Biosynthesis of glucohydrolase I, a glycoenzyme from Aspergillus niger

BIOCHEMICAL

Vol. 36, No. 3, 1969

BIOSYNTHESIS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF GLUCOHYDROLASE I,

John

H. Pazur,

Department

David

A GLYCOENZYME FROM ASPERGILLUS

L. Simpson

and Harvey

of Biochemistry, The Pennsylvania University Park, Pennsylvania,

NIGER*

R. Rnull

State 16802

University

June 13, 1969

Received

Summary: Enzymes which contain carbohydrate units as integral structural TWO aspects of the components are appropriately designated as glycoenzymes. biosynthesis of glucohydrolase I, a glycoenzyme from Aspergillus niger have been investigated. First, the glyco-portion of the glucohydrolase is synthesized via nucleotide diphosphate hexose pathways and the synthesis does Second, data on the properties not seem to be under direct genetic control. of glucohydrolase isolated from inside and outside the cell establish that the carbohydrate moieties become attached to the enzyme inside the cell rather than during transport of the enzyme through the cell membrane and wall.

An increasing drate

moieties

number

in

their

proteins.

Enzymes

oxidase

from Aspergillus

(alpha

amylase)

Aspergillus

galactose Other

from

(Morris Cole,

type

Bacillus

(Pazur, all

or glucosamine

enzymes which

and Hager,

1966),

malic

found

to contain

are

1965),

contain

unique linked

(glucoamylase)

to their

1964), beta

dehydrogenase

*This investigation was supported, Elkhart, Indiana atories, Inc., AM-10822.

1955),

chloroperoxidase

glucuronidase from bovine

polypeptide are

glucose, chains.

B from

liver

in part, by grants from and the National Institutes

394

al -et --,

from Caldariomvces

(Dugan,

from

glucanohydrolase

ribonuclease

from bovine aorta

(Pazur,

of mannose,

shown to be glycoproteins et al.,

glucose

glucanohydrolase

and Rhizopus delemar combinations

glyco-

include

and glucohydrolase

1963)

carbohy-

therefore

laboratory

al -et -.J

et al.,

(Hanafusa,

and Hirs,

in our

(Pazur,

covalently

(Plununer

1967),

studied

subtilis

have been

oryzae

have been

and such enzymes

niger

enzymes

from Aspergillus pancreas

structure

of this

niger These

1967).

of enzymes

fumazo

(Plapp

al -et --,

bovine

and

1967),

the Miles Laborof Health, Grant

Vol. 36, No. 3, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acid phosphatase from rat kidney (Dugan, -et --> al from bovine pancreas (Catley, appropriately vestigating

descriptive

et al.,

1969).

glycoenzyme, seems

The term,

for this unique group of enzymes. Wehave been in-

pathways for the biosynthesis

of the glyco-portion

with the view of assessing a possible function the transport

19671, and deoxyribonuclease

of glycoenzymes

for the carbohydrate units in

In this communication, results

system of the organism.

of our

studies on the biosynthesis of glucohydrolase I, a glycoenzyme from Aspergillus

niper are being reported. 4. niger produces two isoenzymes of glucohydrolase (Pazur and Ando,

1959)

but because of ease of purification

most of our experiments have been

performed with the more abundant isoenzyme, glucohydrolase I. contains approximately

100 moles of monosaccharide residues per mole of

enzyme representing about 15s of the total Mannose constitutes

This isoenzyme

the major fraction

mainder being glucose and galactose.

weight of the enzyme molecule.

of the carbohydrate residues, the reResults from several types of experi-

ments point to nucleoside diphosphate hexose pathways as the route of synthesis for the glyco-portion

of the enzyme. First,

carbohydrates are incorporated little

into the glyco-portion

randomization of the label;

labeled

of the enzyme with

second, pyrophosphorylase activities

several types are present in a cell-free a transfer

specifically

extract

of

from the organism; and third,

of hexose units from nucleoside diphosphate hexoses to a modified

enzyme molecule is effected suggest that,

by a cell-free

since the addition

sequent to protein synthesis,

enzyme extract.

It

is tempting to

of carbohydrate units appear to occur sub-

this aspect of glycoprotein

synthesis is appar-

ently not under genetic control. While specific

functions

of carbohydrate moieties in glycoenzymes are

at present unknown, the suggestion has been made that the carbohydrate units become attached to the glycoenzyme during passage through the cell membrane, thereby facilitating

the transport

of the enzyme. Data from our experiments

show that glucohydrolase I from inside the cell possesses the samemolecular 395

Vol. 36, No. 3, 1969

BlOCHEMlCAL

weight,

mobility,

electrophoretic

position is

membrane ties,

carbohydrate

as the enzyme isolated

completely

synthesized

through

the

of their

culture

the cell

prior

inside

cell

neutral

membrane

has,

content

from the

The possibility

and wall.

because

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Eylar,

and carbohydrate

The glucohydrolase

filtrate. to secretion

1965)

com-

that

through

the cell

the carbohydrate

character,

aid

in

the transport

of course,

not

been excluded

moie-

of the by our

enzyme

findings.

EXPEXIMENTAL Incorporation gillus

of glucose-l-14C

niger

glucose-l-

(NRRL 330)

was grown

14C or mannose-l-14C,

source

(nutrient

broth)

glucohydrolase method

will

of incubation.

filtrate

were

and further

(Pazur,

al -et -a,

with

purified 1963).

I was obtained

materials,

electrophoresis

gradient

columns.

activity

were

located

in

The carbohydrates

and located glucose pure carbon

Leuconostoc

ethyl

mesenteroides

for

volumes

of ethyl

alcohol

on DEAE-cellulose

a high

degree

gel-columns

and ultracentrifugation

the same fractions

hydrolysis,

aliquot

I from cells by paper

the chromatogram

(ATCC 10881)

acid that

the gluco-

on ion-exchange on densityand radio-

grown

on glucose-

chromatography

The labeled

of the labeled

and lactic

for

the columns.

separated

from

and Ando, columns

enzyme activity

by radioautography.

extracted

alcohol

from

the glucohydrolase

by acid

were

the

mannose,

and obtained

in

mannose was degraded

(Gibbs,

by

and the gluco(Pazur

of purity

in

the protein,

of the occurred

on density-gradient

on chromatography

in

assay

in the culture

experiments

An appropriate

dioxide,

glucohydrolases

The

the details

from

on the chromatogram

and galactose form.

four

cases,

1966).

enzyme synthesis

the

by centrifugation

In all

liberated

point

Evidence

a nitrogen

al -et -a>

oxidase;

Maximal

by chromatography

hydrolase

1-14C were

elsewhere.

precipitated

I was isolated

glucose

Asper-

containing

or mannose,

(Lineback,

-

by a new spectrophotometric

with

At this

nlucohydrolase in media

glucose

salts

reaction

be published

86 hours

hydrolase

nonlabeled

was followed

on a coupled

into

in 500 ml cultures

and inorganic

production

based

new method

1959),

and mannose-l-14C

al -et -*,

had been adapted

1963) to grow

to

with on man-

Vol. 36, No. 3, 1969

BIOCHEMICAL

nose.

of the i4C

The distribution

was as follows: Degradation Labeled

86% in Cl,

of the

mannose-lJ4C. mannose

the

was obtained

labeling

pattern

in

old

in

amount

a small

ide,

0.005

tion

x g for was used

cosyl

of the

residue

activities.

Samples

of approximately

hexose-l-phosphates sulting tions

digests times

glucose,

were were

was centrifuged

elaborates

the

for

supernatant for

-

The myceand washed

were

suspended

M magnesium

and then

broken

at 15,000

chlor-

in a Virtis

x g for

from

the

second

pyrophorylase

and gly-

and 1 mg

detected

carbohydrate

enzyme probably

in digests

these

nucleoside

residues

occurs

UDP-

The organism

of synthesizing

of the

reac-

method.

and hexose-l-phosphate.

capable

of the

The reafter

chromatographic were

at

centrifuga-

triphosphates

and GDP-mannose

30

was recentrifuged

of new nucleotides

by a paper

The activation

of the glyco-portion

0.01

detecting

the presence

triphosphate

only

The

in 0.1 ml of the enzyme extract.

pyrophosphorylases

hexoses.

studies.

The cells

The supernatant

DDP-galactose

the appropriate

X-100

1 mg of nucleoside

examined

containing

thesis

mixture

of 1, 3, 8 and 25 hours

TDP-glucose,

diphosphate

M Triton

dissolved

on

was low,

on a filter

containing

and 0.1

enzyme preparation

completed. grown

activities

was collected

buffer

The clear

transferase

degradation

of pH 7.2.

was discarded.

30 minutes. as the

buffer

phosphate

The resulting

and the

20,000

M phosphate

M dithiothreitol

homogenizer. minutes

0.1

niger

experiment

transferase

of 4. niger

been

to above.

and glycosyl

culture

have not yet

for

glycoenzyme

in C4 + C5, and 9s in Ce.

enzyme in this

was similar

a k-day with

of the

the labeled

from Aspergillus

of the mannose

lium

thoroughly

yield

isolated

quantity

activities

from

and galactose

sufficient

Pyrophosphorvlase from

glucose

I was also

Since

in the mannose

2.5% in C2 + C3, 2.54

labeled

glucohydrolase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

syn-

via

these

pyrophos-

for

use as the ac-

phorylases. A partially ceptor

material

glucohydrolase

modified for I with

the

glucohydrolase

glycosyl

alpha

I was prepared

transferase

mannosidase

(Li, 397

experiments 1966).

by treating In the

transferase

pure ex-

Vol. 36, No. 3, 1969

periments,

the

lium

niger,

of 4.

guanosine

contained

into

with

collected content

M sodium

acetate column

glucohydrolase

radioactivity

weight

and 1 mg of

components

for

2 x lo6

the digest

of pH 5.3.

and assayed

I,

the myce-

approximately

at room temperature,

solution

from

was

on a Sephadex

Fractions

G-25

of 3 ml were

enzyme activity,

protein

and radioactivity.

tions

in these

cpm.

Unreacted

active

basis

of results

from

diphosphate likely

to 24)

exhibited

several

such experiments glucohydrolase

by a transfer

to the partially

transfer

of galactosyl

units

et al.,

1966)

and glucosyl

ponding

sugar

nucleotides

preparations

from

Comparison

of intra-

liters

and extracted

different

with

Each extract

the mycelium

(Bosmann

types

several

from

the seventh

was broken

controls, from

guanosine

In mammalian

most

diphosphate systems,

xylosyl 1968)

was

the guanosine

The synthesis

from the

1965),

it

units from

the

(Grebner,

the correswith

enzyme

of tissue. glucohvdrolase

-

The mycelium

of 4. niner

was collected

of 0.05

M citrate-phosphate

aliquots

was assayed

frac-

On the

has been demonstrated

and extracellular culture

These

The inter-

and adequate

and Eylar,

to glycoproteins

of a 46 hour

the sample

units

radio-

of radioactivity.

glucohydrolase. al -et -.,

weight

2'7 to 33.

glucohydrolase.

(McGuire,

was 1,600

radioactivity.

units

concentra-

fractions

was synthesized

of mannosyl

modified

these

traces

fractions

in comparable

fractions

only

in

and low molecular

of the initial

mannose- 14C and the modified

mannose

in

in

was located

were in

mannose

located

portion

radioactive

occurred

pH 4.0.

were

the major (18

the digest

radioactivity

diphosphate

products

fractions

that

Total

guanosine

vening

from

and enzyme activity

fractions.

contained

concluded

recovered

The protein

hydrolytic

tions

point

6 hours

the Sephadex

14, 15 and 16.

vity

hydrolyzed

and low molecular

The glucohydrolase

eight

0.4 ml of the enzyme extract

14C (total

for

high

0.05 from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 mg of partially

incubation

fractionated column

digests

diphosphate-mannoseAfter

cpd .

BIOCHEMICAL

for

glucohydrolase extraction

in a Virtis 398

by decantation buffer

activity.

was virtually

homogenizer

from

and the

of

Enzyme actinil.

At this

intracellular

Vol. 36, No. 3, 1969

glucohydrolase lated

BIOCHMCAL

was liberated.

and purified

fic

activities Suitable

The glucohydrolase

by the usual are

recorded

samples

AND BlOf’HYSlCAL

procedures.

in Table

filtrate

were

trophoresis

on paper

and carbohydrate

tained

for

rule

out

the

portion

through

the

the

is

cell

synthesized

the

was isoand speci-

values

measurements.

carbohydrate

units

passage

through

inside

cell

and from

centrifugation,

Identical

of these

completely

inside

gradient

analysis.

by all

that

mixture

on enzyme yields

I from

to density

of the enzyme during

glycoenzyme

Data

this

I.

subjected

the two preparations the possibility

protein

I in

of the glucohydrolase

the culture

RESEARCH COMMUNICATIONS

elecwere

These

obfindings

become attached

to the

the cell

Rather,

the cell

wall.

and then

secreted

wall.

TABLE I Data on glucohydrolase

isolated

from a 46 hr. Total units/l

Extracellular

glucohydrolase

1st

extract

7th

extract

Buffer

Intracellular

+ II)

precipitation

Density-gradient

activity of culture

of _A. niger Specific unitslmg

(I (I

+ II)

chromatography centrifugation

glucohydrolase

+ II)

(I) (I)

I and glucohydrolase

activity protein

15,000

--

1,400

--

80

--

70

me

--

(I) glucohydrolase

DEAE-cellulose

*Representing

+ II)*

blank

Glucohydrolase

Alcohol

(I

(I

culture

2,300

5,500

15

5,100

477

4,000

2,290

1,300

2,310

II,

respectively.

References Bosmann, l-l. B. and Eylar, E. H., Nature, 218. 582 (1968). Catley, B. J., Moore, S. and Stein, W. H., 2. Biol. Chem. && 933 (1969). B. and Berenson, G. S., EnzYmologia, B Dugan, F. A., Radhakrishnamurthy, 215 (1967).

Vol. 36, No. 3, 1969

Eylar, Gibbs,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E. H., 2. -Theoret. l& -Biol. M., Kindel, P. K. and Busse, \

.

89 (1965). M., --Methods

in Carbohyd.

w.

II,

456

c,

Grebner,

E. E., Hall, C. W. and Neufeld, E. F., --Arch. Biochem. Biophvs. 116, 591 (1966). Hanafusa, l-l., Ikenaka, T. and Akabori, S., 2. Biochem. (Japan) 4e, 55 (1955). Li, Y. T., II. Biol. Chem. 2& 1010 (1966). Lineback, D. R., Georgi, C. E. and Doty, R. L., 2. a. w. Microbial. l& 27 (1966). McGuire, E; J. .Jourdian, G. W., Carlson, D. M. and Roseman, S., 2. w. a. & P&112 (1965). Morris, D. R.. and Hager, L. P., J. Biol. Chem. 2& 1763 (1966). Pazur, J. H. and Ando, T., 2. Biol. Chem. 2& 1966 (1959). Pazur, 3. H., Kleppe, K. and Andersop. S., Biochim. Bionhys. Acta 65, 369 ( ww Pazur, J. Ii., Kleppe, K. and Ball, E. M., -Arch. Biochem. Biophvs. a 515 (1943). Kleppe, K. and Cepure, A., -Arch. Biochem. Biophys. 111, 351 Pazur, J. H. and Okada, S., Carbohyd. Res. 4, 371 (1967). Plapp, B. V. and Cole, R. D., m.z 3676 (1967). Pluamrer, T. H., Jr. and Hirs, C. H. W., 2. Biol. Chem. a

400

2530

(19%).