N-linked Carbohydrate Research Articles

Abrogating effect of N-linked carbohydrate modifiers on the stem cell factor and endothelin-1-stimulated epidermal pigmentation in human epidermal equivalents

BackgroundWe previously demonstrated that the hyperpigmentation that occurs in UVB-melanosis as well as in solar lentigos is associated with the increased production of melanogenic cytokines, such as endothelin (EDN)-1 and stem cell factor (SCF), by keratinocytes in those areas of the skin. ObjectiveWe developed a model for these hyperpigmentary disorders in EDN1+SCF stimulated human epidermal equivalents (HEEs) and characterized the effects of the N-linked carbohydrate core synthesis inhibitor glucosamine or N-linked carbohydrate processing inhibitors deoxynojirimycin or monensin on the stimulated HEE pigmentation. MethodsThose effects were assessed by melanin analysis, real-time RT-PCR and Western blotting. ResultsThe addition of these N-linked carbohydrate modifiers (NCMs) markedly abolished the EDN1+SCF-elicited increase in HEE pigmentation over 14 days. Real-time RT-PCR and Western blotting of these NCM-treated HEEs unexpectedly revealed that the EDN1+SCF-stimulated steady-state levels of tyrosinase (TYR), TYR-related protein-1, dopachrome tautomerase and PMEL17 as well as microphthalmia-associated transcription factor (MITF) were significantly attenuated at the transcriptional and translational levels without any cytotoxic effects on keratinocytes and melanocytes in the HEEs. Pre-treatment of cultured normal human melanocytes with the NCMs interrupted the EDN1+SCF-induced stimulation of steady-state levels of MITF at the transcriptional and translational levels and TYR activity without any direct inhibitory effect on the catalytic activity of TYR in vitro. ConclusionThis study provides evidence that NCMs have a potential to attenuate the EDN1+SCF-stimulated pigmentation of HEEs by abrogating the increased steady-state levels of MITF mRNA, which results in the attenuation of the increased steady-state levels of these melanocyte-specific proteins.

‘a’-Position-Mutated and G4-Mutated Hemagglutinin-Neuraminidase Proteins of Newcastle Disease Virus Impair Fusion and Hemagglutinin-Neuraminidase-Fusion Interaction by Different Mechanisms

Objectives: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. Methods: We mutated six ‘a’ residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. Results: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. Conclusions: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures.

At last: erythropoietin as a single glycoform.

At last: erythropoietin as a single glycoform.

Neutralizing Capacity of Monoclonal Antibodies That Recognize Peptide Sequences Underlying the Carbohydrates on gp41 of Simian Immunodeficiency Virus

Extensive glycosylation of the envelope spikes of human and simian immunodeficiency virus (HIV and SIV) is an important factor for the resistance of these viruses to neutralization by antibodies. SIVmac239 gp41 has three closely spaced sites for N-linked carbohydrate attachment. Rhesus macaques experimentally infected with mutant versions of SIVmac239 lacking two or three of these carbohydrate sites developed strong serum reactivity against mutated peptide sequences at the site of these glycosylations, as well as high titers of neutralizing activity to the mutant viruses (E. Yuste et al., J. Virol. 82:12472-12486, 2008). However, whether antibodies that recognize these underlying peptides have neutralizing activity has not been directly demonstrated. Here we describe the isolation and characterization of three gp41-specific monoclonal antibodies (4G8, 6G8, and 7D6) from one of these mutant-infected monkeys. All three antibodies reacted with mutant gp41 from viral particles and also with peptides corresponding to mutated sequences. Slight differences in peptide specificities were observed among the three antibodies. Sequence analysis revealed that the heavy chains of all three antibodies were derived from the same germ line heavy-chain segment (IGHV4-59*01), but they all had very different sequences in complementarity-determining region 3. The light chains of all three antibodies were very closely related to one another. All three antibodies had neutralizing activity to mutant viruses deficient in gp41 carbohydrate attachment, but they did not neutralize the parental SIVmac239. These results demonstrate unambiguously that antibodies with specificity for peptide sequences underlying gp41 carbohydrates can effectively neutralize SIV when these carbohydrates are absent. However, the presence of these gp41 carbohydrates effectively shields the virus from antibodies that would otherwise neutralize viral infectivity.

Involvement of dopaminergic neuronal cystatin C in neuronal injury‐induced microglial activation and neurotoxicity

Factors released from injured dopaminergic (DA) neurons may trigger microglial activation and set in motion a vicious cycle of neuronal injury and inflammation that fuels progressive DA neurodegeneration in Parkinson's disease. In this study, using proteomic and immunoblotting analysis, we detected elevated levels of cystatin C in conditioned media (CM) from 1-methyl-4-phenylpyridinium and dieldrin-injured rat DA neuronal cells. Immunodepletion of cystatin C significantly reduced the ability of DA neuronal CM to induce activation of rat microglial cells as determined by up-regulation of inducible nitric oxide synthase, production of free radicals and release of proinflammatory cytokines as well as activated microglia-mediated DA neurotoxicity. Treatment of the cystatin C-containing CM with enzymes that remove O- and sialic acid-, but not N-linked carbohydrate moieties markedly reduced the ability of the DA neuronal CM to activate microglia. Taken together, these results suggest that DA neuronal cystatin C plays a role in the neuronal injury-induced microglial activation and neurotoxicity. These findings from the rat DA neuron-microglia in vitro model may help guide continued investigation to define the precise role of cystatin C in the complex interplay among neurons and glia in the pathogenesis of Parkinson's disease.

Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus

Glycoprotein H (gH) is an envelope protein conserved in the Herpesviridae. Together with glycoprotein B (gB), the heterodimeric complex of gH and glycoprotein L (gL) mediates penetration and direct viral cell-to-cell spread. In herpes simplex and pseudorabies virus (PrV), coexpression of gH/gL, gB, and gD induces membrane fusion to form polykaryocytes. The recently determined crystal structure of a core fragment of PrV gH revealed marked structural similarity to other gH proteins (M. Backovic et al., Proc. Natl. Acad. Sci. U. S. A. 107:22635-22640, 2010). Within the membrane-proximal part (domain IV), a conserved negatively charged surface loop (flap) is flanked by intramolecular disulfide bonds. Together with an N-linked carbohydrate moiety, this flap covers an underlying patch of hydrophobic residues. To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry.

The N-linked carbohydrate moiety of male reproductive tract CD52 (mrt-CD52) interferes with the complement system via binding to C1q

Antisperm antibody detected in infertile female patients’ sera has been shown to correlate with reduced fertility. The antibody showed strong complement-dependent cytotoxicity as determined by the sperm immobilization test (SIT). CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen present in lymphocytes and male reproductive tracts (mrt), including mature sperm and seminal plasma. Recently, purified mrt-CD52 from human seminal plasma has been reported to interfere with the classical complement pathway, but not lectin binding or alternative pathways of the complement system. The purpose of this study is to determine which stage of the classical pathway mrt-CD52 regulates. mrt-CD52 was purified from human seminal plasma or intact sperm membrane. Immunoprecipitation assay was performed with the reaction of mrt-CD52, human complement and mAb H6-3C4. Immunoprecipitate was formed by the carbohydrate moiety of mrt-CD52, but not by the GPI-anchor peptide. The C1q molecule (29kDa) was detected in the immunoprecipitates by Western blotting analysis probed with anti C1q antibody, indicating that the carbohydrate moiety of mrt-CD52 binds to C1q. Also, the complement-dependent SIT revealed that purified CD52 inhibited sperm immobilization activity by antisperm antibody. These results suggest that mrt-CD52 protects sperm function from complement attack if antisperm antibody is generated in the female reproductive tracts.

Analysis of neofructooligosaccharides production mediated by the extracellular β-fructofuranosidase from Xanthophyllomyces dendrorhous

The extracellular β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous mainly synthesizes the neo-fructooligosaccharides (neo-FOS) neokestose and neonystose. This enzyme is a glycoprotein with a content of 59–67% N-linked carbohydrates and an estimated molecular mass of 160–200kDa. The extent level of glycosylation affects the thermal behaviour of the enzyme but not its hydrolase and transferase activities, which are optimal at 60–70°C. The neo-FOS yield of this enzyme was increased from 40 to 168g/L when the sucrose concentration increased from 420 to 600g/L and when the reaction was carried out at 60°C. The neo-FOS levels obtained (168g/L) in this work are the largest reported for any microbial β-fructofuranosidase.

Production and fragmentation of negative ions from neutral N‐linked carbohydrates ionized by matrix‐assisted laser desorption/ionization

Although negative ion fragmentation mass spectra of neutral N-linked carbohydrates (those attached to Asn in glycoproteins) provide much more structural information than spectra recorded in positive ion mode, neutral carbohydrates are reluctant to form negative ions by matrix-assisted laser desorption/ionization (MALDI) unless ionized from specific matrices such as nor-harmane or adducted with anions such as chloride. This paper reports the results of experiments to optimize negative ion formation from adducts of N-linked glycans with respect to ion abundance and fragment ion production. The best results were obtained with 2,4,6-trihydroxyacetophenone (THAP) as the matrix with added ammonium nitrate as the salt providing the anion. This approach is demonstrated to be applicable for a wide range of N-linked glycan structures. Phosphate adducts, analogous to those that are usually encountered in electrospray spectra from N-glycans released by protein N-glycosidase F, were produced by addition of ammonium phosphate to the matrix but in relatively low yield allowing competitive ionization of endogenous anionic compounds leading to complex spectra. Fragmentation of the nitrate adducts, which were formed in higher yield, generally paralleled that seen by collision-induced dissociation following ionization by electrospray, with the first stage of the dissociation being the elimination of the nitrate with a proton from one of the hydroxyl groups of the sugar. The spectra of the resulting [M-H](-) species displayed very specific fragment ions, mainly cross-ring and C-type glycosidic cleavage products, that revealed more structural (linkage and branching) information of the compounds than the mainly glycosidic cleavage products that dominated the positive ion spectra.

N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20-70% higher activity (p<0.05) within pH 6-9, but 15-45% lower activity (p<0.05) at 45-70°C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (T(d)) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower K(m) vaules, but higher k(cat)/K(m) ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29kDa under reducing conditions and non-reducing ∼25kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65Da) and exhibited high specificity for BAρNA, Michaelis–Menten behavior with Km 5.4×10−1 M and the Vmax 7.9×10−1 nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60°C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF).The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom.TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF).In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical–scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.

A Critical Role for N- and O-Linked Carbohydrates In Modulating Von Willebrand Factor Clearance In Vivo

Abstract 382▪FN2▪This icon denotes a clinically relevant abstractVWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF−/− mice.Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p<0.01). Digestion of pdVWF with α2–3 neuraminidase to remove predominantly O-linked sialic acid (which constitutes less than 20% total VWF sialylation) was also sufficient to markedly enhance VWF clearance (t1/2 = 13.1 min; p<0.05). In the presence of the asialoglycoprotein receptor (ASGPR)-antagonist ASOR, the mean residence time of Neu-VWF was identical to that of pd-VWF. Recent studies have shown that macrophages may be important in VWF clearance. Since the ASGPR is expressed on both hepatocytes and macrophages, the effect of macrophage depletion on VWF clearance was assessed. Pre-treatment with liposome-encapsulated clodronate depleted F4/80+CD11b+ murine macrophages by 75%, and significantly prolonged Neu-VWF survival. However Neu-VWF survival was not corrected to that observed in the presence of ASOR. For example, plasma Neu-VWF survival after 5 mins was corrected from 30±6% to 92±7% in the presence of ASOR, compared to 78±10% following clodronate macrophage-depletion. Cumulatively, these findings demonstrate that both N- and O-linked sialylation are critical in protecting VWF against ASGPR-mediated clearance. Moreover, ASGPR-modulated clearance is at least in part macrophage-dependent. ß-galactose residues exposed following removal of capping sialic acid are recognised by the ASGPR. To further define the role of specific sugars in regulating VWF clearance, the effect of terminal sialic acid and sub-terminal galactose removal by sequential neuraminidase and galactosidase digestions was studied. Surprisingly, VWF exposed to sequential neuraminidase and galactosidase digestions (NeuGal-VWF) was cleared rapidly from the plasma in a monophasic fashion (t1/2 = 4.8 min). Moreover, treatment with PNGase F to completely remove N-linked carbohydrate structures also markedly decreased the plasma half-life (PNG-VWF; t1/2 = 2.1 min). In keeping with their lack of exposed galactose residues, the enhanced clearance of NeuGal-VWF and PNG-VWF were not mediated via the ASGPR (ASOR had no significant effect). In contrast, macrophage depletion by liposomal clodronate significantly inhibited the enhanced clearance of both NeuGal-VWF and PNG-VWF respectively. These data suggest that the ASGPR is not the only macrophage receptor involved in modulating VWF clearance, which is consistent with the relatively minor prolongation in VWF survival previously reported in Asgpr1−/− mice.These novel data demonstrate that variation in the N- or O-linked carbohydrate structures significantly modulate VWF half-life in vivo. Moreover, VWF clearance is not mediated solely through the ASGPR, but may also require additional as yet unidentified macrophage receptors for full clearance. Therefore, qualitative and quantitative variation in VWF glycosylation represents a key regulator of VWF clearance, and as such is likely to be of direct pathophysiological significance. Disclosures:No relevant conflicts of interest to declare.

Specific N- and O-Linked Carbohydrate Structures Mediate Von Willebrand Factor Interaction with Galectins -1 and -3

Abstract 2234Von Willebrand Factor (VWF) is extensively glycosylated with both N- and O-linked carbohydrates. Moreover, these complex glycan structures influence VWF functional properties, including susceptibility to ADAMTS13 proteolysis, and plasma clearance. The molecular mechanisms through which VWF glycosylation (including ABO blood group antigens) act to influence VWF physiology remains unexplained. However, recent data suggest that VWF circulates in normal plasma bound to various carbohydrate-binding proteins, including specific members of the galectin family. In addition, galectin-3 binding has been reported to influence VWF cleavage by ADAMTS13. In this context, we sought to elucidate the role of specific VWF glycan determinants in modulating galectin interaction.VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. VWF glycosylation was then modified using exoglycosidases and quantified by specific lectin ELISAs. Blood group specific VWF was also purified from pooled group AB, O, or Bombay plasmas. Galectins-1 and -3 were transiently expressed in competent E-coli cells with an N-terminal histidine tag, and purified by nickel chromatography. Finally, binding interactions were characterized via modified immunosorbant assay.In keeping with the previous report of Lenting et al, human pdVWF bound to both galectin-1 and galectin-3 in a dose-dependent manner. Enzymatic desialylation of pdVWF with α2-3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced binding to galectin-1 (231±6%, p<0.0001). Similarly, removal of terminal sialic acid also increased binding to galectin-3, albeit to a lesser extent (136±6%, p<0.05). To further define the role of VWF glycans in regulating galectin binding, pdVWF was exposed to sequential neuraminidase and galactosidase digestions to remove terminal sialic acid and sub-terminal galactose residues (NeuGal-VWF). In contrast to the enhanced binding of Neu-VWF, binding of NeuGal-VWF to both galectin -1 and -3 was significantly reduced (51±5% and 52±6% compared to pdVWF; p<0.005). Cumulatively these findings suggest that loss of capping sialic acid and exposure of sub-terminal galactose critically regulates VWF-galectin binding. Treatment with PNGase F to completely remove N-linked carbohydrate structures (PNG-VWF) markedly decreased binding to galectin -1 and -3 (13±1% and 57±2%, p<0.001). Moreover, combined PNGase F and O-glycosidase digestions further attenuated galectin-3 binding (21±1%, p<0.001), suggesting that both the N- and O-linked glycans are involved in mediating the VWF-galectin interaction.ABO(H) blood group antigens are expressed on both the N-linked and O-linked glycans of human VWF. Moreover, ABO(H) determinants influence VWF susceptibility to ADAMTS13 proteolysis and plasma VWF half-life, through unknown mechanisms. Purified VWF from normal group AB individuals bound to both galectin-1 and galectin-3 significantly better than group O VWF (146±8% and 483±19%; p<0.01). Conversely, no significant difference in binding was observed between Group O and Bombay VWF. Consequently, although terminal A (GalNAc) and B (Gal) sugar moieties promote galectin binding, expression of terminal α1–2 fucose residues is not important. The glycosylation profile of platelet-VWF differs from that of pdVWF. In particular, platelet-VWF expresses reduced levels of both capping sialic acid and sub-terminal galactose residues (∼50%), and lacks AB blood group antigens. To characterize the effects of this differential sugar expression on galectin binding, platelet-derived VWF was isolated and purified (platelet freeze-thawing followed by immuno-affinity chromatography with monoclonal CLB-Rag20). In keeping with the reduction in Gal and AB blood group antigen expression, platelet VWF bound less well to galectin-1 and galectin-3 (72±6% and 67±7% versus pdVWF; p<0.05).These novel data demonstrate that both the N- and O-linked oligosaccharide structures of VWF are involved in mediating galectin binding. In particular, expression of terminal AB blood group antigens, and expression of sub-terminal galactose moieties following loss of capping sialic acid, both markedly enhance galectin binding affinity. Further studies will be required to define how galectin binding is involved in mediating the functional consequences of variation in VWF glycans. Disclosures:No relevant conflicts of interest to declare.

Specific Enzyme Complex of β-1,4-Galactosyltransferase-II and Glucuronyltransferase-P Facilitates Biosynthesis of N-linked Human Natural Killer-1 (HNK-1) Carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.

Tissue- and development-specific glycosylation states of the natriuretic peptide receptors guanylyl cyclase-A (GC-A) and GC-B

Background The natriuretic peptides (NPs) atrial NP (ANP), B-type NP (BNP) and C-type NP (CNP) elicit cellular effects by binding to transmembrane proteins with guanylyl cyclase (GC) activity, referred to as GC-A (representing the common receptor for ANP and BNP) and GC-B (the CNP receptor). GC-A and GC-B are structurally closely related proteins with very similar cDNA-deduced molecular masses in the range of 115 kDa, but their native molecular weights are significantly (by up to 20 kDa) higher due to glycosylation. Differential glycosylation of cell surface receptors can affect folding, trafficking, ligand binding and/or agonistinduced signal transduction. Moreover, specific temporal expression patterns of carbohydrate structures are frequently involved in developmental processes. Hitherto, however, the glycosylation states of GC-A and GC-B in vivo are largely unknown, since currently available data are based to a great extent on studies with receptor-transfected cell lines. This investigation aimed to characterize the sizes and posttranslational modifications of native GC-A and GCB in different tissues of adult rats and mice. Based on increasing evidence for specific activities of NPs and their receptors in the developing brain (1-3), we examined the glycosylation of GC-A and GC-B during postnatal brain development. Methods and results Western blot (as well as receptor/ligand crosslinking) experiments and treatments with carbohydrate-digesting enzymes served to investigate the sizes and posttranslational modifications of the two receptors. Due to marked tissue-specific differences in N-linked glycosylation, mean apparent molecular masses of both rat and mouse GC-A range from 119 kDa in brain to values between 124 (lung) and 132 (heart) kDa in peripheral tissues. The reduced glycosylation in brain seems to be central nervous system (CNS)-specific, since the pituitary, which contains both CNS and CNS-unrelated tissue, co-expresses the CNS-typical (119 kDa, reference: olfactory bulb) and the peripheral (up to 130 Da) forms of GC-A. GC-B sizes differed in the same manner between peripheral organs, indicating equal tissue-dependent influences on oligosaccharide processing at GC-A and GC-B. A higher (by 3 kDa) total amount of N-linked carbohydrates suggests usage of an additional consensus N-glycosylation site (7 instead of 6 in the case of GC-A). The existence of region-specific GC-B size variants in cerebellum and olfactory bulb discriminates this receptor from GC-A in the adult brain. Both GC-A and GC-B (but not two other glycosylated membrane proteins examined) are hyperglycosylated at N-linked sites during early postnatal brain development. At postnatal day 1, the vast majority of GC-B (but not GC-A) contain additionally an O-linked carbohydrate modification.

The carbohydrate ligands on the host embryo mediate intercellular migration of the parasitic wasp embryo

Invasive stage embryos of the parasitic wasp Copidosoma floridanum transmigrate through the epithelium of phylogenetically distant host embryos in a manner that is similar to mammalian leukocyte infiltration. Host embryonic cells appear to recognize the invading wasp embryo by components on the cell surface. We developed an in vitro wasp entry inhibition assay and found that C-type lectins of the wasp embryo bound to N-linked carbohydrate chains with fucose residues on the surface of host embryo. This is the first report showing a receptor–ligand interaction between heterologous multicellular organisms.

M-Ficolin Interacts with the Long Pentraxin PTX3: A Novel Case of Cross-Talk between Soluble Pattern-Recognition Molecules

Ficolins and pentraxins are soluble oligomeric pattern-recognition molecules that sense danger signals from pathogens and altered self-cells and might act synergistically in innate immune defense and maintenance of immune tolerance. The interaction of M-ficolin with the long pentraxin pentraxin 3 (PTX3) has been characterized using surface plasmon resonance spectroscopy and electron microscopy. M-ficolin was shown to bind PTX3 with high affinity in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by N-acetyl-D-glucosamine, indicating involvement of the fibrinogen-like domain of M-ficolin. Removal of sialic acid from the single N-linked carbohydrate of the C-terminal domain of PTX3 abolished the interaction. Likewise, an M-ficolin mutant with impaired sialic acid-binding ability did not interact with PTX3. Interaction was also impaired when using the isolated recognition domain of M-ficolin or the monomeric C-terminal domain of PTX3, indicating requirement for oligomerization of both proteins. Electron microscopy analysis of the M-ficolin-PTX3 complexes revealed that the M-ficolin tetramer bound up to four PTX3 molecules. From a functional point of view, immobilized PTX3 was able to trigger M-ficolin-dependent activation of the lectin complement pathway. These data indicate that interaction of M-ficolin with PTX3 arises from its ability to bind sialylated ligands and thus differs from the binding to the short pentraxin C-reactive protein and from the binding of L-ficolin to PTX3. The M-ficolin-PTX3 interaction described in this study represents a novel case of cross-talk between soluble pattern-recognition molecules, lending further credit to the integrated view of humoral innate immunity that emerged recently.

Major isoform of zebrafish P0 is a 23.5 kDa myelin glycoprotein expressed in selected white matter tracts of the central nervous system.

The zebrafish mpz gene, encoding the ortholog of mammalian myelin protein zero, is expressed in oligodendrocytes of the zebrafish central nervous system (CNS). The putative gene product, P0, has been implicated in promoting axonal regeneration in addition to its proposed structural functions in compact myelin. We raised novel zebrafish P0-specific antibodies and established that P0 is a 23.5 kDa glycoprotein containing a 3 kDa N-linked carbohydrate moiety. P0 was localized to myelin sheaths surrounding axons, but was not detected in the cell bodies or proximal processes of oligodendrocytes. Many white matter tracts in the adult zebrafish CNS were robustly immunoreactive for P0, including afferent visual and olfactory pathways, commissural and longitudinal tracts of the brain, and selected ascending and descending tracts of the spinal cord. P0 was first detected during development in premyelinating oligodendrocytes of the ventral hindbrain at 48 hours postfertilization (hpf). By 72 hpf, short segments of longitudinally oriented P0-immunoreactive myelinating axons were seen in the hindbrain; expression in the spinal cord, optic pathways, hindbrain commissures, midbrain, and peripheral nervous system followed. The mpz transcript was found to be alternatively spliced, giving rise to P0 isoforms with alternative C-termini. The 23.5 kDa isoform was most abundant in the CNS, but other isoforms predominated in the myelin sheath surrounding the Mauthner axon. These data provide a detailed account of P0 expression and demonstrate novel P0 isoforms, which may have discrete functional properties. The restriction of P0 immunoreactivity to myelin sheaths indicates that the protein is subject to stringent intracellular compartmentalization, which likely occurs through posttranslational mechanisms.

Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein

The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2 kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity.