From the Institute of Biological Chemistry, Academia
Sinica, Taipei 115-29, Taiwan and the § Institute for
Biological Sciences, National Research Council of Canada, Ottawa,
Ontario K1A OR6, Canada
Received for publication, December 16, 2002
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ABSTRACT |
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Polysialic acid (PSA)1
is a unique cell surface homopolymer that is expressed by clinically
important serogroups B and C Neisseria meningitidis in
2,8-PolyNeu5Ac is expressed on neural cell
adhesion molecules during embryogenesis and also re-expressed on
certain tumors. PolyNeu5Ac is therefore an oncodevelopmental antigen,
has important regulatory effects on the adhesive and migratory behavior
of neural cells, and is thus crucial to synaptic plasticity. Until now,
2,9-polyNeu5Ac, a linkage isomer of
2,8-polyNeu5Ac, has long been
thought to occur only in capsules of neuroinvasive Neisseria meningitidis group C bacteria. Here we report the unexpected
discovery of
2,9-polyNeu5Ac in a new cell adhesion-related
glycoprotein on the membrane of C-1300 murine neuroblastoma cells
(clone NB41A3). We also report the expression of
2,9-polyNeu5Ac was
affected by cell growth and retinoic acid-induced
differentiation. Occurrence of the linkage isomer of
2,8-polyNeu5Ac has been left unrecognized by conventional methods
using biological diagnostic probes for
2,8-polyNeu5Ac. Thus,
our discovery may change contemporary views of biology and
pathology of polysialic acid and open new avenues for the development
of anti-neural tumor drugs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2,8- and
2,9-linked isomers respectively (1) (Fig.
1). These molecules encapsulate the
organisms, which are responsible for a dominant portion of all cases of
meningococcal meningitis, and are critical to their
pathogenesis. One of these linkage isomers,
2,8-polyNeu5Ac, which
also forms the capsule of neuroinvasive Escherichia coli K1,
is poorly immunogenic (2), probably due to the fact that it is a
self-antigen occurring as an oncodevelopmentally regulated mammalian
antigen in the neural cell adhesion molecule (NCAM) and certain tumors.
In contrast
2,9-polyNeu5Ac is immunogenic enough to be used as a
vaccine against meningitis caused by group C meningococci and is
expressed by these organisms in both O-acetylated and
O-deacetylated forms (2). Since the 1980s, the molecular
biology, structural biology, and physiological and pathophysiological
functions of
2,8-polyNeu5Ac on NCAM have already been the subject of
major reviews (3-7). Usually detection and identification of
2,8-PSA in NCAM studies are made using monoclonal antibodies (mAbs)
specific to
2,8-polyNeu5Ac. Although such immunological reagents are
useful for studies of developmentally regulated dynamic expression
of
2,8-polyNeu5Ac, they cannot be used to define DP or to
detect either changes in the distribution of glycoforms differing in DP
or the presence of PSA differing in sequences or inter-residue
linkages. Prior to the identification of
2,8-polyNeu5Ac-NCAM, we
discovered
2,8-polyNeu5Gc by chemical and biochemical methods,
which was the first example of animal PSA (8, 9). Later we also
unveiled divergent forms of PSA, such as an
2,8-poly(Neu5Ac, Neu5Gc)
co-polymer,
2,8-polyKDN, and
2,5-Oglycolyl-linked
oligo/polyNeu5Gc (10, 11). These PSA-glycoproteins (gp) were shown to
play biological functions in fertilization and early embryogenesis, and
until now, except for the last example found in sea urchin eggs, all
inter-residue linkages in PSA chains found in vertebrate sources were
2,8.
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Fig. 1.
Depicts the two linkage isomers of
polyNeu5Ac, i.e. 2,9-polyNeu5Ac, which was only
known to occur in the N. meningitidis group C bacterial
capsular polysaccharide until it was discovered in this study to be
expressed by a C-1300 murine neuroblastoma clone (NB41A3)
(A), and
2,8-polyNeu5Ac, which is expressed by N. meningitidis group B bacteria, the embryonic form of NCAM of
chicken, Xenopus laevis, and mammals, including
neuroblastoma cells such as human neuroblastoma cell lines IMR-32 and
CHP-134 (B).
In view of the fact that the presence of a large number of
glycoforms for a single functional protein is important for the delicate and fine regulation of "protein-based functions" in higher animals, we thought that the NCAM of neuroblastoma cell lines would be
an excellent model system to demonstrate how the DP of PSA chains and
the structure of the core N-glycan are modulating the
adhesive and migratory behavior of neural cells. To define DP and the
change in distribution of glycoforms differing in DP we developed an
ultrasensitive fluorescent labeling method using a fluorogenic probe
DMB (1,2-diamino-4,5-methylenedioxybenzene), which selectively reacts
with any type of oligo/poly--ulosonic acids to introduce a strong
fluorescent group at the reducing termini of these chains
(DMB/HPLC-FD method) (12-17). This method permits us to achieve
the same level of sensitivity as that attained by using antibodies.
Here we report the serendipitous discovery of 2,9-linked polyNeu5Ac
in a new cell adhesion-related glycoprotein on the membrane of C-1300
murine neuroblastoma cells (clone NB41A3).
2,9-PolyNeu5Ac has long
been thought, until now, to be a homopolymer only found as capsular
polysaccharide of N. meningitidis group C bacteria.
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EXPERIMENTAL PROCEDURES |
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Antibodies Used--
Sources for rat anti-mouse embryonic
PSA-NCAM (CD56) mAb (12F8, IgM) and anti-rat NCAM mAb (5B8, IgG) were
given previously (16). Serum-pooled mouse polyclonal antisera against
de-O-acetyl group C meningococcal polysaccharide
(GCMP)-tetanus toxoid conjugate Ab against 2,9-polyNeu5Ac was
prepared as described previously (18).
Cell Lines and Their Culture, Growth, and Harvest-- C-1300 murine neuroblastoma (clone NB41A3) cell line was obtained from American Type Culture Collection (ATCC) and actually purchased from Food Industry Research and Development Institute, Hsinchu, Taiwan. Cells were cultured in Ham's F-10 medium, supplemented with 15% horse serum, and 2.5% fetal bovine serum and kept in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were transferred upon reaching confluence, and those within 5 passages after purchasing from ATCC were used for analysis and harvested by trypsination after washing with phosphate-buffered saline. The cells were collected by centrifugation at 3,000 rpm for 10 min. To examine growth stage-dependent change of polysialylation, NB41A3 cells were inoculated at the concentration of 5 × 105/ml and were collected at different stages of growth (5-, 6-, and 7-days). The same amount of cells (250 mg wet weight) was subjected to PSA analysis to facilitate direct comparison. The cells harvested at different stages of growth and differentiation were examined for their morphological features under a phase-contrast microscope (Olympus, IX70; Olympus, Tokyo, Japan). All-trans-retinoic acid (RA; Sigma) was used at a final concentration of 10 µM in the culture medium.
Analysis of PSA and Total Neu5Ac in the Solubilized Glycoproteins-- PSA and total Neu5Ac in the glycoproteins solubilized with Triton X-100 were analyzed by DMB/HPLC-FD method (16). Peaks of oligo/polysialic acids were monitored with a fluorescence detector set at 372 nm for excitation and 456 nm for emission. Because each PSA can acquire only one DMB molecule, the total peak area for DMB-oligo/polysialic acid peaks (DP > 4) represents the total number of PSA chains and is used as a value comparing the level of PSA in the cells.
ELISA was also applied to evaluate quantitatively the PSA expression
level in the cells using mouse polyclonal antisera against de-O-acetylated GCMP (2,9-polyNeu5Ac)-tetanus toxoid
conjugate Ab and alkaline phosphatase-conjugated goat antimouse IgM as
the primary and secondary antibody, respectively. The absorbance at 405 nm (p-nitrophenol) of the reaction mixture represents the amount of PSA epitope. All procedures used in SDS-PAGE and Western blot
were essentially similar to those previously described (16).
Isolation of 2,9-PolyNeu5Ac-glycopeptides from NB41A3
Cells--
The membrane fraction of NB41A3 cells was first delipidated
with a mixture of chloroform-methanol-10 mM Tris-HCl (pH
8.0), 4:8:3 (v/v). The lipidated residue was washed with cold 80%
ethanol, suspended in 2 volumes of 0.1 M Tris-HCl (pH 8.0)
containing 10 mM CaCl2 and incubated with
Streptomyces griseus nonspecific proteinase (Sigma; 1 mg for
1 g of wet cells) for 3 days at 37 °C under a toluene
atmosphere. The incubation mixture was clarified by centrifugation and
applied to a DEAE-Toyopearl 650-M (Tosoh, Tokyo, Japan)
column (1.2 × 8 cm). Compounds bound to the column were eluted
stepwisely with 4-column volume each of 0.1, 0.2, and 0.4 M
NaCl in 10 mM Tris-HCl buffer (pH 8.0). PSA was found only
in the 0.4 M NaCl fraction.
Periodate Oxidation/Borohydride
Reduction/Mild Acid Hydrolysis for C7/C9
Analysis--
Each sample containing 200 ng of Neu5Ac of NB41A3
2,9-polyNeu5Ac-glycopeptides, authentic
2,8-polyNeu5Ac, and
2,9-polyNeu5Ac was dissolved in 20 µl of 0.1 M NaOH
and left at 37 °C for 4 h to remove possible
O-acetyl group. To a 10-µl aliquot of each sample solution
(pH was adjusted to 5.5 with 1 M acetic acid), 10 µl of
water and 10 µl of 0.15 M sodium metaperiodate were added and incubated at 4 °C. After 24, 48, and 96 h, reaction was
stopped by adding 5 µl of 3% glycerol. Then, 10 µl of freshly
prepared 0.04 M sodium borohydride in 0.01 M
sodium hydroxide was added, and the mixtures were kept at room
temperature for 30 min. Excess borohydride was destroyed with 10 µl
of 0.2 M acetic acid. The products were hydrolyzed in 0.1 M trifluoroacetic acid at 80 °C for 4 h,
derivatized with DMB for 2.5 h at 55 °C, and analyzed for C7
and C9 non-ulosonates by HPLC/FD as described elsewhere (17).
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RESULTS AND DISCUSSION |
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The expression of PSA and NCAM in the solubilized membrane
fractions was analyzed for the murine neuroblastoma by DMB/HPLC-FD and
by Western blot analysis using anti-2,8-polyNeu5Ac mAb (12F8) and
anti-NCAM mAbs (5B8 and VIN-IS-53). For 5 cell lines (4 different neuroblastoma cell lines (IMR-32, SK-N-MC, SK-N-SH, and Neuro-2A) and
one pheochromocytoma cell line (PC-12)) examined in our previous report, these two diagnostic tests gave consistent results (16): IMR-32
and PC-12, PSA-positive/NCAM-positive; neuro-2A,
PSA-negative/NCAM-positive. However, conflicting results were obtained
regarding PSA expression in NB41A3 cells that were found to be NCAM
immunoassay-negative with anti-NCAM mAb (Table
I). NB41A3 cells were
2,8-polyNeu5Ac-negative on Western blots using 12F8 (Fig.
2C) but PSA-positive on the DMB/HPLC-FD analysis (Fig. 2, A and B). PSA
residues in NB41A3 cells were also shown to be completely hydrolyzed to
unsubstituted Neu5Ac monomer by treatment with Arthrobacter
ureafaciens sialidase. These surprising findings provided a hint
about the possible occurrence of a linkage isomer of
2,8-polyNeu5Ac,
i.e.
2,9-polyNeu5Ac in NB413A cells, which has long been
thought, until now, to be a homopolymer only found as capsular
polysaccharide of N. meningitidis group C bacteria (1). This
prompted us to investigate the PSA structure of the NB413A
membrane-bound glycoprotein.
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Usually detection and identification of 2,8-polyNeu5Ac are made
using monoclonal antibodies specific to this form of polymer. However,
it should be emphasized here that if we had used only immunochemical
criteria for examining PSA expression, the present finding of
2,9-polyNeu5Ac in NB413A cells would have definitely been missed.
This emphasizes the importance of having additional analytical methods
to detect PSA. Therefore we recommend that the DMB/HPLC-FD method
should be routinely used as a screening test for PSA chains in
biological samples. Confirmation of the presence of novel
2,9-polyNeu5Ac in the NB413A-derived glycoprotein was obtained by
Western blot analysis using a mouse polyclonal antibody induced by a
protein conjugate of the O-deacetylated group C
meningococcal polysaccharide (18). The cell membrane extract of NB413A
PSA-gp revealed a broad anti-
2,9-polyNeu5Ac-positive band on
SDS-PAGE, centering at an apparent molecular size of ~150,000, because of the presence of numerous glycoforms differing in the DP of
their PSA chain(s) (Fig. 2D).
Structural evidence for the presence of 2,9-polyNeu5Ac was also
obtained from the results of the analysis of C7 and C9 ulosonates obtained after periodate oxidation of the sample, followed by borohydride reduction and mild acid hydrolysis. Oxidation of the
2,9-polyNeu5Ac chain with periodate resulted in its depolymerization and the destruction of all Neu5Ac residues to form their C7 analogs. The time-course of reaction was examined for three samples, NB41A3 PSA-glycopeptide, authentic
2,9-polyNeu5Ac, and
2,8-polyNeu5Ac, by monitoring C7 and C9 monomers using the DMB/HPLC-FD method (Fig.
3, A-C). The
reaction proceeded slowly (19), and the NB41A3 PSA-glycopeptide and
authentic
2,9-polyNeu5Ac samples gave the C7 analog of Neu5Ac as the
only product (Fig. 3, B and C), whereas
2,8-polyNeu5Ac remained unoxidized and gave C9 Neu5Ac (Fig.
3A). These results prove that the inter-residue linkage of
polyNeu5Ac in NB41A3 PSA-gp is
2,9.
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Supporting evidence for the presence of 2,9-polyNeu5Ac was obtained,
using the DMB/HPLC-FD method, by co-injecting NB41A3-derived PSA-gp
with each of the authentic samples, which resulted in a subtle
retention time difference between homologous
2,8-oligo/polyNeu5Ac and
2,9-oligo/polyNeu5Ac labeled with
fluorescent quinoxalinone at their reducing termini. The results are
reproduced in Fig. 3, D and E, and as expected,
NB41A3 PSA exhibited a simple profile when co-chromatographed with
authentic standard
2-9-polyNeu5Ac (Fig. 3D),
whereas, when mixed with
2,8-polyNeu5Ac, a series of doublet
peaks was seen on the co-chromatogram (Fig. 3E).
Interestingly, although 2,9-polyNeu5Ac has been known for well over
a couple of decades as a constituent of the capsule of N. meningitidis group C (1), its occurrence in animal glycoproteins had yet to be identified. Indeed, to the best of our knowledge, there
has been only one report concerning the finding of an
2,9-diNeu5Ac residue, Neu5Ac
2
9Neu5Ac
2
, in human ovarian teratocarcinoma (PA-1) cells (20), but we failed to detect PSA in PA-1
cells by the DMB/HPLC-FD method.
2,8-PolyNeu5Ac is almost
exclusively associated with NCAM in animal, where it can modulate the
homotypic adhesive properties of this member of the immunoglobulin
superfamily. Based on its immunoreactivity, the newly identified
2,9-PSA in NB41A3 neuroblastoma cells is expressed on a distinct
glycoprotein species other than
2,8-polyNeu5Ac-bearing NCAM, but its
biological function has yet to be defined. However, by analogy these
two cell surface molecules are probably functionally equivalent,
2,9-polyNeu5Ac playing a similar role to
2,8-polyNeu5Ac as a cell
adhesion regulatory molecule during cellular development and
differentiation. This can be indicated by the observed change of
2,9-polyNeu5Ac expression profiles with culture time in the presence
or absence of RA as determined by the DMB/HPLC-FD method (Fig.
4), and this can be deduced from
coordinated behavioral changes in both the expression level of PSA and
cellular adhesive nature of NB41A3 cells (Fig. 5). The growth
stage-dependent change in PSA expression was evaluated by
two methods; one as the total peak areas in DMB/HPL-FD, and the other
as the PSA level estimated by ELISA. The results in the absence and
presence of RA, respectively, are shown in Fig. 5, A and
B. The amount of total protein-bound Neu5Ac (ng/mg of wet
cells) was also included. In the absence of RA the PSA level increased
with cell culture time and reached the maximum values at confluence
(day 7) (Fig. 5A). By contrast, in the presence of RA the
level of PSA was high on day 5 and gradually decreased during cell
growth until it reached the lowest value at day 7 (Fig. 5B).
We were not able to determine the PSA expression level at stages before
the day 5 post-inoculation time simply because the cells grew so slowly
that not enough of them could be harvested.
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Cellular adhesive properties were examined in cultured NB41A3 cells.
When NB41A3 cells were incubated in the absence of RA, they formed
aggregates at the initial stages of proliferation (day 5 post-inoculation incubation time; Fig. 5C) and underwent dissociation of cell aggregates at later stages (day 7; Fig.
5C). These changes were associated with a parallel increase
in the level of PSA per unit weight of wet cells (Fig. 5A).
In sharp contrast to these observations, in the presence of 10 µM RA NB41A3 cells did not form aggregates at the initial
stages of incubation (day 5; Fig. 5D) and a majority of the
cells differentiated to a neuronal phenotype. They extended long
neuritic processes during incubation, and finally on day 7 of culture a
large portion of the cells formed large aggregates (Fig.
5D). Similar morphological changes were correlated with
changes in the expression level of 2,8-polyNeu5Ac chains in human
neuroblastoma IMR-32 cells and pheochromocytoma PC-12 cells during
growth in the absence and presence of RA (16). Because cell aggregation
can be correlated with change in the expression level of PSA, the
newly identified
2,9-polyNeu5Ac-expressing glycoprotein may be
considered as a molecule closely related to NCAM.
2,8-PolyNeu5Ac
chain was suggested to be associated with the voltage-sensitive
Na-channels in adult rat brain merely by immunohistochemical and
Western blot data (21). However, to our knowledge, there is no report
showing that expression of PSA on Na-channels affects cell adhesion.
Identification and structural determination of the new cell adhesion
molecule is important and is underway. In addition we are pursuing the identification of a specific polysialyltransferase(s) (PST) that catalyze formation of
2,9-polyNeu5Ac in NB41A3 cells.
Over the past decade, there has been a rapid advance in the knowledge
of biochemistry, molecular biology, and genetics of the PST genes and
enzyme proteins. The discovery of a novel 2,9-PSA-glycoprotein on
NB41A3 cells will open up a new avenue of research to characterize the
biosynthesis of
2,9-polyNeu5Ac by identifying its associated enzyme(s), PST, and determining how this unique glycosylation influences the fate and function of this putative new cell adhesion glycoprotein on neuroblastoma cells.
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FOOTNOTES |
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* This work was supported by Academia Sinica, Taiwan, National Science Council, Taiwan Grants NSC 91-2311-B-001-106 (to S. I.) and NSC 90-2311-B-001-140 (to Y. I.) and National Health Research Institutes, Taiwan Grant NHRI-EX90-8805BP (to Y. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Inst. of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. Tel.: 886-2-2785-5696 Ext. 6020; Fax: 886-2-2788-9759; E-mail: syinoue@gate.sinica.edu.tw.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M212799200
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ABBREVIATIONS |
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The abbreviations used are: PSA, polysialic acid; NCAM, neural cell adhesion molecule; DMB, 1,2-diamino-4,5-methylenedioxybenzene; HPLC, high pressure liquid chromatography; mAb, monoclonal antibodies; GCMP, group C meningococcal polysaccharide; RA, all-trans-retinoic acid; ELISA, enzyme-linked immunosorbent assay; gp, glycoprotein; DP, degree of polymerization; FD, fluorescence detection.
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REFERENCES |
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