Glycobiology vol. 22 no. 12 pp. 1721–1730, 2012
doi:10.1093/glycob/cws115
Advance Access publication on July 24, 2012
Glycosphingolipid composition of epithelial cells isolated
along the villus axis of small intestine of a single human
individual
Michael E Breimer1,2, Gunnar C Hansson3,
Karl-Anders Karlsson3, Göran Larson4,
and Hakon Leffler5
2
Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy
at University of Gothenburg, SE-416 85 Gothenburg, Sweden; 3Department
of Medical Biochemistry, Sahlgrenska Academy at University of Gothenburg,
SE-405 30 Gothenburg, Sweden; 4Department of Clinical Chemistry and
Transfusion Medicine, Sahlgrenska Academy at University of Gothenburg,
SE-413 45 Gothenburg, Sweden; and 5Section of Microbiology, Immunology
and Glycobiology (MIG), University of Lund, SE-223 62 Lund, Sweden
Received on January 30, 2012; revised on July 5, 2012; accepted on July 17,
2012
A 6-cm fresh proximal ileum surgical specimen from a
blood group A1Le(a-b+) secretor individual was used for
stepwise isolation of epithelial cells from villus tip to crypt
bottom by gentle washing with ethylenediaminetetraacetic
acid-containing buffer. Acid and non-acid sphingolipids
were prepared from the epithelial cell fractions and the
non-epithelial intestinal residue. Molecular information on
the sphingolipid composition was obtained without further
isolation of individual species by applying thin-layer chromatography using chemical and biological (monoclonal
antibodies, cholera toxin, Escherichia coli) detection
reagents, mass spectrometry and proton NMR spectroscopy of derivatized glycolipids. In this way, the structure
of major and minor saccharides, ceramide components
and their relative amounts were obtained. Epithelial cells
and non-epithelial residue were distinctly different in their
sphingolipid composition. Sphingomyelin was the major
single component in both compartments. Characteristic
for epithelial cells was the dominance of monoglycosylceramides, sulphatides and blood group fucolipids (mainly Leb
hexaglycosylceramides and ALeb heptaglycosylceramides).
The non-epithelial residue had about five times less glycolipids mainly mono-, di-, tri- and tetra-glycosylceramides
and gangliosides, including the GM1 ganglioside. The ceramides were more hydroxylated (1–2 additional hydroxyls)
in epithelial cell glycolipids compared with the non-epithelial
residue. Combined with a separate detailed study on the
glycoproteins of the same epithelial cell preparation, this
1
To whom correspondence should be addressed: Tel: +46-31-3434000;
Fax: +46-31-417631; e-mail: [email protected]
human intestinal sample is the only epithelial cell preparation where both protein- and lipid-linked saccharides are
characterized in detail.
Keywords: cholera toxin receptor / epithelial cells /
glycosphingolipids / human small intestine / lipid rafts /
Norovirus receptor
Introduction
Despite the tremendous progress in identification of the molecular components of cell membranes and their functional
interactions, there is a paucity of quantitative composition
data of specialized cells in authentic tissues. One example is
the small intestinal epithelial cells, that have highly specialized membranes not resembling those of other cells, or
even cultured cells supposed to be models of intestinal epithelial
cells.
The small intestine of various animal species has been of
great importance for our present knowledge of glycosphingolipid structure and diversity. This is in part due to the richness
of complex fucolipids in intestines, in contrast to the low
amounts in the red cells, the earliest source for studies of
histo-blood group fucolipids. One major advantage of intestinal tissues as a source for studies is the very large area of
the epithelial monolayer covering the intestinal villi, as well
as the microvilli extensions of epithelial cells providing a very
large plasma membrane surface area, the major site of
sphingolipid localization (Breimer et al. 1982b; Lingwood
2011). The intestinal epithelial cell is covered with carbohydrates, both loosely bound as secreted mucus (Linden et al.
2008; Johansson et al. 2010) and plasma membrane bound.
The latter may be either protein bound (smaller glycoproteins
or large transmembrane mucins and GPI-anchored glycoproteins) or lipid bound (glycolipids). The intestinal epithelial
cells of several animal species have unique high concentrations of species specific cell surface carbohydrates which limit
extrapolation of information from one species to another. The
glycolipids are major components among the lipids and the
type of glycolipids is different compared with non-epithelial
tissues (Breimer et al. 1982b). Glycolipids have been found
enriched in preparations of brush borders and basolateral
membranes (Forstner and Wherrett 1973; Christiansen and
Carlsen 1981; Breimer et al. 1982b; Hansson 1983) consistent
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1721
ME Breimer et al.
with typical plasma membrane components. A major issue for
understanding the role of glycosphingolipids in epithelial cells
is to obtain data on the molecular organization of sphingolipids and other lipid or protein components in micro-domains
or lipid rafts from non-cultured epithelial cells (Danielsen and
Hansen 2006; Lingwood and Simons 2010).
Available techniques span from preparative and analytical
biochemistry to morphological analysis with nanometer resolution (Nguyen et al. 2006; Wrackmeyer et al. 2006; Hoetzl
et al. 2007). The small intestinal epithelial cells are produced
in the lower part of the crypt from a small number of epithelial stem cells and migrate to the villus tip (Barker et al.
2007). For the rat and other experimental animals it has been
possible to isolate epithelial cells of different phenotypes
(crypt-villus tip) by a successive incubation with ethylenediaminetetraacetic acid (EDTA)-containing solutions (Weiser
1973; Bouhours and Glickman 1976; Breimer et al. 1981b).
This technique was applied on human small (Björk et al.
1987) and large (Holgersson et al. 1991) intestine surgical
specimens with focus on structural characterization of histoblood group ABO and related lipid-bound (Björk et al. 1987;
Holgersson et al. 1991) and protein-bound (Finne et al. 1989)
carbohydrate antigens. The present report is focused on the
alkaline stable sphingolipid composition of the small intestinal epithelial cell fractions isolated along the crypt to villus
tip axis from an A1Le(a-b+) secretor individual. Taken
together, our data provide a basis for understanding the role
of carbohydrates as receptors for pathogenic microorganisms
and bacterial toxins (Hanada 2005) in the human small
intestine.
Results
Isolation of epithelial cells
The results of the enzymatic assays of the epithelial cells
obtained from the small intestine are shown in Figure 1. The
decrease of alkaline phosphatase (a GPI-anchored protein)
activity and the increase of thymidine kinase activity in the sequentially obtained cell fractions gave evidence that cells of different phenotypes were isolated (Weiser 1973). Microscopical
examination confirmed this, revealing mature epithelial cells
with typical brush border in the fractions with high alkaline
phosphatase activity and aggregates of crypt bottom cells
lacking brush border in the fractions having a high thymidine
kinase activity. These results show that the epithelial cells were
sequentially removed from the residual intestinal stroma principally in the same manner as documented for rat small intestine
(Breimer et al. 1981b).
Sphingolipid composition
An outline of the sphingolipid composition is given in
Figure 2A, which shows the total alkali stable polar lipids of
the epithelial cell fractions and the non-epithelial residue.
Yield and molar ratios of different components are listed in
Table I. The major glycosphingolipids of the epithelial cells
were monoglycosylceramides (bands a and b) and histo-blood
group fucolipids (bands d and f ). Sphingomyelin (sm) was
the single major sphingolipid. The non-acid and acid glycolipid fractions of the epithelial cells and residue were isolated and further characterized. The individual glycolipid
structures were determined by mass spectrometry and proton
Fig. 1. Isolation of human small intestine epithelial cells. A 6-cm piece of proximal ileum was first filled with a citrate–dithiothreitol containing buffer (solution
A; Weiser 1973) and incubated at 37°C for 15 min (fraction I). After emptying, the intestine was filled with an EDTA-containing buffer (solution B; Weiser
1973), incubated 5 min and emptied and refilled repeatedly. The process was repeated 15 times (cell fractions 2–16). The intestine was manipulated gently every
min. during incubations (Breimer et al. 1981b). After each emptying, it was washed twice with buffer (solution B). Enzyme activities and protein content were
measured as in Breimer et al. (1981b). Alkaline phosphatase was highest in the mature villus tip cells, whereas thymidine kinase was highest in the crypt cells.
The 16 cell fractions were pooled into four fractions classified as villus tip cells (I), intermediate (II and III) and crypt cells (IV) as marked by bars above the
diagram.
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Epithelial cell glycolipids
NMR spectroscopy of glycolipid mixtures or after partial
subfractionation.
A combined interpretation of thin-layer chromatography,
mass spectrometry and NMR spectroscopy allowed the identification of the structures listed in Table II.
Non-acid compounds a and b of epithelial cells
The thin-layer chromatogram of the non-acid glycolipid fractions revealed bands with mobility as monoglycosyl ceramides (Figure 2B). Using borate impregnated thin-layer
chromatography (TLC) plates (Figure 2C), bands with mobility as GlcCer and GalCer and with heterogeneous ceramide
compositions were observed.
The permethylated and permethylated-reduced derivatives
of the non-acid glycolipid mixture were analyzed by direct
inlet mass spectrometry. The glycolipids were gradually “distilled” off the probe while spectra were collected (Breimer
et al. 1979). The spectra recorded at lower temperatures (150–
225°C) were typical for HexCer. They showed ceramide parts
containing hydroxy fatty acids combined with mainly dihydroxy base (d18:1) and a smaller part with trihydroxy base
(t18:0). This agreed with the ceramide composition revealed
by thin-layer chromatography (Figure 2C). The hydroxy fatty
acid chain length distribution was bimodal with C16:0, C24:0
and C24:1 fatty acids as major species while the C18–C20
homologues were minor species. The bimodal distribution of
chain lengths explains the double band TLC appearance of
both GlcCer and GalCer (hydroxy fatty acids-dihydroxy and
trihydroxy base (h–d) and (h–t)) in Figure 2C.
Non-acid compounds c to h of epithelial cells
The details of this analysis have been reported (Björk et al.
1987) and are only summarized here. Part of the non-acid glycolipids of epithelial fractions I and II were combined. The
monohexosylceramides were removed by silicic acid column
chromatography and the fraction containing the complex
glycolipids was analyzed by mass spectrometry and NMR
spectroscopy of the permethylated and permethylated-reduced
derivatives. Compounds identified were blood group Lea pentaglycosylceramides (compound c), Leb hexaglycosylceramides
(d), blood group A hexaglycosylceramides (e) and ALeb heptaglycosylceramides (f). The difucosylated compounds d and f
were the predominant complex glycolipids while c and e were
only minor components in all fractions. The ceramide parts
contained hydroxy fatty acids combined with di- and
Fig. 2. Thin-layer chromatograms of the sphingolipid and glycosphingolipid
fractions isolated from human small intestine epithelial cells (fractions I–IV)
and non-epithelial residue (NE). (A) Total alkali stable lipids. Letters to the
sides indicating compound identity refer to Table II and text. Sphingomyelin
(sm) was identified by the mobility and characteristic blue color with the
anisaldehyde reagent (Breimer et al. 1981b). The amounts applied
corresponded to 0.6 mg of cell protein for lanes I–IV and to 2 mg dry weight
for lane NE. Solvent: CHC13–CH3OH–H2O, 60:35:8, by vol. (B) Total
non-acid glycolipids. Conditions of analysis were as in (A). The amounts
applied corresponded to 3 mg of cell protein for lanes I–IV and to 50 mg dry
weight for lane NE. (C) Analysis of monoglycosylceramides on a borate
impregnated thin-layer chromatography (Holgersson et al. 1991). The total
non-acid glycosphingolipid fractions were applied. The complex glycolipids
remained at the origin or slightly above for di- and triglycosylceramides (two
bands for lane NE). The monoglycosylceramides were separated according to
sugar and ceramide composition. The identification, by comparison with
reference monoglycosylceramides and by mass spectrometry, is given to the
left. The ceramide type is given in brackets by n for non-hydroxy fatty acids,
h for hydroxy fatty acids, d for dihydroxy base and t for trihydroxy base. The
GlcCer (h–d) and GalCer (h–d) appear as two bands each corresponding to
long-chain (mainly C24) and short-chain (mainly C16) fatty acids for the
upper and the lower band, respectively (see text). Lane S is the desulphated
product of the acidic glycolipids of cell fraction II (see text). It shows the
release of GalCer from sulphatide and its ceramide composition. Solvent:
CHC13–CH3OH–H2O, 100:30:4, by vol. Detection: anisaldehyde (Breimer
et al. 1981b). The amounts applied in each lane were as in (B).
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ME Breimer et al.
Table I. Yield and molar ratios of glycosphingolipids and sphingomyelin in human small intestinal epithelial cells from a single individuala
Epithelial cell fraction
Yield of glycolipids (mg; mg/g protein)
Compoundb
a,b
c–f
i
k
m
n
p
q
o–s
Sphingomyelin
g,h
Non-epithelial residue
I
II
III
IV
1.5 (0.3)
Molar ratiosc
20
10
10
d
<3 × 10−1
n.d.
n.d.
n.d.
1.5 × 10−3
n.d.
60
5 × 10−1
1.8 (0.3)
0.7 (0.2)
0.12 (0.2)
0.6
20
10
10
<4 × 10−1
<1.6 × 10−2
<0.4 × 10−2
1.10−1
3 × 10−3
n.d.
60
5 × 10−1
20
5
5
<3 × 10−1
n.d.
n.d.
n.d.
1.5 × 10−3
n.d.
60
5 × 10−1
20
5
5
<3 × 10−1
n.d.
n.d.
n.d.
1.5 × 10−3
n.d.
60
5 × 10−1
2.5
—
n.d.
1.5
1.5
1.5
0.4
0.1
1
90
n.d., not determined.
a
The amounts of the different components were estimated by comparing the staining intensity of bands on thin-layer chromatograms. The unknown samples were
compared with known reference glycolipids of similar structure applied in series of known amounts. For the quantification of compounds a–k of epithelial cells,
a, b, k–t of non-epithelial cells and sm the CuAc reagent was used (Karlsson et al. 1973; Breimer et al. 1981b). The data were supported by staining with
anisaldehyde (Breimer et al. 1981b) or resorcinol (Svennerholm 1963). For compound p of epithelial cells the staining with a monoclonal antibody (Nudelman
et al. 1982; Brodin et al. 1985) was used and for q cholera toxin (Magnani et al. 1980). The upper limit for compounds m and n in epithelial fraction II was
calculated from the detection level with staining by bacteria (Bock et al. 1985). No staining was observed.
b
Letters refer to structures in Figures 2 and 3 and Table II.
c
Total sphingolipids are set as 100.
d
When given with the inequality sign the figure indicates an upper limit to the amount. The compound was not detected.
trihydroxy base as for monoglycosylceramides (a, b) and had
similar fatty acid carbon chain distributions.
Compounds g and h were identified only by mass spectrometry of the permethylated-reduced derivatives (Breimer
et al. 1979). Fragments containing the complete saccharide
chain and the fatty acid defined the number and type of
sugars as given in Table II.
Acid compound i of epithelial cells
The major acidic glycolipid of the epithelial cells migrated as
GalCer sulphate on thin-layer chromatography both as native
fraction (Figure 3A) and acetylated derivative (Breimer et al.
1983). Part of the acidic glycolipids of epithelial fraction II
was treated with HCl in CH3OH for desulphation (Breimer
et al. 1983).The product was analyzed by thin-layer chromatography on borate impregnated plates (Figure 2C, lane S) which
revealed the formation of GalCer with a ceramide composition
similar to that of the non-acid glycolipids (lanes I–IV). In addition, a minor band migrating as cholesterol sulphate (cs) was
seen in all epithelial cell fractions.
Non-acid compounds a, b, k, m and n of the non-epithelial
residue
Thin-layer chromatography of the non-epithelial residue
non-acid glycolipids revealed bands with mobility as mono-,
di-, tri- and tetraglycosylceramides (Figure 2B, lane NE).
Mass spectrometry established the presence of HexCer,
HexHexCer, HexHexHexCer and HexNAcHexHexHexCer.
The anomeric region of the proton NMR spectrum of the
permethylated-reduced derivative showed signals in accordance of Hexβ1Cer, lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer)
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Table II. Glycosphingolipid structures identified in human small intestine
epithelial cells and non-epithelial residue and discussed in the present report
Compound
identity in
text
a
b
c
d
e
f
g
h
i
k
m
n
o
p
q
r
s
t
Shorthand
designation
a
Le
Leb
A-6-1
A-7-1
Sulphatide
LacCer
Gb3
Gb4
GM3
GD3
GM1a
Structure
Glcβ1Cer
Galβ1Cer
Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer
Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer
GalNAcα3(Fucα2)Galβ3GlcNAcβ3Galβ4Glcβ1Cer
GalNAcα3(Fucα2)Galβ3(Fucα4)
GlcNAcβ3Galβ4Glcβ1-Cer
(dHex)3(HexNAc)2(Hex)4Cer
(dHex)3(HexNAc)3(Hex)4Cer
03S0-Galβ1Cer
Galβ4Glcβ1Cer
Galα4Galβ4Glcβ1Cer
GalNAcβ3Galα4Galβ4Glcβ1Cer
NeuAcα3Galβ4Glcβ1Cer
NeuAcα8NeuAcα3Galβ4Glcβ1Cer
Galβ3GalNAcβ4(NeuAcα3)Galβ4Glcβ1Cer
NeuAcα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer
NeuAcα3Galβ3GalNAcβ4(NeuAcα3)
Galβ4Glcβ1Cer
NeuAcα8NeuAcα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer
The letters are referred to in the text and Figures 2 and 3.
(Falk et al. 1979c). Overlay of a thin-layer chromatogram with
uropathogenic Escherichia coli specifically binding Galα4Gal
(Bock et al. 1985) stained the tri- and tetraglycosylceramide
bands confirming the globo-series identity of these compounds. Borate impregnated thin-layer chromatography
(Figure 2C) identified the monoglycosylceramides as mainly
containing galactosylceramides with hydroxy fatty acids and
di- and trihydroxylated long-chain base together with less
Epithelial cell glycolipids
(Holmgren et al. 1975). Here only partial structures were
established by thin-layer chromatography (Figure 3A and B)
and by mass spectrometry of the permethylated and the
permethylated-reduced-thrimethyl derivatives of the total
non-epithelial fraction (not shown). These results were in accordance with published structures. Thus, mass spectrometry
identified (NeuAc)2HexHexCer (Holm et al. 1977) and
(NeuAc)2HexHexNAcHexHexCer (Karlsson 1974) as major
components. Rearrangement fragments from the latter showed
that a major part was NeuAcHexHexNAc(NeuAc)HexHexCer
(Karlsson 1974) agreeing with compound s, but NeuAc
NeuAcHexHexNAcHexHexCer, agreeing with compound t,
could also be present. In addition, NeuAcHexHexCer (compound o) and (NeuAc)1HexHexNAcHexHexCer (compounds
q and r) were identified as minor components. Thin-layer
chromatography in several solvent systems showed major
bands corresponding to compounds o, p, s and minor bands
corresponding to r, q and t (Figure 3A and B and not shown).
Thin-layer chromatography overlay analysis using cholera
toxin (Figure 3C) showed staining of a band with mobility as
GM1 reference ganglioside corresponding to compound
q. Staining with the 4.2 monoclonal antibody against human
melanoma specific for NeuAcα8NeuAcα3Gal- (Nudelman
et al. 1982; Brodin et al. 1985) showed staining corresponding
to compounds p and t (not shown). Staining with a monoclonal antibody specific for NeuAcα2-3Gal showed bands corresponding to structures o, r and s (not shown). In addition to
the gangliosides, a band corresponding to cs was also detected
in the non-epithelial fraction (Figure 3A).
amounts of glucosylceramides (mainly non-hydroxy fatty
acids and dihydroxy long-chain base).
Gangliosides of epithelial cells
The epithelial cells contained very small amounts of gangliosides. The only clearly identifiable resorcinol positive band corresponded to the mobility of compound p found in the
non-epithelial residue (Figure 3B) and staining with the 4.2
monoclonal antibody supported the presence of this ganglioside.
A thin-layer chromatogram of the acidic glycolipid fractions was
stained with 125I cholera toxin (Magnani et al. 1980) using conditions as in (Hansson et al. 1985) are shown in Figure 3C. A
single band corresponding to the mobility of GM1 reference
ganglioside (compound q in Table II) was found. The staining
was strong for the non-epithelial residue fraction but weak for
the epithelial cell fractions of which fractions II showed strongest staining. The amount of the cholera toxin receptor was estimated by comparing the staining by cholera toxin with a
dilution series of known amounts of a brain GM1a ganglioside
reference compound q (not shown). As given in Table I the
amount of the cholera receptor ganglioside was very low compared with other glycolipids especially in the epithelial cells;
0.01% of all glycosphingolipids and about 1% of all lipid-bound
sialic acid. Human small intestine mucosa scraping was reported
to contain 3% GM1, of the lipid-bound sialic acid (Holmgren
et al. 1975). This slightly higher content is probably due to contamination of subepithelial tissue.
Acid compounds o–t (gangliosides) of non-epithelial residue
The structures o–t, listed in Table II, are those earlier reported
for gangliosides of whole human small intestine (Keranen
1975, 1976a, 1976b) and mucosal scraping/muscularis tissue
Changes of epithelial cell sphingolipids in the crypt to villus
tip cell axis
No qualitative differences between the crypt and villus cell
glycosphingolipid components were detected. A semi
Fig. 3. Thin-layer chromatograms of acid sphingolipid fractions isolated from
human small intestine epithelial cells (I–IV) and non-epithelial residue (NE).
(A) Total alkali stable acid lipids. Cholesteryl sulphate (cs) was identified by
mobility and its characteristic violet staining with anisaldehyde (Björkman
et al. 1972). Other non-glycolipid contaminants are marked with x. solvent:
CHC13–CH3OH–H2O–CH3COOH, 60:35:10:8, by vol. Detection:
anisaldehyde (Breimer et al. 1981b). The amounts applied corresponded to
6 mg of cell protein for lanes I–III, 4 mg of cell protein for lane IV and
75 mg of dry weight for lane NE. (B) Total acid glycolipids. Solvent:
CH3OCOCH3–CH3CHOHCH3–CaC12 in H2O (8 mg/mL)—NH3 in H2O
(5 M) 45:35:15:10, by vol. Detection: anisaldehyde (Breimer et al. 1981b).
Bands staining purple with resorcinol (Svennerholm 1963) in a parallel
chromatogram (not shown) are marked with +. The amount applied
corresponded to 30 mg of cell protein for lane II and 200 mg of dry weight
for lane NE. (C) Detection of cholera toxin receptor in the total acidic
glycolipid fractions. Solvent: CHC13–CH3OH–H2O, 60:35:8, by vol.
Detection: iodinated cholera toxin and autoradiograhy (Magnani et al. 1980).
1725
ME Breimer et al.
quantitative estimate of the relative amounts of the different
sphingolipids was done based on staining intensity of thinlayer chromatograms (Table I). The relative ratio of glycolipids to sm and to total cell protein remained roughly constant.
The ceramide compositions were significantly different for
crypt cells compared with villus cells as revealed by mass
spectrometry (not shown) and supported by thin-layer chromatography for monohexosylceramides (Figure 2A and C). This
showed an increase in the proportion of long-chain fatty acids
(C22–C24) and decrease of short chain fatty acids (C16–C18)
in the crypt to villus tip cell gradient. This change of fatty
acid chain length is seen in Figure 2A and B (lanes I–IV) for
monoglycosylceramides (compounds a and b) migrating as a
pair of bands corresponding to short (lower band) and long
(upper band) chain fatty acids. The proportion of the bands
was 1/1 in the crypt cells while in the villus cells the upper
band was more abundant. The proportion of non-hydroxy/
hydroxy fatty acids or dihydroxy/trihydroxy base remained unchanged in contrast for that found in rat where hydroxy fatty
acids were increased in the villus tip (Breimer et al. 1982a).
Discussion
Glycosphingolipids and membrane glycoproteins are found in
the plasma membrane and in membranes related to their biosynthesis and export (e.g. ER and Golgi). The carbohydrate
chains are typically facing the interior of the subcellular structures or the outer surface of the plasma membrane where they,
together with the secreted mucins, contribute to the glycocalyx of individual cells and epithelia. Therefore, the present
sphingolipid analysis, together with the analysis of the cellbound glycoproteins from the same cellular samples (Finne
et al. 1989), gives a tentative picture of carbohydrate architecture at the human small intestinal epithelial surface. Most of
the carbohydrate is protein bound. Most of the complex saccharide chains carry histo-blood group determinants, and the
major part of these determinants is also protein bound. The
histo-blood group determinants of the glycolipids are based
on type 1 core chains (Björk et al. 1987), while protein linked
saccharides carried histo-blood group determinants mostly of
the type 2 chain (Finne et al. 1989). Besides the histo-blood
group type saccharides, the lipids carried the monosaccharides
Glc, Gal and O3SO.Gal. Only minute quantities of sialic acid
were found in the glycolipids (e.g. GM1a) and practically no
sialic acid was detected bound to the glycoproteins. Instead,
the sulphate group of the 3-O-sulphated GalCer give a major
contribution to the negatively charge and together with cs it
will give a strong negative charge just at the cell membrane
(Figure 2A). The N-linked glycans are largely neutral (Finne
et al. 1989), but we do not know if the O-glycans of the
heavily glycosylated transmembrane mucins are sulphated or
sialylated. Thus, it is possible that the lipid-bound sulphate
groups contribute heavily to a negatively charged cell surface
of human small intestine. The O-linked glycans are especially
abundant on the membrane bound mucins that in the small intestine are made up of the mucins MUC3, MUC12 and
MUC17 (Johansson et al. 2010; Kim and Ho 2010). Although
formally not proven, these molecules make up the major part
of the dense glycocalyx sometimes called the fuzz when
1726
observed by electron microscopy (Ito 1969). The MUC3,
MUC12 and MUC17 all have a large extracellular mucin
domain, a juxtamembrane sea-urchin sperm protein, enterokinase and agrin (SEA) domain, a transmembrane and a small
cytoplasmic domain are in total comprised of between 4500
and 5500 amino acids (www.medkem.gu.se/mucinbiology/
databases/). The best sequenced and most abundant of these
mucins is MUC17 that has more than 1800 serines and threonines in its mucin domain. All of these are potential glycosylation sites. However, today we have no information on how
many of these sites are glycosylated, how many mucins of this
type are present on each cell and not even any information on
glycan structure of these mucins. Thus it is not possible to
make any estimate on the contribution of these components to
the glycan repertoire of the human small intestine, although
they obviously make a major contribution to the intestinal
glycan profile.
Considerable insight in the molecular action of the cholera
toxin has been achieved as reviewed in (Chinnapen et al.
2007). In the initial binding phase, the toxin B-subunit pentamer binds five GM1 receptors localized in lipid raft domains
on the apical cell membrane and is internalized into apical
early endosomes (Chinnapen et al. 2007). Ultrastructural
studies of different cells have shown that the cholera toxin receptor is not uniformly distributed in the plasma membrane
but concentrated in what is believed to be lipid rafts (Parton
1994; Chinnapen et al. 2007). In the human small intestine,
the cholera toxin receptors were found exclusively on the
outside of the plasma membrane and showed a temperature
sensitive distribution (Hansson et al. 1977). The amount of
cholera toxin receptor GM1 identified in the epithelial cells
was extremely low (0.01% of total glycolipids). Therefore,
this suggests that GM1 needs to be concentrated, e.g. in lipid
rafts, to function as cholera toxin receptor in the enterocytes.
Alternatively, its site of action is not the enterocytes, but
perhaps mucosal nerve cells (Chambers et al. 2005) or other
cells. Indeed, even the long held idea that cholera diarrhea is
due to fluid secretion emanating from the intestinal epithelial
cells has been challenged (Lucas 2010).
Early studies of human small intestinal mucosa scrapings
(Keranen 1975, 1976a, 1976b) indicated a much higher
content of gangliosides of the epithelial cells when compared
with our findings. However, the ceramide composition
reported for these gangliosides (Keranen 1976b) resembled
that for the non-epithelial glycolipids (non-hydroxy fatty acids
and dihydroxy base) and differed from those we found for the
epithelial glycolipids (hydroxyl fatty acids combined with dior trihydroxy base). Therefore, the gangliosides reported
earlier (Keranen 1975, 1976a, 1976b) probably originated in
part from non-epithelial tissue obtained in the mucosa scraping. In the present report a more selective method for separation of epithelial cells from the residue was used as shown
by the absence of Gb3Cer and Gb4Cer (Figure 2B; Björk
et al. 1987) in the epithelial cells.
The glycolipid carbohydrate pattern was similar for the
epithelial cells of different phenotype along the crypt to villus
tip axis. However, a few clear differences in amounts of individual bands were observed for the glycolipids c–f and i,
between villus and crypt cells. These alterations can largely be
Epithelial cell glycolipids
attributed to alterations in their lipophilic parts with an
increased proportion of long-chain fatty acids in the villus
cells. Thus the repertoire of complex glycolipids was already
fully developed in the crypt cells. This is in accordance with
the enterocytes maturing from the intestinal stem cells within a
very short distance in the crypt, reviewed in van der Flier and
Clevers (2009). Previous more thorough studies on the glycolipid turnover along the crypt-villus axis suggested that the
lipophlic part of the glycolipids turned over faster than the
cells (Breimer et al. 1982a). The intestinal brush border membrane is actively turned over by endocytosis followed by degradation or recycling and by vesicles shedding into the lumen
(Donowitz and Li 2007; McConnell et al. 2009). Although
there are no modern studies of glycolipid turnover of the
brush border membrane and in the intestinal epithelial cells
during their transition from stem cells to their shedding into
the lumen at the villus tip, there are thus reasons to believe
that the glycolipids are continuously biosynthesized and
turned over.
The glycosphingolipids of small intestinal epithelial cells
have been studied for several species (Forstner and Wherrett
1973; McKibbin 1978; Breimer et al. 1979, 1981a, 1983;
Christiansen and Carlsen 1981) and some features seem to be
general. The concentration of glycolipids in relation to sm and
other lipids is higher than for non-epithelial cells. The ceramide parts are mainly of the hydroxylated type (containing trihydroxy base and or hydroxy fatty acids). Such structural
factors may, through the possibilities of increased hydrogen
bonding of sphingolipid components (Löfgren and Pascher
1977; Pascher and Sundell 1977; Karlsson 1982) contribute
to an increased membrane stability and impermeability. In
contrast to the general features, there is a striking variation in
the structure of the carbohydrate chains between species,
which includes neutral, sialylated and sulfated glycolipids
(Breimer et al. 1981a, 1983).
Lipid rafts are nanoscale membrane microdomains enriched
in glycosphingolipids, cholesterol and proteins that function
in membrane signaling and trafficking (Lingwood and Simons
2010). Initially lipid rafts were defined as detergent-resistant
membrane subfractions and their biological relevance has
been questioned although cell biology data have shown their
existence also in living cells. Sphingomyelin is often only a
minor component of lipid rafts. Rafts of brush border membranes of pig enterocytes were shown to be highly enriched in
glycosphingolipids (Hansen et al. 2001; Danielsen and Hansen
2006). Rafts of human small intestinal epithelial cells have not
yet been analyzed, but for the epithelial glycosphingolipids
listed in Table II many of them are probably present in rafts.
The possible functional importance of hydrogen bonds involving ceramide was first established from the crystal structure of
GalCer (Pascher and Sundell 1977) and is now included in the
raft concept of self-association (Lingwood and Simons 2010).
Each GalCer molecule of the crystal (composed of dihydroxy
base and 2-hydroxy fatty acid) was shown to participate in
eight hydrogen bonds. Two of these were intramolecular and
the remaining intermolecular. It was also shown that the presence of a 2-hydroxy group of the fatty acid or a 4-hydroxy
group of the base ( phytosphingosine) promoted a condensation
effect on a surface monolayer film compared with species
without these groups (Löfgren and Pascher 1977). Therefore,
the extra hydroxyl group of the fatty acid and the long-chain
base in the epithelial glycolipids compared with the nonepithelial glycolipids probably confer additional stability of the
raft in the varying environment of intestinal contents, including
dietary lipid and bile salts. The higher level of ceramide hydroxylation in various cells is apparently related to the extent
of physicochemical stress on the surface membrane (Karlsson
1982).
During the last ten years significant progress has been
made clarifying the biosynthesis of ceramides (based on
d18:1) and phytoceramides (based on t18:0) from dihydroceramides through the action of two mammalian desaturatases,
i.e. DES1 and DES2, respective (see references in Merrill
2011). DES2 is highly expresses in the small intestine,
kidneys and skin where phytoceramides are typically present.
A DES1 −/− mouse with highly elevated levels of dihydroceramide, low levels of ceramide, multi-organ dysfunction and
failure to thrive has been described but up to now no such
model has been presented for the DES2 gene (Gault et al.
2010). Additionally, the fatty acid C2-hydroxylase (FA2H)
was recently identified as the NAD(P)H-dependent enzyme
that catalyzes the C2-hydroxylation of fatty acids to generate
2-hydroxy sphingolipids or ceramides (Alderson et al. 2004;
Eckhardt et al. 2005). Such 2-hydroxy fatty acids are known
as important constituents of plasma membrane lipid rafts critical for signaling and trafficking events. A series of mutations
in the human FA2H gene are associated with late onset
complex neurodegenerative phenotypes;, e.g. complicated
spastic paraplegia (SPG35), leukodystrophy with spastic
paresis and dystonia, neurodegeneration with brain iron accumulation and leukodystrophy with a mixed phenotype
(Garone et al. 2011). The C2 hydroxylation introduces an
asymmetric carbon and very recently it was shown that
human FA2H, overexpressed in CHO cells, selectively produces the (R)-enantiomers, which have quite different biological functions as the (S)-enantiomers (Guo et al. 2012). In
FA2H knock-down adipocytes, the diffusional mobility of
raft-associated lipid was increased, leading to reduced GLUT4
protein levels, an effect that independently from caveolin-1
levels, could be reversed by treatment with exogenous
(R)-2-hydroxy palmitic acid but not with the corresponding
(S)-enantiomer. Interestingly, the (R)-enantiomer was enriched
in the monohexosylceramides whereas the (S)-enantiomer was
preferentially incorporated into free ceramides suggesting different routes for biosynthesis of sphingolipids containing the
two 2-hydroxy fatty acid enantiomers. FA2H is preferentially
expressed in brain, skin, stomach, kidney and testis and not in
the small intestine. However, the presence of other enzymes
with overlapping substrate specificity with FA2H was recently
indicated by studies on sm profiles in various cells of patients
with a deleterious FA2H mutation (Dan et al. 2011). In our
material sm of epithelial cells had mainly non-hydroxy fatty
acid and dihydroxy base (not shown). Further studies of
sphingolipids in plasma membranes, with special focus on intestinal epithelial cells of single individuals, are expected to
improve our understanding of raft function and create new
possibilities for interfering with membrane associated signaling and uptake mechanisms.
1727
ME Breimer et al.
Glycosphingolipids are membrane attachment sites of
various microbes and toxins (Karlsson 1989; Hanada 2005),
and there is growing evidence that rafts are sites of infections
(Waheed and Freed 2009; Hartlova et al. 2010). The dominating glycolipid of the epithelial cells was GalCer (Table I).
When infected through the intestine, there is evidence that
GalCer is mediating transfer of HIV-1 through large intestine
epithelial cells to reach the target cells of lamina propria by
transcytosis (Meng et al. 2002; Lingwood 2011). Glycoside
analogs of GalCer have been synthetized that inhibited HIV-1
fusion and infection of cells (Garg et al. 2008). The same substances inhibited vesicular stomatitis virus infection (Garg
et al. 2008), and earlier a number of viruses belonging to the
families adenoviridae, herpetoviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae and reoviridae all were shown to
interact with monoglycosylceramides, and were inhibited in
binding or infection by synthetic analogs (Karlsson KA,
Norrby E, et al. United States Patent 1990; Number 4, 980,
462). Human norovirus, the cause of winter-vomiting disease
which affects millions of people and causes 200,000 deaths
each year, has been shown to bind specifically, in a strain specific manner, to histo-blood group antigens typically found on
saliva mucins and on glycosphingolipids of just the kind
expressed in human small intestine (Rydell et al. 2011).
Additionally, we have recently shown that the human Dijon
strain norovirus, which belongs to the globally dominating
genotype GII.4, also binds to GalCer aggregated in domains
of solid supported lipid membranes (G. Larson et al. in preparation). The significance of the very close apposition of the
virus to the host membrane for penetration into the cell
remains to be shown. Of general interest for penetration at
rafts, Shiga toxin, which recognizes Gb3Cer, was shown to
induce protein-independent tubular invaginations in human
and mouse cell membranes and model membranes (Romer
et al. 2007). There is thus a need for deeper analyses of the
microbe to gut epithelial cell interaction as one important port
of entry of human infections.
Material and methods
Tissue specimen
The intestinal specimen was collected from 47 year old
woman undergoing elective surgery for a leiomyoma located
in the proximal part of the ileum. A 25 centimeter part of
the gut was removed, and a 6 centimeter segment proximal to
the tumor was collected for analysis. The blood group status
of the patient, A1Le(a-b+)Secretor, was established by
routine typing of the erythrocytes and saliva at the laboratory
of Transfusion Medicine, Sahlgrenska University hospital,
Gothenburg. The Ethical Committee, University of Gothenburg,
approved the study.
Isolation of epithelial cells
The intestinal epithelial cells were isolated by the method of
(Weiser 1973) with slight modifications as described in detail
elsewhere (Breimer et al. 1981b). The epithelial cells were
released in a sequential manner from the villus tip to the crypt
bottom by repeated incubations with an EDTA-containing
1728
buffer. Cell fractions obtained were analyzed for thymidine
kinase activity, alkaline phosphatase activity, protein content
and the cell purity and morphology were evaluated in a phase
contrast microscope (Breimer et al. 1981b). The different cell
fractions were pooled according to the results of enzyme
assays and visual appearance in the microscope as shown in
Figure 1 corresponding to villus tips cells (fraction I), intermediate cells (II and III) and crypt cells (IV), respectively.
Glycolipid preparation
Alkaline stable polar non-acid and acid lipid fractions were
prepared from the epithelial cells and the residual nonepithelial intestinal stroma as described (Angstrom et al.
1981; Breimer et al. 1981b; Björk et al. 1987).
Glycolipid characterization
TLC was performed using micro analytical plates (HPTLC,
Si-60, Merck, Darmstadt, Germany) with different solvents
and detection reagents (details described in legends to
figures). Permethylation of the glycolipids was done according
to the procedure of Hakomori (Hakomori 1964) and part of
the permethylated fraction was reduced with LiA1H4
(Karlsson 1974). Permethylated and permethylated-reduced
glycolipid fractions were analyzed by direct inlet mass spectrometry and by proton NMR spectroscopy. Details about the
technical conditions, interpretation and reference spectra for
mass spectrometry (Breimer et al. 1979; Björk et al. 1987)
and NMR spectroscopy (Falk et al. 1979a, 1979b, 1979c)
have been reported. Direct binding of biological ligands
(cholera toxin, monoclonal antibodies and E. coli) to thinlayer chromatograms was done according to (Magnani et al.
1980; Hansson et al. 1983, 1985; Bock et al. 1985) using aluminum backed HPTLC Si-60 plates (Merck). After developing, the plates were immersed in 0.5% poly-isobutyl
methacrylate in diethylether for 1 min. (Hansson et al. 1985).
Anti-melanoma ganglioside monoclonal antibodies 4.2 and
13-M1 were gifts from Drs Hellström, Seattle, WA (Nudelman
et al. 1982; Brodin et al. 1985). The uropathogenic E. coli
strain 26692 was donated by Dr C. Svanborg and labeled as
in (Hansson et al. 1985). Cholera toxin was obtained from
Sigma and labeled with 125I using the Iodo-Gen™ reagent
(Hansson et al. 1985).
Funding
The work was supported by grants from the Swedish
(Medical) Research Council.
Acknowledgement
The experiments of this work on human intestine were initiated
in the late 1970s when we extensively studied intestinal epithelial tissues of various species. With this publication we former
PhD students (MEB, GCH, GL and HL), gratefully acknowledge our scientific mentor Prof Karl-Anders Karlsson.
Epithelial cell glycolipids
Conflict of interest
None declared.
Abbreviations
The abbreviations of known glycosphingolipid or carbohydrate structures follow the recommendations by IUPAC and
IUB. 1977. Eur J Biochem. 79:11–21. For partially known
structures, deduced by mass spectrometry, the following carbohydrate designations are used: dHex for deoxyhexose, Hex for
hexose and HexNAc for N-acetylhexosamine. The ceramide
type is given by n for non-hydroxy fatty acids, h for hydroxy
fatty acids, d for dihydroxy base and t for trihydroxy base. cs,
cholesterol sulphate; EDTA, ethylenediaminetetraacetic acid;
FA2H, fatty acid C2-hydroxylase; Gb3Cer, globotriaosylceramide; Gb4Cer, globotetraosylceramide; LacCer, lactosylceramide; sm, sphingomyelin; TLC, thin-layer chromatography.
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