(Received for publication, May 10, 1995; and in revised form, June 13, 1995)
From the
The properties and developmental regulation of vertebrate
polysialyltransferase (PST), an enzyme activity responsible for
extension of 2,8-linked sialic acid homopolymers (PSA) associated
with the fifth Ig domain of the neural cell adhesion molecule (NCAM),
have been studied. The assay for PST used exogenous NCAM as a
substrate, with a PSA-specific endoneuraminidase as a control for
specificity. Optimal conditions for PST activity at 37 °C were
found to be pH 6.0 in the presence of divalent cations
(Mn
, 20 mM). The enzyme K
was found to increase with increasing polymer length,
ranging from 0.7 to 0.07 µM. The developmental regulation
both of PST activity and of the addition of PSA to NCAM were studied in
chick whole brain, tectum, and cerebellum and found to be precisely
coordinated. In each tissue PSA and PST were highest during early
stages of morphogenesis, followed by a decrease as development reached
completion. The insertion of the VASE exon in the fourth Ig domain of
NCAM was also found to parallel closely the developmental
down-regulation of PSA, and on this basis could be considered a
potential determinant in the specific polysialylation of NCAM. However,
in direct tests of this hypothesis in transfected cells the presence of
VASE did not markedly alter the level of NCAM polysialylation or alter
the affinity of PST for the NCAM substrate.
Polysialic acid (PSA) ()is a linear homopolymer
composed of negatively charged N-acetylneuraminic acid
residues in an
2, 8-linkage(1) . In vertebrates, PSA is
attached to the neural cell adhesion molecule (NCAM), and its regulated
addition to NCAM results in an attenuation of cell-cell
interactions(2) , probably via steric and/or charge effects at
the cell surface(3) . The regulation of PSA on NCAM during
development is known to affect a variety of cell-cell interactions that
are important in the pathfinding and target innervation of
axons(4, 5) , development of skeletal
muscle(6) , formation of the kidney(7) , and migration
of neuronal precursors in the brain(8) .
Because of the unusual biochemistry of PSA and its specific addition to NCAM, as well as its significance as a developmental regulator of cell-cell interactions, it has become important to characterize in more detail the properties of the substrate and enzyme activity involved. The polysialyltransferase (PST) activity responsible for the biosynthesis of PSA on NCAM was first detected by McCoy et al.(9) in fetal rat brain. More recently we have carried out an extensive study on the structural features of NCAM that are involved in polysialylation(10) , and a cDNA for the putative PST has been cloned(11) . The present study, using a novel enzymatic assay to characterize PST activity more rapidly and easily, complements those findings by providing a description of the properties of the endogenous enzyme activity, and evidence that levels of this enzyme are closely coordinated with changing levels of NCAM polysialylation during development of a variety of neural tissues. In addition, we have used cell transfection studies to test directly whether the developmentally regulated insertion of the VASE exon (12) into the Ig domain adjacent to the site of NCAM polysialylation might be a determinant in control of PSA biosynthesis.
These primers result in the amplification of a 300-bp product when VASE sequence is absent and a 330-bp product when VASE is present. Amplified product was separated by electrophoresis in 1.5% agarose, and ethidium bromide-stained gels were photographed using a UV transilluminator.
Amplification resulted in a 498-bp fragment in the absence of VASE and a 528-bp fragment in the presence of VASE. Primers were designed up and downstream of the VASE sequence such that the flanking sites, NdeI (upstream) and HindIII (downstream), in the chicken cDNA sequence could be utilized to clone the amplified VASE sequence into the intact chicken NCAM cDNA sequence. The VASE-containing fragment was gel purified and cloned into the Srf1 site of the pCR-Script cloning vector to produce the plasmid, pCR-Script-VASE. The DNA sequence predicted amino acid sequence of chicken VASE is identical to that of mouse, rat, and human. The sequence of chicken VASE compared to mouse or rat NCAM is also well conserved: nucleotide sequence, 5`-GCT TCG TGG ACC CGG CCC GAG AAG CAA GAG-3`; amino acid sequence, Ala Ser Trp Thr Arg Pro Glu Lys Gln Gly.
An NdeI fragment encoding the entire upstream
coding sequence of NCAM and some non-coding plasmid sequence was
excised from the expression plasmid, pCMVchNCAM (10) and
ligated into the NdeI site upstream of the VASE sequence in
the pCR-Script-VASE plasmid. After restriction analysis to determine
proper insert orientation, this plasmid was treated with HindIII, and the approximately 1700-bp fragment (which
contains the VASE sequence) was used to replace the analogous sequence
in pCMVchNCAM-Intra(10) (which lacks VASE
sequence and intracellular domain coding sequences).
Chicken NCAM expressed by AtT20 cells was solubilized from cell membranes using Nonidet P-40 extraction buffer (150 mM NaCl, 50 mM HEPES, 1 mM EDTA, 100 KIU/ml aprotinin, 2 mM PMSF, 1% Nonidet P-40, pH 7.4) and purified by immunoaffinity adsorption to Sepharose-4B beads (Pharmacia) derivatized with monoclonal antibodies specific to chicken NCAM. After washing with phosphate-buffered saline + aprotinin, PSA was removed from adsorbed proteins by adding 0.5 µl of purified Endo N to 250 µl of bead slurry (in phosphate-buffered saline + aprotinin, pH 7.4), then incubating for 4-6 h at 4 °C. Equivalent amounts of Endo N-treated and untreated proteins isolated by immunoadsorption were separated by 7% SDS-PAGE. Affinity resin slurry with bound chicken NCAM was divided into 2 equal volume aliquots. Immunoadsorbed protein was released from beads by denaturation with Laemmli sample buffer. After electrophoresis, proteins were electrotransferred to nitrocellulose and detected by immunoblotting using anti-chicken NCAM polyclonal antibody. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody and detection of antigen carried out by the ECL method.
PST activity was proportional to the time of incubation up to 3 h,
after which a plateau was reached (Fig. 1A). Activity
was also proportional to the protein concentration up to 60 µg, the
maximal concentration tested (Fig. 1B). Under optimal
assay conditions (see below) using stage 33 chick brain as the enzyme
source, more than 80% of the incorporated radioactivity released by
Endo N treatment was incorporated into exogenous NCAM, as the
difference between total NCAM (exogenous + endogenous) and
exogenous NCAM was less than 20% (Fig. 1B). Moreover,
70% of the radioactivity incorporated into exogenous NCAM was released
by Endo N, indicating its incorporation into 2,8-linked PSA
chains. No enzymatic activity was detected when asialofetuin was used
as an acceptor, indicating that NCAM was serving as a specific acceptor
for the PST activity studied.
Figure 1: Dependence of PST activity on the incubation time and protein concentration. A, the enzymatic reaction mixture were incubated for various times under the assay condition described under ``Experimental Procedures.'' B, PST activity assay was carried out using various amounts of the enzyme source material expressed as micrograms of protein, and activity assays were run in parallel in the presence or absence of exogenous purified NCAM. To assess the PST activity into endogenous NCAM (Endo), the assay was run in the absence of exogenous NCAM (Exo), and the PST activity for total NCAM (Total, endogenous plus exogenous NCAM) was measured from the radioactivity obtained in the presence of exogenous NCAM. Finally, PST activity for exogenous NCAM was obtained from the difference of activity between total NCAM minus endogenous NCAM.
Figure 2:
Assessment of optimal conditions for assay
of polysialyltransferase activity. The effect on PST activity of pH (A), divalent cations (B), and varying concentrations
of Mn (C) were investigated using Nonidet
P-40 extracted membrane proteins from stage 33 chick brains. PST assays
were carried out as described under ``Experimental
Procedures.''
To test whether the addition of PSA to NCAM in this assay was dependent on tertiary protein structure, the exogenous NCAM substrate was preheated for 5 min at 100, 80, and 65 °C. No degradation of PSA or NCAM, as detected by SDS-PAGE analysis, was apparent after these treatments (data not shown). However, the ability of NCAM to serve as a PST substrate dramatically decreased even after treatment at 65 °C (data not shown), suggesting that the addition of PSA by PST requires higher order protein structure in addition to a specific carbohydrate acceptor.
To investigate the effect of PSA polymer length on PST/NCAM K values, NCAM was treated with Endo N to
remove the PSA prior to the enzymatic assay. The K
value for highly polysialylated NCAM purified at stage
37-38 was 0.71 µM (Fig. 3A). After
Endo N treatment of this substrate, then K
was about 10 times lower than that for Endo N-untreated NCAM (Fig. 3C). Thus increasing polymer length appears to
decrease the affinity of PST for its natural substrate. For comparison,
the K
value for CMP-NeuAc was found to be
48 µM.
Figure 3: Dependence of PST activity on the NCAM concentration. Nonidet P-40-extracted membrane proteins from stage 33 chicken whole brain were used for the enzymatic assay. NCAM was purified from stage 37-38 (A) and stage 45 (B), and digested with Endo N as described under ``Experimental Procedures'' (C and D). NCAM concentration was calculated using the average molecular mass of 160 kDa. The inset shows the data analyzed with a Lineweaver-Burk plot.
Figure 4: Developmental changes of PSA expression and PST activity in chick whole brain. A, SDS-PAGE immunoblot stained with an anti-chicken NCAM antibody that recognizes all NCAM isoforms. Lanes - and + indicate Endo N-untreated and -treated samples, respectively. The top of the polydisperse material (>200 kDa) detected at stage 30 and stage 35 is indicated by a solid line. B, developmental change of PST activity. PST activity is expressed as a percent of the maximal specific activity detected (24 pmol/mg total protein/h at stage 30) normalized to the relative amount of total NCAM quantified by densitometry from the immunoblots (see ``Experimental Procedures'').
To determine the levels of PST activity relative to substrate at each developmental stage, enzyme assay values were normalized to the amount of NCAM expressed in the tissue, as quantified by densitometric scans of SDS-PAGE immunoblots (Fig. 4B). By this criterion, the highest PST specific activity in whole brain was detected at stage 30. Thereafter, the activity gradually decreased and by the end of embryonic development (stage 45), little activity was detected (Fig. 4B). The decrease in PST activity (stage 37) preceded the decrease in PSA expression (stage 40), as would be expected if the levels of PSA are controlled by the activity of the enzyme.
To study more precisely the correlation between PSA expression and PST activity during brain embryonic development, the degree of polysialylation of newly synthesized NCAM was studied by pulse-labeling experiments in two brain regions, tectum and cerebellum. In the tectum (Fig. 5), a decrease in the PSA content at stage 40 was revealed by immunoblot analysis. At this stage, a faint NCAM-180 band was noticed prior to Endo N treatment, and by stage 45 non-polysialylated NCAM-140 and NCAM-180 were both detected (Fig. 5A). When the polysialylation of newly synthesized NCAM was studied, a reduction in PSA addition to NCAM-180 isoform was detected already between stage 37 and 39. A substantial decrease in PST activity was also observed at stage 37 (Fig. 5C). However, the NCAM-140 isoform, which constituted the only isoform expressed in the tectum at late developmental stages, continued to be synthesized and polysialylated. After stage 39, a sharp decrease in the polysialylation of NCAM-140 was also observed, (Fig. 5B).
Figure 5:
Developmental changes of PSA expression
and PST activity in chick tectum. A, SDS-PAGE immunoblot
stained with an anti-chicken NCAM antibody that recognizes all NCAM
isoforms. Lanes - and + indicate Endo N-untreated
and -treated samples, respectively. The top of the position of
polydisperse material (>200 kDa) detected at stage 33 and stage 35
is indicated by a solid line. B, fluorograph of
immuno isolated NCAM from tectum obtained from various embryonic
developmental stages and metabolically labeled with
[S]methionine. NCAM was isolated from the
homogenates by immunoprecipitation, separated by SDS-PAGE, and
fluorograph obtained by suitable film exposure. C,
developmental change of PST activity. PST activity is presented as the
percent of maximal specific activity (12.6 pmol/mg total protein/h at
stage 33) normalized to the relative amounts of total NCAM obtained by
densitometry from the immunoblots.
In cerebellum, the immunoblot analysis of PSA expression showed a similar pattern, with a reduction in the PSA content of NCAM at stage 43 and no detectable PSA by stage 45 (data not shown). However, a decrease in newly synthesized NCAM polysialylation was observed already by stages 40 and 41, and by stage 43 there was significantly less polysialylation of NCAM. Thus, the decrease in newly synthesized PSA coincided even more closely with the decline in PST activity observed from stage 40-43.
In the
course of these studies, it was noticed that the amount of Endo
N-sensitive [C]sialic acid incorporated into
exogenous NCAM varied with respect to developmental stage. For brain at
stage 30, 77% of incorporated [
C]sialic acid was
removed by Endo N; however, at stage 45 only 30% of the incorporated
counts were endo N-sensitive. This finding suggests that at later
developmental stages there is an up-regulation of other enzymes that
can add [
C]sialic acid into NCAM in non-PSA
forms.
Figure 6: Testing the correlation between VASE expression and PSA down-regulation. A, RT-PCR reactions were carried out on RNA derived from whole brain, tectum, and cerebellum at the stages indicated. VASE expression is indicated by the appearance of a 330 bp band (+VASE), which is 30 bp larger than product lacking VASE. Vertical arrows denote stages at which PSA down-regulation was first observed. B, chicken NCAM either lacking (-VASE) or containing VASE (+VASE) was expressed in the AtT20 cell line, which expresses all enzymes required for polysialylation of NCAM. The polysialylation state of the introduced chicken NCAM was assessed by SDS-PAGE separation and Western blotting with antibodies specific to chicken NCAM. Arrows represent the non-polysialylated or poorly polysialylated component of expressed chicken NCAM. The polydisperse staining above these bands represents PSA, as it is specifically removed by treatment with Endo N (data not shown).
Unlike the PSA/PST relationship, in which there is direct enzymatic evidence that the two components are functionally related, the VASE/PSA relationship is purely a temporal correlation. Therefore, a more direct test was devised to determine the effect of VASE expression on NCAM polysialylation in vitro. AtT20 cells were transfected with expression plasmids encoding chicken NCAM with and without the VASE sequence. AtT20 cells were chosen for use because they are known to express all enzymes required for polysialylation of NCAM(18) . In this assay, chicken NCAM containing VASE was only slightly less polysialylated than chicken NCAM without VASE (Fig. 6B). Thus, it would appear that VASE insertion does not play a dominant role in the down-regulation of NCAM polysialylation.
A possible effect of VASE on the affinity between
NCAM and PST was also examined. K values
were determined for NCAM purified from chick brains of different
developmental stages that do (stage 45) and do not (stage 37-38)
express VASE (see Fig. 6A). Brains from stage 33 were
used as the enzyme source, and the concentration of NCAM in the
solution was calculated using an average molecular mass of 160 kDa. The K
value estimated for the NCAM without
VASE was 0.71 µM, which was almost the same K
value detected for NCAM with VASE (0.65
µM) (Fig. 3, A and B), indicating
that the presence of VASE did not affect the binding of PST to NCAM.
The same difference in K
values was found
when Endo N-treated NCAM was used as substrate (Fig. 3, C and D).
The expression of PSA on NCAM is subject to tight developmental control, a process that is critical in the role of PSA as a permissive regulator of cell interactions during the formation and remodeling of tissues. In order to clarify further how this regulation occurs, this study has sought to determine if the regulation of PSA expression in vivo is tightly linked to changes in the levels of specific polysialyltransferase activity, to define more clearly the properties of that enzymatic activity, and to explore whether alternative splicing of the NCAM polypeptide might affect its ability to serve as an acceptor for polysialylation.
Little is known about how polysialylation of NCAM is regulated in nervous tissue. However, two potential factors for regulation of PSA expression have emerged: changes in the level of polysialyltransferase activity(18) , and the insertion of VASE exon between NCAM exon 7 and 8 that changes the fourth Ig domain from a C-type to a V-type(12) .
Three
different kinds of sialyltransferases are involved in the biosynthesis
of the non-NCAM PSA found in trout eggs(19) , but it is not
known how many sialyltransferases(s) are involved in the biosynthesis
of PSA on NCAM. The enzymatic assay used here uses pre-existing PSA
chains as an acceptor, and thus is likely to represent the PST activity
responsible for elongation of the linear homopolymer. Using this assay,
developmental changes of PSA expression were compared with PST
activity. Changes in the level of PST activity were closely correlated
with changes in the degree of polysialylation of newly synthesized
NCAM, suggesting that the developmental changes observed in PSA
expression are at least in part regulated by the level of PST activity.
The developmental down-regulation of previously synthesized PSA as
detected by immunoblot analysis occurred about 2 days after the
shut-down of the polysialylation of the newly synthesized NCAM, which
corresponds well with the 1 day half-life of NCAM found for cultured
ciliary ganglion neurons. ()
The expression of the VASE
exon is also correlated with the level of NCAM polysialylation in
tectum and cerebellum, although analysis of whole brain was less well
linked, implying that some brain regions probably do not exhibit this
relationship. In any case, a direct analysis of the effect of VASE on
the ability of NCAM to became polysialylated in vitro showed
that the insertion of VASE sequence does not serve as a major regulator
of PSA synthesis. Moreover, the affinity between PST and NCAM was not
affected by the presence of VASE, and in studies on the ciliary
ganglion we have found that developmental down-regulation of PSA occurs
in the absence of detectable VASE expression. Nevertheless,
the temporal correlation between VASE and PSA down-regulation in some
tissues is remarkable, and the expression of this spliced exon may well
be a consequence of or even contribute to a signaling pathway that
triggers changes in PST activity.
At early stages of embryonic
development (stage 30-37), more than 70% of the
[C]sialic acid enzymatically incorporated into
exogenous NCAM was released by Endo N treatment. However, only
30-50% of the radioactivity was released by Endo N at later
stages (stage 40-45). One possible explanation for this
phenomenon might be that during the embryonic development of the
nervous system, monosialyltransferases(s) are induced, and the
resulting carbohydrate structure cannot be digested by Endo N. In this
situation, potential polysialylation sites could also be blocked during
the biosynthesis of carbohydrate chains of NCAM. For example, if the
first sialic acid residue of a potential polysialylation site, which is
transferred to a galactose residue, is substituted by an
2,6-linkage instead of the
2,3-linkage, then the PST would
not be able to elongate that chain. In fact, it has been reported the
expression of a
-galactoside
2,6-sialyltransferase in Xenopus embryos blocks the synthesis of PSA(20) .
In this context, glucuronyltransferase, one of the key enzymes in the biosynthesis of the HNK-1 carbohydrates that are commonly expressed on cell adhesion molecules including NCAM(21, 22, 23, 24, 25) , could be considered as a potential regulator of PSA synthesis. Glucuronyltransferase can catalyze the transfer of glucuronic acid to a galactose residue of NCAM, substituting the first sialic acid residue by a glucuronic acid residue and thus blocking polysialylation. This enzymatic argument is bolstered by the fact that in brain the up-regulation of glucuronyltransferase is developmentally correlated with the decrease in PSA expression.
In addition to the initiation and elongation of PSA chains, it is also possible that the chains are terminated by an additional enzymatic step, as has been noted in fish eggs(26) . In considering chain termination mechanisms, it should be noted that polysialylation of NCAM always results in very broad and diffuse bands in SDS-PAGE, suggesting a wide range of polymer lengths. In our studies, we found that PST has about a 10-fold lower affinity for highly polysialylated embryonic brain NCAM than for the same NCAM after treatment with Endo N. Thus PST would tend to bind to short PSA chains and dissociate from long chains. It is possible, therefore, that termination is produced simply through the reduced ability of the enzyme to bind to long polymers and that the range of PSA chain lengths represents the increasing probability that the enzyme will fall off the polymer as it grows.