©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Properties and Developmental Regulation of Polysialyltransferase Activity in the Chicken Embryo Brain (*)

(Received for publication, May 10, 1995; and in revised form, June 13, 1995)

Shogo Oka Juan L. Brusés Richard W. Nelson (§) Urs Rutishauser (¶)

From the Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The properties and developmental regulation of vertebrate polysialyltransferase (PST), an enzyme activity responsible for extension of alpha2,8-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.


INTRODUCTION

Polysialic acid (PSA) (^1)is a linear homopolymer composed of negatively charged N-acetylneuraminic acid residues in an alpha 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.


EXPERIMENTAL PROCEDURES

Materials

CMP-[^14C]NeuAc (286 mCi/mmol) was purchased from Amersham. Fertile White Leghorn chicken eggs were obtained from a local poultry farm and incubated in a forced-draft incubator at 37 °C under a humidified atmosphere, until the desired developmental stage was reached according to Hamburger and Hamilton (13) . NCAM was purified from stage 37-38, 40, and 45 embryonic chick brains by immunoaffinity chromatography using Sepharose conjugated with an anti-chicken NCAM monoclonal antibody (5e) that recognizes all NCAM isoforms(14) . To prepare NCAM without PSA, the affinity-purified protein was enzymatically digested overnight at 4 °C with endoneuraminidase N (Endo N), which specifically cleaves alpha2,8-linked polysialic acid chains(15) . Endo N-treated NCAM was then separated from the enzyme by immunoadsorption (overnight, 4 °C) to 5e-conjugated Sepharose, washing 10 times with 1 ml with 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1% Nonidet P-40, 1 mM EDTA, and 5 times with 1 ml of 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% Nonidet P-40, and 1 mM EDTA, and elution with 50 mM diethylamine, pH 11.5 containing 0.1% Nonidet P-40. Ultraspec RNA isolation solution was purchased from Biotecx (Houston, TX). Oligonucleotides were purchased from DNA International. pCRScript plasmid was purchased from Stratagene (La Jolla, CA). Eukaryotic plasmids encoding intact chicken NCAM-180 (pCMV-chNCAM) and NCAM lacking its intracellular domain were constructed as described previously(10) . Lipofectamine and Geneticin was purchased from Life Technologies Inc. The AtT20 cell line was purchased from ATCC (Rockville, MD).

Preparation of an Enriched Polysialyltransferase-containing Fraction

All procedures were carried out at 4 °C. Embryonic chick brains from various developmental stages were homogenized with a Teflon-glass homogenizer in 10 volumes of 0.32 M sucrose in 10 mM MES buffer, pH 6.0, containing 1 mM EDTA, 1 mM PMSF, and 4.6 units/liter aprotinin. The homogenates were centrifuged at 1,000 g for 10 min, and the supernatant was further centrifuged at 17,000 g for 30 min. The pellet was resuspended in two volumes of 10 mM MES buffer, pH 6.0, containing 1 mM EDTA, 1 mM PMSF, 4.6 units/liter aprotinin, and 1% Nonidet P-40. The suspension was stirred for 30 min and then centrifuged at 17,000 g for 30 min. The supernatant obtained was used as the enzyme source. To stabilize the enzyme, glycerol was added to the supernatant to a final concentration of 30%.

Polysialyltransferase Assay

The reaction solutions at pH 6.0 contained the following components in a total volume of 50 µl: 10 µg of NCAM, 20 mM CMP-NeuAc (2.8 10^5 disintegrations/min), 100 mM MES buffer, 20 mM MnCl(2), 2.5 mM ATP, and the enzyme preparation. The final concentration of Nonidet P-40 was 0.4%. After incubation for 3 h at 37 °C, the reaction was terminated by addition of EDTA to a final concentration of 50 mM. To determine whether the incorporation of [^14C]NeuAc into NCAM was in the form of alpha2,8-linked polysialic acid, samples were divided into 2 aliquots, Endo N was added to one of them, and both aliquots were incubated for 1 h at 37 °C. Thereafter, the samples were spotted onto 2.5-cm Whatmann DE81 paper discs, rinsed three times in 20 mM MES buffer, pH 6.0, containing 250 mM NaCl, rinsed once in 95% ethanol, and air-dried. The radioactivity in the discs was measured using a scintillation counter. The difference in the radioactivity measured between control samples and Endo N-treated samples was taken as a measure of the amount of sialic acid that had been enzymatically incorporated into alpha2,8-linked polysialic acid chains.

SDS-PAGE and Immunoblots

Tissue samples were prepared as described in the preparation of the enzyme and extracted with Nonidet P-40. The samples were divided in 2 aliquots, one being treated with Endo N, and both aliquots were incubated for 30 min at 37 °C. After addition of Laemmli sample buffer and heating at 80 °C for 5 min, the samples were loaded on a 7% SDS-polyacrylamide gel. After electrophoresis, proteins were blotted onto nitrocellulose filters, and the remaining protein-binding capacity of the nitrocellulose filters was blocked by incubation with Blotto (Tris-HCl buffer, pH 8, containing 50 g/liter of nonfat dry milk) for 1 h. The blots were incubated overnight at 4 °C with a rabbit polyclonal anti-chicken NCAM antibody, rinsed with Tris-buffered saline containing 0.1% Tween 20, and incubated with horseradish peroxidase-conjugated anti-rabbit IgG. The peroxidase product was developed either with 0.05% 4-chloronaphthol and 0.01% H(2)O(2) or with the enhanced chemiluminescence method (ECL kit; Amersham); luminescent blots were exposed to Hyperfilm-ECL (Amersham) for between 30 s and 10 min. Relative quantitation of the amount of NCAM isoforms in the blot was carried out by densitometric analysis using a Shimadzu CS-930 TLC scanner.

Pulse Labeling

Cerebella or tecta were dissected from chick embryos at various developmental stages, chopped into small pieces (<1 mm^3) with a razor blade, collected in Dulbecco's modified Eagle's medium-HEPES culture medium without methionine, and incubated for 30 min at 37 °C. The medium was then replaced with the same medium containing 300 µCi/ml of L[S]methionine (Amersham, specific activity 1000 Ci/mmol) and pulsed at 37 °C for 60-90 min, followed by several washes with ice-cold culture medium containing 3 mM non-radioactive methionine. The label was then chased for 4 h at 37 °C in medium containing 3 mM non-radioactive methionine, and the tissue homogenized by sonication in extraction buffer (25 mM HEPES, pH 7.6, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 4.5 units/liter aprotinin), centrifuged at 100,000 g for 30 min in an airfuge, and the supernatant adsorbed overnight at 4 °C with anti-chicken NCAM antibody (5e) conjugated to Sepharose beads (CNBr-activated, Pharmacia). The immunobeads were then washed three times with a washing buffer (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1% Nonidet P-40, 1 mM EDTA), 3 times with a second washing buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS), and resuspended in extraction buffer. Each sample was split into 2 aliquots, Endo N added to one of them, and both aliquots were incubated for 1 h at 37 °C. The same amount of radioactivity from each sample was then loaded on a 7% SDS-polyacrylamide gel. After the electrophoresis, the gel was fixed for 30 min in propanol/water/acetic acid (25:65:10) and incubated for 30 min in Amplify (Amersham) before drying. Fluorographs were obtained by suitable exposure of preflashed Hyperfilm-MP (Amersham) at -70 °C with an intensifying screen.

Evaluation of Stage-specific VASE Expression

Total RNA was isolated from chicken embryo brain tissues at various developmental stages. This RNA was used in RT-PCR reactions using the GeneAmp Kit (Perkin Elmer). Primers sequences utilized in this reaction were: 5`- AAA CCC AAA ATC ACA TAT GTG-3` (forward) and 5`-T GGG AGC ATA CTG CAC-3` (reverse).

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.

Cloning of Chicken VASE

Chicken cDNA containing the VASE sequence was amplified using RT-PCR using the GeneAmp Kit (Perkin Elmer). Total RNA was derived from E17 chicken brain using Ultraspec reagent. Primer sequences were: 5`-AAA CCC AAA ATC ACA TAT GTG-3` (forward) and 5`-ATT TTC AGA GTC TGG TGT-3` (reverse).

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).

Analysis of Polysialylation State of Chicken NCAM Expressed in AtT20 Cells

Plasmids encoding chicken NCAM containing or lacking the VASE sequence were transfected into AtT20 cells using Lipofectamine (Life Technologies, Inc.). Geneticin-resistant cells were grown in Dulbecco's modified Eagle's medium/F12 +10% fetal bovine serum at 37 °C under 5% CO(2) in 10-cm tissue culture plates. Cells were transfected with 10 µg of plasmid DNAs using Lipofectamine. Twenty-four h after introduction of lipidbulletDNA complexes, medium was removed, and cells were grown for 2 days in normal culture medium. Cells were then passaged 1:1 and stably transfected cells were selected using 200 µg/ml (active) Geneticin. The stably transfected cell population was expanded for use in subsequent analysis.

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.


RESULTS

Polysialyltransferase Activity Assay

To evaluate quantitatively the role of PST in the developmental regulation of NCAM polysialylation, an assay was developed to measure the incorporation of [^14C]NeuAc into alpha2,8-linked PSA chains on exogenous NCAM purified from embryonic brain. The removal of the incorporated radioactivity by Endo N treatment was used to identify the specific incorporation of [^14C]NeuAc into alpha2,8-linked PSA chains. Short chains of PSA, as might be found on endogenous NCAM from more mature tissues, might not be susceptible to Endo N(16) . However, the exogenous NCAM substrate has longer chains of PSA, and thus the incorporation of label, after correction for endogenous incorporation, should be completely sensitive to Endo N.

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 alpha2,8-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.



Properties of the PST Activity

When membrane proteins extracted with Nonidet P-40 from embryonic chick brain were used as the enzyme source, PST activity was highest at pH 6.0 (Fig. 2A). EDTA (50 mM) inhibited PST activity completely (data not shown), suggesting a requirement for divalent cations. Several metal ions were tested, the highest activity being obtained detected with 20 mM Mn (Fig. 2C), with Mg, Ca, and Co being partially effective, and Zn and Cu having no effect (Fig. 2B).


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.



Developmental Regulation of PSA Expression and PST Activity in Chick Brain Tissues

To study the relationship between PSA expression and PST activity, the pattern of regulated tissue expression of PSA during embryonic development was compared with that of PST activity (Fig. 4). The expression of PSA was evaluated by SDS-PAGE immunoblot analysis using a polyclonal anti-chicken NCAM antibody. Removal by Endo N treatment of polydisperse material with low electrophoretic mobility (that migrating above the 200 kDa standard), together with the appearance or intensification of distinct higher mobility polypeptide bands characteristic of NCAM polypeptide isoforms, was taken as an indicator of the presence of PSA. As shown in Fig. 4A, from stage 30 to stage 37 all of the NCAM-140- and NCAM-180-kDa isoforms were polysialylated, and no significant developmental change in the amount of PSA was detected in whole brain. At stage 40 a detectable decrease in PSA occurred, as indicated by the higher mobility of the polydisperse material and the presence of NCAM-140 and NCAM-180 bands prior to Endo N treatment. These changes were more pronounced by stage 45, suggesting that polysialylation of NCAM may have ceased, with only residual PSA being detected.


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 [^14C]sialic acid incorporated into exogenous NCAM varied with respect to developmental stage. For brain at stage 30, 77% of incorporated [^14C]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 [^14C]sialic acid into NCAM in non-PSA forms.

Developmental Regulation of VASE Expression and Its Effect on NCAM Polysialylation

Previous reports have implicated Ig4 in NCAM polysialylation (10) and have noted an overall correlation between the alternative splicing of the VASE exon into the fourth Ig domain of NCAM and the down-regulation of PSA(12, 17) . In order to evaluate this relationship in more detail, the relative proportion of NCAM mRNA containing the VASE sequence was determined in whole brain, cerebellum, and tectum at various stages of embryonic development using RT-PCR (Fig. 6A). In both tectum and cerebellum, the appearance of VASE was closely correlated with down-regulation of PSA. For tectum, VASE was first faintly detectable at stage 37, with a higher level of expression at stage 40, thus being approximately simultaneous with down-regulation of PSA (Fig. 6). For cerebellum, VASE expression was first detectable at stage 40, the same time that a decrease in the amount of PSA on NCAM could be identified. However, for whole brain the expression of VASE did not precisely follow down-regulation of PSA. That is, VASE was first detectable at stage 40, with higher levels of expression detected at stage 45, whereas down-regulation of PSA occurred slightly earlier (Fig. 4).


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).


DISCUSSION

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. (^2)

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.^2 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 [^14C]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 alpha2,6-linkage instead of the alpha2,3-linkage, then the PST would not be able to elongate that chain. In fact, it has been reported the expression of a beta-galactoside alpha2,6-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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HD18369 and EY06107 (to U. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Training Grant HD07104.

To whom correspondence should be addressed: Dept. of Genetics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4955. Tel.: 216-368-2428; Fax: 216-368-3182; uxr{at}po.cwru.edu.

(^1)
The abbreviations used are: PSA, polysialic acid; NCAM, neural cell adhesion molecule; Endo N, endoneuraminidase-N, an alpha 2, 8-polysialic acid-specific neuraminidase; PST, polysialyltransferase; VASE, the variable alternatively spliced exon between NCAM exons 7 and 8; bp, base pair(s); RT-PCR, reverse transcriptase-polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride.

(^2)
J. L. Brusés and U. Rutishauser, unpublished results.


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