Investigation of Shiga-like toxin binding to chemically synthesized oligosaccharide sequences.

Shiga-like toxin (SLT)-I and SLT-II/IIc bound to Synsorbs containing synthetic alpha Gal(1-4)beta Gal (P1 disaccharide), alpha Gal(1-4)beta GlcNAc (P1 trisaccharide), or alpha Gal(1-4)beta Gal(1-4)beta Glc (Pk trisaccharide) sequences but not to Synsorbs containing alpha Gal(1-3)beta Gal, alpha Gal(1-3)beta Gal(1-4)beta GlcNAc, or the hydrophobic oligosaccharide linkage arm. SLT-I had a preference for Synsorbs containing trisaccharides, whereas SLT-II/IIc binding was less selective. 125I-labeled SLT-I remained bound to Pk trisaccharide Synsorb in the presence of lactose, galactose, or EDTA but was partially released by acetic acid, guanidine HCl, or a 10% solution of SDS. Vero cells coincubated with Pk trisaccharide Synsorb and SLT I extract were protected from this toxin, whereas Pk trisaccharide Synsorb was much less efficient at neutralizing SLT-II/IIc activity in Vero cell coincubation experiments. The SLT-IIc component was not responsible for the inefficient neutralization. Results suggest that synthetic oligosaccharide sequences related to the P blood group antigens coupled to inert matrices could be useful for rapid diagnosis or possibly therapeutic intervention in enterohemorrhagic Escherichia coli infections.     

A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats.

We previously reported that short-term immobilization stress of rats causes increased colonic mucin release, goblet cell depletion, prostaglandin E2 secretion, and colonic mast cell activation, as well as increased colonic motility. The purpose of this study was to investigate whether neurotensin (NT), a peptide expressed in both brain and digestive tract, participates in these responses. Rats were pretreated with SR 48692 (1 mg/kg, i.p.), an NT antagonist, 15 min before immobilization (30 min). The administration of the antagonist significantly inhibited stress-mediated secretion of colonic mucin, prostaglandin E2, and a product of rat mast cells, rat mast cell protease II (P < 0.05), but did not alter the increase in fecal pellet output caused by immobilization stress. Immobilization stress also resulted in a quantifiable decrease in the abundance of NT receptor mRNA in rat colon compared with that in colonic tissues from nonimmobilized rats as measured by densitometric analysis of in situ hybridization studies (P < 0.03). We conclude that the peptide NT is involved in colonic goblet cell release and mucosal mast cell activation after immobilization stress.     

Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum

The purpose of this study was to compare the effects of Clostridium difficile toxin A and cholera toxin on fluid secretion, intestinal permeability, and arachidonate metabolites in rabbit ileum. Injection of 25 micrograms of either purified toxin into 10-cm ileal loops caused significant increases in fluid secretion and intestinal permeability to mannitol as well as release of prostaglandin E2 into the lumen. Toxin A, but not cholera toxin, caused a severe inflammatory reaction of the lamina propria and necrosis of enterocytes as well as increased release of leukotriene B4. The toxin A-mediated increases in prostaglandin E2 and leukotriene B4 could be blocked by prior instillation of 10 mg of 5-aminosalicylic acid into ileal loops. 5-Aminosalicylic acid also significantly diminished the expected increase in mannitol permeability after both toxins, but had no significant inhibitory effect on fluid secretion or, in the case of toxin A, intestinal inflammation. Our results indicate that C. difficile and cholera enterotoxins differ substantially in their effects on the rabbit intestine. Clostridium difficile toxin A, an inflammatory toxin, produces a striking infiltration of the lamina propria with neutrophils that is associated with increased release of leukotriene B4. In contrast, cholera toxin does not cause inflammation or leukotriene B4 release. Increased release of prostaglandin E2 occurs after exposure to both toxins and appears to be correlated with increased intestinal permeability.     

CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.

Toxin A from Clostridium difficile mediates acute inflammatory enterocolitis in experimental animals, while cholera toxin causes noninflammatory secretory diarrhea. The purpose of this study was to investigate whether an antagonist to the peptide substance P, a constituent of primary sensory neurons known to participate in inflammatory responses, would inhibit toxin A-mediated enteritis in the rat ileum. Pretreatment of rats with CP-96,345 (2.5 mg per kg of body weight), a substance P antagonist, dramatically inhibited fluid secretion (P < 0.01) and mannitol permeability (P < 0.01) in ileal loops exposed to toxin A. The protective effects, which were dose dependent, caused a significant reduction of inflammation in the lamina propria, reduction of the necrosis of intestinal epithelial cells, and complete inhibition of toxin A-mediated release of rat mast cell protease II, a specific product of rat mucosal mast cells. An inactive enantiomer of the substance P antagonist, CP-96,344, had no effect. In contrast, pretreatment with CP-96,345 had no inhibitory effect on the intestinal effects caused by administration of cholera toxin into the ileal loops. From these data, we conclude that the peptide substance P is involved in the secretory and inflammatory effects of toxin A but not of cholera toxin.     

Regulation of intestinal mast cells and luminal protein release by cerebral thyrotropin-releasing hormone in rats

BACKGROUND & AIMS: Intestinal mast cell activity is modulated by the central nervous system, but the mechanisms are not well established. The aim of this study was to investigate whether cerebral thyrotropin- releasing hormone (TRH) activates intestinal mast cells and to elucidate the mechanisms involved, specifically, the contribution of mast cells to vagally stimulated luminal protein release. METHODS: In anesthetized rats, mast cell activation was assessed by measuring the release of the specific mucosal rat mast cell protease II (RMCP II) and prostaglandin (PG) D2 into the intestinal lumen. Luminal protein release was measured as an index of epithelial permeability to macromolecules. RESULTS: Intracisternal injection of the TRH analogue RX 77368 (30 ng) induced a transient increase in intestinal release of RMCP II and PGD2 that was abolished by dox-antrazole. RX 77368- stimulated RMCP II release was also abolished by vagotomy and reduced by atropine by 65%. However, both systemic capsaicin and indo-methacin enhanced RMCP II release. RX 77368-stimulated luminal protein release was abolished by vagotomy and reduced by doxantrazole. CONCLUSIONS: Central vagal activation by TRH stimulates intestinal mast cell secretion, in part via peripheral muscarinic receptors, and is modulated by PGs and capsaicin-sensitive afferent innervation. Intestinal mast cell activation contributes to the TRH analogue- stimulated luminal protein release. (Gastroenterology 1996 Dec;111(6):1465-73)     

Dexamethasone prevents visceral hyperalgesia but not colonic permeability increase induced by luminal protease-activated receptor-2 agonist in rats

BACKGROUND: Low-grade inflammation may play a role in the pathogenesis of irritable bowel syndrome (IBS). Although corticosteroids are potent inhibitors of inflammatory processes, only one study with corticosteroids in patients with postinfectious IBS exists, which suggests that prednisolone is not an effective treatment for IBS symptoms. AIM: To evaluate whether dexamethasone treatment prevents protease-activated receptor-2 (PAR-2) activation-induced visceral hyperalgesia and increased permeability in rats, and to determine whether the effects involve colonic mast cells. METHODS: Abdominal contractions provoked by rectal distension were recorded in rats equipped with intramuscular electrodes. Changes in visceral hypersensitivity provoked by intracolonic administration of PAR-2-activating peptide (SLIGRL; H-serine-leucine-isoleucine-glycine-arginine-leucine-OH), changes in colonic mucosal rat mast cell protease-II (RMCP-II) content, mast cell count and PAR-2 expression were measured after a 4-day treatment with dexamethasone (1 mg/day/rat intraperitoneally) or its vehicle (water). The effect of mast cell stabiliser (doxantrazole, 1 mg/kg intraperitoneally, 2 h before and 6 h after intracolonic infusion of SLIGRL) on SLIGRL-induced visceral hyperalgesia was also assessed. The effects of SLIGRL and a mast cell degranulator (compound 48/80) on the permeability of colonic strips from vehicle- or dexamethasone-treated rats were investigated in Ussing chambers. RESULTS: 4 days of dexamethasone as well as doxantrazole diminished the SLIGRL-induced hyperalgesia for all volumes of distension. This effect of dexamethasone was accompanied by a reduced responsiveness of colonic permeability to compound 48/80, and decreased RMCP-II content and mast cell number. Dexamethasone treatment did not influence colonic mucosal PAR-2 expression and permeability responsiveness to SLIGRL. CONCLUSIONS: Dexamethasone treatment improves PAR-2 agonist-induced visceral hypersensitivity but does not prevent PAR-2 agonist-induced increase in colonic permeability in rats. This effect is coupled with a reduction of colonic mast cell number and RMCP-II contents.     

Pathogenesis of shigella diarrhea: evidence for a developmentally regulated glycolipid receptor for shigella toxin involved in the fluid secretory response of rabbit small intestine

Shigella toxin reproduces the major manifestations of shigellosis in ligated intestinal loops from adult rabbits and binds to a microvillus membrane (MVM) glycolipid receptor, globotriaosylceramide (Gb3). Because neonatal human shigellosis is uncommon, we used the animal model for obtaining MVMs from rabbits of different ages to determine the presence of toxin receptors and Gb3 and to measure the fluid secretory response to toxin in ligated ileal loops. A single class of MVM receptors for 125I-labeled shigella toxin, first detected at 20 d of age, reached adult levels by 24 d (n = 1.7-23.8 X 10(10)/micrograms of protein; K = 1.1-3.8 X 10(9) M-1). Binding was specific for toxin subunit B. A toxin binding MVM glycolipid, identified as Gb3, was detected in animals greater than or equal to 16 d of age by high-performance thin-layer chromatography and autoradiography. Fluid secretion in response to shigella toxin in ligated small bowel loops occurred in temporal relation to the appearance of Gb3, a result thus indicating the involvement of Gb3 in mediating the toxin effects.     

Is the secretagogue effect of deoxycholic acid mediated by the adenylate cyclase-cAMP system

The role of the adenylate cyclase (Ac)-cAMP system in mediating deoxycholic acid (DOC)-induced fluid secretion was studied in the rat jejunum and colon in vivo using the AC inhibitor, RMI 12 330 A. A potent inhibitory effect of RMI 12 330 A on fluid secretion induced by cholera toxin was demonstrated in ligated rat jejunal loops. On the contrary, the changes of fluid movement in jejunal and colonic loops caused by DOC could not be influenced by RMI 12 330 A, and mucosal cAMP levels of colonic loops were not increased. Colonic mucosal permeability estimated by the 14C-erythritol clearance increased significantly during a 45-min exposure to 3 mmol DOC, and was not affected by RMI 12 330 A. These results do not support the theory that the AC-cAMP system plays an important role in DOC-induced intestinal fluid secretion and suggest that an increase in mucosal permeability is the predominant factor responsible for the secretagogue effect.     

Immunoglobulin G glycosylation and clinical outcome in rheumatoid arthritis during pregnancy

OBJECTIVE: To determine whether clinical outcome during pregnancy in rheumatoid arthritis (RA) is associated with changes in the levels of exposed immunoglobulin G (IgG) terminal sugars. METHODS: Serum IgG glycosylation from 23 pregnant patients with RA was analyzed during the prenatal, antenatal, and post-partum periods. Patients were randomly selected on the basis of whether they achieved spontaneous remission (n = 11) or did not remit (n = 12); of the latter group 6 patients experienced a relapse in disease activity. Levels of exposed terminal IgG sugars, galactose (Gal), N-acetylglucosamine (GlcNAc), and sialic acid (SA), were estimated in a lectin binding assay using Ricinis (communis, Bandeiraea simplicifolia II, and Sambucus nigra, respectively. RESULTS: Exposed Gal levels increased (p<0.02) and GlcNAc levels decreased (p<0.05) in the antenatal period, and returned to preconception levels during post-partum. GlcNAc rebound was instantaneous (p<0.005), whereas Gal remained high for a further 10 weeks. SA did not undergo any major changes. Remission was associated with an earlier and significantly greater antenatal reduction in GlcNAc (2nd and 3rd trimester; p<0.02) in comparison to the groups that did not experience a decrease in disease activity. Analysis of individual IgG samples during the first trimester revealed a significant negative correlation between Gal and GlcNAc in the remission group (r = -0.80; p<0.05), which was opposite to that found in the relapse group (r = +0.87; p<0.03). There was no significant difference between the groups with regard to the timing and/or incidence of a post-partum flare of disease. CONCLUSION: Temporal changes in the levels of IgG terminal sugars, in particular exposed GlcNAc, are integrally associated with the clinical manifestation of RA in pregnancy. Generation of IgG sugar micro-heterogeneity is complex and understanding it may help unravel pathogenic features associated with RA.     

Role of 5-hydroxytryptamine type 3 receptors in rat intestinal fluid and electrolyte secretion induced by cholera and Escherichia coli enterotoxins.

Cholera toxin and Escherichia coli heat labile toxin (LT) induced intestinal secretion has in the past been attributed exclusively to an increase in intracellular cAMP whereas E coli heat stable toxin (ST) induced secretion is mediated through cGMP. Evidence is accumulating on the importance of 5-hydroxytryptamine (5-HT) in cholera toxin induced secretion, but its role in LT and ST is not well established. This study therefore investigated in vivo the effect of 5-HT3 receptor antagonist, granisetron, on intestinal fluid and electrolyte secretion induced by cholera toxin, LT, and ST. Granisetron (30, 75, 150, or 300 micrograms/kg) was given subcutaneously to adult male Wistar rats 90 minutes before instillation of 75 micrograms cholera toxin or 50 micrograms LT in isolated whole small intestine. In situ small intestinal perfusion was performed with an iso-osmotic plasma electrolyte solution (PES) to assess fluid movement. In a second group of animals, granisetron (300 micrograms/kg) was given subcutaneously and two hours later small intestinal perfusion with PES containing 200 micrograms/l ST was performed. Cholera toxin induced net fluid secretion (median -50.1 microliters/min/g (interquartile range -59.5 to -29.8)) was found to be dose dependently decreased or abolished by granisetron (plateau effect at 75 micrograms/kg: 18 (-7.8 to 28), p < 0.01). Granisetron in high dose (300 micrograms/kg), however, failed to prevent LT or ST induced secretion (-52 (-121 to -71) v -31 (-44 to -18), and (-39 (-49 to 17) v (-22 (-39 to -3)), respectively). Sodium and chloride movement paralleled that of fluid. In conclusion, these data show that 5-HT and 5-HT3 receptors play an important part in cholera toxin induced secretion but are not involved in E coli heat stable or heat labile toxin induced secretion.     

A major serine protease in rat skeletal muscle: Evidence for its mast cell origin

The physical, chemical, and immunologic properties of a protease from rat skeletal muscle, proposed to function in the degradation of certain intracellular enzymes, are identical to those of a chymotrypsin-like serine protease isolated from peritoneal mast cells. The results of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea indicate that the two rat proteases have identical mobilities corresponding to a molecular weight of 26,000. The relative amino acid compositions of the proteases are nearly identical. Immunodiffusion tests for crossreaction between the muscle protease and antisera directed toward mast cell protease indicate that the former is immunologically identical to mast cell protease. The first 35 amino-terminal residues of the two enzymes are identical and indicate homology of these proteins to other mammalian serine proteases. The sequence analysis of the protease from muscle was extended for an additional 16 positions, and comparison of this amino-terminal sequence with that of a similar enzyme from small intestine showed approximately 75% sequence identity. In contrast, only 40% of the residues in this region of bovine chymotrypsin A were found at corresponding loci in rat muscle protease. It is concluded that the protease from muscle or mast cells is closely related to the enzyme from small intestine which recently was localized in the "atypical" mast cells of gut mucosa [Woodbury, R. G., Gruzenski, G. M. & Lagunoff, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2785-2789].     

Neonatal maternal deprivation triggers long term alterations in colonic epithelial barrier and mucosal immunity in rats

BACKGROUND: Stressful events in the early period of life (for example, maternal deprivation) have been shown to modify adult immune and gastrointestinal tract functions. The present study aimed to establish whether maternal deprivation affects colonic epithelial barrier and the development of an experimental colitis in adult rats. METHODS: Male Wistar rat pups were separated during postnatal days 2-14 or left undisturbed with their dam. At 12 weeks of age, we assessed colonic paracellular permeability, bacterial translocation, myeloperoxidase (MPO) activity, mucosal mast cell density, cytokine (interleukin (IL)-1 beta, IL-2, IL-4, IL-10, and interferon gamma (IFN-gamma)) mRNA expression, and macroscopic damage. Total gut permeability, MPO activity, and macroscopic damage were also assessed four days after intracolonic administration of 2,4,6-trinitrobenzenesulphonic acid (TNBS). RESULTS: Maternal deprivation triggered a significant increase in colonic permeability associated with bacterial translocation into the mesenteric lymph nodes, liver, and spleen. These alterations were associated with some macroscopic damage and an increase in colonic MPO activity, mucosal mast cell density, and cytokine mRNA expression. Intracolonic infusion of TNBS induced a significantly higher inflammatory reaction in separated animals, as judged by enhanced MPO colonic levels, total gut permeability, and macroscopic lesions. CONCLUSIONS: Maternal deprivation promotes long term alterations in the colonic epithelial barrier associated with an exaggerated immune response to an external immune stimulus. This suggests a role for early psychological factors in the regulation of colonic mucosal barrier in later life.     

Mucin and nonmucin secretagogue activity of Entamoeba histolytica and cholera toxin in rat colon

Depletion of colonic mucus occurs before invasion of the colonic mucosa by Entamoeba histolytica trophozoites. It is hypothesized that E. histolytica releases a mucus secretagogue; this was studied in a rat colonic loop model. In colonic loops exposed to live amebae, mucus secretion was quantitated by release of acid-precipitable [3H]glucosamine-labeled luminal glycoprotein and by specific immunoassay. Mucus secretion increased in dose-dependent fashion in response to greater than or equal to 1 X 10(5) trophozoites; cholera toxin (20 micrograms per loop), a known mucus secretagogue, elicited a similar response. Thin-section histological analysis of amebae and cholera toxin-exposed loops showed increased mucus release and streaming from mucosal goblet cells with cellular cavitation compared with control loops. Sepharose-4B chromatography of amebae and cholera toxin-stimulated glycoproteins demonstrated secretion of mucins and an 80%-90% increase in low-molecular-weight proteins. E. histolytica trophozoites and cholera toxin enhanced the secretion of preformed and newly synthesized mucin glycoproteins and stimulated colonic glycoprotein synthesis. The level of mucus secretion elicited by axenic E. histolytica strains correlated with their virulence in vivo and in vitro. The amebic secretagogue was released into the culture medium and was heat stable. Mucus secretagogue activity of E. histolytica may contribute to depletion or alteration of the protective mucus blanket, facilitating pathogenesis of invasive amebiasis.     

Differential effects of Clostridium difficile toxins A and B on rabbit ileum

The pathogenesis of Clostridium difficile enterocolitis appears to involve colonization of the bowel followed by release of toxin A, an enterotoxin, and toxin B, a cytotoxin. The purpose of this study was to determine the effect of purified toxins A and B on intestinal secretion, epithelial permeability, and morphology in perfused rabbit ileal loops. Intestinal permeability after toxin exposure was assessed by blood-to-lumen clearance of [3H]mannitol. Toxin A at doses of 5-100 micrograms/10 cm ileal loop caused a threefold to fivefold increase in [3H]mannitol permeability (p less than 0.001) vs. equal concentrations of toxin B or buffer control. In addition, perfusate from toxin A-exposed loops contained significantly more neutrophils (p less than 0.001) than toxin B or control loops. Toxin A caused severe epithelial cell necrosis with destruction of villi and polymorphonuclear infiltration. Electron microscopy of mucosa subjected to a low dose of toxin revealed widespread nonspecific dilatation of endoplasmic reticulum and mitochondrial swelling. In contrast to these effects of toxin A in ileal loops, in vitro experiments with ileal explants in short-term organ culture revealed that toxin A had no effect on epithelial cell permeability, protein synthesis, release of alkaline phosphatase, or morphology. Our results show that purified toxin A but not toxin B causes severe inflammatory enteritis in rabbit ileal loops, but has no discernable effect on rabbit ileum in vitro. We speculate that toxin A may contribute significantly to intestinal damage in C. difficile-associated colitis and diarrhea.     

Systemic release of mucosal mast cell protease during infection with the intestinal protozoal parasite, Eimeria nieschulzi. Studies in normal and nude rats

The systemic secretion of rat mucosal mast cell protease (RMCPII), a major product of rat mucosal mast cells (MMC), was examined during primary infections with the protozoan parasite, Eimeria nieschulzi in CFH/B, athymic (rnu/rnu) and euthymic (rnu/+) rats. Release of RMCPII into the blood stream (2.9 micrograms/ml of serum) of normal rats occurred within 1 day after infection. This response developed 3-6 hours after inoculation with oocysts, was dose-dependent, and was found in both naive and immune rats. Maximal release of RMCPII (4.5 micrograms/ml of serum) in naive rats occurs 9 days after primary infection, whereas the numbers of MMC and concentrations of mucosal RMCPII were maximal 14 days after infection, by which time the systemic RMCPII response had begun to decline. The numbers of MMC and concentrations of mucosal RMCPII in uninfected nude rats were similar to those in the heterozygous (rnu/+) litter-mates. After infection, the numbers of MMC and concentrations of mucosal RMCPII increased in the heterozygotes but not in nude rats. Similarly, RMCPII was detected systemically only in the heterozygotes.     

Early functional effects of Clostridium difficile toxin A on human colonocytes

BACKGROUND & AIMS: Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function. The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells. METHODS: Two model systems were used: the HT29-CI.16E colonic goblet cell line and primary cultures of human normal colonocytes. RESULTS: Toxin A exerted a rapid and dose- related inhibition of stimulated mucin exocytosis without altering baseline (constitutive) mucin exocytosis from HT29-CI.16E cells. Toxin A was also able to induce the secretion of IL-8 from both HT29-CI.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation. CONCLUSIONS: The results show that while toxin A is able to down-regulate stimulated mucin exocytosis, it is able to up- regulate the secretion of an important chemoattractant chemokine, IL-8. These modifications illustrate the ability of colonocytes to recruit inflammatory and immune cells that will eventually bring about major mucosal damage. (Gastroenterology 1997 Jun;112(6):1887-94)     

Colonic and jejunal motor disturbances after colonic antigen challenge of sensitized rat

BACKGROUND & AIMS: Inflammation in the colon may alter motility in the proximal gut and potentiate clinical symptoms. The aim of this study was to characterize the effect of colonic anaphylaxis on local (colonic) and remote (small intestinal) motility and identify the mechanism and mediators involved. METHODS: Rats were sensitized by intraperitoneal injection of 10 microg egg albumin and surgically prepared with electrodes in jejunum and colon and a colostomy tube. Colonic and jejunal myoelectric activity were recorded in fasted animals before and after colonic antigen challenge without and then after pretreatment with specific antagonists. RESULTS: Colonic antigen challenge of sensitized rats was associated with significant (1) increase in colonic myoelectric spike activity, (2) disruption of fasting jejunal motility and initiation of aborally propagating spike complexes, and (3) increase in plasma rat mast cell protease II levels with a decrease in granulated mast cells in colon but not jejunum. The myoelectric disturbance in both colon and jejunum was inhibited significantly by pretreatment with atropine and hexamethonium, doxantrazole, cyclooxygenase, and lipoxygenase inhibitors. Methysergide inhibited only the jejunal disturbance. CONCLUSIONS: Colonic antigen challenge of sensitized animals results in local mast cell activation and the release of mediators that modulate neural pathways to initiate both a local colonic and a remote jejunal myoelectric disturbance. (Gastroenterology 1997 Jun;112(6):1996-2005)     

Immune regulation by the ST6Gal sialyltransferase

The ST6Gal sialyltransferase controls production of the Siaα2-6Galβ1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.     

Fringe modulation of Jagged1-induced Notch signaling requires the action of β4galactosyltransferase-1

Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a beta 3N-acetylglucosaminyltransferase (beta 3GlcNAcT) that transfers GlcNAc to O-fucose in epidermal growth factor-like repeats of Notch. Here we use five different Chinese hamster ovary cell glycosylation mutants to identify a key aspect of the mechanism of fringe action. Although the beta 3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAc beta 3Fuc, is not elongated. The trisaccharide, Gal beta 4GlcNAc beta 3Fuc, is the minimal O-fucose glycan to support fringe modulation of Notch signaling. Of six beta 4galactosyltransferases (beta 4GalT) in Chinese hamster ovary cells, only beta 4GalT-1 is required to add Gal to GlcNAc beta 3Fuc, identifying beta 4GalT-1 as a new modulator of Notch signaling.