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Sialic acids (SAs) often exist as the terminal sugars of glycan structures of cell surface glycoproteins and glycolipids. The level and linkages of cell surface SAs, which are controlled by both sialylation and desialylation processes and environment cues, can dramatically impact cell properties and represent different cellular status. In this study, we systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation to M0 macrophages, and polarization to M1 and M2 macrophages by the combination of LC-MS/MS, flow cytometry and confocal microscopy. Interestingly, both α2-3- and α2-6-linked SAs on the cell surface decreased after monocytes were differentiated to macrophages, which was in accordance with the increased level of free SA in the cell culture medium and the elevated activity of endogenous Neu1 sialidase. Meanwhile, the siaoglycoconjugates inside the cells increased as confirmed by confocal microscopy and the total SA inside the cells increased as determined by LC-MS/MS. Western blot analysis showed higher expression levels of sialyltransferases, including ST3Gal-I, ST3Gal-V, ST6Gal-I and ST6GalNAc-II. Further, upon polarization, the cell surface sialylation levels of M1 and M2 macrophages remained the same as M0 macrophages, while a slight decrease of cellular SAs in the M1 macrophages but increase in the M2 macrophages were confirmed by LC-MS/MS.

Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNgamma) were treated using short interfering RNA (siRNA) and short-hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNgamma was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology., ((c) 2009 Wiley Periodicals, Inc.)

trans-Sialidases (TSs) are virulence factors that allow some trypanosomatids to incorporate sialic acid from host molecules. Trypanosoma cruzi bears a complex gene family coding for TS members, which can be broadly divided into two groups: one translated in stages present in the mammalian host (trypomastigote TS, tTS) and one translated in the insect vector stages (epimastigote TS, eTS). The molecular basis underlying the expression of different, nonoverlapping sets of TS proteins in either host is poorly understood, particularly because of the lack of transcription initiation control in this organism. Here we show that 3' untranslated regions (3'UTRs) of tTS and eTS are highly conserved within each gene group but completely different between both groups. Importantly, tTS-3'UTR but not eTS-3'UTR promoted high expression of the green fluorescent protein reporter gene in the mammalian-dwelling stages. In epimastigotes, both 3'UTRs lead to a comparatively low expression of the reporter gene, although eTS-3'UTR was more efficient than tTS-3'UTR. These results stress the importance of posttranscriptional events, mainly driven by specific 3'UTRs, in gene expression regulation in T. cruzi.

Entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles (IJs) employ multiple strategies to combat with insect innate immune system after invasion. We employed suppressive subtractive hybridization (SSH) technique to study the gene expression in the IJs upon contact with the insect hemolymph in vitro. We identified two clones having higher expression levels in the IJs than IJs treated with insect hemolymph. The differential expression levels were confirmed by Northern blot hybridization with reference to the constitutive expression level of Heterorhabditis bacteriophora actin2 gene. Clone HbGPS11.C1G02 encoded a phosphofructokinase (PFK) with a 2.5 kb transcript and clone HbGPS11.C4C08 corresponded to a 2.1 kb transcript encoding a protein that had weak similarity to trans-sialidase from Trypanosoma cruzi. The differential expression of PFK in H. bacteriophora IJs before and during recovery process represented a switch from active movement in search of insect hosts to a state of combating insect innate immune system. This first report of H. bacteriophora differential gene expression provides a glimpse at the gene expression profile of H. bacteriophora IJ recovery process.

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.

Trypanosoma cruzi, the agent of Chagas' disease, presents an enzyme that catalyzes the transfer of sialic acid among glycoproteins and glycolipids known as trans-sialidase (TS), displaying some interesting features: 1) It differs from all other eucaryotic sialyltransferases, both kinetically and in substrate specificity and 2) it is involved in the parasite's mechanism of mammalian host cell invasion. We report here the production and purification to homogeneity of an enzymatically active recombinant TS (rTS) lacking the C-terminal amino acid repeats, using iminodiacetic metal affinity chromatography. Initial ratios of non-fusion recombinant versus total protein were very low in several expression systems tested, mainly due to high degradation rate. However, after purifying 1,330 times, we were able to obtain an essentially homogeneous preparation of rTS with a final yield of 29%. After minor changes, a modified protocol for a medium scale production was designed obtaining 0.5 mg of homogeneous rTS per liter of bacterial culture. The purified rTS behaved as a homogeneous protein in silver-stained denaturing gels, isoelectrofocusing and N-terminal sequencing having identical pH and temperature optima as the natural enzyme. Conditions to keep the rTS for long periods without a significant loss of activity were identified.

The expression of developmentally regulated sialidase and trans-sialidase activities in kinetoplastid protozoa was investigated. The occurrence of these enzymes was found not to be a common feature among the Kinetoplastida, but to be restricted to distinct developmental life cycle stages of only a few species. While sialidases without trans-sialylating activities were demonstrated in Trypanosoma vivax and T. rangeli, trans-sialidase activity is expressed throughout the brucei-group and in T. congolense. Neither T. evansi, nor T. equiperdum express sialidases or trans-sialidases. Furthermore, the absence of both, sialidase and trans-sialidase activities was proven in the Leishmania, Crithidia, Herpetomonas, Leptomonas and Phytomonas, respectively. In all species tested, the occurrence of sialic acids coincides with the expression of trans-sialidase activity. Those parasites, which lack trans-sialidases or only display regular sialidases, also lack cell-bound sialic acids. The regular sialidase activity from bloodstream form T. vivax was characterized. The trans-sialidase from T. congolense is restricted to the procyclic culture forms and is shed into the culture medium. The enzyme has a pH-optimum at pH 7.0, displays sensitivity towards chlorides and is resistant against commonly used sialidase inhibitors. T. congolense trans-sialidase transfers preferentially alpha(2-3)-linked sialic acids onto terminal beta-galactose residues. Also hydroxylated sialic acids (Neu5Gc) are transferred. The major glycoprotein GARP from procyclic T. congolense was identified as one potential natural sialic acid acceptor on the parasite's surface. In order to facilitate the characterization of trans-sialidases a novel, fluorimetric trans-sialidase assay was developed.

Infective trypomastigote forms of Trypanosoma cruzi express an oligomeric trans-sialidase that contains a long stretch of 12-amino-acid repeats at the C terminus, while the insect epimastigote forms having a monomeric trans-sialidase without repeats. Here we show that messenger RNAs encoding trans-sialidases containing the repeats are not present in epimastigotes but are abundant in trypomastigotes. In contrast, mRNA species encoding the conserved N-terminal domain are detected in epimastigotes. A cDNA clone derived from epimastigote mRNA was isolated and characterized. It predicts a repeat-minus amino-acid sequence that has 84% identity to the conserved N-terminal domain of trypomastigote trans-sialidase, and contains some of the necessary amino acids for the catalytic activity, as shown by fusion experiments. Transcripts corresponding to this clone were detected in epimastigotes and in trypomastigotes by reverse-transcriptase and polymerase chain reaction. In addition, the lack of repeats is not due to RNA processing because the corresponding gene without repeats was amplified from the parasite DNA. These results suggest that a distinct set of genes encode the repeat-minus trans-sialidase, and only these trans-sialidase genes are expressed in epimastigote forms.

We have studied the trans-sialidase from insect forms of Trypanosoma cruzi growing in axenic culture. Log phase epimastigotes expressed little or no trans-sialidase activity, and were unable to incorporate exogenous sialic acid. Transsalidase started to be expressed at the late logarithmic phase, with specific activity increasing steadily as the culture reached the stationary phase. Trans-sialidase was purified from the late log phase epimastigote culture, which contained less than 2% of metacyclic forms, yielding a glycoprotein that migrated as a single 90-kDa band in sodium dodecyl sulfate gels. This enzyme features: (1) no reaction with antibodies against the peptide repeats present in the carboxy-terminal of trypomastigote trans-sialidase; (2) positive reaction with antibodies raised against a fragment of trypomastigote trans-sialidase that contains the active site; (3) similar kinetic properties and identical acceptor-donor specificity when compared to the trypomastigote enzyme; and (4) neuraminidase activity in the absence of acceptors. Upon differentiation into metacyclic forms, a trans-sialidase activity containing the carboxy-terminal repeats of the trypomastigote enzyme was released into the medium. These results suggest that epimastigotes express a developmentally regulated trans-sialidase that contains the same catalytic site but lacks the tandem amino acid repeats typical of trypomastigote trans-sialidase.

Fractionation by polyacrylamide gel electrophoresis (PAGE) demonstrated that in the infected cells the newly synthesized influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase (NA), differ from mature proteins of virus particles. After some time of life in the cells the differences are levelled. Since this phenomenon was demonstrable only in an analysis under the conditions favourable for the retention of disulphide bonds, it was designated as "disulphide maturation" of glycoproteins. Two causes of disulphide maturation of HA are considered: posttranslational folding of molecules conducive to drawing closer of the oxidizable thiol groups, and gradual loss of sensitivity to endogenous reducing agents. As for NA, the observed maturation here is the result of disulphide dimerization of monomers. Some factors affecting disulphide maturation of glycoproteins have been studied.