(Received for publication, May 9, 1995; and in revised form, May 30, 1995)
From the
A rapid, sensitive, and facile method for screening and
characterizing anti-polysialic acid (polySia) antibodies using
lipid-conjugated oligo/polysialic acids (oligo/polySia) was developed,
which is based on an enzyme-linked immunosorbent assay.
Homooligo/polymers of 2
8-linked N-acetylneuraminic
acid (Neu5Ac), N-glycolylneuraminic acid, and
2-keto-3-deoxy-D-galacto-nononic acid (KDN) were
conjugated with phosphatidylethanolamine dipalmitoyl (PE) by reductive
amination to prepare neo-oligo/polysialoglycolipids (oligo/polySia-PE).
Using this method, the anti-polySia equine antibody, H.46, bound to
(
8Neu5Ac
2
)
-PE, where n = 9 or more residues, a result in confirmation of previous
binding studies using radiolabeled oligo/polyNeu5Ac. The antigenic
specificity and sensitivity of two monoclonal anti-poly/oligoNeu5Ac
antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody
(mAb.kdn8kdn), were also determined. mAb.12E3 could detect as little as
25 pg/well of oligo/polyNeu5Ac-PE, while mAb.5A5 and polyclonal
antibody H.46 required at least 0.4 ng/well of oligo/polyNeu5Ac-PE to
be detected. mAb.kdn8kdn detected as little as 12 ng/well of
oligoKDN-PE. Using a series of oligo/polySia-PE with defined degrees of
polymerization (DP), the minimum chain length for immunoreactivity of
the anti-polySia antibodies was determined to be: DP 5 for mAb.12E3; DP
3 for mAb.5A5; DP 2 for mAb.kdn8kdn; and DP 8 for H.46. Thus, mAb.12E3
and mAb.5A5 recognize shorter oligomers of Neu5Ac than H.46, a finding
that is of practical value for identifying shorter oligoSia chains in
glycoconjugates. Because mAb.12E3 and mAb.5A5 also recognize extended
polySia chains, these antibodies cannot be used, however, to
differentiate between short and long chains of polySia when both are
expressed on the same molecule.
The polysialic acid (polySia) ()glycotope represents
a group of carbohydrate chains consisting of N-acetylneuraminic acid (Neu5Ac), N-glycolyneuraminic
acid (Neu5Gc), and deaminoneuraminic acid (KDN;
2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid) residues(1) . These sugar polymers have a remarkable
structural diversity resulting from different modifications of the
sialic acid (Sia) residues including different N-acyl
substitutions at C-5 (Neu5Ac, Neu5Gc), deamination at C-5 (KDN), and
the presence of O-acetyl or Olactyl
substituents(1) . Our recent finding of a novel type of
polyNeu5Gc structure consisting of
(
5-O
-Neu5Gc
2
)
chains in a Sia-rich glycoprotein from the jelly coat of sea
urchin eggs extends further the structural diversity of the polySia
glycotope(2) . Homopolymeric structures of
2
8-linked
Neu5Ac (polyNeu5Ac) chains are known to be expressed in certain fish
egg polysialoglycoprotein (PSGP)(1) , on neural cell adhesion
molecules (N-CAM) from a wide variety of animal species ranging from
insects to humans(3, 4, 5) , on some
postnatal tissues(6, 7) , and in a number of human
tumors(8, 9, 10) . PolySia is also recognized
as an oncodevelopmental antigen and is known to function as a regulator
of cell adhesion and cell migration (11, 12, 13) . In neuropathogenic Escherichia coli K1 and Neisseria meningitidis serogroup B, the
2
8-polyNeu5Ac glycotope is a
neurovirulent determinant(11) . A similar function has been
proposed for the polySia glycotope that is expressed on several types
of neuroinvasive human tumors, including malignant
lymphoma(14) , acute myeloid leukemia(15) , and head
and neck cancers(16) .
2
8-Linked homooligomeric
chains of KDN, oligoKDN, were first reported to occur in a KDN-rich
glycoprotein (KDN-gp) of the vitelline envelope (17) and
ovarian fluid (18) of rainbow trout. Because of the high
affinities of these novel polyanions to bind Ca
, we
postulated that they may function to maintain calcium ion around eggs
and developing embryos(19) . Recently, we developed a
monoclonal antibody, mAb.kdn8kdn, that specifically recognizes the
oligoKDN glycotope, although the chain length required for recognition
remained unspecified. Using mAb.kdn8kdn, we discovered that
KDN-containing glycoconjugates were expressed in rat
tissues(20) , a finding that had not been previously described.
The importance of this finding is that it demonstrates that oligoKDN
structures are not restricted to fish egg glycoproteins but, in fact,
are also expressed on mammalian cells.
As noted, the
2
8-linked polyNeu5Ac glycotope is a neurotropic factor in
neuroinvasive E. coli K1 and N. meningitidis serogroup B, and many efforts have been made to develop poly- and
monoclonal antibodies against this structure. Because of structural
mimicry, the
2
8-linked polyNeu5Ac chains are poorly
immunogenic in human and other
animals(21, 22, 23, 24, 25) .
Under special conditions, however, several anti-polySia have been
developed, including (a) an equine polyclonal IgM, designated
H.46(22, 26) , (b) a monoclonal antibody
mAb.735(25) , (c) an IgM
(27) , (d) a monoclonal antibody 2-2B (24, 28) , (e) monoclonal antibody
mAb.12E3(13, 29) , and (f) monoclonal
antibody mAb.5A5(30, 31) . H.46 (22, 26) and mAb.735 (25) are well
characterized and have been shown to be highly specific for recognizing
the oligo/polyNeu5Ac glycotope. These antibodies are not reactive with
oligo/polyNeu5Gc, hybrid chains consisting of oligo/polyNeu5Ac and
Neu5Gc residues or oligo/polyKDN chains(1) . The minimum chain
length recognized by these two antibodies is about 8-10 Sia
residues (DP 8-10) which is larger than the usual antibody
binding sites(32, 33) . The longer polySia chain is
required because H.46 and mAb.735 recognize an extended helical epitope
consisting of about 10 Sia residues, the inner 6 of which comprise the
helical domain(32, 33, 34, 35) .
Recently, we were successful in preparing another anti-polySia
antibody, mAb.12E3, which has a high avidity for embryonic rat
forebrain, but not adult forebrain(29) . This antibody appears
to recognize the 2
8-linked polyNeu5Ac chains on N-CAM,
because
2
8-linked sialyl oligomers (colominic acid)
inhibited the immunoreaction on embryonic rat brain. The usefulness of
this antibody in immunohistochemical studies in determining the
expression and distribution of polysialylated N-CAM has already been
verified(13, 29) , but the specificity and chain
length recognized by this antibody has not been thoroughly studied.
A determination of the glycotope that is bound by these anti-polySia antibodies, particularly the minimum chain length that is recognized, is important because the length of the polyNeu5Ac chains on N-CAM changes markedly during development. The change in DP appears to be inversely correlated with the strength of the N-CAM-mediated homophilic binding of neural cells(36) . Binding and inhibition studies in solution, using radiolabeled free oligo/polyNeu5Ac, have been used to approximate the chain length specificity of H.46 and mAb.735(32, 33, 37) . These experiments usually require relatively large amounts of antibodies, free oligo/polySia chains, and can be time consuming and imprecise. Because of their hydrophilicity, oligo/polySia chains are difficult to non-covalently immobilize on plastic or membrane filters. Previous studies have shown that lipidation of free glycan chains can greatly facilitate their non-covalent immobilization on the solid surfaces(38, 39) . Immobilization of oligo/polySia chains on the solid surface was anticipated to permit us to immunodetect the oligosaccharide antigen rapidly and with high sensitivity.
In this study, we have developed a rapid, sensitive and facile method for detecting the oligo/polySia glycotope, using oligo/polySia chains that are conjugated to phosphatidylethanolamine dipalmitoyl. Using a series of lipidated oligoSia with defined DP has also allowed us to determine the minimum chain length required for binding of the two new anti-Neu5Ac monoclonal antibodies, mAb.12E3, mAb.5A5, and an anti-KDN antibody, mAb.kdn8kdn.
For preparation of the series of
(8Neu5Ac
2
)
oligomers with defined
DPs, about 2.5 mg of colominic acid was applied on a Mono-Q HR5/5
anion-exchange column (0.5
5.0 cm, Pharmacia, Sweden) and
separated on an Irika high performance liquid chromatography system.
Sodium chloride gradients were generated using a Sigma 870 system
controller. The sample was loaded on the column and eluted with 5
mM Tris-HCl (pH 8.0), followed by NaCl gradient (0-320
mM) in 5 mM Tris-HCl (pH 8.0). The flow rate was 500
µl/min, and fractions were collected every minute. The elution
pattern was monitored by the resorcinol method. Oligomers with DPs
ranging from 1 to 16 thus obtained were desalted by Sephadex G-25
chromatography (1.7
140 cm, 5% ethanol) and lyophilized. For
preparation of the series of (
8KDN
2
)
oligomers, about 30 mg of oligoKDN, obtained from KDN-gp
(100 mg as KDN) by the procedure described above, were applied to a
DEAE-Toyopearl 650 M column (1.4
22 cm) and eluted
with a linear gradient of NaCl (0.0-0.3 M) in 10 mM Tris-HCl (pH 8.0). The elution profile was monitored by the
thiobarbituric acid method. Oligomers with DPs ranging from 1 to 7 were
desalted as described above and lyophilized.
Figure 1:
Thin layer chromatography of free and
lipid-conjugated oligo/polySia. The partial acid hydrolysates of
colominic acid (oligoNeu5Ac), rainbow trout PSGP (oligoNeu5Gc), and
rainbow trout KDN-gp (oligoKDN) were run in lanes 1, 3, and 5, respectively. In lanes 2, 4, and 6 are shown the lipid-conjugated oligoSia, i.e. oligoNeu5Ac-PE, oligoNeu5Gc-PE, and oligoKDN-PE,
respectively. The numbers represent the corresponding n in (Sia)-PE for each
sample.
A series of homooligomers of
Neu5Ac and KDN, prepared by Mono-Q and DEAE-Toyopearl 650 M anion-exchange chromatographies of both colominic acid and the
partial acid hydrolysates of KDN-gp, were conjugated separately with
PE. Each of the lipidated oligoSia were examined for purity by TLC. The
major component of each
(8Sia
2
)
-PE fraction contained n-1
intact Sia residues originally present in the parent (
8Sia
2
)
chain (see Structure I). The higher
oligoSia fractions, however, contained a small amount of lower oligoSia
chains (results not shown). These data indicate that some partial
hydrolysis of the oligoSia chains occurred during conjugation, probably
because of being held at 60 °C for 16 h(44) . The
contamination of such lipid-linked sialyl oligomers with smaller DPs
did not affect the semi-quantitative analysis by ELISA, as described
below.
Figure 2:
ELISA of polyclonal H.46 binding to
lipidated oligoSia-PE. Each well was coated with oligoNeu5Ac (),
oligoNeu5Ac-PE (
), oligoNeu5GcPE (
), oligoKDN-PE (
),
and PE (
) at 0.4 ng-47 ng/well as Sia, or 11 ng-1.4 µg/well
as PE, and determined for H.46 binding activity as described under
``Experimental Procedures.'' a, H.46 polyclonal
antibodies were used as primary antibody. b, horse normal
serum was used as control. c, analyses of chain length
specificity of H.46 as a function of DP of oligo/polySia using
lipidated oligo/polySia by the ELISA analysis. The plastic plate was
coated with a series of lipidated (Neu5Ac)
, n = 1-16 (
, 75 pmol/well;
, 38 pmol/well;
, 19 pmol/well).
Figure 3:
Determination of immunospecificity of
mAb.12E3 by the ELISA procedure. a, the plastic wells were
coated with various amounts of oligoNeu5Ac-PE (), oligoNeu5Gc-PE
(
), oligoKDN-PE (
), and PE (
) (25 pg to 12 ng/well
as Sia or 0.75 ng to 360 ng/well as PE). b, the plastic wells
were coated with a set of lipidated (Neu5Ac)
, n = 1-16 (
, 75 pmol/well;
, 38
pmol/well;
, 19 pmol/well).
Figure 4:
Determination of immunospecificity of
mAb.5A5 by the ELISA procedure. a, the plastic wells were
coated with various amounts of oligoNeu5Ac-PE (), oligoNeu5Gc-PE
(
), oligoKDN-PE (
), and PE (
) (0.45 ng to 190
ng/well as Sia or 14 ng to 5.7 µg/well as PE). b, the
plastic wells were coated with a set of lipidated
(Neu5Ac)
, n = 1-10 (
,
75 pmol/well;
, 38 pmol/well;
, 19
pmol/well).
Figure 5:
Determination of immunospecificity of
mAb.kdn8kdn by the ELISA procedure. a, the plastic wells were
coated with various amounts of oligoNeu5Ac-PE (), oligoNeu5Gc-PE
(
), oligoKDN-PE (
), and PE (diao]) (6.0 ng to 750
ng/well as Sia or 180 ng to 23 µg/well as PE). b, the
plastic wells were coated with a set of lipidated
(KDN)
, n = 1-7 (
, 28
pmol/well;
, 14 pmol/well;
, 7
pmol/well).
Because the polySia glycotope is often expressed in minute amounts and is spatiotemporally restricted in development, it is often difficult to detect by chemical methods(1) . Anti-polySia antibodies have been powerful reagents for detecting, localizing, and elucidating the functional involvement of different polySia structures. As noted, the polySia-containing molecules occur in diverse molecular forms that appear to be species, organ, and developmentally specifically expressed in a wide variety of human and other animal species(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 45, 46, 47, 48, 49) . Furthering our knowledge of the possible physiological roles of the polySia glycotopes involves discovering new polysialylated structures with possibly different functions in divergent species. One approach to this analysis is to develop anti-polySia monoclonal antibodies specific for recognizing the different polySia structures. Although the development of immunological probes with defined specificities was anticipated to be essential to further our understanding of the occurrence, structure, localization, and function of various polySia glycotopes, several difficulties have had to be overcome. First, polySia has long been known to be poorly immunogenic(21, 22, 23, 24, 25) . Second, many of the anti-polySia antibodies are IgM, and generally show weak immunoreactivity(22, 27, 50) . Third, the failure to immobilize polySia on plastic or silica gel TLC plates has prevented the development of a highly sensitive detection method.
In this study, we examined four anti-polySia antibodies to determine sensitivity and specificity for identifying the component nonulosonate in different oligo/polySia structures, and their chain length specificity, using as antigens a series of lipid-conjugated oligo/polySia of known structure and DP. Using these neoglycolipids, we have developed a highly sensitive method for identifying the different polySia structures and to determine the chain length dependence of the antibodies. Lipid-conjugated oligo/polySia samples with increasing DPs were found to migrate in a ladder-like fashion on TLC. In contrast to the free sialyl oligomers, the lipid-conjugated oligo/polySia remained bound to the solid surfaces after washing with water, thus allowing immunodetection to be carried out directly on ELISA or TLC plates. The lowest limit of detection of oligo/polySia-PE was determined to be 0.4 ng/well (as Neu5Ac) for H.46 and mAb.5A5, 25 pg/well (as Neu5Ac) for the monoclonal antibody 12E3, and 12 ng/well (as KDN) for mAb.kdn8kdn. Because the background color in the ELISA method was low, this procedure is ideal for screening and characterizing anti-polySia antibodies. A further advantage is that the method is applicable for the immunodetection of any mono-, oligo-, or polysaccharide that can be lipid conjugated with retention of the immunological epitope.
Only 7-75 pmol/well of lipidated antigens (as Sia) were required to determine the minimum chain length that is recognized by these antibodies. In contrast, much larger amounts of free oligo/polySia chains are required to carry out inhibition studies to generate the same information.
A problem of particular importance has been to
determine the minimum DP that can be recognized by the monoclonal
antibodies, mAb.12E3, mAb.5A5, and mAb.kdn8kdn. The antigen
specificities of four different antibodies characterized in this study
are summarized in Table1. Evidence showing that antibodies can
bind 2
8-linked oligoSia chains containing 5 and 3 Neu5Ac
residues, and 2 KDN residues, respectively, has been provided by the
present study. In contrast, H.46 and mAb.735 required about 8 Neu5Ac
residues for binding. The high specificity of these antibodies and
their ability to bind to shorter oligoNeu5Ac chains make them
potentially useful reagents detecting shorter oligoSia chains that may
be expressed on some glycoproteins and glycolipids. mAb.12E3 and
mAb.5A5 cannot be used, however, to distinguish between short and long
chains of oligo/polySia when both are expressed on the same
glycoconjugate (e.g. N-CAM). This follows because antibodies
also detect full-length polySia chains. Unlike antibodies against
oligo/polyNeu5Ac, only 2 KDN residues are required to be bound by
mAb.kdn8kdn. Maximal binding was thus attained for
(
8KDN
2
)
-PE and then decreased with more
KDN residues.
An increasing number of reports on the use of specific
antibodies to detect the 2
8-linked polyNeu5Ac glycotope in a
variety of animal species have
appeared(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
However, the occurrence of other polySia structures such as polyNeu5Gc
and polyKDN has not yet been described except for our original finding
of oligo/polyNeu5Gc and KDN chains in salmonid fish egg
polysialoglycoproteins and
KDN-glycoproteins(1, 45, 48) . Thus, the
importance of the present findings is that it describes the development
of a specific, sensitive, and facile method to identify new polySia
structures, and to determine the minimum DP in their structures that is
recognized by the different anti-polySia antibodies. The use of this
new set of monoclonal antibodies with different specificities not only
increases our experimental approaches to discover new polysialylated
structures, but it also allows us to study how their structural
diversity may relate to the myriad of functions that the polySia
glycotopes have been implicated in.