THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 23, Issue of October 15, pp. 12695-12699 1985 Printed in C.S.A.

CMP-NeuNAc:Poly-a-2,8-sialosyl Sialyltransferase and the Biosynthesis of Polysialosyl Units in Neural Cell Adhesion Molecules*

(Received for publication, April 15, 1985)

Ronald D. McCoy, Eric R. VimrS, and Frederic A. Troy4 From the Department of Biological Chemistry, University of California School of Medicine, Davis, California 95616

Prokaryotic derived probes that specifically recog- nize a-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expres- sion of these unique carbohydrate moieties in devel- oping neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sei. U. S. A. 81, 1971-1975). These poly- sialosyl units cap N-linked oligosaccharides of the com- plex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltrans- ferase that catalyzes the incorporation of [14C]N- acetylneuraminic acid ([14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-’ h-l were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuramini- dase specific for polysialic acid was used to demon- strate that at least 20-30% of the [14C]NeuNAc was incorporated into a-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material con- sisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained a- 2,Slinked polysialosyl chains, thus showing that N- CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indi- cating that the poly-a-2,8-sialosyl sialyltransferase ac- tivity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-a-2,8-sialosyl sialyl- transferase and the trivial name poly-a-2,8-sialosyl sialyltransferase be adopted.

The central involvement of a class of high molecular weight surface sialoglycoproteins that function in neural cell-cell interaction and brain development has been described by several laboratories (2-6). These molecules, designated neural cell adhesion molecule (N-CAM1), D2-cell adhesion molecule

* This investigation was supported by Research Grant AI-09352 from the National Institutes of Health. A preliminary report of this work has been presented (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Present address: Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL 61801.

To whom correspondence should be addressed. The abbreviations used are: N-CAM, neural cell adhesion mole-

cule (E, embryonic; A, adult); NeuNAc, N-acetylneuraminic acid; DP, degree of polymerization; D2-CAM, D2-cell adhesion molecule; MES, 4-morpholineethanesulfonic acid.

(D2-CAM), or brain cell surface protein-2, appear to be struc- turally related if not identical (5, 7, 8). An interesting struc- tural feature of these molecules emerged when it was shown that the embryonic (E) form of N-CAM contained a high sialic acid content that underwent a postnatal conversion to a low sialic acid content in the adult (A) (9-11). The devel- opmental reduction in sialic acid was postulated to modulate cell-cell adhesive properties of neuronal cells and to mediate their specific organization into adult brain tissue (reviewed in Refs. 12 and 13). The fact that in a vesicle aggregation assay the binding efficiency of the A-form was 3.5-fold greater than the E-form and that exoneuraminidase treatment of the E- form increased its binding efficiency 4-fold (14) supported this supposition.

Because sialic acid rarely exists internally to form oligo- or polysialosyl chains, we became interested in the possibility that the high sialic acid content in the E-form might be oligomerized. If so, we reasoned that this would then permit us to extend our studies to delineate the molecular mechanism regulating synthesis and surface expression of the polysialic acid capsule in Escherichia coli K1 to a eukaryotic system. The K1 capsular antigen in E. coli K-235 is a well-charac- terized polysialic acid that contains about 200 sialyl residues joined by a-2,8-ketosidic linkages (15). The capsule can render cells resistant to phagocytosis and confer on some cells the ability to invade and colonize the meninges of neonates (16- 18). Biogenesis of the membranous sialyltransferase complex and the synthesis, intramembranous translocation, and as- sembly of the K1 capsule have remained a major focus of our research efforts (19-20). The first evidence showing that the E-form of N-CAM contained internal sialic acid residues on N-linked oligosaccharides was provided by Finne (10).

Knowledge is rapidly accumulating regarding the molecular topography, surface orientation, binding domain, and carbo- hydrate attachment sites in N-CAM (12, 13,21). In addition, recent changes including post-translational glycosylation, sul- fation, phosphorylation, and intracellular transport of D2- CAM have been reported by Lyles et aE. (8). The interesting possibility that the function of D2-CAM may be develop- mentally regulated in rat forebrain explant cultures by changes in synthesis of the different polypeptide chains and by glycosylation and sulfation will be important to follow up with in vitro studies. In contrast, nothing is known about the biosynthesis or factors regulating the temporal expression of the polysialosyl units in N-CAM or in the mechanism that mediates the developmentally regulated reduction of poly- sialic acid during the embryonic to adult conversion. In antic- ipation of studies to examine these aspects in developing rat and chick brains, we developed three prokaryotic derived probes that are specific for either recognizing or synthesizing polysialic acid containing a-2,8-ketosidic linkages. We used these probes in combination with structural studies to prove

12695

12696 Biosynthesis of Polysialosyl Carbohydrate Units

the presence of polysialosyl carbohydrate chains in mem- branes of embyronic chick and rat brains (22). For reasons discussed in our earlier studies (22), we believe that polysialic acid detected in brain membranes is associated with N-CAM. One of the probes described was a bacteriophage-derived endoneuraminidase specific for a-2,8-linked polysialosyl units.

In the present investigation, we have studied the biosyn- thesis of polysialosyl units in fetal rat brain. We have used the endoneuraminidase to quantitate the amount of sialic acid incorporated specifically into polysialic acid containing a-2,8- ketosidic linkages. The results show that a membrane-asso- ciated CMP-NeuNAc:poly-a-2,8-sialosyl sialyltransferase' from 20-day-old fetal rat brains catalyzes the polysialylation of a high molecular weight protein that appears to be N-CAM. Purified chick N-CAM served as an exogenous acceptor for sialylation in uitro. Adult rat brain membranes do not appear to catalyze similar polysialylation, suggesting that the poly- a-2,8-sialosyl sialyltransferase is restricted to an early stage of development.

EXPERIMENTAL PROCEDURES

Membrane Preparation-Fetal rats were killed in utero at 20 days of gestation by CO, asphyxiation. Ten brains were quickly removed and homogenized in 5 ml of ice-cold MET buffer (50 mM MES, 1 mM dithiothreitol, pH 6.0) using a Dounce homogenizer (B pestle) at 7- 10 strokes. Newborn rats were killed by decapitation within 24 h of birth, and their brains were processed identically to the fetal brains. Adult rats, greater than 3-months old, were killed by cervical dislo- cation, and the brains were processed as described above. Golgi- enriched membrane fractions were prepared essentially as described by Morri. (23). MET buffer was used in all steps to isolate the Golgi- enriched fraction.

Poly-a-2,8-sialosyl Sialyltransferase Assay-Incorporation of ["C] NeuNAc from CMP-['4C]NeuNAc was determined by incubating membrane fractions in MET buffer at 33 "C as previously described (24). To determine the amount of [14C]NeuNAc incorporated into polymeric products, samples were removed at various times and spotted onto Whatman 3MM paper. The papers were subjected to descending chromatography in ethanol, 1 M ammonium acetate, pH 7.5 (73). Radioactivity remaining at the origin was quantitated by liquid scintillation counting. Specific activity was calculated after protein was determined by the modified Lowry protein assay (25).

Endoneuraminidase-The endoneuraminidase specific for cleaving a-2,8-linked polysialic acid was obtained from a bacteriophage, des- ignated KlF, as previously described (22). The "soluble" form of the enzyme in phage lysates of E. coli K1 was purified about 120-fold by standard chromatographic procedures on DEAE-Sephadex and Se- phacryl S-200.3 Endoneuraminidase digestions were carried out in incubation mixtures containing 23 units of enzyme, unless otherwise stated. One unit of enzyme degraded 1% of the U-"C-labeled poly- sialic acid substrate in 1 min at 37 "C. The radiolabeled substrate was prepared as described (26).

Determination of the Amount of P'CINeuNAc Incorporated into a- 2&Linked PolysiuEic Acid-The per cent of [14C]NeuNAc incorpo- rated into polymeric products that was in a-2,8-linked polysialic acid was assessed by determining its susceptibility to depolymerization by the endoneuraminidase. After completion of the sialyltransferase reaction, membranes containing radiolabeled sialyl polymers were divided into two aliquots. One aliquot received endoneuraminidase, while the other received an equal volume of buffer. After incubation at 37 "C, samples were removed at different times, spotted on What- man 3MM, and chromatographed in Et0H:ammonium acetate as described above. The difference in [l'C]NeuNAc remaining at the origin between control samples and the endoneuraminidase-treated samples represented the amount of sialic acid in a-2,8-linked poly-

* We recommend that the enzyme be designated CMP-NeuNAc: poly-a-2,8-sialosyl sialyltransferase and that the trivial name poly- a-2,8-sialosyl sialyltransferase be adopted. The possible involvement of a sialyl lipid-linked intermediate in this reaction has not been investigated.

B. L. Bassler, E. R. Vimr, and F. A. Troy, unpublished results.

sialic acid. The product of the endoneuraminidase (DP 3) is chro- matographically mobile under these conditions.

Characterization of f4C]NeuNAc-labeled Sialyl Oligomers Synthe- sized in Vitro and Released from Brain Membranes by Endoneura- minidase-Radiolabeled sialyl oligomers synthesized in vitro and released by treatment with endoneuraminidase were characterized by DEAE-Sephadex A-25 chromatography. A modification of the pro- cedure of Nomato et al. (27), as previously described, (22) was carried out. Authentic oligomers of sialic acid were prepared from colominic acid (Sigma) after hydrolysis at pH 2.0 and 80 "C for 1 h by ion- exchange chromatography (22).

Isolation of Embryonic Chick Brain N-CAM-N-CAM was purified from 100 14-day-old embryonic chick brains as described by Hoffman et al. (28). Purification was carried out up to the octyl-agarose chromatography. The presence of N-CAM was confirmed at each step by monitoring for polysialic acid by Western blot analysis using the antibody against a-2,b-linked polysialic acid, as previously de- scribed (22). This antiserum, designated H.46, was graciously pro- vided by Dr. John Robbins (National Institutes of Health, Bethesda, MD) .

RESULTS

Distribution of Poly-a-2,8-sialosyl Sialyltransferase in Mem- brane Fractions from Fetal Rat Brains-Poly-a-2,8-sialosyl sialyltransferase in fetal rat brain was enriched in a membrane fraction (Table I). Using a standard method to enrich in Golgi apparatus (23), the specific activity was nearly 3-fold higher in this fraction (84 pmol of NeuNAc/mg of protein/h) than in the homogenate (30 pmol/mg of protein/h). Approximately two-thirds of the sialyltransferase activity were recovered in the Golgi-enriched fraction. The data in Table I also show that about one-third of the [14C]NeuNAc incorporated into polymer was present as a-2,8-linked polysialic acid. Our con- clusion regarding the polysialic acid nature of the product was based on the susceptibility of the radiolabeled sialyl polymers to depolymerization with an endoneuraminidase specific for cleaving poly-a-2,8-linked sialosyl units (22). A membrane fraction isolated from an adult rat brain by the identical

TABLE I Localization of CMP-NeuNAc.poly-a-2,8-sialosyl sialyltransferase in

rat brain membranes Sialyltransferase activityb NeuNAc

in

Homogenate Total membranes

Supernatant 1 Membrane pellet

Golgi-enriched

(P-1)

(P-2)

(P-3)

Golgi-enriched from Adult

pmol NeuNAc % pmol NeuNAcj %

6300 100 30 33 4400 70 31 11

<50 <1 <5 0 100 2 26 30

4200 67 84 2 1

incorporated mg proteinlh

960 NDd 16 0

a Refers to fraction designation as described in by Morri. (23). * Brains from 13 newborn rats were homogenized and fractionated

as described under "Experimental Procedures." The adult rat used in this experiment was >3 months old and was the mother of the newborn rats. Sialyltransferase activity was determined in standard incubation mixtures containing 112 nmol of CMP-[14C]NeuNAc (7.9 X lo3 dpm nmol-*) in 0.32 ml of MET buffer. Protein content was about 3.9 mg/incubation mixture.

e Designates the per cent [14C]NeuNAc in polymeric products that was sensitive to digestion by the endoneuraminidase specific for a- 2,8-linked polysialosyl units, as described under "Experimental Pro- cedures."

ND, not determined.

Biosynthesis of Polysialosyl Carbohydrate Units

procedure contained only about 20% of the sialyltransferase activity of fetal brain (Table I). Importantly, no detectable [ 14C]NeuNAc incorporated by the adult brain was in polysialic acid, since the polymeric products were not sensitive to en- doneuraminidase treatment (Table I). We conclude from this result that the poly-a-2,8-sialosyl sialyltransferase responsi- ble for catalyzing polysialylation appears restricted to an early developmental epoch.

Effect of Endoneuraminidase on the Sodium Dodecyl Sul- fate-Polyacrylamide Gel Electrophoretic Profile of /'4CJPoly- sialic Acid Synthesized Endogenously by Fetal Rat Brain-To determine the molecular weight profile of ["C]NeuNAc in- corporated into polysialic acid by fetal rat brains, Golgi- enriched membranes were allowed to incorporate maximal levels of [ 14C]NeuNAc into endogenous acceptor molecules (see below). The radiolabeled membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis be- fore and after treatment with endoneuraminidase. As shown in Fig. 1, the major peak of radioactivity existed as a broad band suggestive of molecular weight heterogeneity ranging from about 120,000 to 240,000. N-CAM shows a similar mo- lecular weight heterogeneity (8, ll, 13, 22). Control experi- ments (Fig. 1, inset) showed that immunoreactivity to anti- polysialosyl antibodies was in the same high molecular weight region as the 14C-radiolabeled peak (lane I ) and that this immunoreactivity was abolished by pretreatment with endo- neuraminidase ( l a n e 2). We conclude from these results that glycoproteins containing the endoneuraminidase-sensitive ['4C]polysialic acids synthesized by fetal rat brains are poly- disperse in size and have molecular weight values expected for N-CAM.

Stimulation of Polysialic Acid Synthesis by the Exogenous Addition of N-CAM-Golgi-enriched sialyltransferase from fetal rat brain catalyzed the incorporation of [14C]NeuNAc from CMP-[14C]NeuNAc into endogenous acceptor molecules, as shown in Fig. 2 (bottom). In confirmation of the results described above, about 20-30% of the sialic acid incorporated was in polysialic acid, based on its sensitivity to treatment with endoneuraminidase (Fig. 2). At pH 6.0, 84 pmol of NeuNAc/mg of protein/h were incorporated. Polysialic acid synthesis was stimulated about %fold in these membranes by the addition of embryonic chick brain N-CAM as an exoge- nous acceptor (Fig. 2, top). These results also show that at least 60-70% of the product of this reaction was sensitive to endoneuraminidase, suggesting that N-CAM can serve as an exogenous acceptor for polysialylation in uitro. The relation- ship between the degree of oligo- or polysialylation of N-CAM and its efficacy as an acceptor is under investigation. Colom- inic acid, a homooligomer of sialic acid containing 10 units linked a-2,8 did not serve as an exogenous acceptor. This oligosialic acid is effective as an exogenous acceptor for the membranous sialyltransferase from E. coli K1 strains that synthesize a-2,S-linked sialyl polymers containing about 200 sialyl residues (26).

The endoneuraminidase did not remove all of the poly-a- 2,s-linked [I4C]NeuNAc from the exogenous N-CAM (Fig. 3) because the enzyme cannot cleave sialyl oligomers containing less than 3-5 residues (22, 29). After treating brain polysialo- syl glycopeptides with a similar endoneuraminidase, Finne and Makela (29) showed that 5 sialyl units still remained attached to the glycopeptide. For this reason, our results with both the endogenous and exogenous acceptors give only a minimum estimate of the per cent of sialic acid in poly-a-2,8 linkage.

Endoneuraminidase Treatment of Fetal Membranes Incu- bated with CMP-r4CJNeuNAc and Exogenous N-CAM Re-

XIO-3 I 2 Mr

FRONT DYE

I

12697 1 I I I

STACKING 200 116 92 66 45 31 22 14 GEL MOLECULAR WEIGHT x IO-^

FIG. 1. Effect of endoneuraminidase on the sodium dodecyl sulfate-polyacrylamide gel electrophoretic profile of ["C] polysialic acid synthesized endogenously by fetal rat brain. The Golgi-enriched fraction (10.5 mg of protein) isolated as described under "Experimental Procedures" was incubated with 280 nmol of CMP-['4C]NeuNAc (7.9 X lo3 dpm nmol") in 1.0 ml of MET buffer a t 33 "C. After 3 h, the membranes were sedimented (17,000 X g for 30 min), and residual substrate was removed by washing three times in MET buffer. Washed membranes in 0.9 ml of MET buffer were divided in equal aliquots. T o one aliquot was added 58 units of endoneuraminidase (A), and to the second was added an equal volume of MET buffer (0). Both samples were incubated a t 28 "C for 1.5 h and then sedimented a t 17,000 X g for 30 min and washed once. Membranes were then resuspended in 90 pl of Laemmli sample buffer (32). Ten pl of each sample containing about 300 pg of protein was electrophoresed in 5-15% polyacrylamide gradient gels (1.5 mm) and electrophoretically transferred to nitrocellulose, as described by Tow- bin et al. (33). Electroblotting was carried out a t 200 mA for 16 h. Immune blotting using the anti-polysialic acid antibody H.46 was carried out as previously described (22). Inset, lane 1 is the control sample that was not treated with endoneuraminidase before immune blotting, while lane 2 was pretreated with endoneuraminidase. The amount of ["CINeuNAc in polysialic acid was quantitated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the re- maining 80-pl portions of each sample by slicing the gel into 0.5 x 2.5-cm sections and determining the radioactivity by scintillation counting, as previously described (26).

leased 14C-Labeled Sialyl Oligomers-Structural confirmation that endoneuraminidase treatment of Golgi-enriched mem- branes incubated with CMP-["CINeuNAc and N-CAM re- leased oligomers of sialic acid is shown in Fig. 3. In this experiment, [I4C]NeuNAc from CMP-[14C]NeuNAc was in- corporated into fetal rat brain membranes in the presence of exogenous N-CAM, as described in the legend to Fig. 2. After washing to remove excess substrates, the membranes were treated exhaustively with endoneuraminidase. [ 14C]Sialyl ol- igomers were recovered after centrifugation and fractionated by chromatography on DEAE-Sephadex A-25 as previously described (22). As shown in Fig. 3, the endoneuraminidase- solubilized [I4C]sialyl oligomers eluted predominately with a

12698 Biosynthesis of Polysialosyl Carbohydrate Units

- I Plus ENDO

2 I f I - v) 40'A t h A L o - 20 40 '

rnin

FIG. 2. In vitro synthesis of polysialic acid in fetal rat brains is stimulated by the exogenous addition of N-CAM. Standard incubation mixtures containing 1.38 mg of Golgi-enriched membrane protein and 113 nmol of CMP-[14C]NeuNAc (15,000 cpm nmol") were incubated at 33 "C in the absence (H) and presence (A) of 66 pg of partially purified N-CAM prepared from 14-day-old chick embryo brains, as exogenous acceptor. After a 4-h incubation, the amount of [14C]NeuNAc incorporated into a-2,8-linked polysialic acid was determined by quantitating the amount of radiolabeled polymer sensitive to hydrolysis by endoneuraminidase (ENDO) (0) as de- scribed under "Experimental Procedures." The arrows denote the time at which 23 units of endoneuraminidase or buffer was added to each incubation mixture.

DP of 3. This was an expected result because we had previ- ously shown that the primary products of a limit endoneura- minidase digestion of rat brain polysialic acid were oligomers containing 3 or 4 sialyl units (22). Thus, these data support the conclusion that N-CAM can serve as an exogenous accep- tor for sialylation in vitro. Treatment of the sialyl trimer with exoneuraminidase quantitatively converted the label to [ 14C] NeuNAc, thus proving that the oligomer was composed of sialic acid.

DISCUSSION

We have described in vitro conditions that permit detection of a CMP-NeuNAc:poly-a-2,8-sialosyl sialyltransferase activ- ity in a membrane-enriched fraction from fetal rat brains (Table I). The enzyme catalyzes the incorporation of ["C] NeuNAc from CMP-[14C]NeuNAc into polymeric products. We exploited a bacteriophage-derived endoneuraminidase specific for cu-2,8-linked polysialosyl units to prove that at least 20-30% of the [14C]NeuNAc was incorporated into a- 2,8-linked polysialic acid. We believe the polysialosyl chains are associated with the carbohydrate units of N-CAM for three reasons. First, the endoneuraminidase-sensitive ["C] polysialic acid was membrane-bound, polydisperse in size, and showed molecular weight values expected for N-CAM (about 120,000-240,000). Second, immune blotting experiments em- ploying an anti-polysialosyl antibody showed immunoreactiv- ity with apparent molecular weight values of N-CAM. This immunoreactivity was abolished by endoneuraminidase treat- ment. Third, like N-CAM, the poly-cu-2,8-sialosyl sialyltrans- ferase catalyzing synthesis of these polysialosyl units was correlated with neural development. This conclusion was sub-

DPlO J.

20 X, 40 50 60 70 80 90 1 0 0

FRACTION NUMBER

FIG. 3. Endoneuraminidase treatment of brain membranes incubated with CMP-[14C]NeuNAc and N-CAM releases sialyl oligomers. Golgi-enriched membranes (15.6 mg of protein) from 20- day-old fetal rat brains were incubated with 0.96 mg of purified chick brain N-CAM in 1.0 mi of MET buffer containing 280 nmol of CMP- [14C]NeuNAc (7.9 X lo3 dpm nmol-l). After 6 h at 33 "C, membranes were sedimented (17,000 X g for 30 min), washed in 100 mM MES, ph 6.0, and treated with 23 units of endoneuraminidase at 33 "C. After 5.5 h, an additional 23 units of enzyme were added and incu- bated at 33 "C for 1 h. Endoneuraminidase-treated membranes were sedimented at 17,000 X g for 30 min. Oligomers of sialic acid released by the enzyme were lyophilized, resuspended in 0.35 ml of water, and fractionated on DEAE-Sephadex A-25 with a 0-0.4 M gradient of NaCl in 10 mM Tris, pH 7.6 (X). Authentic oligomers of sialic acid with DP of 2, 3, and 4 (3 mg each), and 10 (0.5 mg) were added as markers (A), and their elution volume was determined by the thio- barbituric acid method (34). A tracer amount of [3H]NeuNAc (0) was used to determine the elution position of sialic acid.

stantiated by the experiment shown in Fig. 2 which illustrates that a preparation of chick N-CAM can serve as an exogenous acceptor for the sialyltransferase in vitro. At least two-thirds of the product of this reaction were also sensitive to endoneu- raminidase. The [14C]sialyl oligomers solubilized from the membrane by the endoneuraminidase were isolated and shown to contain primarily 3 sialyl residues. Exoneuramini- dase quantitatively converted the labeled trimer to ["C] NeuNAc, thus establishing that the oligomer was composed of sialic acid.

Several key problems related to the biosynthesis of polysi- alosyl carbohydrate units in developing neural membranes remain to be investigated. First, the possible involvement of a sialyl lipid-linked intermediate in the poly-cu-2,8-sialosyl sialyltransferase reaction requires study. The involvement of sialylmonophosphorylundecaprenol as an intermediate in the synthesis of the polysialic acid capsule in E. coli K-235 and Neisseria meningitidis serogroup B has been documented (30, 31). Second, what regulates synthesis and surface expression of the polysialylated N-CAM is unknown. Also unknown are the recognition signals that may determine which N-aspara- ginyl-linked oligosaccharide chains on a glycoprotein are ac- ceptors for the poly-a-2,8-sialosyl sialyltransferase. Third, the mechanism regulating the developmental reduction of poly- sialic acid during the embryonic to adult conversion will need to be determined. Answers to these problems will be important for a more thorough understanding of how the polysialylated E-form of N-CAM may participate inregulating neural em- bryogenesis. The fact that adult rat brains contain substan- tially reduced levels of sialylated N-CAM and also appear to lack detectable levels of poly-~-2,8-sialosyl sialyltransferase activity (Table I) leads us to conclude that expression of this enzyme is restricted to an early stage in development. The relationship between the biosynthetic pathway and the mech-

Biosynthesis of Polysialosyl Carbohydrate Units 12699

anism regulating the developmental reduction in polysialosyl units during the E + A conversion will be important to understand. Our preliminary studies to investigate the effect of chick N-CAM as an exogenous acceptor in a homologous system (embryonic chick N-CAM plus embryonic chick brain membranes) may be relevant to this point. These studies indicate that under some conditions, the exogenous addition of N-CAM can inhibit the incorporation of [14C]NeuNAc into endogenous acceptors and that the inhibition appears to be specific for the poly-u-2,8-sialosyl sialyltransferase.' This sug- gests that the extent of polysialylation of N-CAM, or its state of aggregation (28), may determine its efficacy as an acceptor in the homologous system. This possibility is suggested be- cause no inhibition is observed in the heterologous system (embryonic chick N-CAM plus embryonic rat brain mem- branes), as shown in Fig. 2. Accessibility of the membranous sialyltransferase to N-CAM added exogenously may also be different between rat and chick membrane preparations. Thus, it will be important to investigate further this new aspect of N-asparaginyl-linked biosynthesis.

Acknowledgments-We thank Dr. Charles C. Sweeley for consul- tation regarding the enzyme nomenclature proposed in this paper. We are indebted to Linda Troy for excellent editorial and secretarial assistance in preparing this manuscript.

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