We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

The structure and biosynthesis of poly-Nacetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Le x . Herein we describe the isolation and functional expression of a cDNA encoding ␤-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-Nacetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by f luorescenceactivated cell sorting because introduced plasmids containing Epstein-Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAc␤1 3 3Gal␤1 3 4GlcNAc 3 R structure and poly-N-acetyllactosamine extension.

We showed previously that the alternative splicing of chondroitin sulfate attachment domains (CS alpha and CS beta) yielded multiforms of the PG-M core protein in mouse. A transcript encoding a new short form of the core protein PG-M(V3) was found in various mouse tissues using polymerase chain reaction. DNA sequences of the polymerase chain reaction products suggested that PG-M(V3) had no chondroitin sulfate attachment domain. PG-M(V3) was also detected in various human tissues. The presence of a transcript for PG-M(V3) was further supported by Northern blot analysis. Southern blot analysis confirmed that multiforms of the PG-M core protein, including PG-M(V3), were derived from a single genomic locus by an alternative splicing mechanism. Because PG-M(V3) has no chondroitin sulfate attachment region, which is the most distinctive portion of a proteoglycan molecule, this form may have a unique function.

Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Le x . Poly-N-acetyllactosamines in mucintype O-glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 ␤-1,6-Nacetylglucosaminyltransferase.Using a ␤-1,4-galactosyltransferase (␤4Gal-TI) present in milk and the recently cloned ␤-1,3-N-acetylglucosaminyltransferase, the formation of poly-N-acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAc␤136-(Gal␤133)GalNAc␣3 R. Since the majority of synthesized oligosaccharides contained N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the ␤4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant ␤4Gal-Ts, ␤4Gal-TIV was found to be most efficient in the addition ofasinglegalactoseresiduetoGlcNAc␤136(Gal␤133)GalNAc␣3 R. Moreover, ␤4Gal-TIV, together with ␤-1,3-Nacetylglucosaminyltransferase, was capable of synthesizing poly-N-acetyllactosamine in core 2 branched oligosaccharides. On the other hand, ␤4Gal-TI was found to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans. In contrast to ␤4Gal-TI, the efficiency of ␤4Gal-TIV decreased dramatically as the acceptors contained more N-acetyllactosamine repeats, consistent with the fact that core 2 branched O-glycans contain fewer and shorter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that ␤4Gal-TIV is responsible for poly-Nacetyllactosamine synthesis in core 2 branched O-glycans.Mucin-type O-glycans are present in a wide variety of cells and play various roles in different cells. Mucin-type glycoproteins are also present in the plasma membrane, and they are often involved in cell-cell interaction (1). For example, O-glycans present in eggs were shown to be a receptor for both mouse and sea urchin (2, 3). In granulocytes, monocytes, and certain T lymphocytes, mucin-type O-glycans can carry sialyl Le x , NeuNAc␣233Gal␤134(Fuc␣133)GlcNAc3 R, at their termini (4 -6). Sialyl Le x and its sulfated form are ligands for E-, P-, and L-selectin (7-11). Importantly, these selectins, in particular P-and L-selectin, preferentially bind to sialyl Le x in a limited number of mucin-type glycoproteins such as PSGL-1 (for P-selectin) and GlyCAM-1 and CD34 (for L-selectin) (12-14). As shown previously, sialyl Le x and its derivatives of O-glycans in blood cells can be only formed on core 2 branches, Gal␤134GlcNAc␤136(Gal␤133)GalNAc␣3 R (4, 5). Recent studies demonstrate that sialyl Le x and sialyl Le a in core 2 branches are highly correlated to tumor invasion and vessel invasion of colon carcinomas (15), probably because tumor cells utilize selectin-carbohydrate interaction for their adhesion.In patients with immunodeficiency such as Wiskott-Aldrich syndrome, AIDS, and leukemia, leukocytes in the peripheral blood...

We previously showed not only the presence of multiple RNA transcripts of different sizes encoding the core protein of mouse PG-M, but also their tissue-dependent expression. Major causes for the multiple forms were found to be due to alternative usage of the two different chondroitin sulfate attachment domains (alpha and beta). In this study, genomic DNA analysis has revealed that these domains are encoded by two large exons, exon VII (2880 base pairs) and exon VIII (5229 base pairs). The splice sites of these two exons were consistent with the occurrence of alternative splicing without frameshift. Furthermore, the mouse PG-M gene was shown to have four distinct polyadenylation signals and three candidates for the transcription initiation site as well. These genomic structural variations may contribute to the multiplicity of PG-M transcripts. Northern hybridization analysis showed that at least three different transcripts were generated by different usage of the distinct polyadenylation signals.

Poly-N-acetyllactosamines are attached to N-glycans, O-glycans, and glycolipids and serve as underlying glycans that provide functional oligosaccharides such as sialyl Lewis X . Poly-N-acetyllactosaminyl repeats are synthesized by the alternate addition of ␤1,3-linked GlcNAc and ␤1,4-linked Gal by i-extension enzyme (iGnT) and a member of the ␤1,4-galactosyltransferase (␤4Gal-T) gene family. In the present study, we first found that poly-N-acetyllactosamines in N-glycans are most efficiently synthesized by ␤4Gal-TI and iGnT. We also found that iGnT acts less efficiently on acceptors containing increasing numbers of N-acetyllactosamine repeats, in contrast to ␤4Gal-TI, which exhibits no significant change. In O-glycan biosynthesis, N-acetyllactosamine extension of core 4 branches was found to be synthesized most efficiently by iGnT and ␤4Gal-TI, in contrast to core 2 branch synthesis, which requires iGnT and ␤4Gal-TIV. Poly-N-acetyllactosamine extension of core 4 branches is, however, less efficient than that of N-glycans or core 2 branches. Such inefficiency is apparently due to competition between a donor substrate and acceptor in both galactosylation and N-acetylglucosaminylation, since a core 4-branched acceptor contains both Gal and GlcNAc terminals. These results, taken together, indicate that poly-N-acetyllactosamine synthesis in N-glycans and core 2-and core 4-branched Oglycans is achieved by iGnT and distinct members of the ␤4Gal-T gene family. The results also exemplify intricate interactions between acceptors and specific glycosyltransferases, which play important roles in how poly-Nacetyllactosamines are synthesized in different acceptor molecules.Poly-N-acetyllactosamines are unique glycans having N-acetyllactosamine repeats (Gal␤134GlcNAc␤133) n in one side chain (1). Poly-N-acetyllactosamines are attached to Nglycans (2-4), O-glycans (5-7), and glycolipids (8 -10). Poly-Nacetyllactosamines are often modified to express differentiation antigens and functional oligosaccharides. One of those oligosaccharides is sialyl Le X , 1 NeuNAc␣233Gal␤134(Fuc␣133)-GlcNAc3 R discovered in human granulocytes and monocytes (11,12). Sialyl Le X and its sulfated forms, such as 6-sulfo sialyl Le X , NeuNAc␣233Gal␤134[Fuc␣133(sulfo36)]GlcNAc3 R in mucin-type glycoproteins, have been shown to be ligands for E-, P-, and L-selectin (13-15).Since these O-glycans are present as clusters in mucin-type glycoproteins, mucin-type glycoproteins can present multiple ligands to a selectin. In mucin-type glycoproteins of blood cells, sialyl Le X can be found in core 2-branched oligosaccharides (5, 6, 16). Similarly, 6-sulfo sialyl Le X in L-selectin ligands found in high endothelial venules are synthesized in core 2-branched oligosaccharides such as NeuNAc␣233Gal␤134[Fuc␣133-(sulfo36)]GlcNAc␤136(Gal␤133)GalNAc␣13serine/threonine (17-19).The enzyme responsible for core 2 branching is called core 2 ␤1,6-N-acetylglucosaminyltransferase (C2GnT), and its cDNA has been cloned (20). When C2GnT was inactivated by gene targeting, leukocytes...

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I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching ␤1,6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewis x , which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching ␤1,6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers ␤1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various ␤1,4-galactosyltransferases (␤4Gal-Ts), ␤4Gal-TI is most efficient in adding a galactose to linear and branched poly-Nacetyllactosamines. When a ␤1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of ␤4Gal-TI and i-extension ␤1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Gal␤134GlcNAc␤133 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that ␤4Gal-TI adds a galactose poorly to ␤1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while ␤1,3-N-acetylglucosaminyltransferase and ␤4Gal-TI efficiently add N-acetyllactosamine to linear poly-Nacetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.