The MUC1 membrane mucin was first identified as the molecule recognised by mouse monoclonal antibodies directed to epithelial cells, and the cancers which develop from them. Cloning the gene showed that the extracellular domain is made up of highly conserved repeats of 20 amino acids, the actual number varying between 25 and 100 depending on the allele. Each tandem repeat contains five potential glycosylation sites, and between doublets of threonines and serines lies an immunodominant region which contains the epitopes recognised by most of the mouse monoclonal antibodies. The O-glycans added to the mucin produced by the normal breast are core 2 based and can be complex, while the O-glycans added to the breast cancer mucin are mainly core 1 based. This means that some core protein epitopes in the tandem repeat which are masked in the normal mucin are exposed in the cancer associated mucin. Since novel carbohydrate epitopes are also carried on the breast cancer mucin, the molecule is antigenically distinct from the normal breast mucin. (Changes in glycosylation in other epithelial cancers have been observed but are not so well documented.) Immune responses to MUC1 have been seen in breast and ovarian cancer patients and clinical studies have been initiated to evaluate the use of antibodies to MUC1 and of immunogens based on MUC1 for immunotherapy of these patients. The role of the carbohydrates in the immune response and in other interactions with the effector cells of the immune system is of particular interest and is discussed.

Glycosylation plays a key role in a wide range of biological processes. Specific modification to a glycan's structure can directly modulate its biological function. Glycans are not only essential to glycoprotein folding, cellular homeostasis, and immune regulation but are involved in multiple disease conditions. An increased molecular and structural understanding of the mechanistic role that glycans play in these pathological processes has driven the development of therapeutics and illuminated novel targets for drug design. This knowledge has enabled the treatment of metabolic disorders and the development of antivirals and shaped cancer and viral vaccine strategies. Furthermore, an understanding of glycosylation has led to the development of specific drug glycoforms, for example, monoclonal antibodies, with enhanced potency.

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In breast cancer, the O-glycans added to the MUC1 mucin are core 1-rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal

The synthesis of the common and well-documented Siaalpha 2,6 to Galbeta 1,4GlcNAc structure (Sia6LacNAc) is principally mediated by the sialyltransferase ST6Gal I, which is particularly highly expressed in liver, lactating mammary gland, intestinal epithelia of newborn animals, and B cells. Multiple independent promoters govern the expression of Siat1, the ST6Gal I gene. In liver, elevation of hepatic and serum ST6Gal is part of the acute phase reaction, the hepatic response to systemic trauma, and is governed by the inducible, liver-specific promoter-regulatory region, P1. A constitutive and nontissue-specific promoter, P3, mediates low-level, basal hepatic Siat1 transcription. We generated a mouse specifically unable to use the transcriptional initiation site uniquely used in P1-mediated ST6Gal I expression. These animals, Siat1deltaP1, are viable and display reduced ST6Gal I mRNA in liver with concomitantly reduced sialyltransferase activities in liver and in serum. Siat1deltaP1 animals are unable to elevate hepatic Siat1 mRNA as part of the inflammatory response induced by turpentine. Surprisingly, serum glycoprotein components exhibit normal extent of sialylation, with no noticeable difference in binding to SNA, the alpha2,6-sialyl-specific lectin. Siat1deltaP1 animals also exhibit an outwardly normal B cell response. On intraperitoneal challenge with the pathogen Salmonella typhimurium, a significantly greater accumulation of neutrophils within the peritoneal space was observed in Siat1deltaP1 animals compared to wild-type mice. Siat1deltaP1 mice also exhibit a greater bacterial burden in liver and spleen, accompanied by more pronounced spleno-/hepatomegaly and greater leukocyte infiltration into affected organs than their wild-type counterparts.

Cleavage and polyadenylation is an essential processing reaction required for the maturation of pre-mRNAs into stable, export-and translation-competent mature mRNA molecules. This reaction requires the assembly of a multimeric protein complex onto a bipartite core sequence element consisting of an AAUAAA hexamer and a GU/U-rich downstream sequence element. In this study we have analyzed 3 end processing of the human melanocortin 1 receptor gene (MC1R). The MC1R gene is an intron-free transcription unit, and its poly(A) site lacks a defined U/GU-rich element. We describe two G-rich sequence elements that are critical for efficient cleavage at the MC1R poly(A) site. The first element is located 30 nucleotides downstream of the cleavage site and acts as an essential closely positioned enhancer. The second G-rich region is positioned more than 440 nucleotides downstream of the MC1R processing site and is instrumental for optimal processing efficiency. Both G-rich sequences contain clusters of heterogeneous nuclear ribonucleoprotein binding motifs and act together to enhance cleavage at the MC1R poly(A) site.The expression of eukaryotic protein-encoding genes requires that the primary RNA transcripts undergo three major processing reactions. The very 5Ј end of each emerging premRNA is cotranscriptionally capped, intron sequences are excised via a complex splicing mechanism, and 3Ј ends of almost all genes are generated by a two-step mechanism involving an endonucleolytic cleavage reaction followed by the polymerization of a poly(A) tail.In mammals, the cleavage and polyadenylation reaction requires the cotranscriptional assembly of a large complex consisting of five multimeric factors on a core cis element embedded in the 3Ј end of the pre-mRNA (41). The core poly(A) recognition site is a bipartite sequence encompassing a conserved hexamer, AAUAAA or AUUAAA, and a lesser conserved U-and/or GU-rich region of variable length located up to 60 nucleotides further downstream (59). Both of these cis elements directly interact with subunits of the two major cleavage and polyadenylation factors, the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulatory factor (CstF), in a cooperative manner (13, 59). After the assembly of additional essential factors, including cleavage factors I (CFI) and II (CFII) and in most cases the poly(A) polymerase, cleavage occurs cotranscriptionally, probably catalyzed by the CPSF-73 subunit (45).The cleavage efficiency at canonical poly(A) sites is determined by the sequence composition of both the hexamer and the U/GU-rich downstream sequence element (DSE). Mutation of each individual nucleotide of the optimal AAUAAA sequence severely reduces the efficiency of cleavage at the mutant sites (24,46,54). The strength of the DSE is dependent on its U and GU composition but also on how far it is positioned from the cleavage site (20,35). The excision of introns has also been associated with the stimulation of the poly(A) cleavage reaction in spliced genes, and the artifici...

Several oncogenic proteins are known to influence cellular glycosylation. In particular, transfection of codon 12 point mutated H‐Ras increases CMP‐Neu5Ac: Galβ1,4GlcNAc α2,6‐sialyltransferase I (ST6Gal I) activity in rodent fibroblasts. Given that Ras mediates its effects through at least three secondary effector pathways (Raf, RalGEFs and PI3K) and that transcriptional control of mouse ST6Gal I is achieved by the selective use of multiple promoters, we attempted to identify which of these parameters are involved in linking the Ras signal to ST6Gal I gene transcription in mouse fibroblasts. Transformation by human K‐Ras or H‐Ras (S12 and V12 point mutations, respectively) results in a 10‐fold increase in ST6Gal I mRNA, but no alteration in the expression of related sialyltransferases. Using an inducible H‐RasV12 expression system, a direct causal link between activated H‐Ras expression and elevated ST6Gal I mRNA was demonstrated. The accumulation of the ST6Gal I transcript in response to activated Ras was accompanied by an increase of α2,6‐sialyltransferase activity and of Neu5Acα2,6Gal at the cell surface. Results obtained with H‐RasV12 partial loss of function mutants H‐RasV12S35 (Raf signal only), H‐RasV12C40 (PI3‐kinase signal only) and H‐RasV12G37 (RalGEFs signal only) suggest that the H‐Ras induction of the mouse ST6Gal I gene (Siat1) transcription is primarily routed through RalGEFs. 5′‐Rapid amplification of cDNA ends analysis demonstrated that the increase in ST6Gal I mRNA upon H‐RasV12 or K‐RasS12 transfection is mediated by the Siat1 housekeeping promoter P3‐associated 5′ untranslated exons.

Hepatic expression of CMP-NeuAc:Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase (ST6Gal I) is induced as part of the acute phase response in mammals by mechanisms that remain poorly understood. Previous work suggests that murine liver ST6Gal I mRNA contains an additional and novel region that is not found on ST6Gal I mRNA from human HepG2 hepatoma cells and from rat liver. This novel region, residing 5' of the common Exon I sequence, is encoded by a discrete upstream exon, Exon H. Here we provide evidence that the Exon H-containing transcript is the murine counterpart of the human and rat ST6Gal I mRNAs transcribed from the hepatic-specific promoter, P1. Exon H-containing ST6Gal I mRNA is expressed in all three mice strains examined: balb/c, C57B46, and 129Sv. Furthermore, murine RNA tissue survey indicates that presence of Exon H-containing transcripts is restricted to the liver. When mice are subjected to subcutaneous injection of turpentine to elicit the hepatic acute phase response, greater than 4-fold elevation in liver ST6Gal I mRNA was observed. Consistent with the view that Exon H-containing transcripts is regulated by the murine P1 promoter, 5'-RACE analysis indicates that the majority of these transcripts contains the Exon H sequence. This is consistent with the view that Exon H-containing transcripts are regulated by the murine P1 region. To assess the mechanism of ST6Gal I response in the hepatic acute phase reaction, mice harboring lesions in both alleles of the IL-6 gene were examined. IL-6(-/-) animals expressed normal levels of ST6Gal I mRNA in liver, with Exon H-containing transcripts remaining the predominant mRNA isoform. However, hepatic ST6Gal I is not elevated upon turpentine injection in the IL-6(-/-) animals. These results indicate that ST6Gal I induction in mouse liver during the acute phase reaction is mediated predominantly by the IL-6 pathway, and results in the induction of the Exon H-containing class of ST6Gal I mRNA that is specific to the liver.