Neoplastic transformation results in a wide variety of cellular alterations that impact the growth, survival, and general behavior of affected tissue. Although genetic alterations underpin the development of neoplastic disease, epigenetic changes can exert an equally significant effect on neoplastic transformation. Among neoplasia-associated epigenetic alterations, changes in cellular glycosylation have recently received attention as a key component of neoplastic progression. Alterations in glycosylation appear to not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Many of these changes may support neoplastic progression, and unique alterations in tumor-associated glycosylation may also serve as a distinct feature of cancer cells and therefore provide novel diagnostic and even therapeutic targets.

Human core 1 ␤3-galactosyltransferase (C1␤3Gal-T) generates the core 1 O-glycan Gal␤1-3GalNAc␣1-Ser͞Thr (T antigen), which is a precursor for many extended O-glycans in animal glycoproteins. We report here that C1␤3Gal-T activity requires expression of a molecular chaperone designated Cosmc (core 1 ␤3-Gal-T-specific molecular chaperone).

The expression of ABO(H) blood group antigens causes deletion of cells that generate self anti-blood group antibodies, but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against these pathogens, given such limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectins-4 and -8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing E. coli, while failing to alter viability of other E. coli strains or other gram-negative or gram-positive organisms both in vitro and in vivo. Killing by both galectins-4 and -8 resides within their C-terminal domains, occurs rapidly and independently of complement, and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that display blood group self-antigens on their surface.

Glycoproteins in animal cells contain a variety of glycan structures that are added co- and/or posttranslationally to proteins. Of over 20 different types of sugar-amino acid linkages known, the two major types are N-glycans (Asn-linked) and O-glycans (Ser/Thr-linked). An abnormal mucin-type O-glycan whose expression is associated with cancer and several human disorders is the Tn antigen. It has a relatively simple structure composed of N-acetyl-D-galactosamine with a glycosidic α linkage to serine/threonine residues in glycoproteins (GalNAcα1-O-Ser/Thr), and was one of the first glycoconjugates to be chemically synthesized. The Tn antigen is normally modified by a specific galactosyltransferase (T-synthase) in the Golgi apparatus of cells. Expression of active T-synthase is uniquely dependent on the molecular chaperone Cosmc, which is encoded by a gene on the X chromosome. Expression of the Tn antigen can arise as a consequence of mutations in the genes for T-synthase or Cosmc, or genes affecting other steps of O-glycosylation pathways. Because of the association of the Tn antigen with disease, there is much interest in the development of Tn-based vaccines and other therapeutic approaches based on Tn expression.

Neoplastic lesions typically express specific carbohydrate antigens on glycolipids, mucins, and other glycoproteins. Such antigens are often under epigenetic control and are subject to reversion and loss upon therapeutic selective pressure. We report here that two of the most common tumor-associated carbohydrate antigens, Tn and sialyl Tn (STn), result from somatic mutations in the gene Cosmc that encodes a molecular chaperone required for formation of the active T-synthase. Diverse neoplastic lesions, including colon cancer and melanoma-derived cells lines, expressed both Tn and STn antigen due to loss-of-function mutations in Cosmc. In addition, two human cervical cancer specimens that showed expression of the Tn/STn antigens were also found to have mutations in Cosmc and loss of heterozygosity for the cross-linked Cosmc locus. This is the first example of somatic mutations in multiple types of cancers that cause global alterations in cell surface carbohydrate antigen expression. [Cancer Res 2008;68(6):1636-46]

Altered intestinal O-glycan expression has been observed in patients with ulcerative colitis and colorectal cancer, but the role of this alteration in the etiology of these diseases is unknown. O-glycans in mucin core proteins are the predominant components of the intestinal mucus, which comprises part of the intestinal mucosal barrier. Core 3–derived O-glycans, which are one of the major types of O-glycans, are primarily expressed in the colon. To investigate the biological function of core 3–derived O-glycans, we engineered mice lacking core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT), an enzyme predicted to be important in the synthesis of core 3–derived O-glycans. Disruption of the C3GnT gene eliminated core 3–derived O-glycans. C3GnT-deficient mice displayed a discrete, colon-specific reduction in Muc2 protein and increased permeability of the intestinal barrier. Moreover, these mice were highly susceptible to experimental triggers of colitis and colorectal adenocarcinoma. These data reveal a requirement for core 3–derived O-glycans in resistance to colonic disease.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO 3 ) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe x ) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO 3 or C2-O-sLe x do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6, which contains sLe x on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K d of ϳ350 nM. GSP-6 (<5 M) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe x (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.The interactions between selectins and their carbohydratebased ligands initiate adhesion of leukocytes to the vascular wall during inflammation. Although L-, E-, and P-selectin can bind a simple glycan containing sialyl Lewis x (sLe x ) 1 (NeuAc␣233Gal␤134[Fuc␣133]GlcNAc␤13 R) in a Ca 2ϩ -dependent manner, each selectin binds with higher affinity to a limited number of macromolecular ligands expressing sialylated and fucosylated glycans (1-4). P-selectin, which is expressed by activated platelets and endothelial cells, demonstrates the most discriminating ligand specificity of any selectin. It interacts predominantly with a disulfide-bonded dimeric mucin on leukocytes termed P-selectin glycoprotein ligand-1 (PSGL-1) (subunit mass ϳ120 kDa) (5).Each 120-kDa subunit of human PSGL-1 contains numerous sialic acids and approximately 70 extracellular Ser and Thr residues, which are potential sites for O-glycosylation, plus three potential sites for N-glycosylation (6, 7) (Fig. 1). These features suggested that the large amount of carbohydrate on the mucin might promote high avidity binding to P-selectin. However, indirect evidence suggests that the extreme N-terminal extracellular region of mature PSGL-1, which begins at residue 42, is important for high affinity binding to P-selectin (reviewed in Ref. 3). Specifically, tyrosine sulfate residues and O-glycans within that region have been considered essential for binding (Fig. 1). A monoclonal antibody directed to a peptide ep...

Mucin-type O-glycans (O-glycans