Expression of a Differentiation Antigen and Poly-N-acetyllactosaminyl 0-Glycans Directed by a Cloned Core 2 ~-1,6-N-Acetylglucosaminyltransferase*

California9.2037-Chinese hamster ovary (CHO) cells do not contain detectable amounts of core 2 p-l,6-N-acetylglucosaminyl- transferase, C2GnT, and thus lack various modifications in their branched 0-linked oligosaccharides. In the pre- sent study, the 0-linked oligosaccharides and the occur-rence of a differentiation antigen were analyzed in CHO cells stably transfected with cDNA encoding human leukosialin alone (CHO-leu) or with cDNAs encoding both leukosialin and C2GnT (CHO-leu-CZGnT). The analysis of 0-glycans, released from [sH]glu-cosamine-labeled cells, revealed that CHO-leu cells synthesize 0-glycans with a Galpl-rSGaINAc backbone, whereas CHO-leu-C2GnT cells synthesize in addition 0-glycans with a Galpl+3(Gal~1+4GlcNAc~l+6)GalNAc backbone. Moreover, CHO-leuC2GnT cells express poly-N-acetyllactosaminyl extensions from the GlcNAcpl+6 branch in 0-glycans, while CHO-leu cells express no detectable amount of poly-N-acetyllactosaminyl 0-glycans. It was also demonstrated that leukosialin in CHO-leuX2GnT the monoclonal the not In the expression cloning using the a of cDNA leukosialin. These results indicate that C2GnT determines the expression of poly-N-acetyllactosamines in 0-glycans and together with leukosialin, an onco-differentiation anti- gen recognized by the T305 antibody.

Chinese hamster ovary (CHO) cells do not contain detectable amounts of core 2 p-l,6-N-acetylglucosaminyltransferase, C2GnT, and thus lack various modifications in their branched 0-linked oligosaccharides. In the present study, the 0-linked oligosaccharides and the occurrence of a differentiation antigen were analyzed in CHO cells stably transfected with cDNA encoding human leukosialin alone (CHO-leu) or with cDNAs encoding both leukosialin and C2GnT (CHO-leu-CZGnT).
The analysis of 0-glycans, released from [sH]glucosamine-labeled cells, revealed that CHO-leu cells synthesize 0-glycans with a Galpl-rSGaINAc backbone, whereas CHO-leu-C2GnT cells synthesize in addition 0glycans with a Galpl+3(Gal~1+4GlcNAc~l+6)GalNAc backbone. Moreover, CHO-leuC2GnT cells express poly-N-acetyllactosaminyl extensions from the GlcNAcpl+6 branch in 0-glycans, while CHO-leu cells express no detectable amount of poly-N-acetyllactosaminyl 0-glycans. It was also demonstrated that leukosialin in CHO-leuX2GnT cells is recognized by the T305 monoclonal antibody, while the same antibody did not react at all with CHO-leu cells. In addition, the transient expression cloning scheme using the T305 monoclonal antibody as a selectin marker and COS-1 cells, which endogenously express C2GnT as recipient cells, resulted in the isolation of cDNA encoding leukosialin.
These results indicate that C2GnT determines the expression of poly-N-acetyllactosamines in 0-glycans and together with leukosialin, an onco-differentiation antigen recognized by the T305 antibody.
Leukosialin (CD43) is a major cell surface sialyoglycoprotein of T-lymphocytes, granulocytes, monocytes, and platelets (1,2). I t is also expressed on hematopoietic stem cells but is absent from erythrocytes (3,4). Resting B-lymphocytes do not express leukosialin, but antibody-forming B-lymphocytes and myeloma cells express leukosialin (5). It was shown that the addition of anti-leukosialin antibody apparently activates lymphocytes (6) and monocytes (7,8), due to the modulation of phosphorylation through protein kinase C (9).
The amino acid sequence based on isolated cDNAs and the chemical composition of purified leukosialin indicate that its (NeuNAca2-3Galpl-.4GlcNAc~1-6)GalNAc. Similarly, leukocytes from patients with leukemia (14) and immunodeficient syndromes such as Wiskott-Aldrich (15,16) and AIDS (14) express the hexasaccharides in leukosialin. The hexasaccharides are formed only when core 2 p-1,6-N-acetylglucosaminyltransferase, C2GnT,l is present. Leukosialin having the hexasaccharide exhibit a larger molecular weight than leukosialin carrying the tetrasaccharide. The high molecular weight form can be selectively detected with the monoclonal antibody T305 (15,16), which was originally raised using T-lymphocytic leukemia cells a s immunogenes (17).
It was shown recently that a soluble form of leukosialin is present in plasma (18). The carbohydrate composition of this soluble leukosialin is the same as those found on granulocytes (19) and activated T-lymphocytes (131, and this soluble form was shown to be the result of proteolytic shedding from those cells (20,21). The shedding of leukosialin from granulocytes is necessary for spreading of activated granulocytes and serum albumin in the plasma apparently counteracts the shedding (20). Leukosialin was also shown to interfere with the adhesion of lymphocytes (221, suggesting that shedding of leukosialin from activated lymphocytes may play some role in T-and Blymphocyte interaction. The pl-6 branching by C2GnT is an important control point in the biosynthesis of polylactosamine in 0-glycans provided that the /3-1,4-galactosyl-and p-1,3-Nacetylglucosaminyltransferases essential for polylactosamine synthesis are also present (23,24). This is particularly noteworthy since these polylactosaminoglycans may carry oligosaccharide determinants involved in cell-cell adhesion, e.g. the sialyl Lex, NeuNAca2+3Gal~1-+4(Fucal-3)GlcNAcp1~R structure (25)(26)(27). Since granulocytes have been shown to contain 0-glycans with sialyl Le" termini (191, it is likely that the leukosialin shedded from these cells also contains this structure. Altogether this suggests that the 0-glycans attached to leukosialin might play a role in cell-cell interaction of granulocytes and lymphocytes. Recently, we have isolated a cDNA clone from a HL-60 cDNA library that encodes C2GnT (28). In order to understand the The abbreviations used are: C2GnT, UDP-GlcNAc: GalP1-3GalNAc-R (GlcNAc to GalNAc) ~-l,6-N-acetylglucosaminyltransferase; CHO-leu and CHO-leu.C2GnT, Chinese hamster ovary cells stably expressing leukosialin alone and leukosialin and C2GnT, respectively; PBS, phosphate-buffered saline. 4473 effect of C2GnT on the 0-linked oligosaccharides in general and on leukosialin in particular, we have expressed this enzyme, together with human leukosialin, in Chinese hamster ovary (CHO) cells that were shown to lack C2GnT (28,29). Our results indicate that the unique differentiation antigen detected by the T305 monoclonal antibody is determined by the expression of both leukosialin and C2GnT. Moreover, the expression of C2GnT results in the formation of poly-N-acetyllactosamine extensions in 0-glycans.

EXPERIMENTAL PROCEDURES
Plasmids-pcDNAI-C2GnT, encoding full-length C2GnT, was isolated from a HL-60 cDNA library as described previously (28). The plasmid vector pZIPNEO-leu was constructed by introducing the EcoRI insert of Peer-3 cDNA (lo), which contains the complete coding sequence for human leukosialin, into the unique EcoRI site of plasmid pZIPNEO.
Establishment of CHO Cells Stably Expressing C2GnT-CHODG44 cells were transfected either with pZIPNEO-leu alone or with pZIP-NEO-leu and pcDNAI-CaGnT using a calcium phosphate procedure (30) and subsequently selected for G418 resistance. Clonal cell lines were obtained by limiting dilution, and each clonal cell line was examined by immunofluorescence using anti-leukosialin antibodies or T305 monoclonal antibody. Clonal cell lines expressing either a significant amount of leukosialin alone (CHO-leu) or both leukosialin and T305 (CHO-leuC2GnT) were selected.
Expression of Leukosialin a n d the T305 Antigen in CHO-leu a n d CHO-leu-C2GnT Cells-Expression of leukosialin and the T305 antigen was examined by both immunofluorescence and Western blotting. CHOleu and CHO-leu.C2GnT were cultured on glass coverslips and then fixed with 0.05% formaldehyde in PBS. The cells were stained with rabbit anti-leukosialin antibodies (4), followed by fluorescein-conjugated (Fab)z fragment of goat anti-(rabbit IgG). The cells were separately stained with mouse T305 monoclonal antibody (IgG,) followed by fluorescein-conjugated (Fab)2' fragment of goat anti-(mouse IgG). The cells were then examined under a Zeiss Axioplan microscope as described previously (31).
For Western blotting analysis, the different CHO cells were lysed in PBS containing 1% Nonidet P-40 and protease inhibitors as described (32) and the supernatants were recovered by brief centrifugation. After boiling in the gel loading buffer, SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (33) and proteins were electroblotted onto nitrocellulose membranes. The Western blots were then incubated with mouse monoclonal anti-human leukosialin antibody (Leu22, Beckton Dickinson) or mouse monoclonal T305 antibody followed by incubation with colloidal gold conjugate goat anti-mouse IgG (Bio-Rad). Since the staining by T305 was not strong enough, the signal was further enhanced using the procedure recommended by Bio-Rad. Rabbit anti-human leukosialin antibodies (4) and mouse T305 monoclonal antibody (17) were kindly donated by Drs. Sven Carlsson (University of Umeb, Sweden) and Robert Fox (Scripps Research Foundation), respectively.
Analysis of 0-Glycans from CHO-leu and CHO-leuC2GnT Cells-The CHO cells (-1 x lo7 cells) were metabolically labeled with [3H]glucosamine (10 pCi/ml) in a-minimal essential medium supplemented with 10% fetal calf serum for 24 h. The labeled cells containing approximately 6-7 x lo6 cpm were harvested with a rubber policeman, washed with PBS, and collected by Centrifugation. The cell pellets were then extracted with PBS containing 1% Nonidet P-40 and protease inhibitors (32). The supernatants, obtained after brief centrifugation, were then incubated with rabbit anti-leukosialin antibodies followed by Staphylococus aureus protein A conjugated to Sepharose as described previously (32). The protein A-Sepharose was recovered by centrifugation and the supernatant aRer centrifugation represented glycoproteins from the total lysates (except for the majority of leukosialin). Leukosialin was recovered from the protein A-Sepharose by boiling in 0.1% SDS.
The samples of leukosialin and the total cell glycoproteins were digested with Pronase for 24 h at 60 "C in a toluene atmosphere. The digest was then boiled for 5 min to inactivate the remaining Pronase activity. After centrifugation, the supernatant was applied to a column (1.0 x 110 cm) of Sephadex G-50 (superfine) equilibrated with 0.1 M NH,HCO,. High molecular weight glycopeptides were pooled, desalted, and subjected to p-elimination in 0.05 M NaOH, 1 M NaBH, at 45 "C for 16 h. The released 0-linked oligosaccharides were separated from the remaining glycopeptides by gel filtration using the same Sephadex G-50 column.
Expression Cloning ofLeukosialin by Using T305 Monoclonal Antibody-The monoclonal antibody T305 was used as a selection marker in a transient expression cloning scheme (37) in order to isolate a cDNA determining the T305 phenotype. COS-1 cells were thus transiently transfected by a Lipofectin procedure (28) with the pcDSRa-2F1 cDNA library, which was constructed from poly(A)+ RNA isolated from the human T cell line 2F1, which had been activated with concanavalin A, anti-CD3 antibody, and phorbol 12-myristate 13-acetate (38). This cDNA library was kindly donated by Dr. Naoko Arai (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA).
After a 64-h expression period, the transfected cells were detached in PBS, 5 mM EDTA, pH 7.4, collected by centrifugation, and subsequently incubated with T305 antibody as ascites fluid i n 1:200 dilution in PBS, 10 r m EDTA, 5% fetal calf serum, pH 7.4. After a 1-h incubation period on ice, the cells were washed and panned i n dishes coated with goat anti-mouse IgG (Sigma).
Plasmid DNA was rescued by the Hirt procedure (39) from transfected COS-1 cells, which were adherent to the panning dishes and transformed into the host Echerichia coli MClOGVP3-DHa. Plasmid DNA was prepared again and used for a n additional round of screening by the same procedure. E. coli transformants thus prepared from this second enrichment were plated to produce several pools, and the subsequent rounds of sibling selection with sequentially smaller active pools identified a single plasmid, pcDSRa-leu, which determined the expression of the T305 antigen on the cell surface.
DNA Sequence and RNA Blot Analysis-The nucleotide sequences were determined (40) using oligonucleotide primers synthesized according to the flanking sequences. The sequence was then extended by using oligonucleotide primers synthesized according to the sequences obtained within the cDNA insert.
Poly(A)+ RNAprepared using a commercial kit (Stratagene, La Jolla, CA) was resolved by electrophoresis in a 1.2% agarose, 2.2 M formaldehyde gel and then blotted onto a nylon membrane (Micro Separation, Inc.). The putative catalytic domain of C2GnT was amplified by polymerase chain reaction (41) as described (28), labeled with L3'P1dCTP by a random priming method (421, and used as a probe. Hybridization was performed a t 42 "C in buffers containing 50% formamide for 24 h, and blots were washed several times in 0.1 x SSPE, 0.1 % SDS at 42 "C for several hours (43), before exposure to Kodak XAR film at -70 "C. leuC2GnT was obtained by limiting dilution. As a control, CHODG44 cell line was transfected only with pZIPNEO-leu, and a clonal cell line expressing leukosialin, CHO-leu, was obtained. In order to confirm that CHO-leu-C2GnT express the mRNA of C2GnT, Northern blots were prepared using poly(A)+ RNAfrom CHODG44, CHO-leu, and CHO-leuG2GnT cells and hybridized with C2GnT-or leukosialin-specific probes. The results clearly show that CHO-leuC2GnT cells express transcripts for both leukosialin as well as C2GnT (Fig. 1, lanes 3), whereas CHO-leu express only transcripts for leukosialin ( Fig.  1, lanes 2 ). Neither of these transcripts is present in the original CHODG44 cells. Lysates prepared from the cloned cell lines were then assayed for P-1,6-N-acetylglucosaminyltransferase activities using various acceptor substrates. As shown in Table  I, the cell lysate from CHO-leu-C2GnT exhibits a significant amount of activity toward the acceptor specific for the formation of the core 2 structure. However, no significant increase over background levels was detected when acceptors were used for the measurement of the core 4 and I branching p-1,6-Nacetylglucosaminyltransferases. No significant increase, compared to the parent cells, was detected for N-acetylglucosaminyltransferase V. These results indicate that the cloned C2GnT is specific for the formation of the core 2 branch and that no other ~-1,6-N-acetylglucosaminyltransferase activity is associated with C2GnT.

Establishment and Characterization
Characterization of 0-Glycans Present in CHO-leuC2GnT and CHO-leu Cells-In order to elucidate the structures of 0-glycans directed by C2GnT, CHO-leuC2GnT and CHO-leu cells were metabolically labeled with [3H]glucosamine and leukosialin was immunoprecipitated. The supernatants of this immunoprecipitation were used as the total cellular glycoproteins. SDS-polyacrylamide gel electrophoresis demonstrated that these immunoprecipitates consist of one band and that leukosialin from CHO-leuC2GnT exhibits a larger molecular weight than leukosialin from CHO-leu cells (Fig. 2).
The above structural analysis was performed on the samples obtained from the total cellular glycoproteins. However, almost identical oligosaccharide profiles were obtained with the immunoprecipitated leukosialin samples (data not shown). These results indicate that leukosialin and other cellular glycoproteins carry identical sets of 0-glycans.
Presence of Poly-N-acetyllactosaminyl 0-Glycans in CHO-leuC2GnT Cells-Previously, we have shown that poly-Nacetyllactosaminyl 0-glycans are present in HL-60 cells, which express C2GnT, but are not present in K562 cells, which do not express C2GnT (23). Since the parental CHO cells do not express C2GnT (28,291, we tested whether the formation of poly-N-acetyllactosaminyl 0-glycans is dependent on C2GnT in CHO cells. In general, the amount of poly-N-acetyllactosaminyl 0-glycans is very low but they can be preferentially enriched by Jacalin-agarose, as shown previously (23). [3HlGlucosaminelabeled glycopeptides obtained from the total cell lysates of CHO-leu and CHO-leuC2GnT cells were thus applied to a cohmn of Jacalin-agarose. The bound glycopeptides were then treated with alkalin borohydride and the released 0-glycans were subjected to Sephadex G-50 gel filtration. Fig. 5 illustrates that CHO-leu and CHO-leuC2GnT produced two major oligosaccharide peaks ( a and b ) . The structure of these oligosaccharides were confirmed to be the same as those shown in Figs. 3 and 4. In addition, CHO-leu.C2GnT produced oligosaccharides larger than the disialosylhexasaccharide, shown by the horizontal bar in Fig. 5A. Upon digestion by endo-p-galactosidase, smaller oligosaccharides were released from these larger oligosaccharides (Fig. 5C). Furthermore, extensive digestion with Newcastle disease virus neuranimidase, diplococcal P-galactosidase and P-N-acetylglucosaminidase yielded Galpl+3GalNAcOH and GlcNAcpl-. 6(Galpl+3)GalNacOH (Fig. 50). These results indicate that CHO-leu.CZGnT express poly-N-acetyllactosamine extensions in 0-glycans. In contrast, the amount of the corresponding 0-glycans from CHO-leu cells was 30 times less than that from CHO-leuC2GnT (Fig. 5B). Those oligosaccharides from CHOleu were not analyzed further.
These results indicate that the presence of C2GnT is essential for the formation of poly-N-acetyllactosaminyl 0-glycans in CHO cells. and CHO-leu cells were metabolically labeled with 3H glucosamine and glycopeptides were prepared. The glycopeptides (6.9 to 8.6 x lo6 cpm) were applied to a column of Jacalin-agarose, and those bound and eluted were then treated with alkaline borohydride. The released 0glycans were then fractionated by Sephadex G-50 gel filtration (A, CHO-leuC2GnT B, CHO-leu). Portions of the high molecular weight oligosaccharides, shown by a bar in A, were then subjected to endo-pgalactosidase followed by Sephadex G-50 gel filtration ( C ) or to extensive exoglycosidase treatment followed by Bio-Gel P-4 gel filtration ( D ) .
Aliquots (10 pl in A and B and 500 pl in C and D ) were taken from each ter. The labeling of the standard oligosaccharides is the same as in Fig. fraction (1 ml) for determination of radioactivity by a scintillation coun-4GlcNAcpl*3Gal; e, GlcNAcpl-3Gal; f, GlcNAcpl-6(Galp1+3)-

T305 Antigen Is Expressed in Leukosialin but
Not in Other Proteins-The cell line expressing both leukosialin and CZGnT, CHO-leu.C2GnT, was stained well by immunofluorescence using rabbit anti-leukosialin antibodies and mouse T305 monoclonal antibody. In contrast, CHO-leu was stained well by immunofluorescence using anti-leukosialin antibodies but not at all with T305 monoclonal antibody (data not shown). The results establish that the expression of the T305 antigen is dependent on C2GnT. In order to understand whether the T305 antigen is expressed in all of the mucin-type glycoproteins or on certain glycoproteins, the cell lysates of CHO-leuC2GnT and CHO-leu were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting.
The Western blots were separately incubated with anti-leukosialin antibody or T305 monoclonal antibody. As shown in Fig. 6, leukosialin was detected in the lysates from both CHO-leuC2GnT and CHO-leu. In addition, reactivity with the T305 antibody was only detected for a protein with the same molecular weight as leukosialin, in the lysates of CHO-leu.CZGnT cells (Fig. 6B, lane 3 ). In contrast, the lysate from CHO-leu did not have any specific band stained by T305 monoclonal antibody.
These results are consistent with the results obtained by immunofluorescence experiments. The results from Figs. 3 and 5 indicated that both leukosialin and other cellular glycoproteins express the hexasaccharides in CHO-leu.C2GnT cells. However, Fig. 6 demonstrates that only leukosialin was recognized by the T305 antibody. The results thus indicate that the T305 antibody recognizes the hexasaccharides only when they are attached to leukosialin. This was confirmed by the fact that CHO cells expressing only C2GnT but not leukosialin were not stained by the T305 monoclonal antibody (data not shown).
Expression Cloning of Leukosialin Using the T305 Mono- clonal Antibody-The above results indicate that T305 preferentially binds to the hexasaccharides attached to leukosialin and its epitope most likely includes both the hexasaccharide and the peptide portion of leukosialin.
This conclusion was confirmed by the transient expression cloning of cDNA encoding leukosialin using the T305 monoclonal antibody. Thus, COS-1 cells were transiently transfected with pcDSRa-2F1 cDNA library, and cells expressing the T305 antigen were enriched by panning using the T305 monoclonal antibody. Further enrichment of plasmids was achieved by repeated panning and subsequent rounds of sibling selection with sequentially smaller active pools. Finally, one plasmid (pcDSRa-leu) was identified that directed the expression of the T305 antigen at the cell surface.
DNA sequencing of the insert clearly established that the isolated cDNA clone encoded human leukosialin. Since COS-1 cells do express a detectable amount of C2GnT activity (results not shown), these and the above results obtained in the previous sections clearly indicate that the T305 monoclonal antibody recognizes both the core 2-based hexasaccharide structure, as well as a part of the peptide moiety of leukosialin.
When the leukosialin cDNA sequence was compared to those previously reported, a difference was noted in the 5"untranslated sequence. Comparison with the genomic sequence revealed that the start site of this new sequence can be located 716 base pairs upstream from the 5'-end of exon-2 (see 1A in Fig. 7). The exon-intron junction of this newly identified exon has a consensus sequence for splicing (44), and in the cDNA this sequence is immediately followed by the authentic exon 2 sequence. The results indicate that different exon 1 sequences are utilized in the leukosialin gene depending on the cell type.

DISCUSSION
The present study demonstrates that in CHO cells C2GnT directs the expression of core 2 structures in 0-glycans, which results in the formation of the hexasaccharides, NeuNAca2+3Ga1/31+3(NeuNAca2+3Gal/31+4G1cNAc/31~ 6)GalNAc. The hexasaccharides are present on leukosialin, as well as on other glycoprotein acceptors. However, the monoclonal antibody T305 binds to the hexasaccharides only when they are attached to leukosialin. In addition, the present study demonstrated that poly-N-acetyllactosaminyl extension in 0glycans are formed on the core 2 GlcNAcP1+6 branch, which is synthesized by C2GnT. The T305 monoclonal antibody was originally raised against a T-lymphocytic leukemia cell line, and its binding to peripheral blood lymphocytes is dramatically increased in patients with leukemia (14) or immunodeficiency such as Wiskott-AIdrich syndrome (15) or AIDS (14) as compared to normal lymphocytes. It was shown that this increase is correlated with an  was found in the cDNA sequence isolated in the present study. Either exon 1A or exon 1B is followed by the authentic exon 2 sequence. The nucleotides are numbered as the initiation of exon 2 as +l. The genomic sequence and the rest of the exon 2 sequence are given in Ref. 59. increase in the hexasaccharides in leukosialin while the lymphocytes under normal conditions have a negligible amount of the hexasaccharide (14,15). These results are consistent with the results obtained in the present study, showing that the T305 binds to the hexasaccharides which are attached to leukosialin.

T T A V Q T P T S G E P L V S T S E P L A C A A C~C A G T G C A G A C A C C C A C C T C C T T T G G
Preliminary studies indicated that the hexasaccharides are present on immature thymocytes but that they are replaced by the tetrasaccharides during thymocyte maturation. In fact, the T305 antibody apparently stains immature thymocytes much more strongly than mature thymocytes (17h2 The establishment of the epitope of the T305 antibody should be helpful to understand the roles of 0-glycans during hematopoiesis in future studies. It has been previously shown that some antibodies recognize both protein and carbohydrate determinants for binding. For instance, anti" and anti-N antibodies do not bind to erythrocytes after sialic acid residues are removed (45). However, M and N blood group antigens are primarily determined by the amino acid sequence of glycophorin A. When the first and fifth residue are serine and glycine, glycophorin A express M antigen, whereas N antigen is expressed when the first and fifth residue are leucine and glutamic acid (for review see Ref. 46). These results indicate that anti" and anti-N antibodies must bind both sialic acid and the underlying polypeptide portion. This is similar to the binding by T305. It will be of interest in future studies to determine which portion of the leukosialin molecule, in addition to the hexasaccharides, is recognized by the T305 antibody.
In the present study, a transient expressing cloning scheme using T305 monoclonal antibody as a selection marker resulted in the isolation of cDNA encoding leukosialin, a camer molecule for the 0-glycan hexasaccharides. The results indicate that it will be possible to clone a camer molecule which presents most efficiently the ligand to binding proteins such as antibodies or selectins. The present study also revealed that leukosialin gene can utilize different upstream exons depending on cell type. Since this novel exon usage was detected in activated T-lymphocytes, further studies will be of significance to determine whether different exon usage in leukosialin might be related to different stages of T-lymphocyte differentiation.
Le", sialyl Le", NeuNAca2-.3Gal~1+3(Fuca1~4)GlcNAc-/31+, was also found to serve as ligand for E-selectin (47). In order to form sialyl Le" and sialyl Le" structures in 0-glycans, it is prerequisite to form N-acetyllactosamine extension. The present study demonstrated that the formation of N-acetyllactosamine unit in 0-glycans is entirely dependent on the presence of C2GnT in CHO cells. In the absence of C2GnT, no poly-N-acetyllactosamine extension was found in 0-glycans although CHO cells are enriched with the extension enzyme, ~-1,3-N-acetylglucosaminyltransferase (48). This also indicates that /3-1,3-N-acetylglucosaminyltransferase in CHO cells does not utilize the core 1 as a substrate to form, for instance,

R+Gal~1+4GlcNAc~1+3Gal~1+3(R+6)GalNAc.
The results obtained in the present study are consistent with the results reported previously. In blood cells, almost all of the poly-N-acetyllactosaminyl extension takes place on the side chain elongated from the core 2 GlcNAcpl-tG branch attached to C-6 of GalNAc (19,23). The present study extended these findings into CHO cells, suggesting that C2GnT controls the formation of N-acetyllactosamine extension in 0-glycans in a wide variety of cells (see also Fig. 10 in Ref. 23). In relation to this conclusion, it has also been demonstrated that metastatic tumor cells contain an increased amount of C2GnT (24). The same cells were also found to have a n increased amount of another ~-l,6-N-acetylglucosaminyltransferase, N-acetylglucosaminyltransferase V. Previous studies indicated that the branch formed by N-acetylglucosaminyltransferase V is a preferential site where poly-N-acetyllactosamine extension takes place inN-glycans (29,(49)(50)(51). It was also shown that this side chain, (Gal/31+4GlcNAc~l+3Gal~l+4)n GlcNAcpl+GMan-a1+6, is preferentially modified to have sialyl Le", NeuNA-ca2+3Gal/3l+4(Fucal+3)GlcNAc~l+, at the termini (52). It has been separately shown that tumor cells express an increased amount of sialyl Le" and sialyl Le" structures compared to their normal counterparts (53, 54). It is thus likely that the increased amount of sialyl Le" and sialyl Le" structures is at least partly due to the increased amount of /3-1,6-GlcNAc branchings in both 0-and N-glycans, which can be extended to form poly-N-acetyllactosamines and to have sialyl Le" and sialyl Le" structures in their termini. These results suggest that tumor cells may utilize carbohydrate-selectin interaction for the attachment to endothelial cells at sites of metastasis. In fact, it has been demonstrated that tumor cells bind to endothelial cells through E-selectin-mediated adhesion (55-57). Moreover, highly metastatic tumor cells were found to adhere more efficiently to endothelial cells, compared to low metastatic tumor cells (58). It will thus be of great significance to test if the formation of ~-1,6-N-acetylglucosaminyl linkages by genetic manipulation results in increased adhesion of tumor cells to endothelial cells and acquisition of metastatic spread of tumor cells.