(Received for publication, November 10, 1995; and in revised form, November 30, 1995)
From the
Polysialic acid (PSA) is a linear homopolymer of
-2,8-linked sialic acid residues whose expression is
developmentally regulated and modulates the adhesive property of the
neural adhesion molecule, N-CAM. Recently, hamster and human cDNAs
encoding polysialyltransferase (PST-1 for the hamster enzyme and PST
for the human enzyme) were cloned, and by using the human cDNA it was
demonstrated that the expression of PSA in N-CAM facilitates neurite
outgrowth (Nakayama, J., Fukuda, M.N., Fredette, B., Ranscht, B., and
Fukuda, M.(1995) Proc. Natl. Acad. Sci. U. S. A., 92,
7031-7035; Eckhardt, M.A., Mühlenhoff, M.,
Bethe, A., Koopman, J., Frosch, M., and Gerardy-Schahn, R.(1995) Nature 373, 715-718.) Although these studies
demonstrated that PST-1 and PST synthesize PSA in cultured cells, it
was not shown that they could catalyze the polycondensation of
-2,8-linked sialic acid on a glycoconjugate template containing
-2,3-linked sialic acid. Here we demonstrate that PSA formation by
PST is independent from the presence of N-CAM in vivo. We then
develop an in vitro assay of PSA synthesis using glycoproteins
other than N-CAM as acceptors and a soluble PST as an enzyme source.
The soluble PST, produced as a chimeric protein fused with protein A,
was incubated with rat
-acid glycoprotein, fetuin or
human
-acid glycoprotein as acceptors together with
the donor substrate CMP-[
C]NeuNAc. Incubation of
fetuin with the soluble PST, in particular, resulted in a high
molecular weight product that was susceptible to PSA-specific
endoneuraminidase. Polysialylated products were not formed when
-2,3-linked sialic acid was removed from the acceptor fetuin
before incubation. These results establish that a single enzyme, PST,
alone can catalyze both the addition of the first
-2,8-linked
sialic acid to
-2,3-linked sialic acid and the polycondensation of
all
-2,8-linked sialic acids, yielding PSA.
Polysialic acid (PSA) ()is a developmentally
regulated glycan composed of a linear homopolymer of
-2,8-linked
sialic acid residues. PSA is mainly linked to the neural cell adhesion
molecule (N-CAM) and is more abundant in embryonic brain than adult
brain. Presence of this large negatively charged carbohydrate modulates
the adhesive property of N-CAM(1, 2, 3) , and
the removal of PSA from N-CAM increases the adhesive capability of
N-CAM (4, 5) . It was also demonstrated that the
presence of PSA affects cell-cell interactions carried by other cell
surface receptors(6, 7) . Since PSA is mainly present
in tissues undergoing synaptic rearrangement and cell migration, PSA is
implicated in reducing N-CAM adhesion and thus perhaps in allowing
increased neurite outgrowth and cell mobility.
It was shown
previously that the reducing terminus of a polysialic acid side chain
is attached to -2,3-linked sialic acid, which in turn, is linked
to a galactose residue in a presumed acceptor such as
N-CAM(3) . It has been suggested that PSA synthesis requires
two distinct
-2,8-sialyltransferases; the first
``initiation'' enzyme, which adds a single
-2,8-linked
sialic acid to
-2,3-linked sialic acid in the acceptor and a
``polymerase,'' which adds multiple
-2,8-linked sialic
acid residues to the NeuNAc
2
8NeuNAc
2
3Gal
R
structure(8, 9) . As a support for this hypothesis,
the recently cloned ST8SiaIII was shown to add a single
-2,8-linked sialic acid residue to a terminal
-2,3-linked
sialic acid in N-glycans, but not to form PSA(10) .
As an alternative biosynthetic pathway, it is possible that a single
enzyme catalyzes all the reactions that form -2,8-sialic acid
linkages. Recently, we and others have cloned a cDNA of
polysialyltransferase (PST for human and PST-1 for hamster enzyme) that
forms polysialic acid attached to N-CAM(11, 12) . The
amino acid sequences of PST and PST-1 are more than 97% identical. The
introduction of PST or PST-1 cDNA together with N-CAM cDNA into various
cell lines, such as COS-1, CHO-2A10, HeLa, and 3T3 cells, resulted in
PSA expression in those cells. More recently, it has been demonstrated
that STX also directs the expression of PSA in small cell lung
carcinoma cell lines(13) . STX was originally cloned as a
sialyltransferase predominantly present in fetal brain(14) ,
and STX and PST have 59% identity in the amino acid
sequences(11, 12) . These results suggest that PST and
STX catalyze polycondensation of
-2,8-linked sialic acid residues.
However, these results did not formally exclude the possibility that
PST or STX adds the first -2,8-linked sialic acid as an initiation
enzyme, and a hitherto unknown enzyme present in those tested cells
catalyzes the polycondensation of polysialic acid. Moreover, it was not
certain whether or not PST or STX adds polysialic acid residues to a
single
-2,8-linked sialic acid residue, which was already attached
by other
-2,8-sialyltransferases such as ST8SiaIII to an
-2,3-linked sialic acid residue in a precursor.
To determine
whether or not PST can catalyze both initiation and polycondensation,
we report here the construction of a PST chimeric protein which
consists of a signal peptide, protein A, and a catalytic domain of PST.
This chimeric protein, released into the culture medium from COS-1
cells, was then incubated with various glycoprotein acceptors and donor
substrate CMP-[C]NeuNAc. By analyzing the
products, we show that PST directly adds an
-2,8-sialic acid
residue on an
-2,3-linked sialic acid in a glycoprotein template,
and in addition catalyzes polycondensation of
-2,8-linked sialic
acid, demonstrating that PST can add all
-2,8-linked sialic acid
residues necessary for PSA formation.
In parallel, HeLa cells were
transfected with pcDNAI-PST and pSVneo, and the transfected
cells were selected with G418 (Life Technologies, Inc.). Clonal cell
lines stably expressing PSA were chosen by immunofluorescent staining
with 735 antibody as described above. Both the transfected COS-1 and
HeLa cells were examined for the presence of N-CAM by immunofluorescent
staining with mouse anti-N-CAM antibody, followed by FITC-conjugated
(Fab`)
fragment of goat anti-mouse IgG antibody.
The cDNA encoding a catalytic domain of PST was prepared by
polymerase chain reaction (PCR) using pcDNAI-PST (11) as a
template. Upstream and downstream primers used were
5`-cgggatccgGGTGAATTGTCTTTGAGTCGGT-3` (BamHI site shown by
underline), and 5`-ggggtaccTCAAAATGTGCTTTATTGCTTTACAC-3` (KpnI
site shown by underline), respectively. The PCR product encompasses the
sequence from nucleotide 118 (codon 40) to nucleotide 1092 (12
nucleotides after the stop codon). In parallel, pAMoA-GD3 (20) was digested with BamHI and KpnI,
resulting in pAMoA containing cDNA encoding only the signal peptide and
protein A. pAMoA-GD3 was kindly provided by Drs. Katsutoshi Sasaki and
Tatsunari Nishi, Kyowa Hakko Kogyo Co. (Machida, Japan). The PCR
product of PST was digested with BamHI and KpnI and
cloned into pAMoA digested with the same restriction enzymes, yielding
pAMoA-PST. From this pAMoA-PST, the cDNA encoding the fusion protein
consisting of the signal peptide, the protein A and the PST catalytic
domain was excised by SalI and Asp718 digestion. The
released cDNA insert was filled in by the Klenew fragment of DNA
polymerase I and cloned into pcDNAI that had been digested with EcoRV, producing pcDNAI-APST. As a control vector,
pcDNAI-A containing only the signal peptide and protein A cDNAs was
similarly constructed.
After confirming the correct orientation by
nucleotide sequencing, pcDNAI-APST and pcDNAI-A were separately
transfected into COS-1 cells using Lipofectamine(11) . After 62
h of transfection, the protein A-PST fusion protein secreted into the
culture medium was adsorbed to IgG-Sepharose 6FF (Pharmacia Biotech
Inc.) essentially as described(21, 22) . The resin was
collected by centrifugation, washed nine times with 50 mM Tris-HCl, pH 7.5, containing 1% bovine serum albumin, and then two
times with 20 mM Tris-HCl, pH 7.5, containing 7.5 mM CaCl
and 0.05% Tween 20, and finally suspended in the
equal volume of a serum free medium of Opti-MEM I (Life Technologies,
Inc.). This procedure is a slightly modified one from that which was
reported previously(21) .
For linkage analysis
of incorporated sialic acid, 90% of ethanol was added to the reaction
mixture and the glycoproteins were recovered by centrifugation. After
washing in 90% ethanol one more time, the sample was digested with
NANase I (0.17 unit/ml), NANase II (5 units/ml), or NANase III (1.7
units/ml), purchased from Glyko, Inc. (Navato, CA), at 37 °C for 19
h according to the protocol provided by the supplier. NANase I, II, and
III specifically cleave -2,3-linked sialic acid,
-2,3- and
-2,6-linked sialic acids, and
-2,3-,
-2,6- and
-2,8-linked sialic acids, respectively. Similarly, the reaction
mixture was digested for 36 h with N-glycanase according to
the manufacturer's protocol (Genzyme, Cambridge, MA) or for 24 h
with endoneuraminidase (endo-N) according to the procedures
described(26) . These digested materials were then subjected to
SDS-polyacrylamide gel electrophoresis followed by fluorography using
the same procedure as described above.
In order to determine the requirement of sialic acid residues in the acceptor glycoproteins, fetuin was digested with NANase I, NANase II, NANase III, or N-glycanase. After the incubation, ethanol (90%, the final concentration) was added to the reaction mixture and the solution was mixed well. The digested substrate was recovered by centrifugation and washed again with 90% ethanol. The recovered desialylated or de-N-glycosylated fetuin was used as acceptors for incorporation of radioactive sialic acid residues under the same conditions described above.
Figure 1: Expression of PSA in transiently transfected COS-1 cells. COS-1 cells were transiently transfected with pcDNAI-PST and examined by immunofluorescent staining using M6703 (A), 12E3 (B), and 735 (C) antibody. Transfected COS-1 cells stained by anti-N-CAM antibody is shown in D. Bar, 50 µm.
Similarly, we established HeLa cells stably expressing PSA in the absence of N-CAM. Immunofluorescent staining of the transfected HeLa cells confirmed that N-CAM was absent in the transfected HeLa cells (data not shown). However, the transfected HeLa cells expressed PSA, which was detected by the 735 antibody, and this expression was abolished by the treatment of endo-N, which specifically cleaves PSA (Fig. 2). These results indicate that PSA can be formed by PST in the absence of N-CAM.
Figure 2: Expression of PSA in HeLa cells stably transfected with PST. HeLa cells stably transfected with pcDNAI-PST were examined by immunofluorescent staining using 735 antibody before (A) and after (B) endo-N treatment. Bar, 50 µm.
As shown in Fig. 3, the
chimeric PST protein directed the formation of a broad and high
molecular band (mass 100-170 kDa) when fetuin was incubated
for 24 h. Although small amounts of broad and high molecular weight
bands were produced from rat and human
-acid
glycoproteins, the majority of the radioactivity incorporated into
-acid glycoproteins migrated close to the position
where untreated glycoproteins migrated (mass
48 and
44 kDa,
respectively). The products from fetuin and human
-acid glycoprotein also contained a band with a high
molecular mass in excess of 200 kDa, which did not enter into the
separation gel. These bands most likely represent insoluble aggregates
of sialylated glycoproteins since they were present when a large amount
of the products were analyzed but absent when only a small amount of
the products were analyzed, shown in Fig. 4.
Figure 3:
Incorporation of sialic acids by a soluble
form of PST into various glycoproteins. Glycoprotein acceptors,
indicated on the top of each lane, were incubated with the soluble form
of PST and donor substrate, CMP-[C]NeuNAc.
Incubations were carried out for 4 h (labeled as 4) or 24 h
(labeled as 24) at 37 °C. The mock transfection with
pcDNAI encoding only a signal peptide and protein A, pcDNAI-A is
labeled as M. The products were subjected to
SDS-polyacrylamide gel electrophoresis followed by fluorography. The
mobility of molecular weight standards are shown at the left. AGP denotes acid glycoprotein.
Figure 4:
Characterization of radiolabeled products
by various glycosidases. The products obtained by the soluble form of
PST, which are shown in Fig. 3, lane 5 and 8 from the left (fetuin and human 1-acid glycoprotein,
respectively), were subjected to NANase II, NANase III, endo-N, or N-glycanase as indicated on the top of the gel under the
conditions described under ``Experimental Procedures.'' The
digested samples were then subjected to SDS-polyacrylamide gel
electrophoresis followed by fluorography.
In order to
characterize the products formed by PST, they were digested with
various enzymes and then subjected to SDS-polyacrylamide gel
electrophoresis. The products were not susceptible to NANase II, which
cleaves both -2,3-linked- and
-2,6-linked sialic acid (NANase
II in Fig. 4). The products were, however, susceptible to NANase
III, which cleaves all of
-2,3-,
-2,6- and
-2,8-linked
sialic acids (NANase III in Fig. 4). They were also susceptible
to endo-N and N-glycanase (Fig. 4). These results
establish that the broad and high molecular weight bands represent
glycoproteins containing polysialic acid side chains in N-glycans. Similarly, the sialic acid residues incorporated
into human
-acid glycoprotein were removed by NANase
III or N-glycanase (Fig. 4), indicating they are also
attached through an
-2,8-linkage(s).
The product from fetuin
after the treatment of endo-N still migrated as a larger molecule (mass
90 kDa) than the untreated glycoprotein (Fig. 4). This is
most likely due to the fact that endo-N can not cleave polysialic acid
chains that are shorter than those consisting of 5 or 6 sialic acid
residues(26, 27) . The product after endo-N treatment
most likely represents fetuin containing polysialic acid chain(s)
consisting of 6 or fewer
-2,8-sialic acid residues in a side
chain. Fetuin contains both N- and O-linked
oligosaccharides(28, 29) . Since N-glycanase
treatment removed almost all of the incorporated sialic acid (Fig. 4), the majority of polysialylation took place in N-glycans.
Figure 5:
Incorporation of sialic acids on
glycosidase-treated fetuin. Fetuin was digested with NANase I, II, III,
or N-glycanase, as indicated on the top of the gel, and then
used as acceptors. After incubation of these acceptors with the soluble
PST and CMP-[C]NeuNAc for 24 h, the resultant
products were subjected to SDS-polyacrylamide gel electrophoresis
followed by fluorography. The experiments shown in Fig. 3and Fig. 5were carried out at the same
time.
It is noteworthy that fetuin
is the best acceptor among glycoproteins tested. Fetuin contains mainly
tri-antennary N-glycans(29) , while rat and human
-acid glycoproteins mainly contain bi-antennary and
tetra-antennary N-glycans(30) , respectively. The
present findings are consistent with the report that PSA containing N-glycans isolated from chicken and bovine fetal brains were
mainly composed of tri-antennary oligosaccharides (31) .
Further studies are, however, required to determine whether or not PST
preferentially adds sialic acid residues on tri-antennary N-glycans.
The present study established an in vitro assay system for PSA synthesis, allowing us to conclude that PST
alone can add the first -2,8-linked sialic acid to a precursor
containing
-2,3-linked sialic acid and then add multiple
-2,8-linked sialic acid residues to the acceptor containing
NeuNAc
2
8NeuNAca2
3Gal
R structure, yielding PSA.
It is unlikely, but possible, that the specificity of PST might be
modified because a protein A is fused with the catalytic domain of PST.
Further studies are thus needed to prove formally that the intact PST
can catalyze the same reactions as demonstrated for the fusion protein.
The results consistent with our conclusion were, however, obtained in
the intact form of both hamster PST-1 (12) and
STX(13) . The studies on hamster PST-1 showed that only one
kind of CHO mutant cell lines defective in polysialic acid synthetase
was obtained. Expression of PSA was restored after the mutant CHO cells
were transfected with hamster PST-1 cDNA(12) . Similar results
were obtained on human STX. In a small cell lung carcinoma, STX, but
not PST, directs the expression of PSA(13) . This PSA synthesis
took place in the absence of other
-2,8-sialyltransferases such as
PST(11) , ST8SiaIII(10) , and GD3
synthase(20, 32) . Taking account of the results
obtained in the present study, these results strongly suggest that
PST-1 or STX alone might be able to form all
-2,8-linked sialic
acid residues in these cell lines. Further studies will be still
needed, however, to prove formally that STX and hamster PST-1 can also
catalyze polycondensation of all
-2,8-linked sialic acid residues,
and to determine if other
-2,8-sialyltransferases such as
ST8SiaIII participate in PSA synthesis in certain cells. The in
vitro assay established in the present study is expected to be
useful to address these questions.