Isolation and characterization of a mannan-binding protein from rabbit liver

BIOCHEMICAL

Vol. 8 1, No. 3, 1978 April 14,197B

ISOLATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1018-1024

AND CHARACTERIZATION

OF A MANNAN-BINDING

PROTEIN

FROM RABBIT LIVER Toshisuke Department

Kawasaki,

of Biological Kyoto

Received

March

2,

Ritsuko

Etoh

Chemistry,

Faculty

University,

Kyoto

and Ikuo

Yamashina

of Pharmaceutical 606,

Sciences,

Japan

1978

SUMMARY: A membrane protein which binds mannan has been isolated from rabbit Upon polyacrylamide gel electrophoresis, liver by affinity chromatography. a single major band was recovered with an estimated molecular weight of 31,000. When assayed as inhibitors, N-acetylmannosamine, N-acetylglucosamine, and mannose were potent inhibitors of mannan binding; N-acetylgalactosamine and Glycoproteins with terminal N-acetylglucosmannose-6-phosphate were inert. amine and/or mannose residues which are cleared rapidly from the circulation On the basis of these results, by the liver were the most potent inhibitors. it is proposed that the mannan-binding protein is the principle mediating the hepatic uptake of glycoproteins with terminal N-acetylglucosamine and/or mannose residues. The role the

of prosthetic

catabolism

sides

of circulating

the well

glycoprotein

systems

clearance

the course glycoproteins, preparations. protein mannose

on a presumptive

Subsequent

which recognizes residues.

for

receptors binding

a remarkable

clearance

protein

studies

have resulted

glycoproteins

with

MATERIALS

in

terminal

include glyco-

and RNase B

extent, for

of

These

these

has not been

binding

obser-

resolved.

In

mannose-terminated

of mannan by rabbit isolation

asialo-

N-acetylglucosamine

the rapid

or to what

Be-

for

and mannose-terminated

of specific

we have observed

responsible

have been described.

whether,

for

much attention.

ahexosamino-orosomucoid

However,

the presence

of studies

receptor

of N-acetylglucosamine-

(5 - 10).

reflect

receptor

modes for

circulation

such as agalacto-orosomucoid,

[EC 2.7.7.161 vations

hepatic

alternative

(4),

from mammalian

for

proteins

mammalian

determinants

has received

(1 - 3) and the avian

glycoprotiens

glycoproteins

as regulatory

glycoproteins

characterized catabolism

terminated

oligosaccharides

liver

of a membrane

N-acetylglucosamine

and

AND METHODS

Nal"I, carrier-free, in diluted NaOH was obtained from the Radiochemical Mannan (430 ug) was iodinated with 1 mCi Nalz51, Center, Amersham, England. by a modification of the procedure of Greenwood --et al. (11). The iodinated mannan, purified by passage through a column of Sephadex G-25, was recovered with specific activities ranging from 0.3 to 0.5 uCi per ug. 0006-291X/78/0813-1018$Ul.O0/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

1018

Vol. 8 1, No. 3, 1978

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Orosomucoid was a gift of the American National Red Cross Laboratory, Asialo-orosomucoid, agalacto-orosomucoid and ahexosaminoBethesda, MD, U.S.A. orosomucoid were prepared as described previously (4). Hog kidney cr-mannosidase [EC 3.2.1.241 purified according to the procedure of Okumura and Yamashina (12) had a specific activity of 7 units per mg protein. Rat preputial gland 8-glucuronidase [EC 3.2.1.311, kindly provided by Dr. K. Kato, Kyushu University, Japan, had a specific activity of 104 units per mg protein. The yeast used, Saccharomyces cerevisiae, was a commercially available Baker's yeast purchased from the Oriental Yeast Co.. Tokyo, Japan. Mannan prepared-from this yeast as described in ref. (13) comprised approximately 3 % of protein. The following materials were kindly provided by Dr. S. Suzuki of Tohoku College of Pharmacy, Japan: A mixture of a series of linear oligosaccharides with al+6 linked mannose from S. cerevisiae mannan (14); (Manal+6Man) with an estimated molecular weight of 8,000, obtained by a-mannosidase (ArtErobatter GJM-1) digestion of 5. cerevisiae mannan (15); Manorl+2Mancrl+ZMan, obtained from Candida albicans A-207 mannan (16). Oligosaccharides, Mano,l+6Man, Manol+6Manol+6Man, and Manctl+6Manol+6Man al+bMan were isolated by gel filtration of a mixture of manno-oligosaccharides with al+6 linkage on the column of Bio-gel P-2. Manctl-+3Manal+ZManol+2Man was isolated from -S. cerevisiae mannan according to the procedure of Lee and Ballou (13). Preparation of affinity resins of Sepharose 4B-asialo-orosomucoid and Sepharose OB-mannan was carried out essentially as described in ref. (2). Asialoorosomucoid (100 mg) or mannan (30 mg) was coupled to 60-ml aliquots of Sepharose 4B which had been activated with cyanogen bromide (17). The binding assay was carried out essentially according to "Assay A" of ref. (2). The standard incubation mixture contained, in a final volume of 0.5 ml, the following components: Tris-HCl at pH 7.8, 25 pmol; NaCl, 500 pmol; CaClz, 20 Dmol; Triton X-100, 0.1 % (W/V); bovine serum albumin, 0.6 % (W/V); "'1-mannan, 300 ng; purified binding protein, 0.2 to 2.0 l.lg or an equivalent amount of crude preparation. The mixture was incubated for 15 min at room temperature and the labeled complex was precipitated by the addition of an equal volume of cold saturated ammonium sulfate that had been adjusted to pH 7.8 with solid Tris. After 10 min, at O“, the precipitate was collected by filtration, washed and counted. The isolation of a mannan-binding protein from rabbit livers was carried Frozen livers (150 g) were homogenized with cold acetone in out as follows. a Waring blendor and the homogenate was filtered under reduced pressure and allowed to dry in air. The acetone powder was stirred for 30 min at 4" in 500 ml of 0.2 M NaCl and centrifuged at 12,000 g for 15 min. The residual pellet was suspended by blending in 400 ml of extracting buffer consisting of 0.01 M Tris-HCl, pH 7.8, 0.4 M KC1 and 2 % Triton X-100. After stirring for 30 min at 4', the crude extract was obtained by centrifugation. The pellet was suspended in the same buffer and extraction was repeated three more times. The combined crude extract (1.6 1) was made 0.02 M in CaClz and applied to the Sepharose 4B-mannan column (60 ml) which had been equilibrated with a loading buffer consisting of 0.01 M Tris-HCl, pH 7.8, 0.02 M CaClz, 1.25 M NaCl and 0.5 % Triton X-100. The column was washed with several additional bed volumes of this buffer prior to elution of the binding protein with an eluting buffer consisting of 0.02 M Tris-HCl, pH 7.8, 1.25 M NaCl, 2 mM EDTA and 0.5 % Triton XThe eluate (crude mannan-binding-protein fractions) was made 0.02 M in 100. CaClz and applied to the Sepharose 4B-asialo-orosomucoid column which had been equilibrated with the loading buffer to remove asialo-glycoprotein-binding protein. The fractions which passed through the column were pooled and then adsorbed onto a second, smaller affinity column of the Sepharose 4B-mannan. The column was washed with the loading buffer and the binding protein was eluted as described above except that the Triton X-100 concentration of the eluting buffer was reduced to 0.05 %. The major portion of the binding activity was recovered

1019

Vol. 81, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Polyacrylamide gel electrophoresis of the isolated binding protein in the presence of sodium dodecyl sulfate. The binding protein (13 ug) freed from detergent by ethanol precipitation was subjected to electrophoresis in 10 % polyacrylamide gel according to the procedure of Weber and Osborn (27). Protein bands were stained with Coomassie Brilliant Blue G-250 in 12.5 % trichloroacetic acid. The arrow denotes the migration of marker dye, Pyronine Y.

The binding protein specific in the second bed volume of the eluting buffer. for asialo-glycoproteins was prepared simultaneously from the pass-through fractions of the first Sepharose 4B-mannan column as described in ref. (3). Protein was determined by the method of Lowry -7 et al. (18). RESULTS AND DISCUSSION From 150 g of frozen was recovered

with

tein,

a figure

cific

hepatic

a specific

closely binding

binding

constituents, major

protein

comparable gel

(2,

"'1-mannan

to that

shown previously in sodium

to a major

in Fig.

and modified to the

binding

for

protein

per ug pro-

galactose-spe-

3).

1.

band with Approximate

band was estimated to be 31,000. In an attempt to determine the specificity of

1 mg of purified

of 50 to 60 ng mannan bound

electrophoresis

gave rise

as illustrated

of mannc+oligosaccharides binding

approximately

activity

protein

Upon polyacrylamide lated

livers,

isolated

molecular

of the binding

mannans

were

binding

protein.

1020

dodecyl

sulfate,

a few slower

assayed

moving weight

protein, as inhibitors

Mannan

digested

the

iso-

minor of the a number of the with

8lOCHEMlCAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Inhibitory

activity

1

of manno-oligosaccharides

Amount 50 % inhibition m-0

to give

Compound

Mannose

41

Manal-+2Manal+2Man

11

Manal+3Manal+2Mana;l+2Man

10

Manal-dMan

10 4

Mana1-+6Manal+6Man

pronase tory its

Manal+6Manal%Manal%Man

4

(Manal-dMan)

0.04

and purified

activity.

n

by gel

filtration

inhibitory

activity

almost

protein

towards

The results

summarized

inhibitor

the molecular

sizes

ty was shown

to vary

Inhibition

subjected

than

towards

in Table

1 revealed

directly

with

of binding

the

by various

al+6

mannose

were size

degradation

findings

that

linked

of the oligosaccharides

full

comparable.

sugars

is

given

in mannan,

mannosamine except

were

at C-2,

these

results

protein

inhibitors

is blind

the conditions

ing

protein

tested.

isolated

of inhibition

from

by simple 3 presents

on the binding

that

chain.

in Table

2.

Where-

which normally

is

a

pres-

Since mannose, N-acetyla common three-dimensional structure, these

sugars

to the C-2 position.

(20 - 22),

activi-

tested.

to be a recognition

in human fibroblasts

der

share

suggest

which

has been postulated

pinocytosis

Table

potent

and M-acetylglucosamine

on the binding which

the most

was a where

Inhibitory

a good inhibitor, N-acetylglucosamine, as, mannose was, as expected, minor component of mannan, and N-acetylmannosamine, which is not ent

rather

mannose

conditions

of the oligosaccharide

simple

lost

the specificity moiety

linked

under

inhibi(19)

imply

the oligosaccharide

of mannan.

or al+3

retained

to Smith

moiety al+2

G-100

These

completely.

to be directed

the polypeptide

more potent

on Sephadex

mannan

In contrast,

of the binding than

Mw 8,000

component was devoid

may share

a common site

Mannose-6-phosphate, on lysosomal of inhibitory

enzymes

for

potency

un-

It is noteworthy that the asialo-glycoprotein-bindthe same livers showed a distinctly different spectrum

sugars. data

of mannan

on the

inhibitory

to the binding

protein.

1021

activities

of various

glycoproteins

Among the derivatives

of oroso-

Vol. 81, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Inhibitory

activity

2 of various

sugars

Inhibition

of binding

Compound Mannan-BPa) 30 mM N-Acetylmannosamine

70

N-Acetylglucosamine

49

1

40

4

L-Fucose

26

37

2-Deoxyglucose

16

7

Glucose

15

10

Galactose

13

64

Lactose

5

81

Glucosamine

0

5

Galactosamine

0

69

N-Acetylgalactosamine Mannosamine

0 0

83 ---

2 mM Mannose-6-phosphate

0

---

Glucose-6-phosphate

0

-

mannan-binding

b) 8.2 ng of purified

asialo-glycoprotein-binding

mucoid

agalacto-orosomucoid was the next,

N-acetylglucosamine er inhibitions consisting

circulation

consistent

with

glycosidases,

protein

inhibitor

of mannose

are

was used.

and ahexosamino-oroso-

the potent

inhibitory

activities

More provocative

o-mannosidase

which

adn FWase B (24),

and to be taken

was used.

respectively.

by lysosomal

exclusively

as are ovalbumin rat

being

and mannose, These

protein

was the strongest

displayed

curonidase. ties

---

Mannose

a) 1.0 ug of purified

mucoid,

ASGP-BPb)

were

and preputial

glycoproteins

up by the liver

with

8-glu-

carbohydrate residues

shown to be rapidly following

the great-

gland

and N-acetylglucosamine

have been

of

cleared

intravenous

moie(12,

23),

from

the

injection

into

isolated

in the

(25). The data

present terminal

study

reported

here

is the principle

N-acetylglucosamine

imply

that

the mannan-binding

mediating and/or

the hepatic

mannose

1022

residues.

protein

uptake While

of glycoproteins this

work

with was in

BIOCHEMICAL

Vol. 81, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Inhibitory

3

activity

of glycoproteins

Glycoprotein

24

Orosomucoid

progress, protein tion

Agalacto-orosomucoid

0.33

9

Ahexosamino-orosomucoid

1.70

52

Ovalbumin

3.30

77

a-Mannosidase

0.12

1

&Glucuronidase

0.38

1

further

evidence

of clearance may participate with

an efficient proteins, tein, the

role

(10).

and mannan

the direct role

precursors

zero

of ligands

of the mannan-binding

protein

lysosomal

with

a significant

enzymes role

from

the

rat is

of serum glycoprotein near

raises

of the mannan-binding

protein

protein.

maintaining

role

They demonstrated

of the mannan-binding

asialo-glycoprotein-binding mechanism

may play

the presumptive

--et al. of agalacto-orosomucoid

nose and N-acetylglucosamine tein

supporting

by Achord

in the regulation

ance of circulating

--et al.

200

Asialo-orosomucoid

was provided

junction

550

8.0

The physiological It

Amount 50 % inhibition (l.w

to give bghl)

plasma. uncertain.

homeostasis

in con-

in

for

in the packaging

The rapid

constituents that

rich

the mannan-binding

mechanism

of

asialo-glyco-

the mannan-binding

may be auxiliary. carbohydrate

the possibility

the presence

of circulating

appropriate

inhibi-

currently

However,

levels

cross

suggested

pro clearin manpro-

by Neufeld

(26). ACKNOWLEDGMENT

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. A part of this work was carried out in the Radioisotope Research Center, Kyoto University. The authors wish to thank Dr. G. Ashwell, NIH, for his kind help in the preparation of this manuscript. REFERENCES 1. 2. 3. 4.

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Vol. 81, No. 3, 1978

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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