Glycoconj J
DOI 10.1007/s10719-016-9684-0
MINI-REVIEW
Changes of glycoconjugate expression profiles
during early development
Kazuko Handa 1 & Sen-itiroh Hakomori 1,2
Received: 12 April 2016 / Revised: 23 May 2016 / Accepted: 24 May 2016
# Springer Science+Business Media New York 2016
Abstract A variety of glycoconjugates, including
glycosphingolipids (GSLs), expressed in mammalian tissues
and cells were isolated and characterized in early biochemical
studies. Later studies of virus-transformed fibroblasts demon-
strated the association of GSL expression profiles with cell
phenotypes. Changes of GSL expression profile were observed
during mammalian embryogenesis. Cell surface molecules
expressed on embryos in a stage-specific manner appeared to
play key roles in regulation of cell-cell interaction and cell
sorting during early development. Many mAbs showing
stage-specific reactivity with mouse embryos were shown to
recognize carbohydrate epitopes. Among various stage-
specific embryonic antigens (SSEAs), SSEA-1 was found to
react with neolacto-series GSL Lex, while SSEA-3 and SSEA-4
reacted with globo-series Gb5 and monosialyl-Gb5, respective-
ly. GSL expression during mouse early development was
shown to shift rapidly from globo-series to neolacto/lacto-se-
ries, and then to ganglio-series. We found that multivalent Lex
caused decompaction of mouse embryos, indicating a
functional role of Lex epitope in the compaction pro-
cess. Autoaggregation of mouse embryonal carcinoma
(EC) F9 cells provided a useful model of the compaction
process. We showed that Lex-Lex interaction, a novel type of
molecular interavction termed carbohydrate-carbohydrate in-
teraction (CCI), was involved in cell aggregation. Similar
shifting of GSL expression profiles from globo-series and
* Sen-itiroh Hakomori
hakomori@u.washington.edu
1
Division of Biomembrane Research, Pacific Northwest Research
Institute, 720 Broadway, Seattle, WA 98112, USA
2
Departments of Pathobiology and Global Health, University of
Washington, Seattle, WA 98195, USA
neolacto/lacto-series to ganglio-series was observed dur-
ing differentiation of human EC cells and embryonic stem
(ES) cells, reflecting the essential role of cell surface
glycoconjugates in early development.
Keywords Glycoconjugate . Glycosphingolipid . Embryonal
carcinoma . Embryonic stem cell . Stage-specific embryonic
antigen . Embryogenesis
Introduction
Biochemical research, during its early stages, was focused on
isolation, purification, and characterization of a wide variety of
biomolecules, including glycosphingolipids (GSLs), from tis-
sues and cells. Pioneering studies during the early 1960s by G.
Blix, E. Klenk, T. Yamakawa, and R. Kuhn led to identification
of such GSLs as cerebrosides (GalCer, GlcCer), sulfatide,
LacCer, brain gangliosides and a tetrasaccharide-containing
GSL (later termed Bgloboside^) [1–3]. GSLs were shown to
be localized primarily in the outer leaflet of the lipid bilayer of
plasma membranes. Over 1000 distinct GSLs have been
identified to date. They display great structural diversity,
but can be classified into four general groups on the
basis of core structure: globo-(Galα4Gal), lacto-
(Galβ3GlcNAcβ3Gal), neolacto- (Galβ4GlcNAcβ3Gal),
and ganglio-series (GalNAcβ4Gal). Lacto- and neolacto-
series core structures are also termed as type 1 and type 2
chains, respectively.
S. Iseki [4], S. Luria, and P. Robbins [5, 6] demonstrated that
structural changes of bacterial surface lipopolysaccharides by
phage-induced lysogenic transformation resulted in striking
phenotypic changes, e.g., in growth behavior and colony shape.
These studies of bacteria led to the novel concept that
cell surface glycans, even in eukaryotic cells, may

determine various phenotypic processes, including
ontogenic development and oncogenic transformation.
Demonstration of striking changes of GSL expression associ-
ated with DNA virus-induced transformation of hamster and
mouse fibroblasts [7–9] provided the first clear illustration of
this concept.
Subsequent studies (detailed below) revealed similar major
changes of glycan epitopes during early embryonic develop-
ment and differentiation of embryonal carcinoma (EC) and
embryonic stem (ES) cells. Various EC cell lines were
established from teratocarcinomas, i.e., malignant tumors
characterized by the presence of EC cells together with vari-
ous differentiated cell types derived from the primary germ
layers. EC cells are the pluripotent stem cells of such tumors,
and show remarkable morphological and biochemical similar-
ities to pluripotent cells of early embryos. Isolated clonal lines
of mouse EC cells retain their pluripotency during in vitro
culture, and their ability to form teratocarcinomas when
injected into histocompatible mice. Clonal EC cell lines
provide useful models for research on embryogenesis
and have been extensively used in studies of mammalian early
development [10].
Table 1 The structures of GSLs described in this mini review
Glycoconj J
Expression changes of glycoepitopes during mouse
embryogenesis
It had been predicted that cell surface molecules showing
stage-specific expression on mammalian embryos play impor-
tant roles in regulation of cell-cell interaction and cell sorting
during early embryonic development and differentiation.
Studies during the early 1970s reported several candidate mol-
ecules based on the use of various antisera [11, 12]. However,
the reliability of such studies was limited because of cross-
reactivity of the antisera. Following the establishment of hy-
bridoma technology in the mid-1980s [13], Solter et al. pre-
pared mAbs that recognized epitopes expressed on the embryo
cell surface at specific stages, through immunization of mouse
EC F9 cells or embryos at various stages. Epitopes that were
defined by such mAbs and expressed on the cell surface in a
temporally and spatially restricted manner were termed collec-
tively as stage-specific embryonic antigens (SSEAs), and indi-
vidually as SSEA-1, −2, −3, etc. Unexpectedly, many SSEAs
were identified as glycoconjugates, i.e., glycans expressed in
the form of GSLs, glycoproteins, or proteoglycans. Anti-
SSEA-1 mAb was established through immunization of F9
Glycoconj J
cells [14], and SSEA-1 was identified as Lex epitope [15–18].
Anti-SSEA-3 mAb (MC631) was derived from rats immu-
nized with mouse embryos at the 4–8 cell stage, and anti-
SSEA-4 mAb (MC813–70) was obtained from mice immu-
nized with human EC 2102Ep cells. Using GSLs prepared
from EC 2102Ep cells, SSEA-3 and −4 epitopes were identi-
fied as globo-series Gb5 and monosialyl-Gb5, respectively
[19, 20]. These findings reflect the functional importance of
glycosylation in defining the developmental process [21, 22].
During preimplantation development, the mouse embryo
passes through a series of stages termed 1-cell, 2-cell, 4-cell,
compaction, morula, blastocyst, etc. The fertilized egg un-
dergoes successive cleavages (e.g., 2-cell and 4-cell stages),
and then the compaction process leading to formation of the
morula. At 2.5 or 3 days after fertilization, the first visible
differentiation occurs. The outer cells of the morula develop
into a single layer (Btrophectoderm^), and the inner cells give
rise to a blastocoelic cavity and inner cell mass (ICM); the
resulting structure is the blastocyst. Mouse embryos express
predominantly globo-series GSLs such as globoside,
Forssman antigen, SSEA-3, and SSEA-4 (see structures in
Table 1). SSEA-3 and −4 are synthesized during oogenesis,
and are present on membranes of oocytes, zygotes, and em-
bryos at early cleavage stages [19, 21]. SSEA-3 disappears at
the early blastocyst stage, but subsequently reappears during
early post-implantation development on primitive endoderm
Fig. 1 Stage-specific shifting of Fertilization
carbohydrate epitope expression Day: 0.5 1.5
from globo-series to
neolacto-series, and then to
ganglio-series, during early
mouse developement. T:
trophectoderm; I: inner cell mass.
: Glc; ○: Gal; : GalNAc; :
SA; : Fuc; ●: GlcNAc.
Modified from Ref. [9] 2 cell
and its derivative, extraembryonic visceral endoderm. After
day 7, SSEA-3 expression is restricted to extraembryonic vis-
ceral endoderm and visceral yolk sac cells [23]. In contrast to
SSEA-3 and −4, Forssman antigen first appears on the embryo
surface after the third cleavage, and during blastocyst forma-
tion its expression is limited to the ICM [24]. The appearance
of Forssman antigen may be coupled to loss of SSEA-3 and
−4 at the 8-cell stage, reflecting changes in the dynamic bal-
ance of glycosyltransferases that compete for globoside as
substrate. Cell staining and complement-mediated lysis assays
have shown that SSEA-1 first appears on blastomeres of 8-cell
stage embryos, is expressed strongly at morula stage, and
declines following compaction, suggesting possible involve-
ment of SSEA-1 in the compaction process. SSEA-1 is also
expressed at the ICM and transiently in trophectodermal cells
[14, 22]. Ley was detected in trophectodermal cells of blasto-
cyst using specific mAb AH6 [25], and shown to play an
important role in implantation [26, 27]. Various ganglio-
series GSLs are expressed in post-implantation mouse embry-
os. A blood group-I-associated antigen, defined by specific
mAb C6 [28], was detected in mesodermal cells and their
derivatives [29] (Fig. 1)
The compaction process is controlled in part by a number
of cell surface molecules, including cadherin (uvomorulin)
[30] and gap junction proteins [31]. The striking change of
Lex expression level at the compaction stage suggested that
Implantation
2 2.5 3 3.25 3.5 7
Compaction
T
I
4 cells 8 cells 16 cells 32 cells 64-100 cells
Globo- Neolacto- Ganglio-
 Glycoconj J

Fig. 2 Involvement of Lex

antigen in mouse embryonic cell
adhesion. Compacted 8- to
16-cell embryos a were cultured
in the presence of 1 mM trivalent
Lex-lysyllysine b or trivalent
Lea-lysyllysine c for 12 h.
Molecular structures are shown at
right. Modified from Ref. [32]

Lex may play a role in stabilizing the compaction process by studies showed that (i) only Lex-expressing cells, but not
maximizing intercellular contacts. To test this possibility, we Lex-negative variant cells, adhered to Lex-coated plates, (ii)
purified Lex and Lea oligosaccharides from human milk and the major carrier of Le x hapten in F9 cells was
examined their effects on compacted mouse embryos in two polylactosaminoglycan with high mass range, (iii) the
molecular forms: monomers and multimers attached to the polylactosaminoglycan fractions autoaggregated in the pres-
primary amino group of lysyllysine. Multivalent Lex conju- ence of Ca2+, and (iv) the autoaggregation was abolished by
gated to lysyllysine had a striking effect: individual blasto- fucosidase treatment [39, 40] (Fig. 3). Taken together, these
meres in the embryos assumed a rounded shape and lost their findings suggested that Lex hapten expressed on one cell rec-
close apposition of membranes. This morphological effect ognized Lex expressed on another cell that Ca2+ was necessary
(termed Bdecompaction^) was not observed for monovalent for the recognition, and that Lex-Lex interaction was involved
Lex oligosaccharide by itself or mixed with lysyllysine, nor for in F9 cell autoaggregation. Lex-Lex interaction was subse-
multivalent Lea conjugated to lysyllysine (Fig. 2). The quently confirmed by other groups using physicochemical
decompacting effect of multivalent Lex was observed at con- techniques such as NMR spectroscopy and atomic force
centrations ranging from 0.1–1.0 mM. The possibility that the
effect was due to cell toxicity was ruled out by trypan blue
exclusion test [32]. Because decompaction was caused by a
multivalent Lex hapten but not by monovalent Lex oligosac-
charide, Lex valency is evidently crucial. Bird and Kimber
made similar observations [33], and T. Feizi’s group demon-
strated the importance of carbohydrate antigens in early de-
velopment [34].
Compaction is the first of many cell-cell interactions that
occur during embryogenesis. Ca2+ is required for compaction
[35], and a molecule involved in Ca2+-mediated cell adhesion
was reported [36, 37]. Our finding that multivalent Lex is in-
volved suggested a role in compaction of some molecule that
recognizes multivalent Lex hapten. To search for such a mole-
cule expressed on the cell surface, we used autoaggregation of
mouse F9 EC cells as a model of compaction. F9 cells, which
were used to establish anti-SSEA-1 mAb, highly express Lex
(SSEA-1) epitope, as well as Gb4 and Forssman antigen, Fig. 3 a Lactosaminoglycan (Bembryoglycan^; LAG) fractions were
which are expressed during mouse embryogenesis [24]. F9 prepared in the presence of EDTA from F9 cells (Lex-positive mouse
autoaggregation was inhibited by trivalent Lex, but not by tri- EC cell line) and PSY-2 cells (Lex-negative mouse EC cell line)
metabolically labeled with [3H]GlcNAc. Radioactivity in precipitates
valent Lea, similarly to mouse embryo compaction. A search (ppt) formed after dialysis against buffer containing 5 mM Ca2+ are
for a Lex-binding protein in F9 cells capable of blocking anti- shown as % total activity. b LAG from labeled F9 cells was digested
SSEA-1 mAb binding to F9 cells led to isolation of a 15–25 kD with pronase to produce LAG-glycopeptide (LAG-GP), and subjected
component that displayed inhibitory activity. The component to gel filtration. Radioactivity was measured for each fraction. Gel
filtration patterns of LAG-GP in the presence of Ca2+ or EDTA, and
did not have detectable protein content, and its inhibitory ac- of LAG-GP treated with bovine liver α-fucosidase, are shown. a and
tivity was unaffected by proteinase digestion, suggesting that b, high molecular weight aggregates. Vo, void volume. Vi, column
the component was a glycoconjugate [38]. Our subsequent volume. Modified from Ref. [40]
Glycoconj J
microscopy [41–43]. It was also demonstrated that Gb4 inter-
acts with SSEA3 or nLc4 using human EC 2102Ep cells,
which highly express these glycans [44]. Studies by our group
and others showed clearly that this novel type of molecular
interaction, termed carbohydrate-carbohydrate interaction
(CCI), occurred between various carbohydrate pairs (e.g.,
GM3-Gg3, GM3-LacCer), and is involved in biological pro-
cesses such as growth factor-induced cell growth and cancer
metastasis [45–51].
Expression changes of glycoepitopes
during differentiation of EC and ES cells
A pluripotent human EC cell line, NTERA-2, was cloned by
P. Andrews’ group from teratocarcinoma-derived cell line
TERA-2 [52]. NTERA-2 cells cultured in the presence of
retinoic acid undergo extensive differentiation into various
somatic cell types, including neuronal cells [53, 54].
Similarly to GSL core structures in mouse embryos, undiffer-
entiated NTERA-2 cells express predominantly globo-series
GSLs such as Gb3, globoside, SSEA-3, SSEA-4, globo-H,
and globo-A [55] (Table 1). Induction of differentiation by
cell culture in the presence of 10−5 M retinoic acid resulted
in a remarkable shift of GSL expression pattern. The differen-
tiated cells showed reduced expression of globo-series GSLs,
and increased expression of lacto/neolacto-series GSLs (Lex,
sialosyl type 2 chain) and of ganglio-series GSLs (GM3,
GD3, 9-O-acetyl-GD3, GM2, GD2). This GSL core
structure switching was dependent mainly on glycosyl-
transferases involved in synthesis of a few key core
structures. During the NTERA-2 cell differentiation, ac-
tivities of the key enzymes β3GlcNAc transferase and α3Fuc
transferase increased 4-fold and 2-fold, respectively, in asso-
ciation with increased expression of GSLs carrying neolacto-
series core structures [56, 57].
In an extensive study of human ES cell lines using immu-
nofluorescence staining and mass spectrometry, Y-J Liang
et al. observed striking changes in GSL expression profiles
during cell differentiation into embryoid bodies (EBs).
Undifferentiated ES cells expressed SSEA-3, SSEA-4,
Gb4Cer, Lc4Cer, fucosyl Lc4Cer, globo-H, and disialyl
Gb5Cer (Table 1). During differentiation into EBs, GSL core
structures shifted from globo- and lacto/neolacto-series to
ganglio-series. This shift resulted from altered expression of
key glycosyltransferases involved in biosynthesis of the core
structures: down-regulation of those related to globo- and
lacto/neolacto-series GSLs, and simultaneous up-regulation
of those related to ganglio-series GSLs [58]. These findings,
in combination with those from EC cell studies as above,
indicate that changes in glycosyltransferase gene expression
mediate changes in GSL expression during differentiation of
human EC cells and ES cells.
Summary
Cell surface molecules shown in early studies to be expressed
in a stage-specific manner on mammalian embryos were pre-
sumed to play important roles in regulation of cell-cell inter-
action and cell sorting during embryogenesis. Studies along
this line were greatly accelerated by development of mAb
technology. Unexpectedly, many mAbs showing stage-
specific reactivity with embryos (e.g., anti-SSEA-1, −SSEA-
3, and -SSEA-4) were found to recognize carbohydrate epi-
topes. The SSEA-1 epitope was identified as neolacto-series
Lex, while those of SSEA-3 and −4 were globo-series Gb5
and monosialyl-Gb5, respectively. GSL expression during the
course of mouse embryogenesis was found to shift rapidly
from globo-series to neolacto/lacto-series, and then to
ganglio-series. Similar shifting of GSL core structures was
demonstrated during differentiation of human EC cells and
ES cells. Multivalent Lex was shown to cause decompaction
in mouse embryos, reflecting the essential role of cell surface
glycoconjugates in embryonic development.
Acknowledgments Original studies by our group described here were
supported by National Institutes of Health R01 CA20026, GM23100,
CA080054, National Cancer Institute Outstanding Investigator Grant
CA42505 (to S.H.), and The Biomembrane Institute. The authors are
grateful to Dr. S. Anderson for English editing of the manuscript, and to
Mylinh Bui for secretarial assistance.
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