Impact of immune complex size and glycosylation on IgG binding to human FcγRs.

IgG molecules are widely used as therapeutic agents either in the form of intact Abs or as Fc fusion proteins. Although efficient binding of the IgG Fc fragment to cellular FcγRs may be essential to achieve a high cytolytic activity, it may be advantageous for other applications to limit or abolish this interaction. Genetic or biochemical approaches have been used to generate these non-FcγR-binding IgG variants. By using soluble versions of FcγRs and monomeric versions of these altered IgG molecules, it was demonstrated that these IgG variants no longer bind to FcγRs. Importantly, however, these assays do not reflect the physiologic interaction of IgG with low-affinity cellular FcγRs occurring in the form of multimeric immune complexes. In this study, we investigated how the size of an immune complex can affect the interaction of normal and various versions of potentially non-FcγR-binding IgG variants with cellular FcγRs. We show that neither the D265A mutation nor EndoS treatment resulting in IgG molecules with only one N-acetylglucosamine and a fucose residue was fully able to abolish the interaction of all IgG subclasses with cellular FcγRs, suggesting that IgG subclass-specific strategies are essential to fully interfere with human FcγR binding.

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast.

BACKGROUND: The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified. METHODS: AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined. RESULTS: AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA∆ (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA∆ was confirmed by co-immunoprecipitation analysis. CONCLUSION: The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates. GENERAL SIGNIFICANCE: Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.

Influenza virus utilizes N-linked sialoglycans as receptors in A549 cells.

Influenza viruses (IFVs) recognize sialoglycans expressed on the host cell surface. To understand the mechanisms underlying tissue and host tropisms of IFV, it is essential to elucidate the molecular interaction of the virus with the host sialoglycan receptor. We established and applied a new monoclonal antibody, clone HYB4, which specifically recognizes the Neu5Acα2-3 determinant at the non-reducing terminal Gal residue of both glycoproteins and gangliosides to investigate the biochemical properties of IFV receptors in A549 cells. HYB4 significantly blocked virus binding to A549 cells in a dose-dependent manner. Virus overlay assay indicated that several glycoproteins with molecular masses of 80-120 kDa of A549 cells were commonly recognized by different subtypes of IFV, such as H1N1 and H3N2. H1N1 virus binding to the glycoproteins was diminished by pretreatment with either sialidase or PNGase F. On TLC-immunostaining experiments with HYB4, GM3 ganglioside was only detected in A549 cells. Interestingly, this antibody bound to GM3 gangliosides on TLC and plastic surfaces, but not on lipid bilayers. In comparison with the recognition of Maackia amurensis lectins, HYB4 exclusively recognized Neu5Acα2-3Galß1-4GlcNAc residues expressed on glycoproteins. These results strongly suggest that N-linked sialoglycans with the Neu5Acα2-3 determinant on several glycoproteins are receptors for influenza virus in A549 cells.

Expression of terminal alpha2-6-linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13.

von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by alpha2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (+/- 14%) by ADAMTS13, compared with 11% (+/- 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O > or = B > A > or = AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.

The identification of N-glycosylated residues of the human 5-HT3B receptor subunit: importance for cell membrane expression.

The 5-hydroxytryptamine 3 (5-HT(3)) receptor is a pentameric ligand-gated ion channel with potential molecular isoforms arising from different subunit combinations and/or different post-translational modifications of the individual subunits. Since N-glycosylation of the 5-HT3A subunit impacts cell surface trafficking, the presence of N-glycosylation of the human (h) 5-HT3B subunit and the influence upon cell membrane expression was investigated. Following transient expression of the h5-HT3B subunit by human embryonic kidney cells (HEK293 cells) stably expressing the h5-HT3A subunit, the N-glycosylation inhibitor tunicamycin reduced the size of the predominant h5-HT3B-immunoreactive protein (∼ 55 kDa reduced to ∼ 40 kDa). Disruption of each consensus N-glycosylation sequences in the h5-HT3B subunit (N31S, N75S, N117S, N147S and N182S) resulted in a reduced molecular weight (by ∼ 2-4 kDa) of each mutant when expressed by HEK293 cells stably expressing the h5-HT3A subunit. Immunocytochemical studies demonstrated that disruption of each of the N-glycosylation sequences (individually or combined) reduced the expression of the mutant h5-HT3B subunit protein in the cell membrane when co-expressed with the h5-HT3A subunit. The present study has identified utilised N-glycosylation sites of the h5-HT3B subunit and demonstrated that they promote subunit expression in the cell membrane; a prerequisite for 5-HT(3) receptor function.

Role of N-linked glycosylation of the 5-HT2A receptor in JC virus infection.

JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine(2A) receptor (5-HT(2A)R). The 5-HT(2A)R contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT(2A)R N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT(2A)R-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT(2A)R to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT(2A)R to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT(2A)R. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT(2A)R resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT(2A)R-expressing cells. These data affirm the importance of 5-HT(2A)R as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT(2A)R.

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells.

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release.

Transgenic α-1-antitrypsin secreted into the bloodstream from salivary glands is biologically active.

OBJECTIVES: Salivary glands are potentially a valuable target for gene therapeutics. Herein, we examined the expression and biochemical activity of human alpha-1-antitrypsin (hA1AT) produced in rodent submandibular glands after gene transfer. METHODS: A serotype 5 adenoviral vector (Ad.hA1AT) was constructed and first characterized by dose response and time course studies using SMIE cells in vitro. hA1AT expression was analysed by ELISA and the biologic activity determined by the inhibition of human neutrophil elastase (hNE) and formation of hA1AT-hNE complexes. Ad.hA1AT was administered to submandibular glands of rats and mice. The levels and activity of hA1AT were analysed in saliva, serum and gland extracts. Treatment with endoglycosidase H and Peptide N-Glycosidase F was used to assess N-linked glycosylation. RESULTS: Transgenic hA1AT, expressed in submandibular glands following Ad.hA1AT administration, was secreted into the bloodstream, N-glycosylated and biochemically active. CONCLUSION: After in vivo gene transfer, rodent salivary glands can produce a non-hormonal, transgenic, secretory glycoprotein exhibiting complex and conformation-dependent biologic activity.

Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China.

The single radial immunodiffusion (SRID) method currently used to determine the hemagglutinin (HA) content of the inactivated influenza vaccines depends on the availability of reference HA antigen and corresponding anti-serum, updated and provided annually by World Health Organization (WHO) collaborative centers. Particularly early in a pandemic outbreak, reference reagents could be the bottleneck in vaccine development and release. Therefore, other reliable tests capable of quantifying HA content could substantially shorten the time needed for vaccine formulation. Here electrophoretic separation of deglycosylated samples in conjunction with densitometry was used to quantify HA contents of H1N1 vaccine at multiple manufacturing sites. We found the overall consistency between the alternative method and traditional SRID was 88-122% in seven lots of vaccine bulks from four subtypes (types) of influenza vaccine, confirming its suitability to quantify HA content. Moreover, we used the alternative method to prepare a national HA antigen reference in China for quality control of 2009 pandemic influenza A (H1N1) vaccines prior to the arrival of the WHO SRID reference standards, subsequently confirming good agreement between both methods. The alternative method for vaccine quantification enabled the Chinese health authority to approve H1N1 vaccine 1 month earlier than otherwise possible.

Amyloid precursor protein 695 associates with assembled NR2A- and NR2B-containing NMDA receptors to result in the enhancement of their cell surface delivery.

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co-immunoprecipitated with assembled NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1-1a, NR1-2a, NR1-4b(c-Myc), or NR2 subunit transfections revealed that co-association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2' cassettes and, the use of an NR1-2a(c-Myc)-trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N-terminal domains. Anti-APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti-NR1 or anti-NR2A antibodies co-immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co-expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer's disease.

Amyloid precursor protein and amyloid beta-peptide bind to ATP synthase and regulate its activity at the surface of neural cells.

Amyloid precursor protein (APP) and amyloid beta-peptide (Abeta) have been implicated in a variety of physiological and pathological processes underlying nervous system functions. APP shares many features with adhesion molecules in that it is involved in neurite outgrowth, neuronal survival and synaptic plasticity. It is, thus, of interest to identify binding partners of APP that influence its functions. Using biochemical cross-linking techniques we have identified ATP synthase subunit alpha as a binding partner of the extracellular domain of APP and Abeta. APP and ATP synthase colocalize at the cell surface of cultured hippocampal neurons and astrocytes. ATP synthase subunit alpha reaches the cell surface via the secretory pathway and is N-glycosylated during this process. Transfection of APP-deficient neuroblastoma cells with APP results in increased surface localization of ATP synthase subunit alpha. The extracellular domain of APP and Abeta partially inhibit the extracellular generation of ATP by the ATP synthase complex. Interestingly, the binding sequence of APP and Abeta is similar in structure to the ATP synthase-binding sequence of the inhibitor of F1 (IF(1)), a naturally occurring inhibitor of the ATP synthase complex in mitochondria. In hippocampal slices, Abeta and IF(1) similarly impair both short- and long-term potentiation via a mechanism that could be suppressed by blockade of GABAergic transmission. These observations indicate that APP and Abeta regulate extracellular ATP levels in the brain, thus suggesting a novel mechanism in Abeta-mediated Alzheimer's disease pathology.

LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses.

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.

Carcinoembryonic antigen carrying SLeX as a new biomarker of more aggressive gastric carcinomas.

Malignant transformation of gastric cells is accompanied by the deregulated expression of glycosyltransferases leading to the biosynthesis of tumor-associated glycans such as the sialyl-Lewis X antigen (SLex). SLex presence on cell surface glycoconjugates increases the invasive capacity of gastric cancer cells and is associated with tumor metastasis. ST3Gal IV enzyme is involved in the synthesis of SLex antigen and overexpressed in gastric carcinomas. Herein, we identified the glycoproteins carrying SLex in gastric cancer cells overexpressing ST3Gal IV enzyme and evaluated their biomarker potential for gastric carcinoma. Methods: SLex modified glycoproteins were identified applying western blot and mass spectrometry. Immunoprecipitation, proximity ligation assay (PLA), E-selectin binding assay and CRISPR/cas9 knockout experiments were performed to characterize the presence of SLex on the identified glycoprotein. Protein N-glycans of the SLex protein carrier were in deep analyzed by porous-graphitized-carbon liquid-chromatography and tandem mass spectrometry glycomics. In silico expression analysis of α2-3 sialyltransferase ST3Gal IV and SLex protein carrier was performed and the conjoint expression of the SLex modified glycoproteins evaluated by immunohistochemistry and PLA in a series of gastric carcinomas. Results: Carcinoembryonic antigen (CEA; CEACAM5) was identified and validated by different methodologies as a major carrier of SLex. N-glycomics of CEA revealed that complex N-glycans are capped with α2-3 linked sialic acid (Neu5Acα2-3Galß1-4GlcNAc). Data set analysis of ST3Gal IV and CEA showed that ST3Gal IV expression was associated with patient´s poor survival, whereas CEA did not show any prognostic value. The co-expression of both CEA and SLeX was observed in 86,3% of gastric carcinoma cases and 74,5% of the total cases displayed the conjoint CEA+SLexin situ PLA expression. This expression was associated with clinicopathological features of the tumors, including infiltrative pattern of tumor growth, presence of venous invasion and patient's poor survival. CEA immunoprecipitation from gastric carcinoma tissues also confirmed the presence of SLex. Conclusion: CEA is the major glycoprotein carrying SLex in gastric carcinoma and the conjoint detection of CEA-SLex is associated with aggressive tumor features highlighting its PLA detection as a biomarker of gastric cancer patient prognosis for theranostic applications.

Characterisation of the endogenous human peripheral serotonin transporter SLC6A4 reveals surface expression without N-glycosylation.

RT-PCR confirmed that human cell lines of diverse peripheral origins express transcripts for the serotonin transporter (sert/slc6a4). Molecular weights reported for the translated protein appear to be quite variable however. Here we compared directly immunoreactive protein generated from cloned sert transfected into HEK293 with that carried endogenously among the cell lines. The dominant glycosylated 85-95 kDa immunoreactive species contained in HEK-sert transfectants was poorly represented in any native cell: instead, discrete 70 and 60 kDa bands were universally detected. Biotinylation of lymphoid cells revealed that the endogenous non-glycosylated 60 kDa but not 70 kDa protein was available at the surface to access exogenous ligands.

Identification of a novel GDNF mRNA induced by LPS in immune cell lines.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types and has wide ranging effects within the central nervous system. To investigate the expression of the GDNF gene in immune cell lines under inflammatory conditions, we pharmacologically estimated the induction of GDNF mRNA in RAW264.7 cells. RT-PCR analysis revealed that LPS-induced GDNF mRNA in RAW264.7 cells does not include exon 3, which encodes the translational start site of this gene. A novel type of GDNF mRNA cloned by 5'-RACE consisted of the previous exon 4 and its flanking 5' upstream region, akin to a single exon gene. A similar type of human GDNF mRNA was also detected in a human neuroblastoma cell line, SH-SY5Y, without any stimulation. This novel (Ex4) GDNF mRNA was also upregulated by LPS in primary cultured rat macrophages, microglia and astrocytes and was found to exist in mouse brain. Ex4 GDNF protein produced by transfected HEK293 cells was mainly detected in cell lysates, but in conditioned medium only after PMA stimulation. Ex4 GDNF protein was found to exist as an unglycosylated form in both the transfected cells and the conditioned medium while full-type GDNF protein is glycosylated. PMA-stimulated augmentation of unglycosylated Ex4 GDNF protein was demonstrably regulated at the post-translational level.

Glutamate receptor trafficking: endoplasmic reticulum quality control involves ligand binding and receptor function.

The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [3H]kainate binding and patch-clamp recording. Each of the binding site mutations was sufficient to reduce [3H]kainate binding to undetectable levels and eliminate functional responses to glutamate or kainate. As with our studies of other nonfunctional mutants (Fleck et al., 2003), immunocytochemical staining and cell-surface biotinylation studies showed that the mutant receptors were retained intracellularly and did not traffic to the cell surface. Endoglycosidase-H digests and colocalization with endoplasmic reticulum (ER) markers demonstrated that the mutant receptors are immaturely glycosylated and retained in the ER. Immunoprecipitation, native PAGE, and functional studies confirmed that the GluR6-binding site mutants are capable of multimeric assembly, indicating their retention in the ER does not result from a gross protein folding error. Together, these results confirm the role of R523, T690, and E738 directly in ligand binding to GluR6 and further support our previous report that nonfunctional GluRs are retained intracellularly by a functional checkpoint in ER quality control.

Distinctive roles of endoplasmic reticulum and golgi glycosylation in functional surface expression of mammalian E-NTPDase1, CD39.

CD39 is a membrane-bound ecto-nucleoside triphosphate diphosphohydrolase that is involved in the regulation of purinergic signaling. It has been previously reported that N-linked glycosylation is essential for the surface localization of CD39 and for its cellular activity. Here we have addressed the roles of different stages of N-linked glycosylation on CD39's activity and surface expression by using various glycosylation inhibitors, glycosylation deficient CHO cells, and oligosaccharide removal enzymes. The results demonstrate that endoplasmic reticulum glycosylation is required for protein folding and essential for functional surface expression of CD39, while Golgi glycosylation is less important. The study has also shown that N-linked glycosylation of CD39 is dispensable for the activity after the protein is properly folded and targeted.

Characterization of N-glycosylation consensus sequences in the Kv3.1 channel.

N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1-S2 linker is topologically extracellular, and that N-glycosylation influences the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrPC to PrPres.

The PMCA (protein misfolding cyclic amplification) technique has been shown to drive the amplification of misfolded prion protein by PrP(Sc) seeds during several cycles of incubation-sonication. Here, we report that cyclic amplification of normal hamster brain homogenates treated with a number of transition metals (manganese [Mn], copper [Cu], and iron [Fe]) leads to conversion of PrP(C) into protease-resistant PrP(res). The efficiency of PrP(res) formation and the glycoforms induced by Mn were different from those obtained by Cu and Fe. Previous results have shown higher Mn and lower Cu levels in the affinity-purified PrP(Sc) from the brain of prion diseases compared with normal hamster brain homogenates. We focused on Mn because we observed higher levels of Mn in whole brain, mitochondria, and scrapie-associated fibril-enriched fractions from the brains of animals with prion disease. In the presence of minute quantities of Mn-induced PrP(res) template with a large amount of PrP(C), PrP(res) amplification is observed. A metal chelater, EDTA reverses the effect of Mn on PrP(res) amplification, suggesting that Mn may play a role in the formation of PrP(res). It has been proposed that metal-catalyzed oxidation of PrP leads to the oxidation of amino acids and extensive aggregation of oxidized PrP. Carboxyl acids such as deoxycholic acid (DA) are oxidized molecules produced by 3' oxidation pathway. In in vitro studies, the potent effect of Mn on PrP(res) amplification is augmented by DA in a dose-dependent manner. On the basis of the evidence of the elevated Mn levels in scrapie-associated fibril (SAF)-enriched preparations from the brains of animals with prion disease, Mn-loaded PrP and oxidized molecules such as carboxyl acids may contribute to the formation of the scrapie isoform of PrP in prion diseases.

Susceptibility to proteases of anti-Tn-antigen MLS128 binding glycoproteins expressed in human colon cancer cells.

Anti-Tn antigen MLS128 monoclonal antibody was produced two decades ago by immunizing mice with "cancerous antigens" derived from LS180 colon cancer cells. Previous studies demonstrated that MLS128 bound to 110 kDa glycoprotein (GP) in colon cancer cells, thereby inhibiting cell growth. Extensive attempts have been made towards understanding the inhibitory action of MLS128 on colon cancer cell growth and solving the primary structure of 110 kDa GP. Since limited proteolysis of 110 kDa GP was observed in microdomain fractions that had been kept frozen for several years, susceptibility of 110 kDa GP to trypsin and other proteases as well as N-glycosidase F has been investigated. Furthermore, 110 kDa GP expression was examined in colon cancer cells independently cultured in Akiyama laboratory. In summary, 110 kDa GP contains N-glycans. It does not contain inter-disulfide bonds but appears to have intra-disulfides. It must contain multiple cleavage sites for trypsin and thermolysin since these proteases digested 110 kDa GP to MLS128-undetectable small fragments. It seems to contain cleavage sites for cathepsin D which could cause limited digestion. LS180 cells derived from Akiyama laboratory produced a limited proteolysis product-like 75 kDa GP. This study provides a structural basis for developing cancer diagnostics and therapeutics.