(Received for publication, August 28, 1995; and in revised form, November 20, 1995)
From the
Calcium has been shown previously to cause aggregation of
phosphatidylcholine/cholesterol liposomes containing galactosylceramide
(GalCer) with similar liposomes containing cerebroside sulfate
(galactosylceramide I sulfate) (CBS), suggesting that it
mediates a carbohydrate-carbohydrate association between these two
glycolipids. In order to determine if such an association occurs, the
noncovalent complexes formed on addition of calcium chloride to GalCer
and CBS in methanol were examined by positive and negative ion spray
mass spectrometry. Monomeric Ca
complexes of both
lipids were observed. In addition, Ca
also caused
oligomerization of GalCer. Oligomerization of CBS anion was not seen,
but dimers would not have been observed, as they would be neutral.
However, Ca
caused heterotypic complexation of GalCer
and CBS. Although these heterotypic complexes were of low abundance in
methanol compared with the other monomeric and homotypic oligomeric
positive ions formed at low declustering potentials, the heterotypic
dimer
[GalCer
CBS
Ca
-H]
had the greatest stability of all oligomers formed and was the
only one to survive at high declustering potentials. Na
did not cause oligomerization of GalCer in methanol indicating
that the complexes of GalCer with Ca
are not caused
by van der Waals interactions between the lipid moieties. GalCer and
CBS are present in high concentrations in myelin. This
Ca
-mediated carbohydrate-carbohydrate interaction,
which can bridge apposing bilayers, may be involved in adhesion of the
extracellular surfaces of the myelin sheath.
Calcium-mediated interactions between cell surface carbohydrates
have recently been implicated as a basis of cell recognition and
adhesion and have therefore been the subject of increasing interest (1, 2, 3, 4, 5, 6, 7, 8) .
Carbohydrate-carbohydrate interactions between free sugars and
polysaccharides have been known for some time(9, 10, 11, 12, 13, 14) and
have more recently been investigated among glycolipids in lipid
bilayers(1, 2, 3, 6, 15, 16, 17, 18, 19) .
The list of glycolipids that have been shown to participate in divalent
cation-mediated carbohydrate-carbohydrate interactions includes the two
major myelin glycolipids glactosylceramide and cerebroside sulfate
(galactosylceramide I sulfate)(6, 18) . In
central nervous system myelin these two glycolipids comprise 27% (by
weight) of the total myelin lipid(20) . Thus an interaction
between these two glycolipids across apposing membrane surfaces might
play a role in the formation of the compacted myelin membrane.
x-ray
crystallography of a number of divalent cation complexes of simple
carbohydrates has provided detailed information concerning the
structure of these complexes (cf. (9) and (10) for
reviews). NMR and infrared spectroscopy have complemented this
information(11, 12) . However, the complex-forming
properties of calcium with carbohydrates attached to lipid moieties
remain largely unexplored. Most of the evidence for complex formation
so far comes from liposome aggregation or lipid binding studies using
solid phase presentation of the lipids, either bound to a solid support
or in liposomes. In addition, Fourier transform-infrared spectroscopy
was used to provide information about the structure of the complex of
Ca with digalactosyldiacylglycerol and the groups on
the carbohydrate that chelate with the divalent cation in a membranous
environment in the presence of water(19) . The polyvalent
nature of presentation in lipid bilayers or on a solid support
increases the affinity of the interaction. The difficulty of observing
the divalent cation-carbohydrate interactions in solution (especially
in aqueous solution) given their weak nature and the solubility
characteristics of lipid-bound carbohydrates makes the study of such
interactions rather challenging.
Electrospray ionization mass
spectrometry (ESI-MS) ()is a relatively new technique, which
can detect the presence of a complex in a solvent in which it is
soluble(21, 22, 23, 24, 25) .
The soft ionization conditions employed allow the transfer of complexes
present in solution to the gas phase with minimal
decomposition(26) . ESI-MS has recently been used to detect the
divalent cation-mediated complexation of the carbohydrate of some
glycolipids, resulting in the homotypic and in some cases heterotypic
oligomerization of these lipids in methanol(21) . This
technique was successful at detecting Ca
oligomerization of the Le
oligosaccharide(21) , while
H-NMR failed to
detect metal binding or evidence of oligomerization of the free sugar
in water(27) .
In this paper we present evidence for the
noncovalent association among galactosylceramide, Ca,
and the anion of cerebroside sulfate in methanol solution using
electrospray ionization mass spectrometry. Positive ion scans showed
that Ca
caused homotypic oligomerization of GalCer
and that it bound to monomers of CBS anion to form a singly charged
positive ion. Dimers of CBS anions with Ca
form a
neutral species and are not detected. However, Ca
caused heterodimerization of GalCer and CBS anion in methanol.
Although the heterotypic complexes were of low abundance compared with
the others at low declustering potentials, the heterotypic dimer had
greater stability than any other complex.
All reagents and solvents used were either analytical grade
or high pressure liquid chromatography grade. Calcium chloride
(CaCl2H
O) was purchased from Fisher
(Fairlawn, NJ). Methanol was from Caledon (Georgetown, Ontario,
Canada), stearic acid from Fluka (Switzerland),
1-
-D-galactosyl sphingosine (psychosine) from Sigma, and
oxalyl chloride from Aldrich.
Galactosylceramide I sulfate was synthesized from lysosulfatide (psychosine sulfate)
prepared from bovine brain sulfatide and stearoyl chloride as described
previously(29) . The structure of the product was verified as
above.
H NMR showed that it was in the
NH
salt form (data not shown). However,
the NH
adduct was not detected in the
presence of excess Ca
, indicating that the
Ca
completely displaced it from the lipid. When it
was converted to the Ca
salt form and examined by
ESI-MS in the absence of excess Ca
, it formed the
Na
salt form due to exogenous Na
present in the system. Therefore, it was necessary to study it in
the presence of excess Ca
.
Electrospray mass spectra were acquired on
a Perkin-Elmer Sciex API III triple quadrupole mass spectrometer. Both
positive and negative ion scans were performed. Concentrations of the
lipids were usually kept at 20-50 nmol/ml and the Ca concentration at 200 nmol/ml for optimum spectral quality. For
CID experiments, higher concentrations of the lipids were used for
greater signal strength. The solutions were introduced at the flow rate
of 5 µl/min.
Figure 1:
Positive ion spray mass spectrum of
GalCer (50 nmol/ml) with Ca (500 nmol/ml) in
methanol. The declustering potential was 80
V.
It is reasonable to assume that the oligomers of GalCer are
formed by interaction of the carbohydrate head groups through
coordination with Ca. As will be discussed later, the
relatively higher stabilities of the oligomers, especially the dimer
compared with the monomer, lend some support to this suggestion, since
the coordination of hydroxyl groups from more than one carbohydrate
moiety with the cation has been shown to be the preferred arrangement
in the crystal structures of the Ca
complexes of many
carbohydrates including galactose. It should also be noted that in the
presence of Na
, the monomeric ion
[GalCer
Na
]
was the
primary ion present in the spectrum with only trace amounts of a
dimeric species,
[2GalCer
Na
]
(not
shown). There was no evidence of any higher oligomers containing
Na
. As will be seen later, Na
appears
to have a high affinity for GalCer to form the monomeric ion, but even
in the absence of Ca
, its ability to promote
oligomerization is negligible. This supports the conclusion that the
oligomers of GalCer formed in the presence of Ca
for
the most part are not due to Van der Waals interaction between the
ceramide moieties. The negative ion spectrum of GalCer in the presence
of excess calcium chloride on the other hand showed predominantly a
peak due to a monomeric ion at m/z = 762.4 (not shown),
which was identified as
[GalCer+Cl
]
. It
persisted at declustering potentials of -80 to -180 V.
Figure 2:
Positive
ion spray mass spectrum of CBS (46 nmol/ml) with Ca (500 nmol/ml) in methanol. The declustering potential was 80
V.
Figure 3:
Positive
ion spray mass spectrum of GalCer (20 nmol/ml) plus CBS (20 nmol/ml)
plus Ca (200 nmol/ml). The declustering potential was
80 V.
Figure 4:
Positive ion spray mass spectrum of GalCer
(20 nmol/ml) plus CBS (50 nmol/ml) plus Ca (200
nmol/ml) at a declustering potential of 180
V.
Although almost all of the
Ca adducts of GalCer dissociated at a declustering
potential of 180 V (Fig. 4), there is a high intensity peak at m/z = 750.6 due to the retention of the Na
adduct. The new peaks at 408.2 and 348.2 are fragment ions, as
confirmed by parent ion scans, indicating that under the conditions of
the experiment significant fragmentation of the lipids occurs.
The
ratio of intensity of the heterodimer to that of the CBS monomer
increases with increase in declustering potential as shown in Table 1. While it is not clear whether there is actually an
increase in the abundance of the heterodimer ion with increase in
declustering potential, since the intensity of the monomer shows a
gradual decrease, the conclusion that the heterodimer
[GalCerCBS
Ca
-H]
is the most stable of all the Ca
adducts of
either lipid, despite its low abundance, seems inescapable.
The low
abundance of this complex in methanol contrasts with liposome
aggregation studies, which indicated that the
Ca-mediated heterotypic aggregation of GalCer with
CBS anion was greater than the Ca
-mediated homotypic
interaction of either. The polyvalent nature of liposomes compared with
the monovalent lipids in solution may contribute to the greater
heterotypic interaction of liposomes. This difference in behavior may
also be due to the relative stability of the Ca
adducts of GalCer in methanol solution compared with water due to
the lower ability of Ca
to chelate methanol compared
with water(31) . The greater solubility of galactose and other
sugars in methanol containing dissolved calcium chloride than in pure
methanol, in which they are virtually insoluble, is well documented (32) and also indicates the formation of
Ca
-galactose complexes in agreement with the ESI-MS
results.
Figure 5:
CID mass
spectrum of the heterodimeric complex
[GalCerCBS
Ca
-H]
(m/z = 1574).