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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: minoru@burnham-inst.org

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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