We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAc␤1-3Gal␤1-4GlcNAc. Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAc␣2-3Gal␤1-4(SO 4 -6)GlcNAc; double transfection with fucosyltransferase IV yielded Gal␤1-4(Fuc␣1-3)(SO 4 -6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N -acetylgalactosamine alpha2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O -glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situ hybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s).

Midkine and pleiotrophin form a family of growth factors. Mice deficient in one of the genes show few abnormalities on reproduction and development. To understand their roles in these processes, we produced mice deficient in both genes; the double deficient mice were born in only one third the number expected by Mendelian segregation and 4 weeks after birth weighed about half as much as wild-type mice. Most of the female double deficient mice were infertile. In these mice, the numbers of mature follicles and of ova at ovulation were reduced compared to numbers in wild-type mice. Both midkine and pleiotrophin were expressed in the follicular epithelium and granulosa cells of the ovary. The expression of these factors in the uterus was dramatically altered during the estrous cycle. The diestrus and proestrus periods were long and the estrus period was short in the double deficient mice, indicating the role of the factors in the estrous cycle. Furthermore, vaginal abnormality was found in about half of the double deficient mice. These abnormalities in combination resulted in female infertility. Therefore, midkine and pleiotrophin, together with their signaling receptors, play important roles in the female reproductive system.

A cDNA encoding a new alpha 2,8-sialyltransferase (ST8Sia III), which exhibits activity toward the Sia alpha 2,3Gal beta 1, 4GlcNAc sequences of N-linked oligosaccharides, was cloned from mouse brain by means of the polymerase chain reaction-based approach. The predicted amino acid sequence of ST8Sia III showed 27.6 and 34.4% identity with those of so far cloned mouse alpha 2,8-sialyltransferases, i.e. GD3 synthase (ST8Sia I) and STX (ST8Sia II), respectively. Transfection of the protein A-fused ST8Sia III gene into COS-7 cells led to alpha 2,8-sialyltransferase activity toward sialylated glycoproteins and alpha 2,3-sialylated glycosphingolipids, such as alpha 2,3-sialylparagloboside and GM3. However, the kinetic properties of ST8Sia III revealed that it is much more specific to N-linked oligosaccharides of glycoproteins than glycosphingolipids. The expression pattern of the ST8Sia III gene was clearly different from those of other alpha 2,8-sialyltransferase genes. The expression of the ST8Sia III gene was tissue and stage specific. The ST8Sia III gene was expressed only in brain and testis, and it appeared first in 20 postcoitum embryonal brain and then decreased. Therefore, the new alpha 2,8-sialyltransferase is closely involved in brain development.

Two cDNA clones encoding NeuAc␣2,3Gal␤1,3GalNAc GalNAc␣2,6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAc␣2, 3Gal␤1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Gal␤1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14 C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[ 14 C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAc␣2,6-sialyltransferase, which requires sialic acid residues linked to Gal␤1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.Sialic acids are key determinants of carbohydrate structures that play important roles in a variety of biological functions, like cell-cell communication, cell-substrate interaction, adhesion, and protein targeting. The transfer of sialic acids from CMP-Sia 1 to the terminal positions of the carbohydrate groups of glycoproteins and glycolipids is catalyzed by a sialyltransferase. Although roles of sialic acids have been proposed in the regulation of many biological phenomena, the purpose of this structural diversity remains largely obscure. To determine the meaning of the diversity of and the regulatory mechanism for the sialylation of glycoconjugates, it is necessary to obtain information on the enzymes themselves and the gene structure of sialyltransferases. Each sialyltransferase exhibits strict specificity for acceptor substrates and linkages (3-6). Although three linkages, Sia␣2,6Gal, Sia␣2,3Gal, and Sia␣2,6GalNAc, are commonly found in glycoproteins (7), and two, Sia␣2,3Gal and Sia␣2,8Sia, occur frequently in gangliosides (8), each of these linkages has been found in both gangliosides and glycoproteins (8 -10).So far, the cloning of three members of the ␣2,6-sialyltransferase family (ST6GalNAc I, II and III) has been reported (11-14). The cDNAs of ST6GalNAc I and II were cloned from both chick (11, 12) and mouse (13,62).2 The overall amino acid sequence identity of chick ST6GalNAc I is 30.5% to chick ST6GalNAc II, 43.2% to mouse ST6GalNAc I, and 33.6% to mouse ST6GalNAc II, and that of mouse ST6GalNAc I is 29.6% to mouse ST6GalNAc II and 28.3% to chick ST6GalNAc II, and that of chick ST6GalNAc II is 57.3% ...