The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: beta-d-galactosyl-1,4-glucosylceramide alpha-1, 3-galactosyltransferase (iGb(3) synthase) from a rat placental cDNA expression library. iGb(3) synthase acts on lactosylceramide, LacCer (Galbeta1,4Glcbeta1Cer) to form iGb(3) (Galalpha1,3Galbeta1, 4Glcbeta1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40-50% identity) to members of the ABO gene family that includes: murine alpha1, 3-galactosyltransferase, Forssman (Gb(5)) synthase, and the ABO glycosyltransferases. In contrast to the murine alpha1, 3-galactosyltransferase, iGb(3) synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb(3) synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb(3) functions, including the globo- and isoglobo-series of glycolipids.

Carbohydrates attached to proteins and lipids characteristically display complex and heterogeneous structures. However, it is becoming increasingly clear that carbohydrates with definite biological functions also exhibit unique structural features. A number of glycoproteins and glycolipids have been shown to bear oligosaccharides containing sulfate. Often, addition of a sulfate moiety turns a relatively common structural motif into a unique carbohydrate with the potential to be recognized by a specific receptor or lectin. This is clearly the case in three systems in which sulfated oligosaccharides have been shown to play a well-defined biological role: 1) control of the circulatory half-life of luteinizing hormone, 2) symbiotic interactions between leguminous plants and nitrogen-fixing bacteria, and 3) homing of lymphocytes to lymph nodes. The rapidly growing list of glycoproteins and glycolipids identified as bearing sulfated oligosaccharides suggests that sulfated carbohydrates play important biological roles in numerous other systems as well.-Hooper, L. V., Manzella, S. M., Baenziger, J. U. From legumes to leukocytes: biological roles for sulfated carbohydrates.

We have cloned Gb 3 synthase, the key ␣1,4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb 3 synthase, the ␣1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Gal␤1,4Glc␤1Cer (LacCer), to produce Gb 3 (Gal␣1,4LacCer) or iGb 3 (Gal␣1,3LacCer), respectively. GalNAc can be added sequentially to either Gb 3 or iGb 3 yielding globoside and Forssman from Gb 3 , and isogloboside and isoForssman from iGb 3 . Gb 3 synthase is not homologous to iGb 3 synthase but shows 43% identity to a human ␣1,4GlcNAc transferase that transfers a UDP-sugar in an ␣1,4-linkage to a ␤-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb 3 synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an ␣1,4-glycosyltransferase, possibly Gb 3 synthase, throughout evolution. The isolated Gb 3 synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb 3 synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb 3 glycolipid, also called P k antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb 3 and are resistant to verotoxin become susceptible to the toxin following transfection with Gb 3 synthase cDNA.

The carbohydrate moieties found on glycoproteins have long been recognized as having great potential to bear biologically important information. However, actual examples of systems in which oligosaccharides play defined physiological roles have remained limited. These oligosaccharides with known biologic functions typically have distinctive structural features and are generally confined to specific glycoproteins. Synthesis of structurally unique oligosaccharides on specific glycoproteins at defined times is essential if these structures are to fulfill their biologic purpose. Since cells produce many distinct oligosaccharides as newly synthesized glycoproteins pass through the endoplasmic reticulum and the Golgi, mechanisms are required to assure that the correct structures are added to the numerous glycoproteins being synthesized. Determining how synthesis of the vast array of oligosaccharides produced by each cell is regulated is essential for understanding the biologic importance of these complex structures.Asn-linked oligosaccharides arise by processing of a common precursor structure, which is transferred en bloc from dolichol to the nascent peptide chain in the endoplasmic reticulum (1). As a result Asn-linked oligosaccharides have a common core region and differ primarily in the number and location of their peripheral branches and terminal modifications. Since all newly synthesized glycoproteins pass through the same subcellular compartments and are exposed to the same transferases, structural differences in oligosaccharides on individual glycoproteins and/or at individual glycosylation sites must in some fashion reflect the influence of the protein moiety on one or more glycosyltransferases. This suggests that key glycosyltransferases recognize features encoded within the peptide as well as the oligosaccharide of the target glycoprotein. Among the three glycosyltransferases thus far demonstrated to display peptide as well as oligosaccharide recognition, UDP-glucose:glycoprotein glucosyltransferase, UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, and UDP-GalNAc:glycoprotein hormone ␤1,4-N-acetylgalactosaminyltransferase (␤1,4-GalNAcT, 1 reviewed in Ref.2), one of the most extensively characterized is the ␤1,4-GalNAcT, which produces the terminal sequence GalNAc␤1,4GlcNAc␤1-R on glycoproteins that contain a specific peptide recognition determinant in addition to an appropriate oligosaccharide acceptor. The product of the ␤1,4-GalNAcT may be further modified by the addition of sulfate, sialic acid, or fucose, thus producing a range of unique oligosaccharide structures defined by the presence of ␤1,4-linked GalNAc as illustrated in Fig. 1. Each of these structures has the potential to be recognized by a specific receptor or binding protein and thus mediate a distinct biological function. As will become apparent below, the ␤1,4-GalNAcT is a key component of a well characterized system, which includes unique oligosaccharide structures, highly specific glycosyltransferases, an...

The asialoglycoprotein-receptor (ASGP-R) located on liver parenchymal cells was originally identified and characterized on the basis of its ability to bind glycoproteins bearing terminal galactose (Gal) or N-acetylgalactosamine (GalNAc); however, endogenous ligands for the ASGP-R have not to date been definitively identified. We have determined that the rat ASGP-R specifically binds oligosaccharides terminating with the sequence Sia␣2,6GalNAc␤1,4GlcNAc␤1,2Man. Bovine serum albumin chemically modified with 10 -15 tetrasaccharides with the sequence Sia␣2,6GalNAc␤1,4GlcNAc␤1,2Man is cleared from the blood of the rat with a half-life of <1 min by a receptor located in the liver. We have isolated the receptor and identified it as the ASGP-R. Furthermore, we have determined that subunit 1 of the ASGP-R accounts for the binding of terminal Sia␣2,6GalNAc␤. Based on the newly defined specificity of the rat ASGP-R we hypothesize that glycoproteins bearing structures that are selectively modified with terminal Sia␣2, 6GalNAc␤ and are released into the blood may be endogenous ligands for the rat ASGP-R.The rapid clearance of glycoproteins from the blood following removal of sialic acid (Sia) 1 residues and exposure of underlying galactose (Gal) residues was first reported by Ashwell and Morell in the early 1970s (1, 2) and led to the discovery of the asialoglycoprotein-receptor (ASGP-R). The specificity and biochemical features of this endocytic receptor have been extensively investigated since that time (3, 4). Although it is clear that the ASGP-R receptor binds oligosaccharides with terminal ␤-linked N-acetylgalactosamine (GalNAc) or Gal and can mediate the rapid clearance of glycoproteins bearing these terminal sugars from the circulation, endogenous ligands for this abundant receptor have not yet been identified.We first described N-linked oligosaccharides containing ␤1,4-linked GalNAc on lutropin (LH) and other members of the glycoprotein hormone family of glycoproteins (5, 6). The GalNAc is found almost exclusively in the form of GalNAc-4-SO 4 , reflecting the sequential action of a protein-specific ␤1,4-Nacetylgalactosaminyltransferse (␤1,4GalNAcT) and a GalNAc-4-sulfotransferase (GalNAc-4-ST1) (7,8). The terminal GalNAc-4-SO 4 is recognized by a receptor in hepatic endothelial cells that regulates the circulatory half-life of LH following its stimulated release into the blood (9 -12). The control of circulatory half-life is important for regulating estrogen production in vivo during implantation of the embryo (13). We subsequently described the presence of N-linked oligosaccharides terminating with Sia␣2,6GalNAc␤ on prolactin/growth (PLP) hormone family members that are synthesized by rat placenta spongiotrophoblasts between mid gestation and birth (14).