Vol. 81, No. 4, 1978 April 28,1978
BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1286-1293
BLOOD GROUP i AND I ACTIVITIES OF "IACTO-N-norHEXAOSYLCERAMIDE" AND ITS ANALOGUES: THE STRUCTURAL REQUIREMENTSFOR i-SPECIFICITIES* Heiner
Niemannf Kiyohiro
Biochemical Oncology, 1124 Columbia,
Watanabe and Sen-itiroh
Hakomori
Fred Hutchinson Cancer Research Seattle, WA 98104, U.S.A.
Center,
and Robert A. Childs and Ten Feizi Clinical Research Centre Watford Road, Harrow Middlesex, England Received
March
13,1978
Summary-A pure straight chain cersmide hexasaccharide ("lacto-N-norhexaosylceramide" Gal8l~~GlcNAc~1-t3Gal81-+~GlcNAc81~3Gal~1-t~Glc-tCersmide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Gal&+)+GlcNAc residues and GlcNAc81+3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-1 antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues. Structural
elucidation
of the molecular disease" tumors;
basis
for the Ii-determinants
anti-1
structure
of the entire
During the course
ation
with
some glycosphingolipids inhibition
(2).
Ii-antigen
with
human
Ii-antigens
of blood group ABH determinants
of the isolation
ABH activities
agglutinin
changes associated
appear in some human tumors
as the precursors
for the understanding
known as "cold
of antigenic
serum (Ma) has been shown to recognize
chemical
that
is important
of the autoimmune disorder
and for the understanding
(l),
been considered
lipids
of Ii-determinants
have
(3) and one
GalS1+4GlcNAcSl%Gal;
however
the
complex has not yet been elucidated. and characterization
from human erythrocyte
membranes (4) we have noticed
show blood group I and i activity
(5) and radioimmunoassay
(6),
of glycosphingo-
by hemagglutin-
and have found strong
i and
I activities in certain bovine erythrocyte ganglioside fractions particularly after * This investigation was supported by the National Cancer Institute Grant CAl9224. Iatrobeads silica gel used in this study was donated by Dr. T. Yamakawa, Tokyo University under the U.S.-Japan Cooperative Science Program supported by FJ-5084. %upported by Deutsche Forschungsgemeinschaft. solvent composition is expressed Abbreviations: C-M-W: chloroform-methanol-water, by volume ratio, TLC: thin-layer chromatography, DEAE: diethylaminoethyl. 0006-291x/78/0814-1286$01,00/0. Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form resewed.
1286
Vol. 81, No. 4, 1978
One such active
desialylation. found
BIOCHEMICAL
to be identical
of a ganglioside The structural studied ceramide
series
rwrhexaosylceramide"
described
requirement
1
fraction
for
of the
including
minus
of the
Ii-activities
by chemical
(7)
(5)
and by Chien
analogues
from the highly
and has been
and the
i and I activities
of eleven
derived
purified
fucose"
by Wiegandt
expression
those
RESEARCH COMMUNICATIONS
was extensively
to "H2-glycolipid
recently
by comparison
AND BIOPHYSICAL
i-active
"asialo-core" et al
(8).
has been of lactoglycosyl"lacto-N-
modification.
MATERIALS
AND METHODS
Isokztion, Preparation and Purification of GZycosphingoZipids: H2-glycolipid (5 ) , paragloboside (ll), sialosylparagloboside (ll), lacto-N-triosylceramide (12), a-gslactosylparagloboside (13), t%galactosylparagloboside (14) have been prepared according to the methods described. The i/I active gangliosides of bovine erythrocyte stroma were prepared by Folch's partition of the total lipid extract followed by successive chromatographies on DEAF,-Sephadex according to a modified method of Momoi et al (15) and Iatrobeads silica gel (16) (see Fig. 1 Desialylation of gangliosides was carried out by hydrolysis in and Legends). 1% aqueous acetic acid at 100° C for one hour or by incubating with Rbrio ChoZerae H2-glycolipid was neuraminidase (Behring Diagnostics, Somerville, N.J. 08876). degraded by fucosidase of ChaZonia lampas (l'i'), purified by affinity chromatoLacto-N-norpentaosylceramide (see Table I) was prepared by treating graphy (18). desialylated ganglioside fraction E by Jack bean meal @-galactosidase (19). Both fraction E ganglioside and the desialylated compound (lacto-N-norhexaosylcersmide) were further purified after acetylation and TLC in dichloroethaneacetone (55:45) or in butylacetate-acetone-water (25:lO:l). The products were homogeneous on TLC in these solvents and in benzene-methanol (16:3). Structural Deteninution of the Active GangZioside E and its DesiaZyZated Derivative flacto-N-norhexaosyIceramide): Analysis of the carbohydrate composition, sequence of carbohydrate by step-wise enzymatic degradation, methylation analysis, and total mass spectrometry of permethylated compounds have been carried out according to the procedures already described (4,5,11,14,20). Structural Modification of La&o-N-norhexaosyZceramide: The terminal !3-galactosyl residue of lacto-N-norhexaosylceramide was modified to C6-aldehydoGa3. by galactose oxidase in tetrahydrofuran according to the method described by Suzuki (21), and by the limited periodate oxidation according to the condition described by Hakomori et al (22). The terminal Gal was eliminated by Jack bean 8-galactosidase (23) and the resulting compound was lacto-N-norpentaosylcersmide (Table I). The N-acetyl group of N-acetylglucosaminosyl residues of lacto-N-norhexaosylceramide was removed by hydrazinolysis (in hydrazine hydrate, 100° C 5 hrs), under the condition no fatty acyl amide linkage was eliminated (unpublished data), In these chemical modifications,the rate of conversion was checked by TLC. Detemnination of Ii-Activities: Radioimmunoassays of Ii-activities were carried out using five anti-1 and six anti-i sera according to the procedure previously described (6,23). Ii-activity was also determined by hemagglutination inhibition 1. Nomenclature adopted according to IUPAC-IUB Commission on Biochemical Nomenclature (9). Lactoglycosyl series denotes glycolipids containing GlcNAcg1+3Gal Bl+kGlcl+lCer as the common structure. Lacto-N-norhexaosylcersmide signifies, in this paper, a glycolipid having a straight chain hexasaccharide containing Gal and GlcNAc. It differs from "lacto-N-hexaose" or 'lacto-N-neohexaose" which has a branched structure (10). These branched oligosaccharides should be called lacto-N-iso-hexaose or lacto-N-iso-neohexaose.
1287
yol. 81, No. 4, 1978
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IA k.3.
DEAE
Sephadex
A25-Column
. . ..I. --w--m-
-
ABC
D
5Gil
EF
I
1500
low Eluate
in
_.___
-
H
G
2ooo
2500
ml
1B
-..m. --we-.... E
-.... w......
. ..I..._
I 100
200
200 Eluate
Fig.
400 in
a00
I 800
ml
1.
Isolation of i/I active fraction E from bovine erythrocyte gangliosides by chromatography on DEAF-Sephadex A25 and Iatrobeads silica gel. Upper panel (IA) shows the separation pattern of gangliosides on DEAE-Sephadex A25: 600 mg of glycolipid mixture (Folch's upper phase) were dissolved in 20 ml of C-M-W (30:15:2) and applied on 290 ml bed volume of DEAE-Sephadex A25 (acetate After eluting neutral glycolipids with the same solform) in the same solvent. vent , gangliosides were separately eluted by a linear gradient of NH4OAc (0.030.27 M) in the same solvent. Of each fraction (15 ml) aliquots were analyzed by TLC in C-M-W (60:35:8). The fractions containing the same kind of gsngliosides were combined, concentrated, dialyzed, and lyophilized; thus fractions A to I were separated. Fraction E was i/I active. There were other Ii-active fractions with longer carbohydrate chains which will be described elsewhere. Lower panel (lB) shows the separation pattern of fraction E on Iatrobead tography. 20 mg of E was dissolved in 1.5 ml C-M (2:1), applied on an column (120 X 1.5 cm, Iatron Lab., Inc., Tokyo), and eluted by a linear of C-M-W (53:45:2) to C-M-W (16:80:4). Each 5 ml fraction was analyzed in C-M-W (60:35:8). The active fraction E yielded 9 mg (4.5% of total side).
chromaIatrobead gradient by TLC ganglio-
Dench, anti-i Hog (donated with 3 hemagglutination doses of anti-1 Step, anti-i by Mrs. M.C. Crookston, Toronto General Hospital), anti-1 Ma (donated by Dr. E. WA), anti-i McC (donated by Giblett, Puget Sound Central Blood Bank, Seattle, Dr. V. Ginsburg, National Institute of Arthritis and Metabolic Disease, National Institutes of Health). 1288
BIOCHEMICAL
Vol. 81, No. 4, 1978
TABLE I.
Structure
of i/I
active
AND 8lOPHYSlCAL
lacto-N-norhexaosylceramide
RESEARCH COMMUNICATIONS
and its
analogues.
H -glycolipid PFucosyllacto-N-norhexaosylceramide ) Fraction "E" (Sialosyllacto-N-norhexaosylceramide) Lacto-N-norhexaosylceramide (LNHC) Lacto-N-norpentaosylceramide (LNHC minus Gal)
La&o-N-neotetraosylceramide (paragloboside) La&o-N-triosylceramide Sialosylparagloboside a-galactosylparagloboside &galactosylparagloboside
RESULTSANDDISCUSSION An Ii-active successive
(E) was isolated
chromatography
"Iatrobeads" This
fraction
silicic
glycolipid
on DEAE-Sephadex
acid
(Fig.
lB),
was homogeneous
solvent
systems
ation.
The structure
(see Material
analysis
and Methods)
Glc and neuraminic and 2 moles atic
of h-linked
hydrolysis
showed
the
adjacent
acid,
alternating
acid
and 1 mole
the
E completely
terminal
by mass spectrometry.
carbohydrate
agreed
with
1289
and
derivative. in three
after
deacetyl-
findings: one mole
3 moles Glc,
each of
of 3-linked
c) sequential
no indication
d) solid the
probe
above
of two
mass spectrometry
structural
assignment.
as N-glycolylneursxninic
E was identified
Gal
enzym-
and $-N-acetylhexosaminidase,
was identified
The desialylated
by
lA),
acetylated
state
of GlcNAc,
of k-linked
residues,
free
following
of Gal and GlcNAc but
GlcNAc
(Fig.
derivative
yielded
&galactosidase,
sequence
Gal or two adjacent
analysis
stroma
as NGNeur2+3Galf31+4GlcNAc
on the
2 moles
of Gal,
elution
acetylated
and in the
based
b) methylation GlcNAc
gradient
E was determined
by neursminidase,
of the permethylated In addition
3 moles
erythrocyte
by TLC of the
on TLC as the
of fraction
showed
with
followed
~1+3Galf31+~GlcNAcg1+3Galg1+~Glc~1+lCer a) sugar
from bovine
as Galf31+4GlcNAc
Vol. 81, No. 4, 1978
TABLE II.
I and i activities five
I and
BIOCHEMICAL
AND BIOPHYSICAL
of "lacto-N-norhexaosylceramide"
six
and its
eleven
RESEARCH COMMUNICATIONS
malogues
determined
with
i antisera. Anti-I
step
Phi
>lOO 12
-----
t
MEL
era
Da
>lOO -__
-----
-----
Anti-i
1
Tho
McDon
McC
Den
Ha3
Gall
-----
-----
-----
>lOO 0.1
>lOO 0.7
-----
(35%) ---
>lOO 3
H2-glycolipid I$ minus fucose
Ii* H
Fraction
PI** (4;;) H
>lOO ---
>lOO >lOO
>lOO ---
>lOO ---
>lOO ---
La&o-N-wrhexaosylcermide(IXBC)
R H
('6%) ---
>lOO >lOO
>lOO ---
50 ---
---
---
La&a-N-norpentaosy1cermide
R H
>lOO >lOO
>lOO ---
,100 >lOO
>lOO ---
,100 ---
,100 ---
,100 ---
,100 ,100
,100 ,100
--,100
,100 ---
Sialosylparaglobaside
H
>lOO
---
>lOO
---
---
---
---
,100
,100
,100
---
a-Galactosylparegloboside
R H
>lOO >lOO
>lOO ---
>lOO >lOO
>lOO ---
>lOO ---
,100 ---
,100 _--
,100 ,100
,100 ,100
--,100
,100 ---
E-Galactosylparagloboside
R H
>lOO >lOO
>lOO ---
>lOO ,100
>lOO ---
>lOO ---
,100 ---
>lOO ---
,100 >lOO
,100 7100
-->lOO
,100 ---
Paragloboside
R H
>lOO >lOO
>lOO ---
>lOO 100
>lOO ---
>lOO ---
SlOO ---
>lOO ---
>lOO 100
>lOO 100
--100
>lOO ---
La&o-N-triosylcermide
R Ii
>lOO ---
>lOO ---
>lOO ---
>lOO ---
>lOO ---
-----
>lOO ---
>lOO >lOO
-->lOO
_->lOO
>lOO ---
R H
20 40
>lOO ---
>lOO >lOO
>lOO ---
(W) ---
23 ---
---
(40%) 23
--2.5
>lOO ---
(25%) 2.5
---
1.2
>lOO ---
Periodate oxidized
E
LNHC
2.3 10
1
2.0
2.3
R
H
2
>lOO ---
>lOO >lOO
>lOO ---
(25%) _--
(30%) ---
IL ---
de-N-acetylated LNHC
R H
>lOO >lOO
>lOO ---
>lOO >lOO
>lOO ---
>lOO ---
ZlOO ---
>lOO ---
0.8 2.5
0.6
6
Galactose oxidase treated-LNHC
>lOO 12
15.0
2.5
30.0 5.0 >lOO >lOO
>lOO >lOO
--3
>lOO ---
0.6
>lOO ---
---
-->lJO
>lOO ---
*H:
activity by hemagglutination inhibition assays, expressed as the minimum mount (ug) per 0.1 ml of sample that inhibited 3 hemagglutination units of antibody. by radioimuhoassays, expressed aa the mncentration pg/ml required t.0 give 50% inhibition of binding of 1251-labeled Ii-active antigen to the anti-1 or i antibodies. With materials giving less than inhibition the figures in parentheses indicate the amount of inhibition given at the highest concentration tested, 100 pgiml. >lOO: no inhibition at 100 ,,g/ml. :---not tested.
l *R: activity
~1~3Gal~1~~GlcNAc~1~3Gal~1~~Glc~1-tlCeramide b) methylation
analysis,
spectrometry
c) enzymatic
of the permethylated
as "lacto-N-norhexaosylceramide"~ lacto-N-norhexaosylceramide these
glycolipids, Ii-activities
inhibition
degradation
on a) sugar
analysis,
and d) solid
probe
The structure
glycolipid.
is
Lacto-N-norpentaosylcersmide by treating
paragloboside, with
based
several
or by radioimmunoassay
with
and other antisera, are
analogues determined
shown
1290
in Table
are
mass
hereby
designated
was derived
S-galactosidase,
from
The structures listed
50%
in Table
of I.
by hemagglutination II.
Strong
i-activity
was
Vol. 81, No. 4, 1978
found
BIOCHEMICAL
in la&o-N-norhexaosylceramide
H2-glycolipid
showed
indicating
the
structure
must
antibodies. anti-1
identity
inhibition. either
the
was found
this
pentaosylcersmide,
by the
like
to paragloboside ations
cold did
(6).
inhibition.
Since
terminal
and penultimate
the
Both abolished N-acetyl
chain
or lack
by 1) removal
no activity
II).
Tsai
et a2 (24)
lacto-N-
was found
specificity showed
reported
(25),
chemical
is
could
GalSl+kGlcNAc with
not
residues;
thus
sera
the
they
same
are
be due to the
structure, anti-1
Step,
but
demonstrated
and no other
&Gal the
1291
residue
activities
were
glycolipids
similarly
and 2) removal
of the
were
reduced,
similarly
in
as the
analysis,
of lacto-N-norhexaosylceramide of the terminal
share
due to contimanants,
homogeneous
prepar-
in hemagglutination
The I-activity is
directed
of anti-i
to assume that
activity
caused
and three
no inhibition
is reasonable
strong
that
antibodies
was observed
repeating
in fucosyl-
hemagglutination
of paragloboside
glycolipid
Ma by
was found
McC, however,
activity
showed
of two GlcNAc
anti-1
in H2-glycolipid,
while
it
of the
by rigorous
with
Some activity
preparation
of this
by hemagglutination
no activity
anti-i
activity
i and I activities
group
Step
Ma and Gra by radioimmunoassay.
be detected
Phi and Da by radioimmuno-
and lacto-N-norhexaosylceramide
structure,
lacto-N-norhexaosylceramide
could
anti-1
anti-1
of patient
paragloboside
length
anti-1
carbohydrate
with
inhibited
A minimum
Lacto-N-norhexaosylceramide not with
activity
paragloboside
The minimum
chain
high
showed
S-galactosylparagloboside,
show typical
purified
by radioimmunoassays
cross-reactive,
agglutinin
not
of extensively
shorter
also
with
(see Table
(lacto-N-neotetraosylceramide)
anti-i
anti-i
demonstrated
sialosyllacto-N-norhexaosylceramide
paragloboside
This various
and la&o-N-triosylcersmide.
lacto-N-norhexaosylcersmide
a-Lfucosidase,
with
showed
were
with
tested.
of i-activities
with
glycolipid
sera
lacto-N-norhexaosylceramide.
a-galactosylparagloboside,
sialosylparagloboside,
treatment
glycolipid
or weak activity
No Ii-activities
method.
with
expression
same pure
activity
However,
after
product
for
and moderate
A moderate
i-activity
of the
be essential
5 of the 6 anti-i
with
a strong
However,
Step
assay.
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No. 4, 1978
BlOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NHAc
Fig.
Possible structural requirement for i and I specificities. Two formulas for the iI-active hexasaccharide are shown; the upper is the classical Haworth's structure, the lower is a possible tertiary structure according to Rees (26). Note that the GlcNAcg1+3Gal disaccharide unit forms a plane (approximately zero rotation) and that two planes are rotated around the f31+4glycosidic linkage. The terminal trisaccharide sequence of GalBl+~GlcNAc g1+3Gal is a necessary but not sufficient requirement for i-activity. The repeat of the penultimate GlcNAcg1-+3Gal disaccharide unit seems to be an additional requirement for the expression of i-specificity. R could be either lipid or protein. I specificity may require this same structure as well as an additional unknown substituent X, which could be a side chain attached to the hexasaccharide.
but
2.
not
ring
abolished
by 3) periodate
and 4) oxidation Thus,
it
by chemical
of Cb-hydroxyl
was impossible methods
used.
It
polysaccharides
Irrespective
planes
twisted
being
according that
the
invariable
to the
structural
is most
these that
I and i activities
the
studies
species,
they
@l-t& linkage
pyranosyl
Gal,
I-activity
The alternating
diffraction
glycosidic
are
g-Gal
1 eq + 3 eq) as found
assignment
groups It
the
modify
reaction,
shown by X-ray
around
terminal
of the terminal
possible
of the pyranose
two acetamido distance.
a cross
(1 eq -f 4 eq,
has been
structures.
group
is therefore is
@l-t3 pyranosides
of the
to differentially
lacto-N-norhexaosylceramide gl-&,
oxidation
in
sequence connective
are built
(26).
probable
1292
on two disaccharide that
this
compact
of tissue
to form helical up of disaccharidm A model
of lacto-N-norhexaosylcersanide
located
of
constructed indicates
planes two-plane
at an (semi-
BlOCklEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No. 4, 1978
helical)
structure
determinants. this
spatial
relationship
is
the
essential
The structures arrangement is
illustrated
requirement
recognized
by anti-1
but may require
additional
in Figure
for
the majority Step,
Phi
unknown
of i-antigenic
and Da may include structures.
This
2.
REFERENCES Grune and J.V. (1962) The Haemolytic Anaemias 2nd ed. Part II. Stratton, New York; Feizi, T,, Taylor-Robinson, D., Shields, M.D., and Carter, R.A. (1969) Nature 222, 1253-1256. C., Westwood, J.H. (1975) The Lancet ;?, 391-393. Feizi, T., Tuberville, Feizi, T., Kabat, E.A., Anderson, B,, Marsh, W.L., and Vicari, G. (1971) J. Immunol. IO6, 1578-1592. Hakomori, S., Watanabe, K., and Laine, R.A. (1977) Pure & Appl. Chem. 49, 1215-1227. Watanabe, K., Laine, R.A., and Hakomori, S. (1975) Biochemistry I.&, 2725-2733. Feizi, T., Childs, R., Hakomori, S., and Powell, M. Biochem. J. in press. Wiegandt, H. (1974) ~urop. J. Biochem. 45, 367-369. (1977) Fed. Proc. g, 731. Chien, J-L., Li, S-C., Laine, R.A., and Li, Y-T. IUPAC-IUB Commission on Biochemical Nomenclature. The Nomenclature of Lipids. (1977) Lipids 12, 455-463. (1972) J. Biol. Chem. 2& 1525-1529; (1972) Kobata, A., and Ginsburg, V. Arch. Biochem. Biophys. 150, 273-281. Siddiqui, B., and Hakomori, S. (1973) Biochim. Biophys. Acta 330, 147-155. Watanabe, K., and Hakomori, S. (1976) J. Exp. Med. 144, 644-653; Ando, S., Kon, K., Isobe, M., Nagai, Y., and Yamakawa, T. (19m J. Biochem. '& 625-632. E-to, T., Ichikawa, T., Nishimura, K., Ando, S., and Yemakawa, T. O-968) J. Biochem. 64, 205-213; Stellner, K., Saito, H., and Hakomori, S. (1973). Arch. Biochem. Biophys. 155, 464-472. Stellner, K., and Hskomori, S. (1974) J. Biol. Chem. &, 1022-1025. Momoi, T., Ando, S., and Nagai, Y. (1976) Biochim. Biophys. Acta 441, 488-497. (1976) Biochim. Biophys. Acta 424, 98-105. Ando, S., Isobe, M., and Nagai, Y. Iijima, Y., Muramatsu, T., and Egami, F. (1971) Arch. Biochem. Biophys. I&
1. Dacie, 2.
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13. 14. 15. 16. 17.
150-154. 18. Jain, R.S., 19. 20. 21. 22. 23. 24. 25. 26.
Binder, R.L., Levy-Benshimol, A,, Buck, C.A., and Warren, L. (1977) J. Chromat. D, 283-290. Li, Y-T., and Li, S-C. (1972) Methods in Enzymol. 8, 702-713. Laine, R.A., Stellner, K., and Hakomori, S. (1974) Methods of Membrane Biol. 2, 205-247. Ed. E. Korn, Plenum Publishing, New York. Suzuki, Y., and Suzuki, K. (1972) J. Lipid Res. l3, 687-689. Hakomori, S., Siddiqui, B., Li, Y-T., Li, S-C., and Hellerqvist, C.G. (1971) J. Biol. Chem. 246, 2271-2277. Wood, E., Lecomte, J., Childs, R., and Feizi; T. (1977) Proc. of the 4th International Symposium on Glycoconjugates, Woodshole, Massachusetts. in press. Tsai, C-M., Zopf, D.A., Wistar, R., and Ginsburg, V. (1976) J. Iimnunol. 117, 717-721. Schwarting, G.A., and Marcus, D.M. (1977) J. Immunol. 118, 1415-1419. Rees, D.A. (1975) MTP International Review of Science, 2, l-42, ed. Whelan, W.J., Butterworths (London) University Park Press (Baltimore); Atkins, E.D.T., Isaac, D.H., Nieduszynski, I.A., Phelps, C.E., and Sheehan, J.K. (1974) Polymer 2, 263-271.
1293