Cell Surface Glycolipids

495 Pages·1980·8.457 MB·English

by  Charles C. Sweeley (Eds.)

Cell Surface Glycolipids
Charles C. Sweeley, EDITOR
Michigan State University
Based on a symposium
sponsored by the
Division of Carbohydrate Chemistry
at the 178th Meeting of the
American Chemical Society,
Washington, D.C.
September 11-14, 1979.
128
ACS SYMPOSIUM SERIES
AMERICAN CHEMICAL SOCIETY
WASHINGTON, D. C. 1980
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
Library of Congress CIP Data
Cell surface glycolipids.
(ACS symposium series; 12
Bibliography: p.
Includes indexes.
1. Glycolipids—Congresses. 2. Membranes (Biology)
—Congresses.
I. Sweeley, Charles Crawford, 1930- . II. Amer
ican Chemical Society. Division of Carbohydrate
Chemistry. III. Series: American Chemical Society.
ACS symposium series; 128.
[DNLM: 1. Cell membrane—Congresses. 2. Glyco
lipids—Congresses. QU85 C393 1979]
QP572.G56C44 574.87'5 80-15283
ISBN 0-8412-0556-6 ACSMC8 128 1-504 1980
Copyright © 1980
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In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
ACS Symposium Series
M. Joa
Advisory Board
David L. Allara W. Jeffrey Howe
Kenneth B. Bischoff James D. Idol, Jr.
Donald G. Crosby James P. Lodge
Donald D. Dollberg Leon Petrakis
Robert E. Feeney F. Sherwood Rowland
Jack Halpern Alan C. Sartorelli
Brian M. Harney Raymond B. Seymour
Robert A. Hofstader Gunter Zweig
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
FOREWORD
The ACS SYMPOSIUM SERIES founded i 1974 t provid
a medium for publishin
format of the Series parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that in order to save time the
papers are not typeset but are reproduced as they are sub
mitted by the authors in camera-ready form. Papers are re
viewed under the supervision of the Editors with the assistance
of the Series Advisory Board and are selected to maintain the
integrity of the symposia; however, verbatim reproductions of
previously published papers are not accepted. Both reviews
and reports of research are acceptable since symposia may
embrace both types of presentation.
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
PREFACE
T
his collection of papers is a record of the proceedings of a symposium
on the chemistry, metabolism, and biological functions of glycolipids.
It is also a review of some topics presented at an earlier meeting on the
same subject, held in Honolulu, Hawaii in October, 1977 under the aus
pices of the Japan-United States Science Exchange Program.
Both meetings were convened with a hope that aspects of glycolipid
biochemistry at the cell
these meetings have provide insigh inspiratio
to understand the role of these interesting substances in nature. The
reader will note substantial progress and new information about the chem
istry and metabolism of the glycolipids, with especially comprehensive
material on their separation and characterization. The antigenic behavior
and the cell surface receptor role of glycolipids are discussed in some
detail, providing a sound basis for future investigations of cell surface
specificity to particular molecules and supramolecular systems.
I am indebted to the foreign speakers; Lars Svennerholm, Karl-
Anders Karlsson, Guido Tettamanti, Robert Murray, and Yoshitaka
Nagai generously consented to participate in this symposium with little
financial support from the organizer or the society. Their contributions
were an especially important part of the symposium. I am grateful as well
to the American participants, who attended the meeting largely with their
own funds and made the meeting a success. Although Professor Egge of
the University of Bonn could not attend the meeting, he kindly provided
an important chapter on high resolution proton NMR of glycosphingolipids,
for which I am grateful.
Finally, I wish to thank Ms. Dorothy Byrne, Ms. Paula Allen, and
Ms. Susan Leavitt for their dedicated assistance in the organization of the
meeting, correspondence with speakers, and preparation of the final manu
scripts for this book.
East Lansing, Michigan CHARLES S. SWEELEY
December, 1979
vii
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1
Preparative and Analytical High Performance
Liquid Chromatography of Glycolipids
R. H. MC CLUER
Eunice Kennedy Shriver Center, Waltham, MA 02154
M. D. ULLMAN
Center for Disease Control, Atlanta, GA 30333
High performance liqui
use of reusable columns
use of pumps for uniform solvent flow operated at high pressures
if necessary and automatic on-line sample detection. The avail
ability of a large variety of microparticulate column packing
materials, efficient column packing techniques, high pressure
-low volume pumping equipment and various types of highly sensi
tive detectors have led to the development of sensitive, rapid
and quantitative methods, analogous to that available for
volatile materials by gas-chromatography, for the isolation and
analysis of a large variety of relatively high molecular weight
substances of biological interest (1).
We have attempted to utilize these tools of modern liquid
chromatography to develop rapid and highly sensitive methods for
the analysis of glycolipids. Our early experience with HPLC
techniques indicated that the analysis of glycolipids becomes
interestingly sensitive and practical if derivatives are
prepared that allow the use of ultraviolet detectors and that
exhibit good chromatographic properties. We have primarily
studied the preparation of the benzoyl derivatives of glycolipids
for the development of analytical and preparative HPLC methods.
The following is a concise review of studies with neutral
glycosphingolipids with emphasis on recent work in which we have
utilized p-dimethylaminopyridine (DMAP) as a catalyst to effect
benzoylation with benzoic anhydride.
Preparation and analysis of benzoylated cerebrosides
We initially demonstrated that brain cerebrosides, galactosyl-
ceramides containing hydroxy fatty acids (HFA) and nonhydroxy
fatty acids (NFA), could be completely derivatized by reaction
with 10% benzoyl chloride at 60OC for 1 hour (2). After removal
of excess reagents by partition between hexane and alkaline
aqueous methanol, the perbenzoyl derivatives were seen to
separate into two completely resolved components (HFA and NFA
0-8412-0556-6/ 80/ 47-128-001 $5.00/ 0
© 1980 American Chemical Society
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
2 CELL SURFACE GLYCOLIPIDS
cerebrosides) by adsorption chromatography on a pellicular silica
column support (Zipax, E.I. DuPont) with methanol in pentane as
the eluting solvent and 280 nm detection. Hexane-ethyl acetate
was subsequently shown to be a superior eluting solvent because
regeneration of the column adsorbant activity was more repro
ducible (3). Attempts to recover the parent cerebrosides by
treatment of the benzoyl derivatives with mild alkali was
successful with the HFA-cerebrosides, but the NFA-cerebroside
derivative gave rise to benzoyl psychosine as well as the parent
NFA-cerebroside. This was demonstrated to result from the diacyl
amine structure of the perbenzoyl NFA-cerebroside. NMR studies
of the two cerebroside derivatives indicated the presence of six
benzoyl groups in each case and the presence of an amide proton
in the HFA-derivative which was absent in the NFA-cerebroside
derivative. As reported by Inch and Fletcher (4) for the
diacylamine derivatives of amino sugars, the N-acyl groups are
randomly removed durin
other sphingolipids whic
acetyl amino sugars cannot be recovered in high yields because
alkaline hydrolysis of the perbenzoyl derivative results in the
formation of N-benzoyl compounds as well as the parent N-acyl
sphingolipid. Benzoylation of cerebrosides with 10% benzoic
anhydride in pyridine was shown to lead only to the formation of
0-acyl derivatives and the parent glycolipids could be recovered
after alkaline methanolysis; however, this reaction was sluggish
and required treatment at 110 C for 18 hours for completion.
Sulfatides were shown to be completely converted to benzoylated
cerebrosides during this anhydride reaction. We chose the benzoyl
chloride reaction for analytical purposes because reaction times
were shorter and sulfatides do not desulfate under conditions
required for cerebroside derivatization. Because sphingolipids
which contain only hydroxy fatty acids as N-acyl substituents
form the same derivative with either the benzoyl chloride or the
anhydride reaction, they can be easily distinguished from non-
hydroxy fatty acid containing sphingolipids which form different
derivatives, distinquishable by HPLC, when benzoylated with the
chloride as compared to the anhydride reaction.
Analysis of ceramides
A quantitative HPLC method for the analysis of sphingolipids
as their perbenzoyl derivatives was first developed for ceramides
(5). Ceramides can be conveniently derivatized with benzoic
anhydride in pyridine (3 hrs at 110°C) and the products formed
have been utilized for the quantitative analysis of NFA and HFA
ceramides in normal and Farber's disease tissue. Iwamori and
Moser also utilized this procedure for the analysis of ceramides
in Farber's disease urine (6). More recently Iwamori and Moser
(7) established that the ceramide derivatives formed by reaction
with benzoyl chloride or benzoic anhydride are analogous to those
formed with cerebrosides. They also characterized the behavior
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. MCCLUER AND ULLMAN Liquid Chromatography 3
of ceramides that contain phytosphingosine and described the use
of estrone as an internal standard. These published ceramide
methods utilized 280 nm detection and the sensitivity of the
procedures could easily be increased to the pmole level by
detection at 230 nm if a variable wave length detector is
utilized. Also, the speed of derivatization could undoubtedly be
greatly increased by the use of the DMAP as a catalyst as
described below for neutral glycosphingolipids. Samuelsson (8)
utilized elegent gas chromatograph-mass spectrometric (GC-MS)
methods for the analysis of ceramide molecular species, but HPLC
methods offer the advantages of non-destructive measurement so
that components can easily be collected for determination of
radioactivity or for further analysis.
Quantitative analysis of neutral glycosphingolipids
An HPLC method for
designed for the analysis of human plasma glycolipids (3), which
consist primarily of glucosylceramide, lactosylceramide, globo-
triaosylceramide and globotetraosylceramide (globoside).
Conditions for the simultaneous derivatization of this group of
compounds, which provided maximal yields for globoside, were
selected to be 37bC for 16 hours in 10% benzoyl chloride in
pyridine, slightly different from those previously utilized for
cerebrosides. Satisfactory chromatographic conditions, which
provided base line resolution of these four derivatives in a
minimum of time, were found with the Zipax column and a gradient
of ethyl acetate in hexane and 280 nm detection. With this
chromatographic system the standard glycolipid derivatives could
be separated and quantitated in less than 20 min and column
activity could be reproducibly regenerated in eight min.. Less
than 20 nmole of each glycolipid could be easily quantitated with
this procedure.
The utility of ethyl acetate in this chromatographic system
was excellent but prevented the use of detection below 260 nm.
Because the xmax of the benzoyl derivatives is at 230nm we sought
chromatographic solvents which could be utilized at this wave
length, still provide adequate chromatographic resolution and
allow rapidly re-equilibration of column adsorbant activity after
gradient elution. A dioxane-hexane solvent system proved adequate
except residual light absorption due to the dioxane produced an
undesirable rising base line during the gradient. The rising
base line was eliminated by directing the solvent flow through a
precolumn pre-injector high pressure reference cell. This path
generates a horizontal baseline with a negative and positive
deflection at the beginning and end of the gradient respectively.
With this system reliable quantitation of less than 50 pmoles of
each of the four major plasma glycosphingolipids can be obtained
(9). For the analysis of plasma glycolipids it is necessary to
first isolate a glycolipid fraction by solvent extraction,
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
4 CELL SURFACE GLYCOLIPIDS
chromatography on small Unisil columns and treatment with mild
alkali as originally described by Vance and Sweeley (10).
Consistent recoveries of the glycolipids is dependent upon
maintaining a fixed ratio between sample size and the quantity of
Unisil employed. Accuracy of the method is improved by the
utilization of an internal standard such as N-acetylpsychosine
which is added to the plasma samples prior to the initial lipid
extraction. One ml plasma samples are now routinely used for
glycolipid analysis although sensitivity of the HPLC procedure
theoretically should allow analysis of less than 0.1 ml. However,
the isolation of such small quantities of glycolipid prior to
derivatization present difficult recovery problems. The high
sensitivity of the detection system employed for the analysis of
pmole quantities also requires precautions so that UV absorbing
contaminates are not introduced during processing of the samples.
All glassware should be scrupulously clean and HPLC grade solvents
should be used for all step
This HPLC procedure has also been utilized for the analysis of
neutral glycosphingolipids from a variety of sources.
Human erythrocytes, peripheral leukocytes and liver have
been satisfactorily analyzed, but it should be recognized that
each different tissue source may require different extraction
conditions and modified solvent gradient elution in order to
obtain maximal recoveries and optimal chromatographic resolution
of the tissue characteristic glycosphingolipids. Fletcher,
Bremer and Schwarting (11) have optimized the procedure for the
analysis of erythrocyte glycolipids and demonstrated that
erythrocytes from blood group P-| individuals contain more globo-
triaosylceramide and less lactosylceramide than erythrocytes from
blood group ?2 individuals. The dramatic sex difference in mouse
kidney glycolipids and the occurrence of large amounts of glyco
lipids in male mouse urine was readily demonstrated by these HPLC
methods. The light ear (le/le) mouse pigmentation mutant was
shown to have storage of glycolipids in their kidneys which is
apparently due to an abnormality in the secretion of multilamellar
lysosomal bodies that contain large amounts of glycosphingolipids
(12) . Thus, the analytical HPLC method for glycolipids is proving
useful for a variety of studies related to glycosphingolipid
function and metabolism.
Other useful analytical HPLC procedures for the analysis of
derivatized glycolipids have been developed. Nanaka and Kishimoto
(13) have devised an HPLC procedure which allows the tissue levels
of NFA cerebroside, HFA cerebroside, NFA sulfatide, HFA sulfatide,
and monogalactosyl diglyceride to be determined simultaneously.
This procedure involves benzoyl ation of total lipid extracts,
desulfation with mild acid and subsequent chromatography with the
radient of isopropanol in hexane. Susuki, Honda and Yamakawa
14) prepared acetylated glycolipid which were subsequently
reacted with p-nitrobenzoyl chloride to form the O-acetyl-N-p-
nitrobenzoyl derivatives which have good chromatographic
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
1. MCCLUER AND ULLMAN Liquid Chromatography 5
properties and can be detected with high sensitivity with a
single wavelength detector at 254 nm. While offering these
advantages, this procedure cannot be utilized with glycolipids
that contain only a-hydroxy fatty acids and no amino sugar. All
of the benzoylated or O-acetyl-N-p-nitrobenzoyl derivatives can
be usefully separated into molecular species by reverse phase
chromatography (11,12).
We have recently shown that the use of the catalyst N-di-
methylaminopyridine (DMAP) with benzoic anhydride greatly
accelerates the derivatization with this reagent (13). Reaction
with DMAP and the anhydride avoids amide acylation, forms single
products with satisfactory chromatographic properties and parent
glycosphingolipids can be regenerated by mild alkaline hydrolysis.
For analytical purposes, this reaction has been utilized for the
analysis of plasma neutral glycosphingolipids. The glycolipids
were reacted with 20% benzoic acid anhydride, 5% DMAP in pyridine
at 37°C for four hours
each gave single reaction products with maximum yields with
reaction times between 2 and 6 hours. Excess reagents were
removed from the products by partition between hexane and aqueous
alkaline methanol as described previously for the benzoyl chloride
products. The products were than analyzed with the Zipax column
and dioxane gradient also as previously described (3). The
chromatographic analysis of the per-O-benzoylated glycosphingo-
lipid standards and plasma glycosphingolipids are shown in Fig. 1
along with the elution pattern of plasma glycolipid derivatives
obtained by reaction with benzoyl chloride.
The derivatives obtained by reaction with benzoic anhydride
have longer retention times when compared to the benzoyl chloride
products. We have previously shown that galactosylceramide which
contains a-hydroxy fatty acids is not N-benzoylated with benzoyl
chloride and reaction with benzoic anhydride or benzoyl chloride
results in an identical product. Similar results have been
obtained with anhydride in the presence of DMAP as illustrated in
Fig. 2. The behavior of peak 11 b" which we have shown to be
derived from a-hydroxy fatty acid containing glucosyl and
galactosylceramides is illustrative. The UV response from each
of the standard GSLs benzoylated by the anhydride and by the
benzoyl chloride method were compared. The relative responses
(chloride/anhydride) for the mono, di, tri and tetra-hexosyl
ceramide were found to be 1.18, 1.15, 0.94 and 1.03 respectively.
These values were not significantly different from calculated
rations 1.20, 1.12, 1.09, and 1.15, based on the assumption that
the anhydride method avoids amide benzoylation. The yields of
the per-O-benzoylated products were similar to those obtained for
the products of the benzoyl chloride method reported previously.
The parent GSLs can be regenerated from their per-O-
benzoylated products by treatment with mild alkali. Globoside
was benzoylated by both methods, the products subjected to HPLC,
and the peaks collected and treated with 0.5N methanolic sodium
In Cell Surface Glycolipids; Sweeley, C.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1980.