Biosynthesis and Catabolism of Glycoproteins - ACS Publications

2 Biosynthesis and Catabolism of Glycoproteins

Downloaded by PENNSYLVANIA STATE UNIV on June 8, 2012 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0080.ch002

HARRY SCHACHTER, SAROJA NARASIMHAN, and JAMES R. WILSON Research Institute, Hospital for Sick Children, Toronto, Canada

Glycoproteins are a complex group of macromolecules and their biosynthesis is, not unexpectedly, a rather complicated process. Although much has been learned about this process in the past ten years, there are many aspects which we do not understand. In the limited space permitted for this article, it will be possible only to outline the major steps in the biosynthesis of some of the gly coprotein types; more detailed reviews are available (Schachter and Rodén, 1973; Montreuil, 1975; Schachter, 1974a,b, 1977, 1978; Waechter and Lennarz, 1976). I. Initiation of oligosaccharides of the asparagine-N-acetyl-Dglucosamine linkage type. The largest group of animal glycoproteins are those which contain only or predominantly oligosaccharides that are linked to the polypeptide back-bone by an N-glycosidic linkage between N-acetyl-D-glucosamine (GlcNAc) and asparagine (Asn). Many secreted glycoproteins (α1-acid glycoprotein and other plasma globulins, immunoglobulins, various gonadotrophins, ovalbumin, various enzymes, etc.) and membrane-bound glycoproteins (red cell membrane glycophorin, rhodopsin, the envelope glycoproteins of certain viruses such as vesicular stomatitis virus and Sindbis virus) contain Asn-GlcNAc linkage type oligosaccharides. It has become clear that the synthesis of the sugar-amino acid linkage pre-determines the general nature of the oligosaccharide that is subsequently assembled, i.e., that the synthesis of this linkage is an important control point. It is therefore important to understand the initiation process. In recent years it has become apparent that many (but not all) Asn-GlcNAc oligosaccharides share a common "core" structure (Montreuil, 1975) : Man-α1,3 Man-al,6

Man-31,4-GlcNAc-31,4-GlcNAc-Asn

0-8412-0452-7/78/47-080-021$06.50/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

22

GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

Further,

the

Hemming,

Heath,

1976,

and

strong

work

is

evidence

in

Soon

after

suggested cells.


that

It

tissues

of

of

mammalian

core

is

scheme

has

Lennarz,

provided

pre-assembled prior

for

role

of

the

to

as

a

incorporation

initiation

p o l y p r e n o l - l i n k e d sugars

polysaccharide

a

process

similar however,

the

1);

be

pioneering

evidence and

nucleotides

(Fig.

might

assembly,

reaction

the

to

work

for

the

of

tissues hen

has

Leloir s

(rat

from

and p i g

oviduct,

mammalian

a

phosphate,

phosphate

shown

l i v e r ,

human

to

their

phosphoryla-

sugars

formed

are,

Man-3-monophosThe

occur

mouse

workers group

1

i n

N-acetylglucosamine

been

been

higher

dolichol

dolichol

now

in

transfer

Glc-3-monophosphate-dolichol.

and

intermediates

had

various

occuring

GlcNAc-a-pyro-phosphate-dolichol,

thyroid,

Jeanloz,

and

production

in

a

myeloma

lymphocytes,

calf

variety tumor,

pancreas,

. Another

covery that

of

rat

glucose

liver

to

thought

to

water,

in

but

the

be

protein.

This

of

study

in

this of

the

product

product and

an

which

was

in

to

dolichol

the

was

of

radioactive

was

at

f i r s t

subsequently

shown

w h i c h was

et

aJU ,

pyrophosphate

ac to

insoluble

chloroform-methanol

(Behrens

dis

noted

endogenous

chloroform-methanol-water

solvent

was It

transfer

glucose

oligosaccharide acid

group

1

effect

acid-insoluble

soluble

The

length

of

the

tissue

and w i t h

reports

from

contain

about

mannose

and

mouse

20

residues

η

assembly

esting

to

formation

the

led

be

in

(2:1,

(10:10:3,

1971)

v/v)

the

oligosaccharides

units

residues. for

oligosaccharide of

GlcNAc

and

attached

conditions

laboratory

microsomes,

;

way in

5-7

used

indicated

to

for

the of

With

tissues

other

appeared

the to

residues

of

varies The

oligosaccharide

consisting example),

dolichol

synthesis.

to

N-acetylglucosamine, (hen

oviduct

predominant

lack

glucose

mannose,

and

as

and

dolichol contain

follows:

-Man-31,4-GlcNAc-31,4-GlcNAc-pyrophosphate-dolichol of

note of

oligosaccharide

glycose

glucose

myeloma

(Man-α-)

the

Leloir's

pyrophosphate

from

could

L e l o i r s

oligosaccharide.

tissues.

with

The

an

from

monophosphate

trichloroacetic was

intense

two

microsomes

pyrophosphate

discovery

many

contribution pyrophosphate

dolichol

form

dolichol

v/v)

major

dolichol

from

ceptor

to

reviews)

intermediate

general

mannose

l i p i d

(Leloir,

Waechter

1.

the

sugar

phate-dolichol

a

The

see

recent

common

glucose,

polyprenol

etc)

this

strong

respectively,

bovine

for

provided

of

laboratories others,

bacterial

was,

respective ted

in

that

first

1978,

that

Figure

established

and

oligosaccharide

polypeptide.

shown

several

Schachter,

lipid-linked into

of

Lennarz

this the

structure

inversions

3-linked

mannose

is of

shown

in

anomeric

from

Fig.

1.

It

is

configuration

GDP-a-Man and

inter

in

α-linked

the mannose

Man-3-monophosphate-dolichol. M.J.

carried

Spiro

out

containing

et

careful dolichol

a l .

(1976a,b)

and

investigations pyrophosphate

on

R.G. the

Spiro

et

formation

oligosaccharides

al of by

(1976)

have

glucoseseveral

In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

2.

Glycoprotein

SCHACHTER ET AL.

tissues.

Working

with

these

workers

phate

oligosaccharide

Biosynthesis

thyroid

demonstrated with

slices

and

rather

than a

23

Catabolism with

the

formation

of

the

tentative

structure:

microsomes,

dolichol

pyrophos

(Man-α-) -(Glc) _ ~(Man-α-) -Man-GlcNAc-GlcNAcβ

1

2

4

PY r o p h o Evidence and

was a l s o

thymus

pyrophosphate preparations


these

large

molecules lack

slices calf

dolichol

which

Parodi

for the of

pyrophosphate only

However, et

It

is

interesting

of

rat liver

calf

pyrophosphate

oligosaccharide

glucose

believed

to

be present

i n α-linked

of

from

cules

so

that

transfer

involves

The

final

single

inversion

step

i n the

i n i t i a t i o n process

work and the to

from

residue

(Waechter

and Lennarz, established

transfer

labelled

of

endogenous

protein

that

acceptors

membrane the

that of

that

the

presence

which

of

ovalbumin

glycoprotein,

by

retory

The

is

It

always

of

attached

pathway

transfer

However

to

the presence

pyrophosphate

of

of

of

further

forms

of

pathway. for

from

that

any amino sequence

the

dolichol

three

which is

sec

under

oligosaccharide

acid

does

A. effects

not

i n the tripeptide

almost this

time

i n

sec

and ribonuclease

polypeptide

some

a

glycoprotein

the transferase

for

rates

evidence

oligosaccharide

into

synthesis

ovalbumin,

the dolichol

denatured

an asparagine X can be

that

i n secretory

α-lactalbumin

recognized to

by

obtained

specificity

(Thr)-where

dolichol

has

tunicamycin,

normal

oligosaccharide

has been

i n h i b i t e d by

almost

appears

synthesis

however,

continued

oligosaccharide

substrate

Asn-X-Ser 1974).

the

suggested

since

i t

to

proteins

i n the

work,

endo

bound

secretory

only

and

The

1977);

at

been

asparagine

i n i t i a l l y

of

cells

intermediate

a l l tightly

recent is

l i p i d

an

catalyzed

reactions.

known

synthesis

have

the d o l i c h o l

showing

More

myeloma

has not yet

between

involved

glycosylated

proteins-ovalbumin,

incorporation stood.

is

(1977)

i t

of

to

Earlier

and Lennarz,

tunicamycin,

and Lennarz

pyrophosphate

the

from

findings

be

transfer

preparations

were as

ovalbumin

(Struck

retory

of

of

blocks

Pless

involvement

these

might

glycoproteins.

unglycosylated

synthesis

study;

the

(Fig. 1).

i n these

identified

pathway

N-acetylglucosamine of

linkage

experiments

mole

configuration.

is

rat l i v e r ,

however,

synthesized

glycosylation

antibiotic

with

oligosaccharide

n o t be

under

dolichol

membrane-bound

shown an

tissues

is

the

i n these

oligosaccharide

chain

microsome

acceptors;

i n these

and could

the

1976) that

glucose-

molecules;

form

anomeric

pyrophosphate

an N - g l y c o s i d i c

N-acetylglucosamine genous

of

i n the polypeptide

hen oviduct

established

of

dolichol

and

et a l . ,

glucose-3-monophosphate-

a

oligosaccharide asparagine

glucose

smaller

(Herscovics

large

dolichol

dolichol

but

(Behrens

pancreas

synthesizing

containing is

microsome

produce

and N-acetylglucosamine

from

and from

capable

kidney dolichol

that

do n o t

oligosaccharides

mannose

microsomes

a l . , 1972) were

by o v i d u c t ,

glucose-containing

t h y r o i d and hen oviduct

contain

a l . , 1977a,b)

formation

similar

oligosaccharides. from

glucose.

1971; et

obtained

tissue

sphate-do1icho1

sequence(Marshall,

not

necessarily


24

GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

result a

i n

glycosylation

protein

may,

cosylated (Thr)The

forms

site

work

but

that

A l l

lactalbumin sequence

completely

the

and

fully

protein A)

must

the

required tripeptide

and

the

Although serve

phate

transfer that

II.

GlcNAc

contain

structures clear

residues by

such

and

not

as yet

which

lack

and

since

1975)

o fa l l

glucose

stomatitis stages in

involve The

t othis by

the

the

The

viral

class

Work

and

Sindbis

assembly

leads

e ta l . , envelope

plasmacytoma processing

Asn-GlcNAc

oligosaccharides

oligosaccharide i n

2.

F i g . may

f o l -

mannose

evidence has

i n d i -

envelope

subsequent 1977;

glyco-

rough

processing

o f mannose Robbins,

glycoproteins processing since

c e l l

(Tabas

line

GlcNAc,

Man a n d

endoplasmic

are

reprehowever,

biosynthesis o f was

e tal_.,

initiation

(and

1977;

i s , also

shown

1978) .

and

I t i spostulated

undergo

containing the

be

enveloped

virus)

t o removal

glycoproteins;

in

containing

must

the

o f the

type

type

residues, i t

reported with

o fAsn-GlcNAc oligosaccharides

summarized

occurs

Asn-

a n d o f some

recently

o fglycoprotein,

a mouse

are

process

o f the

o f mannose

residues;

(Hunt

processing large

large

N-

protein-bound oligosaccharide i s

mannose

oligosaccharide

initiation

have

virus

in

viruses,

rich

1978).

IgG

the

contain

3 mannose

residues

processing".

o fmembrane-bound

confined

oligosaccharides. primarily

oligosaccharide

the

residues

secretory

oligo-

the

Asn-GlcNAc linkage

only

11 r e s i d u e s

laboratories

a tearly

e ta l . ,

i n

than

N-acetyllactosamine

and

glucose)

sentative

the

pyrophos-

effective

acceptors

of

made

dolichol

which

o f the the

contain

probably Tabas

have

type.

o f glucose

and

(1977)

involves

incorporation o fa large

o f these

moieties

glucose.

glycoproteins

This

oligosaccha-

have

incorporation

pyrophosphate

envelope

a

accept

more

protein

oligosaccharides

(vesicular

large

the

9 times

pathway

these

to

were

proteins

oligosaccharide

e ta l .

oligosaccharides

glucose

Several

that

proteins quite

vivo

"oligosaccharide

viruses

a

not

o fp r o t e i n - b o u n d o l i g o s a c c h a r i d e s

removal

residues.

cated

dolichol

(Montreuil,

that

i n

Turco

about

mannose

linkage Since

not

not

tripeptide

sequence

denatured

glucose-containing

are

pyrophosphate

Processing

lowed

that

i n

acetylglucosamine,

of

site.

glycosylation t o

oligosaccharides

small

donors

t o endogenous

glucose-free

suggests

dolichol

relatively

protein,

oligosaccharides

smaller This

into

observation

saccharide

for

this

did

such

acceptors

(ovalbumin, a -

glycosylation

pyrophosphate

carry

asoligosaccharide

important

1-2

for

for

ungly-

-Asn-X-Ser

exogenous

several

and

a tanother

required

without

sequence

requirements

dolichol

and which

oligosaccharide

is

a tone

proteins

1977);

Lennarz,

resolved.

glucose

do

with

the

and

glycosylated

unfolded

however,

ride

can

be

acceptor

with been

(1977)

proteins

asacceptors;

Pless

both

glycosylated

contained

several

exact

i n

unglycosylated

Lennarz

effective

RNase

whereas

ineffective


may

be

three

be

exist

or

and

1974;

(Marshall,

example,

o fPless

indicates occur.

for

by Glc

reticulum

subsequent that

a l l

transfer (Fig. (see


of

2). next

2.

Glycoprotein

SCHACHTER ET AL.

Biosynthesis

25

and Catabolism

UOP-GicNAc Dd-P *UMP G l c N Acc*-PP- - P - D o l U D P ^ G l c N A c J-

(GOP^Mon) (GOP^M •

n 1

n

γυΟΡ

(Dol-P)4

(

n

GlcNAc GlcNAc*P-P-Dol IcNAc^P-P-Dol GDP-Man *GDP

ί I " ^Man^P-Dol)

£

Mon-GlcNAc^GlcNAc^P-P-Dol—^(Man) *Man*GlcNAc*GlcNAc*P-P-Dol n

"

l U 0 1

P

Acceptor

)


Nooi-p-p ( Man\,

Man - GlcN Ac - GlcN Ac * A S N

Figure 1. Initiation of Asn-GlcNAc-type oligosaccharides. Oli gosaccharides are preassembled as lipid intermediates. Abbrevia tions: Dol, dolichol; P, phosphate group.

Peptide ^

(Glc) -(Man) -(GlcNAc) -P-P-Dolichol 2

n

(Glc) -(Man) -(GlcNAc) -Asn2

2

n

2

Oligosaccharide processing

V M-

Biosynthesis and Catabolism of Glycoproteins - ACS Publications

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