High Sialidase Activity Research Articles

Analysis and Control of Viral Infection Mechanisms by Glycobiology

Glycans are present in all living organisms, including viruses, bacteria, and animals, and perform various biological functions. The author has been studying influenza viruses' glycan usage mechanisms, particularly the functional analysis of neuraminidase (NA), a viral sialidase. The authors recently focused on influenza virus NAs' high sialidase activity with the aim of using sialidase activity detection as a virus detection technology. Using the probe BTP3-Neu5Ac, allows fluorescent imaging of sialidase activity, we created a new technique for easy, rapid, and high sensitivity fluorescent imaging of virus-infected cells. The detection of viruses using BTP3-Neu5Ac does not require specific antibodies and can be performed by simply adding reagents. Furthermore, fluorescence imaging of sialidase as a virus detection technology has many advantages, including isolating viruses from fluorescently imaged infected cells. This detection technique is easy to use in basic research and hygiene testing, where viral culture is conducted and is expected to be widely used.

Large-scale characterisation of the pregnancy vaginal microbiome and sialidase activity in a low-risk Chinese population

Vaginal microbiota-host interactions are linked to preterm birth (PTB), which continues to be the primary cause of global childhood mortality. Due to population size, the majority of PTB occurs in Asia, yet there have been few studies of the pregnancy vaginal microbiota in Asian populations. Here, we characterized the vaginal microbiome of 2689 pregnant Chinese women using metataxonomics and in a subset (n = 819), the relationship between vaginal microbiota composition, sialidase activity and leukocyte presence and pregnancy outcomes. Vaginal microbiota were most frequently dominated by Lactobacillus crispatus or L. iners, with the latter associated with vaginal leukocyte presence. Women with high sialidase activity were enriched for bacterial vaginosis-associated genera including Gardnerella, Atopobium and Prevotella. Vaginal microbiota composition, high sialidase activity and/or leukocyte presence was not associated with PTB risk suggesting underlying differences in the vaginal microbiota and/or host immune responses of Chinese women, possibly accounting for low PTB rates in this population.

A Glycomic Approach Towards Identification of Signature Molecules in CD34+ Haematopoietic Stem Cells from Umbilical Cord Blood.

Umbilical cord blood (UCB) is a powerful storehouse for normal CD34+ haematopoietic stem cells (HSCs), often used for allogeneic bone marrow (BM) transplantation in malignant and non-malignant diseases. The glycomic especially the sialoglycomic aspect of these HSCs has been unravelled in this study. Cell surface expression of the glycans with the related enzymatic activities has been compared with the BM of childhood acute lymphoblastic leukaemia, a common BM-associated malignancy. An enhanced cell surface expression of α2,3-linked sialic acid, P- and E-selectins, and intercellular adhesion molecule along with reduced expression of L-selectin distinguishes CD34+ HSCs of UCB from leukaemic samples. More importantly, high expression of O-acetylated sialoglycoproteins, a hallmark of lymphoblasts, is drastically reduced in the CD34+ HSCs of UCB and is substantiated by the low activity of sialylate-O-acetyltransferase and high sialidase activity. In contrast, a significant variation is evident in the expression of sialic acid, α2,6-linked sialic acids, and the sialyltransferase activity. Taken together, these studies indicate a few signature molecules, forming a unique glycomic template, which may be a potential indicator, reassuring the normal profile of these stem cells, to be used for future transplantation.

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response.

Influenza neuraminidase (NA) proteins expressed in TK− cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.

A semi-multifunctional sialyltransferase from Bibersteinia trehalosi and its comparison to the Pasteurella multocida ST1 mutants

Sialic acids are well known for their crucial roles in many physiological and pathological processes. Improvement in the efficacy of protein drugs, an increase in the anti-inflammatory activity of intravenous immunoglobulin, preparation of infant milk and the diagnosis of diseases are examples of why there is a need for efficient in vitro sialylation. Sialyltransferases are crucial enzymes for the synthesis of sialo-oligosaccharides. Here, we introduce a new α2,3-sialyltransferase from bacteria Bibersteinia trehalosi (BtST1), which is homological to sialyltransferase from Pasteurella multocida (PmST1), Pasteurella dagmatis (PdST1) and Haemophilus ducreyi (Hd0053). BtST1 is active in a wide pH range and shows considerable acceptor flexibility. Very good specific activities have been detected with lactose and LacNAc as acceptors, and these activities were comparable to those of efficient multifunctional PmST1 and higher than PdST1, Hd0053 and also PmST1 M144D which was constructed to decrease the high sialidase activity of PmST1. Testing of PmST1 mutant forms revealed that mutations that included S143 caused only the restriction of sialyltransferase activity, whereas mutations including G142 resulted in the loss of activity with lactose. BtST1 possesses only low sialidase and trans-sialidase activities that are comparable to mutant PmST1 M144D, which are detected only in the presence of CMP. The combination of large acceptor flexibility, high activity for lactose and LacNAc and naturally low sialidase activity make BtST1 an attractive enzyme for biotechnological applications.

A novel method for detection of Newcastle disease virus with a fluorescent sialidase substrate

Newcastle disease virus (NDV), belonging to the family Paramixoviridae, causes respiratory and neuronal symptoms in almost all birds. NDV has haemagglutinin-neuraminidase (HN) glycoprotein possessing sialidase activity. HN glycoprotein is highly expressed on the surface of NDV-infected cells, resulting in much higher sialidase activity in NDV-infected cells than in non-infected cells. It was reported that mouse and human cancer cells up-regulating sialidase expression were histochemically stained with a fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-d-glycero-d-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), which deposits water-insoluble fluorescent compound BTP3 on locations of sialidase activity. By using the BTP3-Neu5Ac assay, we showed that NDV-infected cells and HN gene-expressing cells could be simply detected at room temperature after only 5min. Infection of the cells with the virus resulted in apparent green fluorescence, which disappeared with addition of a sialidase inhibitor. Cells that were stained in the BTP3-Neu5Ac assay were immunostained with an anti-NDV antibody. Moreover, BTP3-Neu5Ac staining was applied to a virus overlay binding assay with NDV particles. NDV-bound protein bands on guinea pig red blood cells were easily and rapidly detected by the BTP3-Neu5Ac assay after Western blotting. BTP3-Neu5Ac offers an easy and rapid protocol for fluorescent staining of NDV and virus-infected cells without antibodies.

Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species

Neuraminidases are virulence factors in many pathogenic microorganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodents and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type AT) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to show proteins having NEAC. In M. canis (a field isolate Larissa and the type strain PG14T), M. cynos (isolate 896) and M. molare (type strain H542T) proteins with NEAC had molecular masses of ∼130kDa, 105kDa and 110kDa, respectively. Identification of these neuraminidases could provide the basis for their molecular characterization.

Low expression of Neu2 sialidase in the thymus of SM/J mice—existence of neuraminidase positive cells “Neu-medullocyte” in the murine thymus

We have already reported that the homogenate of the A/J mouse thymus shows a high sialidase activity at the neutral pH region and that in both soluble and membrane fractions optimal pH was 6.5-7 (Kijimoto-Ochiai et al., Glycoconj. J., 20:375-384, 2004). In the present study, we investigated the level of sialidase activities in the thymus of the SM/J mouse, a mouse strain that we know to have a Neu1(a) allele that reveals a low level of sialidase activity in the liver. We found that while in the A/J thymus the soluble sialidase activity at pH 6.5 was high, the SM/J thymus lacked all such activity. A QTL analysis of SMXA recombinant inbred strains showed that soluble sialidase activity correlated well with the D1Mit8/9 marker on chromosome 1. The murine whole DNA-sequence data and the results of our FISH analysis (Kotani et al., Biochem. Biophys. Res. Comm., 286:250-258, 2001) showed that this location is consistent with the position of Neu2 gene. We confirmed that it is hard to detect the Neu2 enzyme of the SM/J mouse thymus by an anti-Neu2 antibody using a Western blot analysis. We also found that while the mRNA expression of Neu2 was quite normal in the SM/J mouse liver, it was very low in the SM/J mouse thymus. We therefore conclude that the lack of soluble sialidase activity in the SM/J mouse thymus is due to the thymus-specific low expression level of the Neu2 gene. We have previously shown that the sialidase positive cell which contains the Mac-1 and immunoglobulin, and which is located sparsely in the corticomedullar region or medullary region of the A/J mouse thymus (Kijimoto-Ochiai et al., Glycoconj. J., 20:375-384, 2004). We showed now in this paper that the detection of this cell in the SM/J mouse thymus at pH 7.0 was difficult. We propose, therefore, to name the cell "Neu-medullocyte".

Combination of vaginal pH with vaginal sialidase and prolidase activities for prediction of low birth weight and preterm birth

The purpose of this study was to assess if easy to measure vaginal fluid biomarkers are predictive for low birth weight (LBW, <2500 g), very LBW (VLBW, <1500 g), spontaneous preterm at <37 weeks' gestation, and total preterm deliveries (at <37, <35, <32 weeks' gestation). Low and high cutoffs for vaginal fluid pH, sialidase, and prolidase activities were examined in a nested case-control study of 579 Danish women (from a study population of 2846 women) with samples collected at mean 17 weeks' gestation. One hundred sixteen LBW (17 VLBW), 117 preterm deliveries (85 spontaneous), and 418 normal term deliveries were analyzed. Vaginal pH >/=4.7 or pH >/=5 by itself was not associated with LBW or prematurity. Conversely, combination of pH >/=5 and high sialidase activity demonstrated OR 17 (CI 1.8-150) for LBW; OR 31 (CI 1.8-516) for VLBW; along with OR 18 (CI 1.6-204) for preterm at <35 weeks'; and OR 31 (CI 1.9-542) for preterm at <32 weeks' gestation. The combination of pH >/=5 and high prolidase activity demonstrated OR 13 (CI 1.3-122) for LBW; OR 33 (CI 2.0-553) for VLBW, as well as OR 9.2 (CI 0.6-150) for preterm at <35 weeks'; and OR 35 (CI 2.0-586) for preterm at <32 weeks' gestation. In this population, no woman having high sialidase and high prolidase activity had a term birth, or a baby weighting >/=2500 g at birth. In this Danish population, mid-gestation findings of vaginal fluid elevated pH with sialidase and/or prolidase were associated with LBW, VLBW, and early preterm at <35 or <32 weeks' gestation.

Impairment of the mucosal immune system: IgA and IgM cleavage detected in vaginal washings of a subgroup of patients with bacterial vaginosis.

The integrity of the immunoglobulins in vaginal washings of patients with bacterial vaginosis was examined to answer the question of the lack of immune response against Gardnerella vaginalis cytolysin. Clinically diagnosed patients (n=100) were recruited and their vaginal washings examined by Western blotting. Many showed IgA and IgM partially or extensively degraded. According to the degradation pattern, the patients were subdivided into 4 subsets, from intact (score 0) to completely degraded IgA (score +3). Statistical analysis of the data showed a correlation between IgA degradation and absence of immune response to G. vaginalis cytolysin. The extent of IgA degradation correlated also with the sialidase (but not with the prolidase) activity level. All women showed intact IgG and human serum albumin and no trypsin-like activity. Patients with bacterial vaginosis having high sialidase activity and extensive IgA degradation in their secretions could incur more dangerous infections and adverse pregnancy outcomes.

Effects of chronic ethanol exposure on cultured cerebellar granule cells.

The aim of this study was to investigate the lipid content and composition of rat cerebellar granule cells grown in the presence of ethanol (40, 55, or 80 mM) during in vitro differentiation. Quantitative analyses showed no effects of 40 mM ethanol, whereas a significant increase of total cholesterol was observed at 55 mM. Cells exposed to the highest ethanol dose (80 mM) were characterized by a higher sialidase activity, and by the modification of the ganglioside pattern and phospholipid fatty acid composition. The observed modifications were accompanied by changes of membrane anisotropy fluorescence assessed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene.

A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GD 1a isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [ 3H]GM 1 was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4–5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM 1, using as low as 10 μg of fibroblast homogenate as protein. The detection limit could be lowered to 0.02–0.03 nmol, adopting conditions that, however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm 2 plastic flasks was 5.8 ∓ 2.5 (SD) nmol liberated GM 1 h −1 mg protein −1. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl- d- N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate. All this supports the hypothesis that human fibroblasts contain sialidases with different subcellular location and substrate specificity. Particularly, the sialidase acting on gangliosides seems to have two sites of subcellular location, a major one at the level of plasma membranes and/or intracellular organelles functionally related with the plasma membranes and a minor one in the lysosomes.

Large-scale preparation of synaptosomes from bovine brain using a zonal rotor technique

A zonal rotor technique for the preparation of synaptosomes in bulk from bovine brain frontal cortex based on an impirical transformation of a small-volume discontinuous sucrose density gradient arrangement is presented in detail. The procedure yields new information concerning synaptosomes prepared in sucrose gradients. Cerebroside analysis and electron microscopy show myelin contamination to be restricted to the leading, less dense edge of the synaptosomal profile, free mitochondria to the trailing, more dense edge. Exclusion of fringe areas yields a highly purified synaptosome preparation which entirely enters the next dense layer beyond the 0.8 : 1.2 M sucrose interface. This interface collects most of the oubain-sensitive (Na+, K+) adenosine triphosphatase activity. The purified synaptosomes display very high intrinsic sialidase activity and are rich in di-, tri-, and tetrasialogangliosides, the preferred substrates for the enzyme. Up to 90% of the cholinesterase activity in the zonal rotor synaptosome preparation is specific acetylcholinesterase.

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid

Radioactive gangliosides, N-[ 14C]-acetylneuraminylgalactosylglucosylceramide ([ 14C]G M3) and N- [ 14C]-acetylneuraminylgalactosyl- N-acetylgalactosaminyl- [ N-acetylneuraminyl]-galactosylglucosylceramide ([ 14C]G D1a), were synthesized from CMP-[ 14C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [ 14C]G M3 and [ 14C]G D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [ 14C]G M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.