Neuraminidase Treatment Research Articles

Abstract 5301: MUC16-Siglec-9 binding leads to adhesion between ovarian tumor cells and specific subsets of NK cells, B cells, and monocytes

Abstract MUC16 is a cell surface mucin expressed at high levels by epithelial ovarian cancer cells. Following proteolytic cleavage, cell surface MUC16 (csMUC16) is shed in the extracellular milieu and is detected in the serum of cancer patients as the tumor marker CA125. csMUC16 acts as an adhesion molecule and facilitates peritoneal metastasis of ovarian tumors. Both sMUC16 and csMUC16 also protect cancer cells from cytotoxic responses of natural killer (NK) cells. In a previous study, we demonstrated that sMUC16 binds to a specific subset of NK cells. Here, we demonstrate that in addition to NK cells, sMUC16 also binds to B cells and monocytes isolated from the peripheral circulation and the peritoneal fluid. The I-type lectin, Siglec-9, is identified as the sMUC16 receptor on these immune cells. The inhibitory receptor Siglec-9 is expressed on approximately 30-40% of CD16pos/CD56dim NK cells, 20-30% of B cells and >95% of monocytes. sMUC16 binds to the majority of the Siglec-9pos NK cells, B cells and monocytes. sMUC16 is released from the immune cells following neuraminidase treatment. While sMUC16 binds to Siglec-9, it has no affinity for another I-type lectin Siglec-7. Experiments with Siglec-9 transfected Jurkat cells and monocytes isolated from healthy donors demonstrate that immune cells can bind to ovarian tumor cells via Siglec-9-csMUC16 interaction. Siglec-9 is an inhibitory receptor that attenuates T cell and NK cell function. Our studies indicate that sMUC16/csMUC16 may each serve as Siglec-9 ligands and mediate inhibition of anti-tumor immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5301.

Deglycosylation of FcαR at N58 increases its binding to IgA

FcalphaR (CD89) is the Fc receptor for immunoglobulin A (IgA) and plays important roles in IgA-mediated immune responses. It is a heavily glycosylated protein with six potential N-linked glycosylation sites. Previous reports showed that abnormal glycosylation of FcalphaR was involved in some diseases, including human immunodeficiency virus infection, alcoholic liver cirrhosis and IgA nephropathy. In this study, we examined the effects of N-glycosylation on interaction between FcalphaR and IgA. We found that depletion of N-glycosylation of FcalphaR transfected in Chinese hamster ovary (CHO) cells by tunicamycin resulted in increased IgA binding. To identify which glycosylation site is responsible for increased IgA binding, we performed site-directed mutagenesis at each N-linked glycosylation site by changing asparagine to glutamine. Flow cytometry analysis of IgA binding to CHO cells transfected with mutated FcalphaR showed that deglycosylation of FcalphaR at individual N44, N120, N156, N165 or N177 site did not affect IgA binding but deglycosylation at N58 resulted in marked increase of IgA binding. Similar result was shown for N58Q-FcalphaR transfected RBL2H3, a rat basophilic leukemia cell line. Furthermore, increased IgA binding was also observed on desialylated FcalphaR after neuraminidase treatment and desialylation of N58 contributed most to the increased IgA binding. These data demonstrated that glycosylation at N58 site influenced FcalphaR binding to IgA.

Exosome-mediated MHCII cross-presentation substantially enhanced by the receptor binding activity of influenza hemeagglutinin (130.10)

Abstract T cell responses to viral infection often depend upon cross-presentation, a process in which antigen is transferred from an infected cell to a professional antigen presenting cell. This process has been extensively studied with respect to MHC class I-restricted activation of CD8+ T cells, with several mechanisms having been reported including transfer of peptide-loaded heat shock proteins, apoptotic bodies and exosomes. In contrast, little work has been done on MHCII-restricted cross-presentation. Using infectious influenza virus and its glycoproteins expressed in antigen presenting cells, we readily observed MHCII cross-presentation, but only for one of the three epitopes that we routinely study, the “S1” epitope of the hemagglutinin glycoprotein (HA). This selectivity was explained by subsequent experiments that identified the process to be exosome-mediated. While exosomes are naturally endocytosed at a basal level, blocking antibodies and neuraminidase treatment of acceptor APCs revealed that cross-presentation was substantially enhanced by the sialic acid binding activity of the HA that had been incorporated into exosomes produced by the donor cells. This mechanism could provide an explanation for the skewed CD4+ responses to influenza that have been reported and may be an important component of the immune response to other enveloped viruses.

Sialic acid is required for nonspecific adherence ofSalmonella entericassp.entericaserovar Typhi on Caco-2 cells

To initiate infection, bacteria must adhere to and colonize host tissues. Specific and nonspecific mechanisms participate in the adherence process. Salmonella enterica ssp. enterica serovar Typhi (S. Typhi) must first adhere to the intestinal epithelium to invade and disseminate throughout the host. In this study, the role of colonic epithelial cell surface sialic acid in the adherence of S. Typhi was defined. Neuraminidase treatment of colonic Caco-2 cells removed 27-58% of surface sialic acid. Thus desialylation diminished the adherence of S. Typhi by 41%. Sialic acid treatment of S. Typhi had no effect on their adherence to neuraminidase-treated or control cells. These results indicate that sialic acid on the surface of colonic cells enhances S. Typhi adherence. These findings may suggest novel therapeutic strategies for S. Typhi infections.

Prediction of the In‐vivo Biological Activity of Human Recombinant Follicle‐stimulating Hormone Using Quantitative Isoelectric Focusing. Optimization of the Model

Follicle-stimulating hormone (FSH) is a glycoprotein consisting of two non-covalently linked subunits, α and β. FSH exists as multiple isoforms due to the attachment of oligosaccharides. The oligosaccharide moiety of these isohormones differs in branching pattern as well as in the sialic acid (N-acetyl neuraminic acid) content. The isohormone-related inherent microheterogeneity can be easily visualized using charge-based separation methods such as isoelectric focusing (IEF). Isohormones of recombinant FSH (rFSH, follitropin-β) display largely differing and isoelectric point (pI)-dependent in-vivo bioactivity. This has formed the basis for correlation between the charge distribution as determined by IEF (amount of isohormones focusing below pI 5) and the in-vivo bioactivity. Here we show that the sialic acid content is responsible for the differences in the in-vivo bioactivity. Gradual removal of sialic acid by in-vitro neuraminidase treatment of rFSH reduces the in-vivo bioactivity and results in a more basic IEF profile. The data allowed the refinement of the IEF in-vivo bioactivity model especially for samples with low in-vivo bioactivity (less than 5000 IU mg−1). In the optimized model the average pI was found to be a better parameter than the percentage of isohormones below pI 5. The average pI model for the in-vivo bioactivity was subsequently used to predict the in-vivo bioactivity of 27 production batches. The mean difference between the experimental and predicted in-vivo bioactivity was—1% (95% confidence interval—13 to 11%). These data further substantiate the use of IEF to accurately predict in-vivo bioactivity and offers an attractive alternative for the in-vivo bioassay currently in use for the potency declaration of rFSH. Similarly, since there is a strong correlation between sialic-acid content and the in-vivo bioactivity, this parameter may also be used to determine the potency of rFSH.

Further screening of Aspergillus species for occurrence of lectins and their partial characterization

Fifteen species of Aspergillus were screened for occurrence of lectins. Nine of them (A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus, A. pulvinus and A. aculeatus) were found to possess lectin activity. None of the species elaborated lectin in culture supernatant. All the lectins agglutinated rat, pig and rabbit erythrocytes. A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus and A. aculeatus lectins agglutinated all human type erythrocytes equally, while A. pulvinus lectin specifically agglutinated human type A and O erythrocytes. Neuraminidase and protease treatment to erythrocytes substantially augmented lectin titres manyfold. Lectins showed specificity to mucin and asialofetuin and all of them were specific to L-arabinose except that of A. carneus. Lectins from A. sydowii, A. ficuum, A. sparsus and A. carneus displayed remarkable specificities to D-xylose. Maximum lectin activity was expressed by 11 day old cultures of A. sydowii (titre 32), A. ficuum (titre 64) and A. sparsus (titre 1024). Lectins from A. aculeatus, A. candidus and A. terricola were expressed by 7-10 days, 6-9 days and 5-11 days old cultures, respectively. A. allahabadi cultures exhibited maximum lectin activity (titre 32) after 8-10 days of cultivation. A. carneus and A. pulvinus expressed optimal titres of 32 and 8, respectively on the 9th day.

Purification and Characterization of a Novel Thermostable Mycelial Lectin from Aspergillus terricola

Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to D-glucose, D-sucrose, N-acetyl-D-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0-10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 degrees C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine-HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.

Characterization of a Novel Monoclonal Antibody (27H2) Recognizing Human CD34 Class III Epitope

BackgroundMonoclonal antibodies (mAbs) recognizing Class III epitope of CD34 are essential for flow cytometric diagnosis of leukemia.Methods27H2 mAb was developed from a mouse alternatively immunized with human acute leukemia cell lines, KG1 and Molm-1. Using flow cytometric analysis of various leukemic cell lines and peripheral blood, immunohistochemical study of frozen tonsil, we characterized 27H2 mAb. Antigen immunoprecipitated with 27H2 mAb immunobloted with anti-CD34 mAb. A case of bone marrow sample of acute lymphoblastic leukemia (ALL) patient was obtained at CBNU Hospital. For epitope identification enzyme treatment with neuraminidase and O-sialoglycoprotein endopeptidase (OSGE) and blocking assay with known classIII mAb (HPCA-2) were done.ResultsOnly KG1 and Molm-1 revealed positive immunoreactivity. Immunohistochemical staining disclosed strong membranous immunoreactivity on high endothelial venules. Antigen immunoprecipitated by 27H2 mAb showed approximately 100 kDa sized band immunoblotted with anti-CD34 under non-reducing conditions. Epitope recognized by 27H2 mAb disclosed resistancy to both neuraminidase and OSGE treatment and completely blocked with known class III mAb preincubation. CD34 positive leukemic cells in BM of pre B cell ALL patient detected by FITC-conjugated 27H2 and HPCA-2 were identified with similar sensitivity.ConclusionA novel murine mAb recognizing class III epitope of human CD34 with high affinity, which is useful for flow cytometric diagnosis of leukemia, was developed.

Involvement of Sialic Acid in Transport of Serotype C1 Botulinum Toxins through Rat Intestinal Epithelial Cells

Clostridium botulinum produces a large toxin complex (L-TC) composed of neurotoxin (BoNT) and non-toxic proteins. In animal botulism, BoNT or L-TC is absorbed via the intestinal epithelium. To establish the cellular mechanisms of botulinum toxin absorption, we used cultured rat intestinal epithelial cells to test the binding and transport of serotype C1 BoNT and L-TC through the cell layers. BoNT and L-TC bound to and passed through the cell layers, with L-TC exhibiting larger binding and transport. Binding and transport of these toxins were inhibited by N-acetyl neuraminic acid or neuraminidase treatment of the cells. These results suggest that binding of serotype C1 BoNT and L-TC to sialic acid on the cells promoted their transport through intestinal epithelial cell layers.

Rotaviruses require basolateral molecules for efficient infection of polarized MDCKII cells

In this work we evaluated the ability of rotavirus strains with different receptor requirements to infect the apical and basolateral surfaces of polarized MDCKII cells. We used neuraminidase (NA)-sensitive (RRV and TFR-1) and neuraminidase-resistant (Wa and UK) viruses that differ in their use of integrins. Regardless of their receptor requirements, all virus strains tested were found to efficiently infect cells from both membrane surface domains, with preference for the basolateral domain, since: (i) disruption of tight junctions of polarized cell monolayers by calcium chelation led to a reversible increase of rotavirus infectivity, (ii) the viruses infected preferentially the cells located at the borders of microcolonies of polarized cells, and (iii) in cells grown on a permeable support all four virus strains were able to start the infection by either plasma membrane domain. Preferential infection (5–11-fold more efficiently) of the basolateral surface correlated with the neuraminidase resistance of the virus strains, but not with their requirement for integrins, which in MDCKII cells seem to be used by all four viruses. The infection of both cell surface domains by RRV was found to depend on the presence of terminal sialic acids, since its infectivity was reduced by neuraminidase treatment of the cells and it was also blocked by incubation of the virus with glycophorin A. The efficient infection through the basolateral membrane surface of polarized cells might be relevant for the pathogenesis of rotavirus, especially given the recent reports of antigenemia and extraintestinal spread of the virus in children and animal models.

Neuraminidase enhances the initial steps of human T-cell leukemia virus type 1 replication

Human T-cell leukemia virus type 1 (HTLV-1) infection is involved in the development of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. A high HTLV-1 proviral load in circulating lymphocytes of HTLV-1 carriers is a risk factor for HTLV-1-related diseases. The virus–cell interaction is linked to viral tropism and pathogenesis. Characterization of the factors that affect HTLV-1 infection is important for preventing HTLV-1 infection. HTLV-1 virions are believed to be weakly infectious under cell culture conditions; however, we found that the treatment of HTLV-1 virions with microbial neuraminidase, an enzyme catalyzing the removal of sialic acid residues from various glycoconjugates, enhanced the number of proviral DNAs in infected cells in a dose-dependent manner. Neuraminidase treatment of virions, but not target cells, enhanced viral binding and entry into cells and viral infectivity; treatment of target cells prior to infection had no effect. Moreover, the number of HTLV-1-mediated syncytia was higher in the presence of neuraminidase. Our results suggest a possible contribution of microbial agents carrying neuraminidase activity to HTLV-1 pathogenesis.

Measurement of electrophoretic mobility of cardiomyocytes

The electrophoretic mobility (EPM) of rat cardiomyocytes with or without the treatment of neuraminidase was studied by cell electrophoresis. The EPM was found to change over a range from 0 to 8.67 microm s(-1)/V cm(-1), depending on ionic strength, transmembrane potential, pH value, and/or surface charges. It is interesting that zero EPM was observed but reverse of the mobility was not. These results suggested that the negative charges carried on the cardiomyocyte surface might comprehensively consist of surface sialic acid, plasmalemma proteins, phospholipids, and transmembrane potential. The aberrant electrical double layer formed between the carried negative charges and adions had a big adsorption layer and a diffusion layer whose sizes changed circularly, making only negative charges be carried on the surface of living cardiomyocytes. The special structures on the surface of cardiomyocytes probably play a considerable role in the process of cardiac electrical activity.

Effect of pharynx epithelial cells surface desialylation on receptor-mediated adherence ofStaphylococcus aureus

To characterize the interaction between cell surface carbohydrates and Staphylococcus aureus. In the present study, in vitro adherence of S. aureus to Detroit 562 cells, amount of cell surface desialylation and effect of subterminal monosaccharides on desialylated glycoproteins on adherence was studied with colony counting, HPLC, fluorescence microscopy and fluorometric techniques. According to our findings, S. aureus adherence to pharynx cells was enhanced (40%) after neuraminidase treatment, and neuraminidase also cleave great amount of Detroit 562 cells surface sialic acid (39-60%). Adherence assay with various monosaccharides-pretreated bacteria, and lectin competitive inhibition, showed that the residual subterminal galactose, fucose and N-acetyl-D-glucosamine remaining on desialylated Detroit 562 cell surface glycoproteins responsible for this binding. The results are the first to show that galactose, fucose and N-acetyl-D-glucosamine remaining on desialylated pharynx cell surface glycoproteins serve as the adhesine receptors for S. aureus. This study may explain the predisposition of severe S. aureus pneumonia complication in respiratory viral infections.

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of α2β1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

Desialylation of TLR4/MD2/CD14 enhances the response to LPS (135.16)

Abstract Objective: Since bacterial neuraminidase (NANase) treatment of intact human PBMCs led to a synergistic increase in cytokine expression, we hypothesized that components of the TLR4 receptor complex were sialylated, and that removal of sialyl residues enhanced LPS-initiated signaling. Methods: HEK293T cells were transfected with TLR4, MD2, CD14 and ELAM-Luc expression vectors. They were treated with NANase, stimulated with LPS, and assayed for NF-kB-driven luciferase expression. Sialylation was assayed by lectin blot after immunoprecipitation from lysed transfected cells. Results: LPS stimulated NF-kB activation and IL-8 expression only in cells transfected with TLR4/MD2/CD14 or in cells transfected with TLR4/CD14 supplemented with supernatants (SNT) of MD2-transfected cells. NANase treatment of TLR4/CD14/MD2 cells led to enhanced NF-kB activation. While NANase treatment of either TLR4/CD14 cells or MD2 SNTs each enhanced the LPS response in the reconstituted culture, optimal responses occurred when both were treated. Lectin blots revealed MD2 to be sialylated with a α2,6 linkage since SNA but not Maackia bound to MD2. Conclusions: We conclude that removal of sialyl residues from MD2 and perhaps from TLR4/CD14 enhances the response to LPS. Thus mobilization of endogenous sialidase in PBMCs could modulate the activity of the TLR4/CD14/MD2 complex and serve as a therapeutic target. Support: NIH grant no. HL086933-01A1

Neuraminidase alters red blood cells in sepsis

To investigate the influence of neuraminidase, an enzyme that cleaves sialic acid from the red blood cell (RBC) membrane, on RBC shape and biochemistry in critically ill patients. Prospective, observational study and in vitro laboratory study. A 31-bed medico-surgical department of intensive care and a university-affiliated cell biology laboratory. Acutely ill patients with and without sepsis and healthy volunteers. Blood sampling in volunteers. Neuraminidase activity was measured using a fluorescent assay. RBC shape was assessed by the second coefficient of dissymmetry of Pearson using a flow cytometry technique at 25 degrees C. Intraerythrocytic 2,3-diphosphoglycerate and lactate contents were also measured. Neuraminidase activity was significantly higher in septic patients compared with nonseptic patients and healthy volunteers (5.42 [4.85-6.00] vs. 4.53 [4.23-5.23] and 1.26 [0.83-1.83] mU/mL; all p < 0.05). Neuraminidase treatment modified the RBC shape in vitro in a dose-response fashion, and most of these alterations were present after 10 hours of incubation. Incubation of RBCs with phosphatidylinositol phospholipase C modified RBC shape and increased sialic acid concentrations in the supernatant, suggesting a leakage of neuraminidase from the RBC membrane. Alterations in shape were associated with increased 2,3-diphosphoglycerate (0.46 +/- 0.25 vs. 0.19 +/- 0.05 mumol/mL; p = 0.006) and lactate content (0.81 +/- 0.07 vs. 0.66 +/- 0.05 mmoL/L; p = 0.002). In sepsis, desialylation under the influence of increased neuraminidase activity may contribute to the alterations in RBC rheology. Inhibition of neuraminidase may represent a new therapeutic option to ameliorate RBC rheology and perhaps oxygen delivery to the cells.

Ganglioside-Linked Terminal Sialic Acid Moieties on Murine Macrophages Function as Attachment Receptors for Murine Noroviruses

Noroviruses are the major cause of nonbacterial gastroenteritis in humans. However, little is known regarding the norovirus life cycle, including cell binding and entry. In contrast to human noroviruses, the recently discovered murine norovirus 1 (MNV-1) readily infects murine macrophages and dendritic cells in culture. Many viruses, including the related feline calicivirus, use terminal sialic acids (SA) as receptors for infection. Therefore, we tested whether SA moieties play a role during MNV-1 infection of murine macrophages. Competition with SA-binding lectins and neuraminidase treatment led to a reduction in MNV-1 binding and infection in cultured and primary murine macrophages, suggesting a role for SA during the initial steps of the MNV-1 life cycle. Because SA moieties can be attached to glycolipids (i.e., gangliosides), we next determined whether MNV-1 uses gangliosides during infection. The gangliosides GD1a, GM1, and asialo-GM1 (GA1) are natural components of murine macrophages. MNV-1 bound to ganglioside GD1a, which is characterized by an SA on the terminal galactose, but not to GM1 or asialo-GM1 in an enzyme-linked immunosorbent assay. The depletion of gangliosides using an inhibitor of glycosylceramide synthase (d-threo-P4) led to a reduction of MNV-1 binding and infection in cultured and primary murine macrophages. This defect was specifically rescued by the addition of GD1a. A similar phenotype was observed for MNV field strains WU11 (GV/WU11/2005/USA) and S99 (GV/Berlin/2006/DE). In conclusion, our data indicate that MNV can use terminal SA on gangliosides as attachment receptors during binding to murine macrophages.

A Mechanism of Sialylation Functionally Silences the Hyaluronan Receptor LYVE-1 in Lymphatic Endothelium

The major lymphatic endothelial hyaluronan receptor LYVE-1, a Link superfamily glycoprotein similar to the hyaluronan-binding/inflammatory leukocyte homing receptor CD44, was initially implicated in hyaluronan (HA)-mediated cell adhesion and lymph-borne hyaluronan metabolism. However, the apparently normal phenotype of Lyve-1 knock-out mice and the recent demonstration that the receptor undergoes cytokine-induced endocytosis independent of HA uptake have cast doubt on such functions. Here we present new data that reconcile these anomalies by showing that LYVE-1 is functionally "silenced" in a cell-specific fashion by autoinhibitory glycosylation. We demonstrate that LYVE-1 transfected in HEK 293T fibroblasts and Jurkat T cells is competent to bind HA, whereas the endogenous receptor in cultured lymphatic endothelial cells or the receptor transfected in Chinese hamster ovary and HeLa cells is not. Moreover, through a combination of mutagenesis and functional analysis in HEK 293T fibroblasts and glycosylation-defective Chinese hamster ovary cell lines, we reveal that the inhibitory mechanism is reversible and is exerted by terminal sialylation, most likely through alpha2-3 or alpha2-6 linkage to O-glycans. Finally, we provide evidence that the mechanism operates in vivo by showing that native LYVE-1 in primary lymphatic endothelial cells is extensively sialylated and that HA binding can be reactivated by neuraminidase treatment of the soluble ectodomain. These results reveal unexpected complexity in the regulation of LYVE-1 function and raise the possibility that this receptor, like CD44, may become active after appropriate unmasking in vivo.

Changes in post-translational modifications of prolactin during development and reproductive cycles in the chicken

Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.

A novel Plasmodium falciparum erythrocyte binding protein associated with the merozoite surface, PfDBLMSP

Proteins on the surface of the merozoite, the invasive form of the malaria parasite Plasmodium falciparum, and those secreted from its apical secretory organelles are promising vaccine candidates against blood stage malaria. In the present study, we have identified a novel parasite protein (PfDBLMSP; Gene ID PF10_0348), that harbors a predicted signal sequence, a central Duffy binding-like (DBL) domain and a secreted polymorphic antigen associated with merozoites (SPAM) domain in its C-terminal half. Transcription and translation of pfdblmsp is up-regulated specifically in schizont stage parasites, similar to other well-chararacterized merozoite proteins involved in invasion of red blood cells (RBCs). PfDBLMSP was localized on the merozoite surface with a GFP targeting approach using schizont-stage specific expression systems, and by immunofluorescence assays of the endogenous protein. PfDBLMSP expressed on the surface of mammalian cells (COS-7) showed binding with human RBCs and this binding was sensitive to trypsin and neuraminidase treatments. The recombinant proteins corresponding to the DBL and SPAM domains showed reactivity with immune sera from individuals residing in P. falciparum endemic areas. Polymorphism in PfDBLMSP sequences from different P. falciparum strains and field isolates suggested that its DBL domain is under natural immune pressure. Our data on localization and functional assays suggest a possible role of PfDBLMSP in binding of merozoites with erythrocytes during invasion.