Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. (33/567)

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.  (+info)

Xenotransplantation: in vitro analysis of synthetic alpha-galactosyl inhibitors of human anti-Galalpha1-->3Gal IgM and IgG antibodies. (34/567)

Pig-to-human xenotransplantation might be an option to overcome the increasing shortage of human donor organs. However, naturally occurring antibodies in human blood against the Galalpha1-->3Gal antigen on pig endothelial cells lead to hyperacute or, if prevented, acute or delayed vascular rejection of the pig graft. The purpose of this study was therefore to evaluate synthetic oligosaccharides with terminal Galalpha1-->3Gal to inhibit antigen-binding and cytotoxicity of anti-alphaGal antibodies against pig cells. Different oligosaccharides were synthesized chemically and by a combined chemico-enzymatic approach. These included monomeric di-, tri-, and pentasaccharides, a polyacrylamide-conjugate (PAA-Bdi), as well as di-, tetra-, and octamers of Galalpha1-->3Gal. All were tested for inhibitory activity by anti-alphaGal ELISA and complement-dependent cytotoxicity tests. PAA-Bdi was the best inhibitor of binding as well as cytotoxicity of anti-alphaGal antibodies. Monomeric oligosaccharides efficiently prevented binding of anti-alphaGal IgG, but less well that of anti-alphaGal IgM, with tri- and pentasaccharides showing a better efficacy than the disaccharide. The two trisaccharides Galalpha1-->3Galbeta1-->4GlcNAc and Galalpha1-->3Galbeta1-->3GlcNAc were equally effective. Oligomers of Galalpha1-->3Gal were more effective than monomers in blocking the binding of anti-alphaGal IgG. However, they could not block IgM binding, nor could they match the efficacy of PAA-Bdi. We conclude that oligosaccharides with terminal Galalpha1-->3Gal, most effectively as PAA-conjugates, can prevent binding and cytotoxicity of human anti-alphaGal in vitro. The PAA-Bdi conjugate might be most suited for use as a Sepharose-bound immunoabsorption material.  (+info)

Membrane-associated mucins in normal human conjunctiva. (35/567)

PURPOSE: To examine the presence of specific membrane-associated mucins in normal human conjunctiva. METHODS: Glycoconjugates were extracted from membranes with two detergents: octylglucoside and Triton X114. Mucins were separated by cesium chloride density gradient centrifugation. Size was assessed by gel filtration on Sepharose CL2B and charge by ion-exchange chromatography on MonoQ. Cross reaction with antibodies against mucin gene products was assessed in blots of electrophoresis gels. RESULTS: Extraction of total tissue membranes yielded material with a buoyant density typical of mucins. Gel filtration showed material reacting with antimucin antibodies in a range of molecular sizes. Agarose electrophoresis confirmed the presence of MUC1 and MUC4 and the absence of MUC2 or MUC5AC. Isolation of membrane mucins by sequential, exhaustive extraction with octylglucoside followed by Triton X114 suggested the existence of mucins in different membrane environments. Reagents to carbohydrate epitopes revealed high mobility material, comigrating with MUC1 and MUC4. Low mobility membrane-bound mucins did not cross-react with any antibodies to mucin genes known to be expressed in human conjunctiva. CONCLUSIONS: Membrane-associated mucins are distinct from secreted mucins in normal human conjunctiva. MUC1 and MUC4 mature products decorate the membranes of conjunctival epithelial cells. Their segregation between octyl glucoside and the detergent and aqueous phases of Triton X114 suggests a variety of membrane anchoring modes.  (+info)

Expression patterns of glycoconjugates in the three distinctive olfactory pathways of the clawed frog, Xenopus laevis. (36/567)

Xenopus laevis has three distinctive olfactory neuroepithelia. We examined the axonal projection from each of these epithelia to the olfactory bulb by Di-I labeling, and confirmed that the Xenopus primary olfactory pathways involve the dorsal pathway from the olfactory epithelium to the dorsal region of the main olfactory bulb, the ventral pathway from the middle chamber epithelium to the ventral region of the main olfactory bulb, and the vomeronasal pathway from the vomeronasal epithelium to the accessory olfactory bulb. We next examined expression patterns of glycoconjugates in the three olfactory pathways by lectin-histochemistry using 21 biotinylated lectins. Fourteen out of 21 lectins stained the Xenopus primary olfactory system. RCA-I stained the three olfactory pathways uniformly. PHA-E stained only the dorsal pathway. LEL, STL, PNA, ECL and UEA-I stained the dorsal pathway more intensely than the ventral pathway, and among them, only UEA-I stained the vomeronasal pathway. In contrast, s-WGA, DBA, SBA, BSL-I VVA, SJA and PHA-L showed intense stainings in the ventral pathway and moderate stainings in the vomeronasal pathway, but faint or weak stainings in the dorsal pathway. These observations suggest that the ventral pathway expresses glycoconjugates shared commonly with either the dorsal or the vomeronasal pathway. In addition, from the binding patterns of the lectins with a binding specificity for N-acetylgalactosamine, glycoconjugates containing this saccharide seem to play an important role for the organization of the olfactory pathways.  (+info)

Cell surface engineering by a modified Staudinger reaction. (37/567)

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.  (+info)

Human neutrophil elastase releases two pools of mucinlike glycoconjugate from tracheal submucosal gland cells. (38/567)

Neutrophil elastase can contribute to the pathogenesis of increased airway reactivity and excess mucus secretion in many pulmonary diseases. Ten nanomolar human neutrophil elastase (HNE) effectively empties airway serous cells, raising the question of why HNE is not equally effective at emptying mucous cells of their stored mucin because total release of mucin granules is not seen in postmortem examination of even the most severe disease. To better resolve the mucus secretagogue action of HNE, we measured secretion of mucinlike glycoconjugates (MGCs) released from freshly isolated swine tracheal submucosal gland cells in fractions of the superfusate acquired every 2 min. Six to fifty nanomolar HNE released a fixed quantity of MGCs at an increasing rate with increasing concentrations of enzyme, an action consistent with the release of cell surface mucinlike molecules. The polycation poly-L-lysine (1 microg/ml) released a similar transient of MGCs. A steady-state doubling of MGC rate of release was seen as long as 100 nM HNE was present, but this stimulus represented less than a 1% release of stored MGCs/min and was consistent with release of mucin vesicles from cell stores. Both actions of HNE were inhibited by the specific inhibitors L-680833 and DMP-777 but not by 30 microM erythromycin. Therefore, HNE release of MGCs from tracheal submucosal glands is limited by both the fixed quantity of the MGCs in the transient pool and by the small steady-state response to the higher concentrations of enzyme.  (+info)

DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex. (39/567)

The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and primase.  (+info)

Isolation and characterization of protoporphyrin glycoconjugates from rat harderian gland by HPLC, capillary electrophoresis and HPLC/electrospray ionization MS. (40/567)

It has been widely reported that the Harderian gland, present in most vertebrates, accumulates high levels of porphyrins, particularly protoporphyrin. The present study describes the extraction, identification and characterization of a group of hitherto unreported protoporphyrin glycoconjugates in the rat Harderian gland using HPLC, capillary electrophoresis, on-line HPLC/electrospray ionization MS and tandem MS. The major glycoconjugate was identified as protoporphyrin-1-O-acyl beta-xyloside with a smaller amount of protoporphyrin-1-O-acyl beta-glucoside also detected. In the Harderian glands studied, 50-70% of the porphyrins present were in the form of protoporphyrin glycoconjugates. This is the first reported occurrence of glycoconjugates of porphyrins in Nature and suggests that previous studies have wrongly identified the major porphyrin in the Harderian gland as the unconjugated protoporphyrin.  (+info)