Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Targeted point mutation that creates a unique Eco RI site within the signal codons of the beta-lactamase gene without altering enzyme secretion or processing

A method has been developed for constructing site-specific mutations by using a strongly selectable marker on which to "piggy-back" a desired mutation that may be phenotypically silent. Using this approach, a new unique Eco RI restriction site has been generated at the beginning of the signal codons of the beta-lactamase gene of the plasmid pBR322. The consequential alteration of the second amino acid of the signal from Ser to Arg has no effect on either the transport or the processing of the beta-lactamase.     

Isoforms of apolipoprotein A-II in human plasma and thoracic duct lymph. Identification of proapolipoprotein A-II and sialic acid-containing isoforms

Proapolipoprotein (apo-) A-II and several isoforms of apo-A-II including sialylated isoforms were identified in human plasma and thoracic duct lymph. Proapo-A-II secreted by HepG2 cells was identified by a combination of immunoblots and [14C]arginine incorporation. Proapo-A-II which contains 2 arginine residues could be readily differentiated from mature apo-A-II which contains no arginine. The pI of proapo-A-II is 6.79, whereas the pI of the major apo-A-II isoform in plasma and lymph is 4.90. Minor apo-A-II isoforms have pI values of 5.17, 4.68, 4.42, and 4.20, respectively. Sialoisoforms of apo-A-II were identified, which had a higher apparent molecular weight on sodium dodecyl sulfate-gel electrophoresis than the major isoform and disappeared following neuraminidase treatment. The relative quantity of proapo-A-II was relatively constant in lymph very low density lipoproteins, lymph high density lipoproteins, and plasma high density lipoproteins, whereas the sialoforms and the other minor isoforms of apo-A-II were greater in lymph very low density lipoproteins and the lowest in plasma high density lipoproteins.     

Inhibition of repulsive guidance molecule,RGMa, increases afferent synapse formation with auditory hair cells

The peripheral fibers that extend from auditory neurons to hair cells are sensitive to damage, and replacement of the fibers and their afferent synapse with hair cells would be of therapeutic interest. Here, we show that RGMa, a repulsive guidance molecule previously shown to play a role in the development of the chick visual system, is expressed in the developing, newborn, and mature mouse inner ear. The effect of RGMa on synaptogenesis between afferent neurons and hair cells, from which afferent connections had been removed, was assessed. Contact of neural processes with hair cells and elaboration of postsynaptic densities at sites of the ribbon synapse were increased by treatment with a blocking antibody to RGMa, and pruning of auditory fibers to achieve the mature branching pattern of afferent neurons was accelerated. Inhibition by RGMa could thus explain why auditory neurons have a low capacity to regenerate peripheral processes: postnatal spiral ganglion neurons retain the capacity to send out processes that respond to signals for synapse formation, but expression of RGMa postnatally appears to be detrimental to regeneration of afferent hair cell innervation and antagonizes synaptogenesis. Increased synaptogenesis after inhibition of RGMa suggests that manipulation of guidance or inhibitory factors may provide a route to increase formation of new synapses at deafferented hair cells. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 457–466, 2014     

Isolation of sulfited oligosaccharides from glycoproteins treated with alkaline sulfite

The oligosaccharide products resulting from treatment of mucin-type glycoproteins with alkali in the presence of the sulfite anion have been investigated. Treatment of fetuin and of tryptic glycopeptides from the human erythrocyte with this reagent resulted in the release of sulfited oligosaccharides identified as N-acetylsulfohexosamine (HexNAcSO3), alpha-NeuAc-(2----6)-HexNAcSO3, and alpha-NeuAc-(2----3)-Gal-(1----3 or 4)-[GlcNAc-(1----6)]-HexNAcSO3. In addition, 2.7 moles of sialic acid were released per mole of alpha-NeuAc-(2----6)-HexNAcSO3 from fetuin. The sulfohexosamine moiety is formed via unsaturated intermediates from a 3-O-substituted 2-acetamido-2-deoxy-D-galactosyl residue at the carbohydrate-peptide linkage site when this residue is not substituted at O-4 by another sugar residue. A reaction mechanism accounting for the release of the sulfited oligosaccharides from a 3-O- and 6-O-substituted hexosamine is proposed in which the oligosaccharide branch attached to O-6 is obtained as a specific fragment terminating in sulfohexosamine.     

Serum inverts and improves the fluorescence response of an aptamer beacon to various vitamin D analytes

A dominant aptamer loop structure from a library of nearly 100 candidate aptamer sequences developed against immobilized 25‐hydroxyvitamin D₃ (calcidiol) was converted into a 5′‐TYE 665 and 3′‐Iowa black‐labelled aptamer beacon. The aptamer beacon exhibited a mild 'lights on' reaction in buffer as a function of increasing concentrations of several vitamin D analogues and metabolites, with a limit of detection of approximately 200 ng/mL, and was not specific for any particular congener. In 10% or 50% human serum, the same aptamer beacon inverted its fluorescence behaviour to become a more intense 'lights off' reaction with an improved limit of detection in the range 4–16 ng/mL. We hypothesized that this drastic change in fluorescence behaviour was due to the presence of creatinine and urea in serum, which might destabilize the quenched beacon, causing an increase in fluorescence followed by decreasing fluorescence as a function of vitamin D concentrations that may bind and quench increasingly greater fractions of the denatured beacons. However, the results of several control experiments in the presence of physiological or greater concentrations of creatinine and urea, alone or combined in buffer, failed to produce the beacon fluorescence inversion. Other possible mechanistic hypotheses are also discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

An improved method to detect β- galactosidase activity in transgenic mice: a post-staining procedure on paraffin embedded tissue sections

The Escherichia coli -galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring -galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 °C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl--d galactopyranoside (X-Gal). This procedure allows both the retention of a high level of -galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection