Antigen of “serum sickness” type of heterophile antibodies in human sera: Identification as gangliosides with N-glycolylneuraminic acid

VoL79,No.

2, 1977

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ANTIGEN OF "SERUM SICKNESS" TYPE OF HETEROPHILE ANTIBODIES IN HUMAN SERA:

IDENTIFICATION

AS GANGLIOSIDES

WITH N-GLYCOLYLNEURAMINIC ACID H. Higashi Department

and

of Biochemistry, Hokkaido University,

Naiki*

M.

Faculty of Veterinary Sapporo 060 Japan

Medicine,

and S. Matuo Department of Laboratory University, School

September

and K. 6kouchi

Services, of Medicine,

The Hospital of Kyushu Fukuoka 812, Japan

26,1977

SUMMARY: Antigen of "serum-sickness" type of heterophile antibodies in pathologic human sera was purified from equine and bovine erythrocyte stroma. The chemical nature of this antigen was glycosphingolipids with N-glycolylneuraminic acid. The antigen of equine erythrocytes was identified as hematoside with N-glycolylneuraminic acid, GlNeu(a,2-3)Gal(B,l-4)Glc(a,l-1) ceramide and the antigen of bovine erythrocytes was N-glycolylneuraminylparagloboside, G1Neu(~,2-3)Ga1(~,1-4)GlcNAc(B,1-3)Gal(~,l-4)Glc(~,l-l) ceramide. The results indicate that “serum-sickness" antibodies react with a common disaccharide moiety of non-reducing end of the both glycosphingolipids.

The production by receiving described

therapeutic

kinds

erythrocytes

of heterophile

mononucleosis and sera

in sera of patients

of

serum was first

foreign (2).

Thereafter,

these

as "serum-sickness

antibodies"

or

The antibodies

differ

antibodies,

(3) and Paul-Bunnell

infectious

antibodies

(1) and Deicher

have been called

"H-D antibodies".

bodies

injection

by Hanganutziu

antibodies

other

of heterophile

in specificity

such as Forssman anti-

antibodies, (4).

"P-B antibodies"

These antibodies

from various

from

animal

species

react

of with

(horse,

sheep,

* Correspondence

should be sent to Dr. M. Naiki. GSL, glycosphingolipid; Gal, D-galactose; Glc, D-glucose; GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; AcNeu, N-acetylneuraminic acid; GlNeu, N-glycolylneuraminic acid; Cer (ceramide), N-acylsphingosine; PBS, 0.01 M phosphate buffer (pH 7.0) containing 0.15 NaCl. Abbreviations:

Copyright AI1 rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

M

388 ISSN

0006-291X

BIOCHEMICAL

Vol. 79, No. 2, 1977

ox and rabbit) kidney

and are

homogenate.

sera

from

goat

anti-human

patients of

properties

of all

almost

from

being

We now present

with

nature

this

N-glycolylneuraminic

been

antigen

r-globulin

of

still

extractable

75% of ethanol

and equine of

kinds

of guinea-pig were detected

(5) and also

has not

the

sediment

recieved

different

heat-stable, in

the

"H-D antibodies"

serum

"H-D antigen"

erythrocytes

with

patients

thymocyte

and precipitable

the

absorbed

Recently,

suffering

nature

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

fraction sera

(6).

unknown,

except

hot

solution

(6).

purification

of

"H-D antigen"

erythrocytes

and demonstrate

is

really

from

diseases

with

glycosphingolipid

in of some The some

ethanol

from first

(7)

bovine that

(GSL)

acid. MATERIAL AND METHODS

The preparation of the standard GSLs used in the experiments were described previously (8-11). The isolation of hematoside with N-glycolylneuraminic acid (GlNeu-hematoside) from equine erythrocyte stroma and N-glycolylneuraminylparagloboside (GlNeu-sialosylparagloboside) from bovine erythrocyte stroma was performed as follows. Total lipids were extracted from acetone powder of erythrocyte stroma with chloroform-methanol (2:1, v/v), and acetylated with pyridine-acetic anhydride (2:1, v/v) at 60°C. The acetylated GSLs were isolated by Florisil column chromatography according to After deacetylation, GlNeu-hematoside of equine Saito and Hakomori (12). erythrocytes was purified by silicic acid column chromatography described previously (8) and GSLs with sialic acid (gangliosides) from bovine erythrocytes were obtained by DEAE-Sephadex column chromatography following Ledeen With regard to bovine gangliosides, after reacetylation, each et al. (13). ganglioside was isolated from each other by a preparative thin-layer chromatography (TLC) on precoated silica gel plates (E. Merck Laboratories, Inc.) developed one time with a solvent system of chloroform-methanol-water (95:5: 0.5, v/v). The gangliosides were eluted from silica gel, deacetylated, neutralized, determined in the hexose content by phenol-sulfuric acid reaction and suspended in PBS containing 0.5 mg/ml sodium taurocholate (Nutritional Biochemical Corp.) for immunological tests according to the previous paper (11). The analytical TLC on a precoated silica gel plate was carried out with twice development of chloroform-methanol-2.5 M ammonia (60:40:9, v/v) and visualized by spraying with resorcinol reagent. The carbohydrate compositions of "H-D antigen"-active GSLs were analyzed by gas-liquid chromatography (GLC) as previously described (14) for determining the molar ratio of each carbohydrates and sphingosine bases and as described in Ledeen's paper (15) for sialic acid composition. Sera from thirteen different patients with different diseases were detected to have "serum-sickness" type of antibodies by determining higher calf, sheep and rabbit erythrocytes and by levels of hemagglutinin to horse, testing complete absorption of the hemagglutinin with guinea-pig kidney sediment. One of thirteen sera was obtained from a patient "J. S." who had

389

Vol.79,

BIOCHEMICAL

No. 2, 1977

AND BIOPHYSICALRESEARCH

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received therapeutic injection of horse anti-snake venom. Parts of "H-D antibodies" were isolated from the mixed sera of two chromatography described in Marcus's patients "C. S. and T. H." by affinity Five ml of "H-D serum" was absorbed at cold room with 10 ml of paper (16). 7.5% polyacrylamide-2.5% bisacrylamide gel containing 10 mg of equine GlNeu= hematoside, 10 mg of egg lecithin and 100 mg of cholesterol. The bound protein was recovered by elution with 3 M KSCN, dialysis against PBS and concentration to approximately 200 ug/ml. The protein solution was diffused in 1% agarose gel with rabbit antisera to different human immunoglobulin chains, r-chain, p-chain, a-chain, K-chain and r-chain (Behring Inst.) and normal rabbit serum. Sera from four different patients with infectious mononucleosis which contain high levels of "P-B antibodies" were kindly donated from Dr. Kano (School of Medicine, State University of New York) and were tested for cross-reactivity of "H-D antigen"-active gangliosides by Dr. Matuhashi (Institute for Medical Science, University of Tokyo). Hemagglutination inhibition was performed as follows. Prior to experi2 units of hemagglutinin ment, 0.2 ml of diluted H-D serum "J. S." containing was mixed with 0.2 ml of each inhibitor solution and kept for 12 hours at 4°C before 0.2 ml of 0.2% suspension of equine erythrocytes was added to. After one hour incubation again at cold room, each tube was centrifuged at 2,000 rpm for 2 min. Hemagglutination was read by gently shaking.

RESULTS Glycolipid neutral

fraction

glycolipids

Then,

and applied

different order

"H-D antigen"

fraction.

reacetylated

fractions

for

was fraction concentration

0.9 pg/ml

the most abundant

glycolipid

erythrocytes

slower

than

and sphingosines respectively. movility

detected

in only

fraction

were isolated reactivity

in equine

was Seven

as shown in Fig.

after

at the molar The GLC analysis

of TLC were identical

at the

The compound was

erythrocytes

AcNeu-sialosylparagloboside

acid,

deacetylation.

hemagglutination

content.

of bovine

TLC.

and moved from human

The compound was detected glucosamine,

ratio

of

390

galactose,

to

glucose

0.8:1.0:2.0:1.0:1.0,

of carbohydrate with

1

erythrocyte-hemagglutination

as hexose

in analytical

have N-glycolylneuraminic

column

TLC plates.

The compound D inhibited

of

slightly

was

the ganglioside

Rf values

inhibitor

D.

activity

into

were named A, B, C, D, E, F and G in

immunological

The most potent

was isolated

by DEAE-Sephadex

on preparative

those

of decreasing

and tested

erythrocytes

and gangliosides

chromatography. ganglioside

of bovine

the major

composition ganglioside

and of

BIOCHEMICAL

Vol. 79, No. 2, 1977

Fig.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 TLC of each acetylated ganglioside of bovine erythrocytes. Total gangliosides (T) were separated into seven different fractions (A to G) by preparative TLC. An analytical TLC was carried out by developing with chloroform-methanol-water (95:5:0.5, v/v) and visualizing with resorcinol reagent.

bovine

erythrocytes

Wiegandt

et --

Fraction

A and C, also

ration

al.

which (171,

that

of equine

and sugar

components

is

hemagglutination

erythrocyte

(18)

by GLC analysis.

too

composition.

The other

by analytical

TLC and gave no inhibition

rig/ml.

identical

small

Fraction

sylparagloboside,

fractions

fraction but

fractions

However,

the fraction

did not

line line,

in carbohydrate only

one band

and AcNeu-sialo-

inhibitors, with

fraction

"H-D serum" and

in agarose-gel

show precipitin

391

C

by GLC to have the

The potent

precipitin

GlNeu=

at the concentration

of AcNeu-hematoside

respectively.

concent-

on TLC analysis

seemed to given

C gave a weak precipitin

the other

in movility

B and E were analyzed

D and A each gave a clear

with

in amount to be analyzed

sugar composition

by

at the

The compound A was identical

was recovered

15.0

established

GlNeu-sialosylparagloboside.

inhibited

of 1.9 yg/ml.

hematoside

had been already

reaction.

diffusion,

of

GalNAc Ga1(~,1-4)GlcNAc(8,1-3)Gal

Paragloboside

at

ganglioside

' Negative

GDla

a concentration

of

125 ug/ml

Gal(~,l-3)GalNAc(B,l-4)Gal(B,l-4~Glc-Cer I (a,2-3) AcNeu Gal(~,1-3)GalNAc(a,l-4)Gal(B,l-4~Glc-Cer 1 (a,2-3) AcNeU ' Weak

inhibition

i3,1-4)Glc-Cer Aa;i;z-3 1

( 8,1-4)Glc-Cer

GM1 ganglioside

GM2 ganglioside

GlNeusialosylparagloboside

( B,l-4)Glc-Cer

8,1-4)Glc-Cer

Gal(B,l-4)GlcNAc(B,l-3)Gal 1 (a,2-3) AcNeu Gal(B,l-4)GlcNAc(B,l-3)Gal I (a,2-3) GlNeu GalNAc(B,l-4)Gal

GlNeu(a,2-3)Gal

GlNeu-hematoside

i3,1-4)Glc-Cer

!3,1-4)Glc-Cer

AcNeusialosylparagloboside

AcNeu(a,2-3)Gal

AcNeu-hematoside

I (a,l-3)

by different

Gal(B,l-4)Glc-Cer

GalNAc(!3,1-3)Gal(a,l-4)Gal(B,1-4)Glc-Cer

structures

inhibition

Forssman

Chemical

Hemagglutination

GalNAc(~,1-3)Gal(a,l-4)Gal(B,l-4)Glc-Cer

1.

Globoside

Lactosylceramide

Glycolipids

Table

2.0

2.0

125'

-1

1252

Lowest cont. that complete inhibition

glycosphingolipids give @g/ml)

BIOCHEMICAL

Vol. 79, No. 2, 1977

Fig.

2 Double diffusion test of "H-D serum" and "P-B sera" with equine 1 to 4, GlNeu-hematoside and bovine GlNeu-sialosylparagloboside. Sera from four different patients with infectious mononucleosis; HD, Sera from a patient "Z. M." containing "H-D antibodies"; G, GlNeu= sialosylparagloboside from bovine erythrocytes (250 ug/ml); H, GlNeu= hematoside from equine erythrocytes (250 ug/ml).

GlNeu-hematoside stroma

as the only

was also

galactose,

glucose

0.9:1.0:0.9,

other

and sphingosines

of bovine

compounds used,

test

at the concentration

and GlNeu-sialosylparagloboside,

precipitin

line

The other Four

The other

GlNeu-hematoside twelve

of

all

in Table except

Only

GlNeu=

each gave a clear

fused

and the

as shown in Fig.

1 gave no precipitin

gave positive

393

The

gave weak

reacted

nor GlNeu-sialosylparagloboside

"H-D sera",

1.

125 ug/ml

employed.

"P-B antibodies"

sera containing

1.0:

strongest

"H-D antibodies"

seemed completely

compounds shown in Table

different

neither

serum containing

of

were given

and AcNeu-hematoside

hematoside

lines

ratio

as GlNeu-sialosyl-

of which

at the maximum concentration

precipitin

acid,

as shown in Table

inhibition

both

The compound

as well

structures

lactosylceramide

with

substance.

at the molar

erythrocyte the

1, gave no inhibition only

erythrocyte

The compound was the

in hemagglutination

paragloboside

from equine

GSL and had N-glycolylneuraminic

respectively.

inhibitor

purified

"H-D antigen"-active

was the most abundant

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reaction

2.

reaction. with (Fig. with

2).

BIOCHEMICAL

Vol. 79, No. 2, 1977

GlNeu-hematoside proteins

in gel

of the

demonstrated

sera

diffusion

(Q-G),

K-chain

precipitin

isolated

with

The GlNeu-hematoside-binding

by affinity

chain)

(IgM)

by positive

were reaction

to human immunoglobulin and A-chain

was obtained

p-chain

chromatography

immunoglobulins

antisera

(Light

reaction

globulin

diffusion.

to be really

in gel

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

and a-chain

(Light

r-chain

chain).

antisera

But no

to human immuno-

(IgA).

DISCUSSION As above mentioned, different

the chemical

"H-D antigen"

communication.

active

that

terminal

disaccharide

animals,

equine,

mouse, hamster lylneuraminic

bovine,

acid

glycoproteins,

of

membrane,

erythrocyte

some of these

been detected

if

site

invade

tion

of microorganisms,

abnormal

into

some substances

intestinal

antibody"

production

is

problem

future

pathologic preparation stroma

absorption

shall

of milk

species

investigators

animals

394

still

activity have never

and many organs such an antigen antisera,

infec-

and meats from scurf, However,

can be elevated

mononucleosis of

fluids

stimulated.

why such antibodies

of infectious

In addition,

of animal

be readily

Other

carbohydrates

acid

foreign

or inhalation

sites,

human sera.

of various

of

of

cat,

"H-D antigen"

containing

human by injection

species

have N-glyco-

secretions.

from body

the

and gangliosides

N-glycolylneuraminic

in any substances

with

dog,

chicken,

compounds are known to have

Therefore,

(20) -

elk,

immunoglobulins serum and/or

But in human being,

react

20) in complex

(19,

in this

Various

pig,

monkey except

residues

such as mucin,

possibly

rabbit,

two

of the two com-

GlNeu(a,2-3)Gal.

sheep,

and ,primate

proved

moiety

"H-D antibodies" part,

of the

GSLs were first

The common disaccharide

pounds suggested

(21) -

structures

reported antigen

"H-D it

in only that

the

from erythrocyte

possessed

"H-D anti-

Vol.79,

BIOCHEMICAL

No. 2, 1977

gen" activity glycolipids

(6, both

21). did

ACKNOWLEDGMENTS: This Ministry of Education, discussion and aid for sera.

:: 3. 2: 6. 7. 8. 9. 10. 1:: 13. 14. 15. 16. 1:: 19. 20. 21.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

But the present

"H-D antigen"

not have any "P-B antigen"

active

activity.

work was supported by a Research Grant from the We thank Dr. T. Matuhashi for valuable Japan. testing cross-reaction of H-D substances with I.

M.

REFERENCES Hanganutziu, M. (1924) Compt. Rend. SOC. Biol. 9l-, 1457-1459. Oeicher, H. (1926) Z. Ryg. Infektionskr. 106, 561-579. Forssman, T. (1911) Biochem. Z. 37, 78-115. W. W. n932) her. J. Med. Sci. 183, 90-104. Paul, J. R., and Bunnell, J. C., and August, A. (1973) Blood 42, Pirofsky, B., Ramirez-Nateos, 385-393. Kasukawa, R., Kano, K., Bloom, M. L., and Milgrom, F. (1976) Clin. EXP. Immunol. 25, 122-132. Schiff, F. (1937) J. Immunol. 2, 305-313. Naiki, M., and Taketomi, T. (1969) hap. J. EXP. Med. 39, 549-571. Naiki, M., Kamimura, H., Taketome, T., and Ichikawa, R. (1972) hap. J. Exp. Med. 42, 205-219. R. W. (1974) J. Immunol. 113, 84-93. Naiki, M., Marcus, D. M., and Ledeen, 14, 4837-4841. Naiki, M., and Marcus, D. M. (1975) Biochemistry S. (1971) J. Lipid Res. 11, 257-259. Saito, T., and Hakomori, Ledeen, R. W., Yu, R. K., and Eng, L. F. (1973) J. Neurochem. 21, 829-839. Naiki, M., Fong, J., Ledeen, R., and Marcus, D. M. (1975) Biochemistry 14, 4831-4836. Yu, R. K., and Ledeen, R. W. (1970) J. Lipid Res. 11, 506-516. Marcus, D. M. (1976) J. Amer. Gil Chem. SOC. 53, 233-245. Wiegandt, H., and Schulze, B. (1969) z. Naturforschg. m, 945-946. Yamakawa, T., and Suzuki, S. (1951) J. Biochem. 38, 199-212. Gottschalk, A. (1960) The Chemistry and Biology of Sialic Acid and Related Cambridge University Press. Sabstances, Gottschalk, A. (1972) Glycoproteins, their Composition, Structure and Function, B. B. A. Library vol. 5, Elsevier Publishing Company, Amsterdam= London-New York. Callahan, H. J. (1976) Int. Archs Allergy ~ppl. Immun. 51, 696-708.

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