

of

added

the

A s s i m i l a -

the

periods

the

3-galactosidase

were

medium

of

hours,

abnormal

enzyme

enzyme

growth

by

12

i n t r a c e l l u l a r

in

of

by

the

of

10%

for

Furthermore,

a s s i m i l a t e d

3-galactosidase

mediated

and

c e l l s .


receptors

As

the

demonstrated

time

approximately

c o r r e c t i o n

amounts

mediated

with

c e l l s

materials

units/ml

l i n e a r

i n t e r i o r

ing

with

Fibroblast/3-Galactosidase

experiments

for

ing

enzyme

was

time

with

the

i t s

the

3-galactosidase

p a r t i c i p a t e d ed

of

Preliminary

INTERACTION

passed


on

the

through

12. DisTLER E T A L .


167

Figure 2. Histochemical stain offibrobfostsfor β-galactosidase. The fibroblasts were plated on glass slides and stained for β-galactosidase with 5-bromo-4-chloro3-indolyl β-galactoside reagent as described by Lake (22). A, normal fibroblasts; B, generalized gangliosidosis fibrobfosts; and C, generalized gangliosidosis fibro blasts exposed to β-galactosidase (50 units/mL medium) for 12 hours prior to staining; the presence of major enzyme activity in a perinuclear region indicates the enzyme does not coat the cell surface but rather is internalized, presumably in in the cell lysosomes.


168


INTERACTION

0-GALACTOSIDASE (units/ml medium) Figure 3. Effect of the concentration of β-galactosidase in the medium on the rate of assimilation of β-galactosidase by generalized gangliosidosis fibroblasts. The different symbols represent experiments performed at different times using the same enzyme preparation and fibroblast cell strain. The specific activity of the enzyme was approximately 4000 units/mg protein. One unit of enzyme is that amount which catalyzes the hydrolysis of 1 ^mole p-nitrophenyl β-gafoctopyranoside per minute at pH 4.3 and 37° (IS).

PEROXIDASE (units/ml medium) Figure 4. Effect of the concentration of horseradish peroxidase on the rate of assimilation of this enzyme by generalized gangliosidosis fibrobfosts. The spe cific activity of the enzyme was approximately 3000 units/mg protein.


12. DisTLER E T A L . the

a f f i n i t y

support

further

p u r i f i e d

e l u t i o n

with

of

used

by

f r a c t i o n

enzymes

purify to

α-mannoside had a

3-galactosidase

lysosomal

to

adsorption

methyl

glycoprotein that


that

the

3-galactosidase

concanavalin

(21_).

The

carbohydrate

(Table were

169

I)

r e s u l t i n g

i n

the

s i m i l a r

several

crude

and

i n h i b i t o r

composition

and contained

present

and was

A-Sepharose

to

other

extract

(Table

II). A

glycopeptide

coprotein digestion

with

p u r i f i e d and

by

eluted

pronase.

gel

a c e t i c

the

concanavalin

of

3-galactosidase f r a c t i o n

and

by

by

was

a l k a l i n e

3-elimination

Table

III.

i s

t i v e

has a

also

by

carbohydrate

The

r e s u l t s

of

derived

from

i n

gave each

residues. terminated ated

from

single No

organic

metric less in

than

the

a - l , 2 - l i n k e d

glycopeptides

Inhibition

i n

concentration

o f

were

methyl

a-

f r a c t i o n

to

the

bonds

(29).

i s

The

presented prepared

and

that

Spiro d e r i v a

found

f o r

have

The

the

i n of

the 10

been of

a

derived

from

separ a

of

by

was

c o l o r i -

such

that

a s p a r t i c

acid

detected.

studies

mannose

to the

by

of

growth

i n h i b i t o r

times

more

These

r e s u l t s

the

are

glyco

summar

3-galactosidase

function

the

Mannose-Rich

u t i l i z i n g

derivatives

i n h i b i t i o n

100

3-mannosides.

glycopeptides

3-Galactosidase

added

was

r e s i d u e s ;

.N-acetylchitobiose.

residues

logarithmic

compound

a-mannosyl

Mannose

the methodology

per

thyro

chains

N.-acetylchitobiosyl

and other as

terminal

the

from

studies

oligosaccharide

to

glyco-

are

from

these

residues.

the

obtained

f r a c t i o n

together

i n h i b i t i o n

percent

of

f r a c t i o n

f r a c t i o n

contained

the

by

residue

of

degradation

glycoprotein

was a t t a c h e d

would

plotted the

of

f r a c t i o n S p i r o ,

Pre undergo

thyroglobulin

s i m i l a r

mannosyl

approximately o r

of

A s s i m i l a t i o n

glycopeptides

f r a c t i o n t i c

6.

the

Taken

s e n s i t i v i t y

r e s u l t s

i s

seen

glycopeptide

was observed

glycoproteins

Figure

seen

of

The

a s s i m i l a t i o n be

the

A

portion

residue

phosphate

Arima,

glycopeptide

unit

portion

peptide

residues

f r a c t i o n

of

enzymatic

r e s u l t s .

one phosphate

peptides,

the

i n

Compounds. ized

5 ;

l e a s t

a n a l y s i s ;

may be

d i d not

attachment

glycopeptide

i n h i b i t o r

glycopeptide

3-mannosyl

A

f i l t r a t i o n

f r a c t i o n .

At the

unit


N-glycosidic

glycopeptide

composition

and the

a

probable by

bound

and analyzed.

and asparagine

the

procedure

the

s i m i l a r

the

that


gel

f r a c t i o n

polypeptide

c o n t r o l l e d

Figure

3-galactosidase, suggest

of As

glycopeptide

summarized

the

the

presented.

peptides

procedures

was

A-Sepharose

the

sequential

glycopeptide

of

Analysis

(30)

globulin

to

composition

thyroglobulin

by

g l y

by

f r a c t i o n

f r a c t i o n

i n h i b i t e d


i n d i c a t i n g

chains

from

i n h i b i t o r

the

i n h i b i t o r

concanavalin

glycopeptide

p u r i f i e d

of acetyl glucosamine

carbohydrate in

showed

the

3-galactosidase)

glycopeptide

on

column

chromatography

studies

C - l

The

generalized

further

liminary

oligosaccharide

r e s u l t i n g

a c i d .

from

from

adsorbed

A-Sepharose

ion exchange

between

The

f i l t r a t i o n ,

with

was prepared

(and subsequently

to

This

f r a c t i o n

f r a c t i o n

of

the

medium.

mannose As

may

glycoprotein

i n h i b i t o r y suggest

than that


synthe at

170


Table CARBOHYDRATE

ANALYSIS

β-GALACTOSIDASE

AND

INTERACTION

I

OF

BOVINE

INHIBITOR

TESTICULAR

GLYCOPROTEINS* Inhibitor

β-Galactosidase

pmoles/mg

glycoproteins pmoles/mg

protein

protein

Mannose

0.27

0.32

N^- A c e t y l g l u c o s a m i n e

0.15

0.17

The Role of Glycosidically-Bound Mannose in the Cellular

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