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Molecular Dissection of the ST8Sia IV Polysialyltransferase:
Distinct Domains Are Required for NCAM Recognition and Polysialylation*
Kiyohiko Angata, Dominic Chan, Joseph Thibault, and Minoru Fukuda§
Glycobiology Program, Cancer Research Center,
The Burnham Institute, La Jolla, CA 92037
§To whom correspondence should be addressed:
The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037.
Tel: (858) 646-3144; Fax: (858) 646-3193; E-mail: [email protected]
Running title: Domains in ST8Sia IV Necessary for NCAM Polysialylation
JBC Papers in Press. Published on April 2, 2004 as Manuscript M401562200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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SUMMARY
Polysialic acid, a homopolymer of α2,8-linked sialic acid expressed on the neural cell adhesion
molecule (NCAM), is thought to play critical roles in neural development. Two highly
homologous polysialyltransferases ST8Sia II and ST8Sia IV, which belong to the
sialyltransferase gene family, synthesize polysialic acid on NCAM. By contrast, ST8Sia III,
which is moderately homologous to ST8Sia II and ST8Sia IV, adds oligosialic acid to itself but
very inefficiently to NCAM. Here, we report domains of polysialyltransferases required for
NCAM recognition and polysialylation by generating chimeric enzymes between ST8Sia IV and
ST8Sia III or ST8Sia II. We first determined the catalytic domain of ST8Sia IV by deletion
mutants. To identify domains responsible for NCAM polysialylation, different segments of the
ST8Sia IV catalytic domain, identified by the deletion experiments, were replaced with
corresponding segments of ST8Sia II and ST8Sia III. We found that larger polysialic acid was
formed on the enzymes themselves (autopolysialylation) when chimeric enzymes contained the
carboxyl terminal region of ST8Sia IV. However, chimeric enzymes that contain only the
carboxyl terminal segment of ST8Sia IV and the amino terminal segment of ST8Sia III showed
very weak activity towards NCAM, even though they had strong activity in polysialylating
themselves. In fact, chimeric enzymes containing the amino terminal portion of ST8Sia IV fused
to downstream sequences of ST8Sia III inhibited NCAM polysialylation in vitro, although they
did not polysialylate NCAM. These results suggest that in polysialyltransferases the NCAM
recognition domain is distinct from the polysialylation domain and that some chimeric enzymes
may act as a dominant negative enzyme for NCAM polysialylation.
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INTRODUCTION
The neural cell adhesion molecule (NCAM)1 is expressed abundantly in brain
development and modified with various N-glycans including polysialic acid and HNK-1 (1-3).
Polysialic acid is a linear homopolymer of α2,8-linked sialic acid found primarily as a
modification of NCAM (4-8). Polysialylated NCAM is highly expressed in embryonic brain,
while most NCAM expressed in adult brain does not contain polysialic acid. However,
polysialylated NCAM is continuously present in the adult hypothalamus, hippocampus and
olfactory bulb, where synapse formation or neuronal generation persists (9). Removal of
polysialic acid by endoneuraminidase provides evidence that some cell migration and axonal
defasciculation require the presence of polysialic acid (10, 11). Based on phenotypes observed in
NCAM-deficient mice, polysialylated NCAM is likely involved in various neural functions such
as axon pathfinding, circadian rhythm, and memory formation through neuronal plasticity, and is
also important for olfactory bulb formation (12, 13).
Two polysialyltransferases, ST8Sia IV (also called PST) and ST8Sia II (also called STX),
have been cloned and shown to synthesize polysialic acid on NCAM (2, 14-18). These enzymes
catalyze transfer of multiple α2,8-linked sialic acid residues to a precursor containing a
NeuNAcα2→3Galβ1→4GlcNAc→R structure without participation of other enzymes (19-22).
The presence of polysialic acid is always associated with expression of ST8Sia II and ST8Sia IV
(23, 24). Similar to phenotypes observed in NCAM-deficient mice, disruption of ST8Sia IV in
mice leads to impaired long-term potentiation and long-term depression in Schaffer collateral-
CA1 synapses of the adult hippocampus (25). Loss of polysialic acid in ST8Sia IV null mice is
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incomplete, presumably because ST8Sia II compensates for loss of ST8Sia IV. These results
suggest that ST8Sia IV and S8Sia II are the key enzymes controlling expression of polysialic
acid.
Importantly, it has been reported that aberrant expression of polysialylated NCAM
correlates with some disease states. Expression of polysialic acid is often associated with tumor
metastasis in both small cell and non-small cell lung carcinomas (26, 27). Polysialic acid is also
detected in neuroblastomas and in Wilms’ tumor (28-30). In addition, capsules of certain
pathogenic bacteria that cause meningitis bear polysialic acid, suggesting that polysialic acid
leads to immune tolerance by facilitating escape from attack by the immune system in vivo (31,
32). In order to both develop therapeutic agents to treat these diseases and understand the roles of
polysialic acid in neural development, it is important to elucidate the mechanisms of polysialic
acid synthesis.
Polysialyltransferases belong to the vertebrate sialyltransferase gene family, including
α2,3-, α2,6- and α2,8-sialyltransferases. Analysis of the amino acid sequences of various
sialyltransferases shows two regions of weak but discernible homology in their catalytic
domains, designated sialylmotifs L and S (33). It has been demonstrated that sialylmotif L is
involved in binding to the donor substrate, CMP-NeuNAc, while sialylmotif S participates in
binding to both donor and acceptor substrates (34, 35). Disulfide bond structures of ST8Sia IV
determined by mass spectrometric analysis indicate that its carboxyl terminal region is close to
the center of the catalytic domain through a unique disulfide bond that is not formed in other
α2,3- and α2,6-sialyltransferases (36). Of six α2,8-sialyltransferases, ST8Sia II, ST8Sia IV, and
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ST8Sia III, which is moderately homologous to ST8Sia II and IV, can polysialylate themselves
(autopolysialylation) (22, 37, 38). The length of polysialic acid made in vitro by these three
enzymes is, however, substantially different in that ST8Sia II and IV form larger polymers of
sialic acids than does ST8Sia III, and only ST8Sia II and ST8Sia IV can add polysialic acid to
NCAM (22). The mechanistic basis for these differences is not yet known. It was reasonable to
hypothesize that differences in acceptor specificity could allow us to identify protein domains
responsible for synthesis of long polysialic acid and/or recognition of NCAM by swapping
various domains, since the gross structure of ST8Sia II, ST8Sia III and ST8Sia IV is similar to
each other (2).
In order to identify domains responsible for NCAM polysialylation, first we used deletion
analysis to determine the catalytic domain of St8Sia IV. Then, the catalytic domain of ST8Sia IV
was divided into seven different domains based on homology of ST8Sia II, III, and IV (Fig. 1).
These domains of ST8Sia IV were swapped with corresponding segments of ST8Sia II or III.
The enzymatic activity of the resulting chimeric proteins was measured by determining
polysialic acid synthesis in transfected cells, and assaying autopolysialylation and NCAM
polysialylation in vitro. We found that the carboxyl terminal region determines the length of
polysialic acid and the amino terminal region of the catalytic domain is required for NCAM
recognition, indicating that different domains underlie these two functions.
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EXPERIMENTAL PROCEDURES
Plasmid DNAs – cDNAs encoding ST8Sia II, ST8Sia III, and ST8Sia IV (pcDNAI-ST8Sia II,
pcDNAI-ST8Sia III, and pcDNAI-ST8Sia IV, respectively) were prepared and used as described
previously (16, 22, 39). The soluble forms of each enzyme fused to a signal peptide and IgG
binding domain of protein A (pcDNAI-A•ST8Sia II, pcDNAI-A•ST8Sia III, and pcDNAI-
A•ST8Sia IV) were also described previously (19, 21, 22). The modified pIG-NCAM•IgG
encoding a soluble NCAM lacking the VASE and muscle specific domain, and which was fused
with hinge and constant regions of human IgG in pIG vector (40), was generated as described
(22).
Deletion mutants of soluble ST8Sia IV – To construct deletion mutants from the amino terminal
region of ST8Sia IV, pcDNAI-A•ST8Sia IV was used as a template for PCR by Pfu DNA
polymerase (Stratagene) using SP6 primer and following primers. PST-50: 5’-
CGGGATCCCAATAGCTCTGATAAAATCATT-3’ (147-168); PST-62: 5’-
GAGGATCCTTCAATCTTCCAGCACAATGTA-3’ (183-204); PST-72: 5’-
CGGGATCCGAAAATCAATTCCTCTTTGGTC-3’ (213-234); PST-82: 5’-
GAGGATCCAAGGAAGAACATACTTCGTTTC-3’ (243-264); PST-103: 5’-
GAGGATCCTAAGCCTGGTGATGTCATACAC-3’ (306-327); PST-127: 5’-
GAGGATCCCCTCCTACCTGAAGTTTCACCA-3’ (378-399); BamHI sites are underlined.
The amplified DNA fragments were cloned into pBluescript II (Stratagene) and sequenced to
select proper clones. The cDNAs digested with BamHI and XbaI replaced wild-type sequences of
pcDNAI-A•ST8Sia IV.
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Construction of chimeric forms of ST8Sia IV and II or III - To swap sequences, we utilized
PCR-based methods using human ST8Sia II, III and IV cDNA as a template. For ST8Sia IV, an
upstream primer (designated IV-N’) includes the first methionine and a HindIII site, and a
downstream primer (designated IV-C’) contains the terminal codon and an XbaI site. Similarly,
primers II-N’ and III-N’ for ST8Sia II and III contain a HindIII site upstream of the initial
methionine codon, and 3’-primers, II-C’ and III-C’, contain an XhoI site after the termination
codons.
To divide the genes into 7 domains (A to G), regions exhibiting that sequences are highly
homologous among the three enzymes were chosen (see Fig. 1). A BsaI site was incorporated
into each internal primer so that the end of amplified fragments after digestion with BsaI would
share sequences in both directions and in different genes. For example, to obtain IVCDII, an
ST8Sia IV cDNA fragment containing regions A to C and an ST8Sia II cDNA fragment
containing regions D to G were amplified by PCR. For amplification of A to C domains of
ST8Sia IV, a primer IV-N’ and a primer IVDC containing the TGAG sequence within the BsaI
restriction site (underline indicates antisense sequence of Ser193) were used. cDNA encoding
regions D to the stop codon of ST8Sia II was amplified using primers II-C’ and IICD having
CTCA within the BsaI site (Ser208 of ST8Sia II is underlined). The amplified fragments were
cloned into pBluescript II and sequenced to select the proper clone. The HindIII- and BsaI-
digested ST8Sia IV fragment and the XhoI- and BsaI-digested ST8Sia II fragment were ligated
and subcloned into the HindIII and XhoI sites of pcDNAI (Invitrogen), resulting in a membrane
form of IVCDII, pcDNAI-IVCDII. To construct pcDNAI-IIIEFIVFGIII, a chimeric cDNA fragment
was amplified by primers IVEF and III-C’ from pcDNAI-IVFGIII. The resultant cDNA was
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ligated with an ST8Sia III fragment containing regions A to E into pcDNAI at the common site
generated by BsaI digestion.
Construction of soluble forms of chimeric cDNAs - The cDNA in pcDNAI-A•ST8Sia IV was
replaced with chimeric cDNAs derived from ST8Sia IV using common restriction sites, either
BstBI or NarI. Similarly, cDNAs of ST8Sia II and III were replaced with chimeric cDNAs using
either the EcoRI or BamHI site in the pcDNAI-A•ST8Sia II sequence and either the XbaI or
XmnI in the ST8Sia III sequence.
To generate a mutant at the third N-glycosylation site (Asn206) of ST8Sia III in
IVBCIIICDIV, the following primer sets were used for PCR reaction. T7 primer and III-N3-PML:
5’-GGCACGTGTTTCTTCCAACATCTCTTTGG-3’ (591-611, PmlI site is underlined); III-N3-
MSC: 5’-GATGGCCAACTTACCACCTTCAACCCC-3’ (619-636, MscI site is underlined and
the mutated site is doubly underlined) and the SP6 primer. After cloning into pBluescript II,
DNA fragments were excised by NarI and PmlI or MscI and XbaI. The purified fragments
replaced the NarI-XbaI fragment of pcDNAI-A•IVBCIIICDIV, resulting in pcDNAI-
A•IVBCIIICDIV-N.
Expression of polysialic acid on cells transfected with chimeric cDNAs – The chimeric cDNAs
encoding membrane or soluble forms of chimeric enzymes were transfected into COS-1, HeLa,
and CHO cells using LipofectAmine PLUS (Invitrogen) as described previously (36). Forty-eight
h after transfection, the cells were fixed and stained with 12F8 (BD Biosciences), a rat
monoclonal antibody specific to polysialic acid (41), followed by FITC-conjugated goat antibody
specific to rat IgM (Cappel).
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Preparation of enzymes and NCAM•IgG chimera proteins - The cDNAs encoding protein A
fused with ST8Sia II, ST8Sia III, ST8Sia IV, truncated forms of ST8Sia IV, or chimeric enzymes
were transfected into COS-1 cells and soluble protein A-fusion enzymes were purified from
spent serum-free media with human IgG-Sepharose (Amersham Biosciences) as described
previously (21). A 50% suspension of enzyme-adsorbed IgG-Sepharose in the serum-free
medium was used as an enzyme source. The amount of the enzymes was estimated by
densitometric analysis after Western blotting using peroxidase-conjugated rabbit
immunoglobulins (Cappel) and an ECL PLUS kit (Amersham Biosciences) as described
previously (21).
Similarly, the fusion protein NCAM•IgG purified from cultured medium by protein A
beads (Pierce) was subjected to Western blotting analysis to measure quantity and quality of
NCAM•IgG using peroxidase-conjugated goat anti-human IgG antibody (Cappel) as described
previously (21, 22).
In vitro polysialyltransferase assays - An equivalent amount of each chimeric enzyme was used
for in vitro sialyltransferase assays. For autopolysialylation assays, the enzyme adsorbed beads
were incubated in 50 mM sodium cacodylate buffer, pH 6.0, containing 2.5 mM MgCl2, 2.5 mM
MnCl2, 1 mM CaCl2, 0.5% Triton CF-54, and 2.4 nmoles (0.7 µCi) of CMP-[14C]-NeuNAc for 2
hrs at 37 °C. After incubation, the radiolabeled enzymes were released from beads and subjected
to SDS-polyacrylamide gel electrophoresis followed by fluorography (22). To measure
polysialylation activity onto NCAM•IgG, 10 pmol of NCAM•IgG was added to the reaction
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mixture. After incubation, NCAM•IgG in the supernatant were obtained by brief centrifugation
and subjected to the fluorography. The degree of polymerization of sialic acid on ST8Sia III,
ST8Sia IVDEIII, or ST8Sia IV was analyzed by HPLC using a Mono-Q anion exchange column
(Amersham Biosciences) after [14C]-NeuNAc labeled N-glycans were released by PNGaesF
(Calbiochem) as described previously (21, 22).
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RESULTS
Determination of the catalytic domain of ST8Sia IV – Previously, we demonstrated that Cys356
at the carboxyl terminal of ST8Sia IV is required for enzymatic activity, and that only 3 amino
acids can be removed from the catalytic domain of ST8Sia IV to maintain its activity (36). To
determine the amino terminal limit of ST8Sia IV catalytic activity, we generated protein A
fusion proteins of truncated ST8Sia IV and assayed enzymatic activity in vitro. Removing 39,
49, and 61 amino acids from the amino terminal (SF-IV (40), SF-IV (50), and SF-IV (62),
respectively) minimally affected NCAM polysialylation (Fig. 2). However, eliminating more
than 61 amino acids from the amino terminal (SF-IV (72)) dramatically reduced both
polysialylation of NCAM and autopolysialylation (Fig. 2B). These results suggest that the
catalytic domain of ST8Sia IV resides between amino acid residues 62-356. Accordingly, a
soluble form of ST8Sia IV encoding residues 40-359 was assayed in the following experiments
to ensure that the catalytic domain is full accessible to NCAM•IgG.
Functional analysis of ST8Sia II/ ST8Sia IV chimeric enzymes – Although ST8Sia II, III, and
IV can all autopolysialylate, ST8Sia II and IV synthesize longer polysialic acid on NCAM than
does ST8Sia III. ST8Sia IV exhibits 59% identity at the amino acid level with ST8Sia II but only
34.8% identity with ST8Sia III (22). Harrplot analysis revealed that the two NCAM
polysialyltransferases, ST8Sia II and ST8Sia IV, are highly homologous along the entire amino
acid sequence except for the extreme amino terminal region (Fig. 1A). On the other hand,
ST8Sia IV and ST8Sia III share significant homology only in sialylmotif L and S regions. Since
ST8Sia III and ST8Sia IV differ in their efficiency in recognizing NCAM, these results suggest
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that regions exhibiting low homology are involved in NCAM (acceptor) recognition and/or
polysialylation activity (Fig. 1). Since all three enzymes conserve similar structural elements
such as size, Cys residues involved in disulfide bridges, and N-glycosylation sites (2), we
assumed that swapping domains among them was a reasonable strategy to determine functional
domains.
To construct a chimeric polysialyltransferase, the domains of ST8Sia IV were replaced
with corresponding domains of ST8Sia II. In our nomenclature, IICDIV corresponds to the
chimeric protein containing A, B, and C domains of ST8Sia II and D, E, F, and G domains of
ST8Sia IV. In these constructs, almost all chimeric proteins (IIABIV to IIEFIV) displayed high
NCAM polysialylation activity (Fig. 3), demonstrating that chimeric enzymes are functional.
Interestingly, IIBCIV, IICDIV and IIDEIV, in which the carboxyl terminal region consists of
ST8Sia IV domains, showed higher polysialylation activity than ST8Sia II or ST8Sia IV (Fig. 3).
The chimeric enzymes IIABIV, IIBCIV and IICDIV, produced in COS-1 cells, showed an increased
amount of a higher molecular weight band upon Western blot analysis while the same band was
less for ST8Sia II and ST8Sia IV. These second bands represent polysialylated enzymes as
reported for autopolysialylation in vivo (38). The results on Western blot analysis are consistent
with the findings that IIBCIV and IICDIV formed NCAM•IgG with much higher molecular
weights (Fig. 3, middle panel).
Unlike the chimeric enzymes described above, replacement of the G domain of ST8Sia II
with the corresponding domain of ST8Sia IV (IIFGIV) resulted in decreased NCAM
polysialylation activity (Fig. 3). This activity was restored when the F domain of ST8Sia IV was
included in the ST8Sia II sequence (IIEFIVFGII), indicating that F or F and G domains contain the
minimal requirement for efficient polysialylation in these chimeric enzymes. It is possible that
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the ST8Sia II G domain make more stabilized intramolecular interactions with ST8Sia II
sequences in the rest of molecule, and that the ST8Sia IV G domain makes weaker
intramolecular connections with ST8Sia II sequences.
Efficient polysialylation requires the carboxyl terminal region of ST8Sia IV – To determine the
roles of different domains in polysialylation, ST8Sia IV sequences were replaced with
corresponding sequences of ST8Sia III, as shown in Fig. 4. In this series of the constructs, the
ST8Sia III sequence can replace the ST8Sia IV A sequence, which contains a stem region, for
autopolysialylation. Replacement of A to D or A to E regions of ST8Sia IV with the
corresponding sequence of ST8Sia III, IIIDEIV or IIIEFIV, did not interfere with
autopolysialylation activity (Fig. 4A). By contrast, IIIBCIV and IIICDIV exhibited reduced
autopolysialylation activity. These chimeric enzymes were produced in equivalent amounts to
those for other chimeric enzymes, indicating that gross conformational change did not take place
in these chimeric enzymes. The results also suggest that the fusing AB or ABC domains of
ST8Sia III to ST8Sia IV (IIIBCIV and IIICDIV) did not lead to a proper conformation of domains
including these regions while the chimeric enzyme containing ABCD domains of ST8Sia III
(IIIDEIV) was highly active (Fig. 4A). In the following experiments, we performed experiments
using the chimeric enzymes that showed activity for autopolysialylation to interpret the results
since those active enzymes are judged to have no gross structural alteration that affect
polysialylation activity. The above results also indicate that the carboxyl terminal half of ST8Sia
IV is required for efficient polysialylation, since the autopolysialylation products of IIIDEIV and
IIIEFIV were larger than those of ST8Sia III (Fig. 4A).
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In contrast to the ST8Sia II/IV chimeric protein (Fig. 3), replacement of only the F
domain in ST8Sia III with the corresponding domain of ST8Sia IV (IIIEFIVFGIII) did not result in
an enhanced activity (compare IIIEFIVFGIII and IIIEFIV). In addition, replacement of only the G
domain in ST8Sia III with that of ST8Sia IV (IIIFGIV, Fig. 4) dramatically reduced
autopolysialylation activity. These results suggest that sequences from F and G must be derived
from the same enzyme for full activity of ST8Sia III/IV chimeric enzymes and that these
sequences are likely responsible for formation of longer polysialic acid by ST8Sia IV.
The above results also suggest that BCD domain may need to come from the same
enzyme, since IIIDEIV was much more active than IVBCIIICDIV-N or IVCDIIIDEIV (Fig. 4A and
B). Together these results combined indicate that the central region (BCD) needs to be
maintained for optimal folding while the F and G region must come from ST8Sia IV to ensure
the formation of long polysialic acid.
The above results clearly indicate that some chimeric enzymes autopolysialylate more
efficiently than ST8Sia III. To determine if the chimeric enzyme IIIDEIV synthesizes long
polysialic acid as does ST8Sia IV, the 14C-labeled product obtained by in vitro
autopolysialylation was subjected to HPLC analysis using a mono-Q column (Fig. 5).
Autopolysialylation of ST8Sia III synthesized 2-30 polysialic acid on one N-glycan, while
ST8Sia IV added 20-50 polysialic acid per N-glycan. IIIDEIV efficiently synthesized oligosialic
acid to polysialic acid (2-50) on N-glycans of the enzyme. Thus, IIIDEIV, which contains only 92
amino acids from the carboxyl terminal region of ST8Sia IV, synthesizes longer polysialic acid
than ST8Sia III. These results are consistent with the results obtained from ST8Sia II/IV
chimeric proteins described above, showing that chimeric enzymes containing the carboxyl
terminal half (D to G) or F region of ST8Sia IV produced larger polysialic acid than does ST8Sia
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II. These results combined indicate that the carboxyl terminal region of these enzymes
determines the efficiency of polysialylation.
We also generated a series of ST8Sia III/IV chimeric enzymes to determine if internal
ST8Sia IV sequences can be replaced by ST8Sia III sequences. Some of the chimeric enzymes
(IVABIIIBCIV and IVBCIIIDEIV, Fig. 4, panel B) did not show autopolysialylation activity
probably due to structural alteration. Some of the other enzymes (IVCDIIIDEIV and IVABIIIDEIV),
however, showed detectable polysialyltransferase activity, although the activity was lower than
that by ST8Sia IV (Fig. 4, panel B). The results again indicate that the carboxyl terminal of
ST8Sia IV is required for efficient autopolysialylation. Since it has been reported that N-
glycosylation of polysialyltransferases influences their activity (38, 42), we constructed a
chimeric enzyme mutated at the N-glycosylation site (Asn206). This mutation makes the chimeric
enzyme similar to ST8Sia II or ST8Sia IV in this region since N-glycosylation at Asn206 is absent
in the corresponding region of ST8Sia II and ST8Sia IV (Fig. 1B). Although IVBCIIICDIV
showed little enzymatic activity, IVBCIIICDIV-N206 in which Asn206 of ST8Sia III is substituted
with Gln partially recovered its autopolysialylation activity, suggesting that N-glycan at Asn206
suppresses polysialyltransferase activity in the chimeric enzyme. It is possible that eliminating
N-glycan at Asn206 may alter the protein structure in a way to enhance enzymatic activity.
NCAM polysialylation by ST8Sia III/ST8Sia IV chimeric proteins – ST8Sia III/ST8Sia IV
chimeric proteins were then tested for NCAM polysialylation activity. Strikingly, IIIDEIV and
IIIEFIV chimeric proteins exhibited significantly reduced NCAM polysialylation activity, despite
the fact that they exhibited strong autopolysialylation activity (Fig. 6). Furthermore, IIIABIV and
IVBCIIICDIV-N206 showed less NCAM polysialylation activity relative to autopolysialylation
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activity. Although the effect was not evident as that for IIIABIV, IVCDIIIDEIV also exhibited a
reduced NCAM polysialylation relative to autopolysialylation (Fig. 6). These results indicate that
the stem region, amino terminal region of the catalytic domain, and D domain of ST8Sia IV may
be critical for NCAM polysialylation. Alternatively, addition of the ST8Sia III stem region or D
domain to chimeric ST8Sia IV proteins may change their conformations to those more closely
resembling intact ST8Sia III.
If the stem region of ST8Sia IV is involved in NCAM recognition, we expect that
chimeric enzymes containing A domain should compete with NCAM polysialylation by ST8Sia
IV. In order to test this hypothesis, ST8Sia IV was incubated together with IVCDIIIDEIV or
IVFGIII to assay both autopolysialylation and NCAM polysialylation activity. Indeed, NCAM
polysialylation was inhibited as the amount of IVCDIIIDEIV or IVFGIII increased, while no
impairment in autopolysialylation of wild-type ST8Sia IV was observed (Fig. 7). By contrast,
NCAM polysialylation was not inhibited when increased amounts of ST8Sia III were added (Fig.
7). These results support the above conclusion that domains A, B and D are involved in NCAM
recognition. On the other hand, IVBCIII inhibited NCAM polysialylation less effectively than
IVCDIIIDEIV or IVFGIII. These results suggest that the domains other than A, B and D are
marginally involved in NCAM recognition. These results combined indicate that domains A to C
or A to B, and domain D of ST8Sia IV are critical for NCAM polysialylation.
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DISCUSSION
Polysialic acid specifically synthesized on NCAM by polysialyltransferases plays unique
and important roles in neural development and cancers such as neuroblastoma and lung
carcinoma. Understanding the mechanism of polysialic acid synthesis is an important step in
manipulating polysialylation in neural or cancer cells to determine the roles of polysialic acid in
these cells. Our first aim was to define the catalytic domain required for polysialic acid synthesis.
We previously reported that in the carboxyl terminal of ST8Sia IV, Cys356, the fourth amino acid
from stop codon, is essential for polysialylation activity (36). This result is consistent with other
reports showing that the carboxyl terminal region is the most important domain for the enzymatic
activity of fucosyltransferase-V (FucT-V) and N-acetylglucosaminyltransferase-V (GnT-V) (44,
45). In the present study we demonstrated that 61 amino acids from the initiation methionine can
be removed without significant loss of polysialyltransferase activity. Thus, ST8Sia IV likely
consists of a 7 amino acid cytoplasmic tail, a 13 amino acid transmembrane domain, a stem
region of approximately 40 amino acids, and a catalytic domain of 295 amino acids residues
from 62 to 356. Approximately, 20 amino acids in the carboxyl terminal region of A domain
(residues 62-82) are also included as a catalytic domain. The size of the ST8Sia IV catalytic
domain is much smaller than that of GnT-V, but is comparable to that of GnT-I, GalT-IV, FucT-
III, FucT-V, and ST6Gal I (44-50)
The previous study demonstrated that intact disulfide bond structures are important for
ST8Sia IV activity and that Cys356 is linked to Cys156 and Cys292 is linked to Cys142. In this
structure, sialylmotifs S and L are brought together, and the carboxyl terminal region is also
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close to the center of the catalytic domain because of the disulfide bond between Cys156 and
Cys356 (Fig. 8). It is likely that bringing the carboxyl terminal to the center of the catalytic
domain by a disulfide bridge is critical for α2,8-sialyltransferases to act as polysialyltransferases
(36). Using autopolysialylation as an assay, our studies showed that chimeric enzymes
containing the carboxyl terminal domain of ST8Sia IV produce polysialic acid as long as that
produced by intact ST8Sia IV (for example, IIIDEIV in Figs. 4 and 5). In NCAM polysialylation,
the carboxyl terminal domain determines the size of polysialic acid and is essential for
polysialylation activity (for example, IICDIV and IIEFIVFGIV in Fig. 3). The results obtained here
thus indicate that the carboxyl terminal domains (most likely F and G) determine the efficiency
of adding multiple α2,8-linked sialic acid. The sequence of the FG region is highly conserved in
two polysialyltransferases, ST8Sia II and ST8Sia IV, compared to that of STST8Sia III as shown
in Fig. 1A. The amino acid sequences of the FG regions are moderately well conserved among
all α2,8-sialyltransferases (2). Since most α2,8-sialyltransferases share the ability to add more
than one α2,8-linked sialic acid to specific acceptor molecules and may have common disulfide
bond structures due to the conserved cysteine residues, it is reasonable to speculate that
differences in amino acid sequences of FG regions determine differences in polysialylation
capability of these α2,8-sialyltransferases.
It has been previously shown that the sialylmotif S in sialyltransferases is involved in
binding to both acceptor substrate and donor substrate, CMP-NeuNAc (35). Examining
sialylmotif sequences of α2,8-sialyltransferases, however, does not allow us to identify amino
acid residues involved in binding to NCAM since the amino acid sequences in sialylmotif S are
highly conserved among ST8Sia II, ST8Sia III and ST8Sia IV. The present study demonstrated
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that domains separate from sialylmotif L and S are critical for efficient polysialylation of both
the enzyme itself (autopolysialylation) and NCAM polysialylation. More interestingly, we found
that different domains are required for NCAM polysialylation and autopolysialylation. First,
replacing the stem region of ST8Sia IV with the corresponding region of ST8Sia III significantly
reduced NCAM polysialylation activity (IV vs. IIIABIV, Fig. 6). IIIDEIV and IIIEFIV chimeric
proteins exhibited stronger autopolysialylation activity than ST8Sia IV, but poorly polysialylated
NCAM (Fig. 6). Moreover, a chimeric enzyme consisting of domains A to B of ST8Sia IV and
the C to G domains of ST8Sia III (IVBCIII) inhibited NCAM polysialylation by ST8Sia IV (Fig.
7). In contrast, intact ST8Sia III did not compete with ST8Sia IV for NCAM polysialylation (Fig.
7). These results as a whole imply that ST8Sia II and ST8Sia IV evolved by adopting the
sequences in the A to B and D domains, which are different from ST8Sia III, to accommodate an
efficient interaction with NCAM.
Our study indicates that ST8Sia IV domain A including the stem region is required for
NCAM recognition and for stabilizing the conformation mediated by the disulfide bridges. More
precisely, we suggest that the amino terminal region (residues 62 to 127) and possibly D domain
(residues 194 to 267) of ST8Sia IV are required for NCAM recognition. These domains are
presumably close to the catalytic domain consisting of both the carboxyl terminal and amino
terminal regions connected by disulfide bonds (Fig. 8). Because these two domains are
connected, this combined structure captures CMP-NeuNAc and catalyzes transfer of sialic acid
(34-36). In supporting this conclusion, we showed that chimeric proteins consisting of the amino
terminal half of ST8Sia IV and the carboxyl terminal half of ST8Sia III, such as IVBCIII, can
inhibit NCAM polysialylation despite the fact that these chimeric enzymes poorly polysialylate
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NCAM. These results suggest that these chimeric molecules can act as a dominant negative
protein toward NCAM polysialylation.
It has been reported from several laboratories that Ig-like domain 5 (Ig 5), possibly Ig-
like domain 4 (Ig 4) and fibronectin type III-like domain 1 (FN 1) are essential for the
recognition by polysialyltransferases (43, 51, 52). It has been also demonstrated that
polysialylation preferentially takes place on the 6th and 5th N-glycosylation sites on NCAM Ig 5
domain (21, 43). Combined these findings together with the present findings, we proposed a
schematic structure of ST8Sia IV (and most likely ST8Sia II) that is bound to NCAM as shown
in Fig. 8. Since the domain C, E, F and G form a core for catalytic activity, these combined
region was suggested to be close to NCAM Ig 5 in this scheme. While we envisioned in Fig. 8
that ST8Sia IV recognizes NCAM through its binding to Ig 4, Ig 5 and FN 1 domains of NCAM,
this may be too simplistic since the whole catalytic domain is likely involved in adding sialic
acid to NCAM. Our previous results demonstrated that protein portions of NCAM are necessary
for efficient polysialylation, and oligosaccharides serve as poor acceptors for ST8Sia II and
ST8Sia IV (22). It is thus possible that NCAM protein domains may be required to present
polysialylation sites for ST8Sia II and ST8Sia IV rather than binding to polysialyltransferases
directly. Further studies including X-ray crystal structure is required to determine the sterical
structures of ST8Sia IV and ST8Sia II, which are critical for NCAM polysialylation.
In recent studies using chimeric enzymes made between highly homologous
glycosyltransferases, amino acid determinants involved in linkage specificity were revealed for
blood group AB glycosyltransferases and fucosyltransferases (44, 49, 53, 54). In the present
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study, we demonstrated that it is possible to construct a chimeric functional enzyme between
ST8Sia III and ST8Sia IV, which share only 34.8% identity at the amino acid level, suggesting
that ST8Sia III, ST8Sia IV and most likely ST8Sia II share functionally homologous domains.
On the other hand, our recent experiments showed that ST8Sia III adds oligosialic acid to all N-
glycosylation sites of NCAM (data not shown), although the efficiency is very low (see Fig. 6).
By contrast, ST8Sia II and ST8Sia IV almost exclusively add polysialic acid to the 5th and 6th
N-glycosylation sites of NCAM (21, 43), indicating that ST8Sia III recognizes acceptor
molecules distinct from those utilized by ST8Sia II and ST8Sia IV. Moreover, we showed that
some of chimeric enzymes such as IIIDEIV and IIIEFIV exhibited higher autopolysialylation
activity than ST8Sia IV. These observations raise a possibility that these chimeric enzymes may
act efficiently on acceptor molecules other than NCAM as shown on CD36 (55). Recently, it has
been proposed that polysialylation of proteins increases the half-life of proteins in circulating
blood and reduces antibody formation potential against drugs and proteins (56). It will be of
interest to use mammalian polysialyltransferases and chimeric enzymes to produce proteins
polysialylated at a specific site. Such proteins may be useful therapeutic agents when they need a
longer half-life and multiple usage without production of antibody against them.
AKNOWLEDGEMENTS
We thank Dr. Misa Suzuki for help with the HPLC analysis, and Drs. Edgar Ong and Elise
Lamar for critical reading of the manuscript.
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FOOTNOTES:
For page 1,
*This work was supported by grant R01 CA33895 from the National Cancer Institute, the
National Institutes of Health.
For page 3,
1. Abbreviations used are: NCAM, the neural cell adhesion molecule; PCR, polymerase chain
reaction; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate
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FIGURE LEGENDS
Fig. 1. Comparison of the amino acid sequences of ST8Sia IV, ST8Sia II, ST8Sia III, and
ST3Gal I. (A) Comparison of the amino acid sequences using a HarrPlot. The plot was obtained
using a 2 amino acid match per 10 amino acids. Domains A-G are based on sequence homology
between ST8Sia III and ST8Sia IV. It is assumed that a minimum breakdown in conformation of
chimeric enzymes takes place if a domain boundary occurs in a region where sequences are
highly homologous to each other. Sialylmotifs L and S are present in domains C and E/F,
respectively, and indicated by boxes. (B) Comparison of the positions of cysteine residues and N-
glycosylation sites. The last amino acid in each domain is shown by the residue number of
ST8Sia IV. N-Glycosylation sites are numbered for ST8Sia IV. The transmembrane domain
(TM), stem region (Stem), and sialylmotifs L (L) and S (S) are indicated. For soluble forms of
the enzymes, the A domain starts at the arrowhead (residue 40).
Fig. 2. NCAM polysialylation and autopolysialylation activity by truncated mutants of
ST8Sia IV. Deletion mutants of ST8Sia IV were generated by a PCR-based method and fused
with a signal peptide (SP) and a part of IgG-binding domain of protein A (ProA) to enable
secretion of the proteins and purification by human IgG-Sepharose. Activity of each enzyme was
measured after transient expression by staining COS-I cells with a 12F8 anti-polysialic acid
monoclonal antibody (A) and assaying polysialyltransferase activity in vitro (B). The levels of
chimeric enzymes produced was estimated by Western blot analysis using rabbit IgG against
human IgG (Western) and equivalent amounts of chimeric enzymes were assayed in different
experiments. The enzymes bound to IgG beads and CMP-[14C]NeuNAc were incubated with
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NCAM•IgG (NCAM•IgG) or without NCAM•IgG (Enzyme). After centrifugation of the reaction
mixture, NCAM•IgG was isolated from the supernatant while the protein A-enzyme chimera was
released from IgG beads. The reaction mixtures were separated by SDS-polyacrylamide gel
electrophoresis and subjected to fluorography.
Fig. 3. NCAM polysialylation activity of chimeras of ST8Sia II and ST8Sia IV. The
nomenclature used in the top panel is as follows: a chimeric protein consisting of the A to C
domains of ST8Sia II and the D to G domains of ST8Sia IV is designated as IICDIV, as indicated
schematically. The enzymatic activity for NCAM polysialylation shown in the lower panel was
measured as described in Fig. 2.
Fig. 4. Polysialylation activity of chimeras of ST8Sia IV and ST8Sia III. The structures of
chimeric enzymes are shown as in Fig. 1. Analysis of autopolysialylation (Enzyme) and Western
blot analysis using rabbit IgG to determine the amount of the enzyme (Western) was carried out
in the manner described in Fig. 2. Two different series of chimeric enzymes are shown in A and
B.
Fig. 5. HPLC analysis of N-glycans released from autopolysialylated enzymes. N-Glycans
were released by N-glycanase from autopolysialylated enzymes shown in Fig. 4A. The released
N-glycans were fractionated by Mono-Q anion exchange column chromatography as described
under “Experimental Procedures”. Open squares, closed circles, and open triangles denote
radioactivity incorporated to ST8Sia IV, IIIDEIV, and ST8Sia III, respectively. The numbers of
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sialic acid residues (DP) are estimated by determining the elution positions of sialic acid
oligomers and polymers obtained by mild hydrolysis of colominic acid.
Fig. 6. Comparison of autopolysialylation and NCAM polysialylation by ST8Sia III/ST8Sia
IV chimeric enzymes. Equivalent amounts of enzyme bound to IgG beads were incubated with
NCAM•IgG (NCAM•IgG) or without NCAM•IgG (Enzyme). The products were separated by
SDS-polyacrylamide gel electrophoresis and subjected to fluorography as shown in Fig. 2.
Relative activity was estimated by densitometric analysis in comparison with ST8Sia IV (100%)
and shown as the average of at least two different experiments.
Fig. 7. Inhibition of NCAM polysialylation by inactive chimeric enzymes. CMP-
[14C]NeuNAc and equivalent amounts of ST8Sia IV were incubated with increasing amounts of
chimeric enzymes or ST8Sia III in the presence (NCAM•IgG) or absence of NCAM•IgG
(Enzyme). The amount of ST8Sia IV and chimeric enzymes were estimated by Western blot
analysis as shown in ref. 51 and adjusted to be the same in each solution. Note that NCAM
polysialylation was mainly due to ST8Sia IV, while autopolysialylation was due to ST8Sia IV,
ST8Sia III and chimeric enzymes. The relative activity of NCAM polysialylation was estimated
as shown in Fig. 6.
Fig. 8. Schematic representations of polysialyltransferase (ST8Sia IV) and ST8Sia III. The
fourth and fifth immunoglobulin-like domains (Ig 4 and Ig 5) and two fibronectin type III repeats
(FN 1 and FN 2) of NCAM are shown at the left. It was reported that the fifth (Ig 5) and most
likely the fourth (Ig 4) immunoglobulin-like domains and the first fibronectin type III repeat (FN
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1) are necessary for recognition by polysialyltransferases (43, 51, 52). The domains A, B and
possibly D in ST8Sia IV are involved in recognition of NCAM acceptor. The catalytic domains
E, F, and most likely G are involved in polysialylation, in addition to domain C which contains
sialylmotif L. Sialylmotif S is contained in domains E and F. The domains E, F and G are close
to domain C through two disulfide-bond bridges. ST8Sia III, on the other hand, lacks NCAM
recognition although domain C is close to domains E, F and G through two disulfide-bond
bridges, equivalent to the two disulfide-bond bridges in ST8Sia IV. Sialylmotifs L and S are
boxed. The cysteine residues forming two disulfide bridges and conserved free cysteine in
sialylmotif L are denoted by ©. �, �� and � denote sialic acid, CMP-NeuNAc and CMP,
respectively. N-glycosylation sites in NCAM are indicated by the Ψ symbol.
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ST8Sia II
ST8Sia IV
ST8Sia III
ST3Gal I
N50 N74 N119 N204 N219
375 aa
359 aa
380 aa
340 aa
C
C
C C C
C C C
C C C
CCC CC C
C
C
C
C
C
C
A B C D E F G
ST8Sia IVS
T8S
ia IIIST8Sia IV
Identity (%)
A B C D E F G
20 29 58 3426 655245 56 70 6365 6992
A
A B C D E GF
L STM
B
Stem
82 127 193 267 292 318 359
ST
8Sia
II
Fig. 1
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C C C C C CST8Sia IV
N50 N74 N119 N204 N219Activity
C C C C C
C C C C C
C C C C C
C C C C C
C C C C C
C C C C C
C C C C C
SF-IV(40)
SF-IV(50)
SF-IV(62)
SF-IV(72)
SF-IV(82)
SF-IV(103)
SF-IV(127)
+
+
+
+
±
-
-
-SP ProA L S
40 50 62 72 82 103 127
NCAM•IgG
Enzyme
Western
KDa
220
KDa
220
46
KDa
46
97.4
66
A
B
Fig. 2
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NCAM•IgG
Western
B
C
D
E
A
FG
NH2
KDa
220
97.4
66
46
COOH
IIIV
IIABIV
IIEFIV
IIFGIV
II BCIV
II CDIV
IIDEIV
II EFIV
FGII
Fig. 3
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IV
IIIA
BIV
IIIE
FIV
IIIF
GIV
IIIB
CIV
IIIC
DIV
IIID
EIV
IIIE
FIV
FGIII
III
Enzyme
Western
B
C
D
E
A
FG
NH2
KDa
220
97.4
66
46
66
46
COOH
IVB
CIII
CDIV
IVC
DIII
DEIV
IVA
BIII
BCIV
IVB
CIII
DEIV
IVA
BIII
DEIV
IV III
IVB
CIII
CDIV
-NFig. 4
A B
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[14 C
] C
PM
Fractions
DP
100 110 120 130 140 150 16090807060504030201000
50
100
150
200
250
300
350
4001 2 3 4 5 10 20 30 40 50
IVIIIDEIVIII
Fig. 5
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Enzyme
KDa220
97.4
66
46
Relative Activity(%) 100 102 43 243 196 151 159
NCAM•IgG
KDa
220
Relative Activity(%) 100 39 13 19 7 18 10
IV
IIIA
BIV
IIID
EIV
IIIE
FIV
IIIE
FIV
FG
III
III
IVC
DIII
DEIV
IVB
CIII
CD
IV-N
35
69
Fig. 6
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IVCDIIIDEIV
Relative Activity (%)
5 5 5 5 5 5 55 10 20 25
10 20 25
KDa220
97.4
66
46
KDa220
100 90 78 47 96 84 61 821
Enzyme
NCAM•IgG
ST8Sia IV
IVBCIII 5
µlµlµl
IVFGIII
Relative Activity (%)
ST8Sia III
5 5 5 5 5 5 55 10 20 25
5 10 20 25
KDa220
97.4
66
46
KDa220
100 96 77 49 91 97 105 105
Enzyme
NCAM•IgG
ST8Sia IV µlµlµl
Fig. 7
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Ig 4
Ig 5
FN 2
FN 1
COOH
NCAM
Sialylmotif LC
C C
C
S
A
BC
F
E GC
D
ST8Sia III
Fig. 8
Sialylmotif LC
C C
C
S
D
F
CGE
BC
A
NCAM Polysialyltransferase(ST8Sia IV)
NH2 NH2
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Kiyohiko Angata, Dominic Chan, Joseph Thibault and Minoru Fukudarequired for NCAM recognition and polysialylation
Molecular dissection of the ST8Sia IV polysialyltransferase: Distinct domains are
published online April 2, 2004J. Biol. Chem.
10.1074/jbc.M401562200Access the most updated version of this article at doi:
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