Potential N-linked Glycosylation Sites Research Articles

Isolation and cDNA Cloning of Ovarian Cortical Rod Protein in Kuruma Prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.

Role of N-linked glycosylation in the secretion and activity of endothelial lipase

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K(m) and increased apparent V(max). Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V(max). Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.

Evolutionary Dynamics of the Glycan Shield of theHuman Immunodeficiency Virus Envelope during Natural Infection andImplications for Exposure of the 2G12Epitope

Elucidation of the kinetics of exposure of neutralizing epitopes on the envelope of human immunodeficiency virus type 1 (HIV-1) during the course of infection may provide key information about how HIV escapes the immune system or why its envelope is such a poor immunogen to induce broadly efficient neutralizing antibodies. We analyzed the kinetics of exposure of the epitopes corresponding to the broadly neutralizing human monoclonal antibodies immunoglobulin G1b12 (IgG1b12), 2G12, and 2F5 at the quasispecies level during infection. We studied the antigenicity and sequences of 94 full-length envelope clones present during primary infection and at least 4 years later in four HIV-1 clade B-infected patients. No or only minor exposure differences were observed for the 2F5 and IgG1b12 epitopes between the early and late clones. Conversely, the envelope glycoproteins of the HIV-1 quasispecies present during primary infection did not expose the 2G12 neutralizing epitope, unlike those present after several years in three of the four patients. Sequence analysis revealed major differences at potential N-linked glycosylation sites between early and late clones, particularly at positions known to be important for 2G12 binding. Our study, in natural mutants, confirms that the glycosylation sites N295, N332, and N392 are essential for 2G12 binding. This study demonstrates the relationship between the evolving "glycan shield " of HIV and the kinetics of exposure of the 2G12 epitope during the course of natural infection.

Adaptive immunity is the primary force driving selection of equine infectious anemia virus envelope SU variants during acute infection.

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infection in horses. The appearance of antigenically distinct viral variants during recurrent viremic episodes is thought to be due to adaptive immune selection pressure. To test this hypothesis, we evaluated envelope SU cloned sequences from five severe combined immunodeficient (SCID) foals infected with EIAV. Within the SU hypervariable V3 region, 8.5% of the clones had amino acid changes, and 6.4% had amino acid changes within the known cytotoxic T lymphocyte (CTL) epitope Env-RW12. Of all the SU clones, only 3.1% had amino acid changes affecting potential N-linked glycosylation sites. In contrast, a much higher degree of variation was evident in SU sequences obtained from four EIAV-infected immunocompetent foals. Within V3, 68.8% of the clones contained amino acid changes, and 50% of the clones had amino acid changes within the Env-RW12 CTL epitope. Notably, 31.9% of the clones had amino acid changes affecting one or more glycosylation sites. Marked amino acid variation occurred in cloned SU sequences from an immune-reconstituted EIAV-infected SCID foal. Of these clones, 100% had amino acid changes within V3, 100% had amino acid changes within Env-RW12, and 97.5% had amino acid changes affecting glycosylation sites. Analysis of synonymous and nonsynonymous nucleotide substitutions revealed statistically significant differences between SCID and immunocompetent foals and between SCID foals and the reconstituted SCID foal. Interestingly, amino acid selection at one site occurred independently of adaptive immune status. Not only do these data indicate that adaptive immunity primarily drives the selection of EIAV SU variants, but also they demonstrate that other selective forces exist during acute infection.

Molecular cloning and sequencing of Japanese pufferfish ( Takifugu rubripes) NADPH oxidase cDNAs

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91 phox and p22 phox, and cytosolic proteins, p40 phox, p47 phox and p67 phox. In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91 phox, p22 phox, p40 phox, p47 phox and p67 phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91 phox, p22 phox, p40 phox, p47 phox and p67 phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91 phox has three potential N-linked glycosylation sites. gp91 phox and p22 phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47 phox and p67 phox have two potential Src homology 3 domains and p40 phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91 phox, p47 phox and p67 phox expression were high only in the blood and kidney, while p22 phox and p40 phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.

Influence of N-linked glycans on intracellular transport of hepatitis C virus E1 chimeric glycoprotein and its role in pseudotype virus infectivity

We have previously reported a functional role associated with hepatitis C virus (HCV) E1 glycoprotein using vesicular stomatitis virus (VSV)/HCV pseudotype. In this study, we have investigated the role of glycosylation upon intracellular transport of chimeric E1-G, and in infectivity of the pseudotyped virus. Interestingly, surface expressed E1-G exhibited sensitivity to Endoglycosidase H (Endo H) treatment, which was similar to full-length E1, suggesting that additional complex oligosaccharides were not added while E1-G was in transit from the endoplasmic reticulum (ER) to the mammalian cell surface. As a next step, each of the four potential N-linked glycosylation sites located at amino acid position 196, 209, 234, or 305 of the E1 ectodomain were mutated separately (asparagine → glutamine), or in some combination. FACS analysis suggested that mutation(s) of the glycosylation sites affect the translocation of E1-G to the cell surface to different extents, with no single site being particularly essential. VSV pseudotype virus generated from glycosylation mutants exhibited a decrease in titer with an increasing number of mutations at the glycosylation sites on chimeric E1-G. In a separate experiment, N-glycosidase F treatment of pseudotype generated from the already synthesized E1-G or its mutants decreased virus titer by approximately 35%, and the neutralization activity of patient sera was not significantly altered with N-glycosidase F-treated pseudotype virus. Taken together, our results suggested that E1-G does not add complex sugar moieties during transport to the cell surface and retain the glycosylation profile of its parental E1 sequence. Additionally, the removal of glycans from the E1-G reduced, but does not completely impair, virus infectivity.

Molecular cloning and sequencing of Japanese pufferfish (Takifugu rubripes) NADPH oxidase cDNAs

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91 phox and p22 phox , and cytosolic proteins, p40 phox , p47 phox and p67 phox . In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91 phox , p22 phox , p40 phox , p47 phox and p67 phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91 phox , p22 phox , p40 phox , p47 phox and p67 phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91 phox has three potential N-linked glycosylation sites. gp91 phox and p22 phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47 phox and p67 phox have two potential Src homology 3 domains and p40 phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91 phox , p47 phox and p67 phox expression were high only in the blood and kidney, while p22 phox and p40 phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.

Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis

AbstractA missense mutation, FV-Ile359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-Ile359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306Gln/Ile359Thr/Arg679Gln, FV-Ile359Thr/Arg506Gln/Arg679Gln, and FV-Asn357Gln/Ile359Thr). The FV-Ile359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-Ile359Thr migrated more slowly, while the FV-Asn357Gln/Ile359Thr was indistinguishable from FV-wild type (FV-WT), indicating that FV-Ile359Thr was expressed with an additional carbohydrate chain. Activated protein C (APC)-mediated inactivation in an FVa degradation assay showed that the Ile359Thr mutation significantly reduced the cleavage at Arg306 both in the presence and absence of protein S, whereas the cleavage at Arg506 was unaffected. When tested in an FVIIIa degradation assay, the FV-Ile359Thr variant exhibited equally low APC cofactor activity as FV Leiden (FVArg506Gln). In conclusion, the Ile359Thr mutation appears to affect anticoagulation by 2 mechanisms, impeding the APC-mediated down-regulation of the FVa molecule and additionally being a poor APC cofactor for the down-regulation of FVIIIa. These findings explain the association of the FV-Ile359Thr mutation with thrombosis. (Blood. 2004;103:3381-3387)

Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic localization and DPIV-like peptidase activity

Dipeptidyl peptidase (DP) IV has a distinct substrate specificity in hydrolyzing a post-proline bond. Here we present novel data on the sizes and tissue distribution of human and rat gene products and the peptidase activity of the DPIV-related gene DP9. A short cDNA of 2589 bp and a long cDNA of 3006 bp of DP9 were cloned. A ubiquitous predominant DP9 mRNA transcript at 4.4 kb represented the short form, whereas a less abundant 5.0-kb transcript present predominantly in muscle represented the long form. Both forms of DP9 have no transmembrane domain and two potential N-linked glycosylation sites. DP9 exhibited post-proline dipeptidyl aminopeptidase activity and was a cytoplasmic, 110-kDa monomer. Thus, the six DPIV gene family members have diverse characteristics: only DP9 and DP8 have exclusively cytoplasmic localization and only DP9, DP8, fibroblast activation protein (FAP) and DPIV have peptidase activity.

N-linked glycosylation sites of the motor protein prestin: effects on membrane targeting and electrophysiological function.

Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N-glycoprotein with three potential N-linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N-glycosylation were investigated by producing single-point (N163Q and N166Q) or double-point mutations (NN163/166QQ and NN163/166AA) at putative N-glycosylation sites. Further, treatment with tunicamycin or glycopeptidase-F was used to determine the consequences of removing N-linked glycosylation in wild-type prestin. We determined the effects of these manipulations on prestin's cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell-types. Data indicate that prestin is a glycoprotein with N-linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N-linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non-linear capacitance, a signature of prestin's motor function. Compared to glycosylated prestin, the fully de-glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.

Characterization of Pig Intercellular Adhesion Molecule-2 and its Interaction with Human LFA-1

Understanding molecular interactions between human leukocytes and porcine endothelium is important for the future success of pig-to-human xenotransplantation. Here we describe the analysis of pig intercellular adhesion molecule-2 (ICAM-2). A 1020-basepair ICAM-2 cDNA generated from pig lung RNA contained an open reading frame (ORF) encoding a 277-amino-acid protein with six potential N-linked glycosylation sites. The mature protein sequence was 55% identical to human ICAM-2, with conservation of five out of six residues critical for binding of the human protein to its ligand LFA-1. Northern blot analysis identified ICAM-2 transcripts of 4.0 and 1.4 kb in cultured pig endothelial cells and mRNA was detected in pig lung, spleen, kidney, liver and heart by RT-PCR. The gene structure and endothelial expression of pig ICAM-2 were strikingly similar to those of its human and mouse counterparts. However, unlike human ICAM-2, expression of pig ICAM-2 on cultured endothelial cells was not down-regulated by treatment with the inflammatory cytokines TNF-α and IL-1β. Pig ICAM-2 expressed on stable transfectants supported firm adhesion of cells expressing human LFA-1. This conservation of function across the species barrier suggests that pig ICAM-2 plays a role in the cellular interactions associated with xenograft rejection.

Cloning, sequencing and structural features of a novel Streptococcus lipase

An expression library generated from genomic DNA of Streptococcus sp. N1 resulted in C 1N 1 Escherichia coli recombinants forming haloes on lipase indicator tributyrin/Tween-80 agar plates. The excised insert in the recombinant C 1N 1 was of 3.489 kb size. Analysis of the nucleotide sequence of the insert showed two significant operons of 1032 bp (ORF1) and 729 bp (ORF3). ORF1 on the (+)-strand is the structural lipase gene codes for a protein of 343 amino acid residues with a theoretical mass of 38.6 kDa, which is close to the value of the lipase expressed in E. coli (39.36 kDa). The primary structure of the lipase deduced from the nucleotide sequence shows a consensus sequence containing the active serine [VAGHSIGG], a conserved H–G dipeptide in the N-terminal part of the enzyme and a potential site for N-linked glycosylation at amino acid residues [129–131]. The sequence, AAGGA at −6 upstream of the start codon ATG seems to be putative ribosomal binding site (rbs) and is similar to the rbs of lip3 of Moraxella sp. TA 144. The putative −10[TTTAT] and −35[AATG] promoter sequences showed similarity to lip3 of Moraxella sp. TA 144. The lipase shared no significant homology with the members of different lipases families except with group V comprising of Psychrobacter immobilis B-10 [28% identical and 56% positive similarity] and Moraxella sp. TA 144, lip3 [28% identical and 54% similarities]. The 729 bp operon present on (−) strand of 3.489 kb C 1N 1 insert was essential for the secretion of the enzyme. The putative protein on BLAST/FASTA search showed 57% identities and 72% positives with ATP-binding component of ABC-transporter. The recombinant lipase was resistant to organic solvents, non-ionic surfactants, metal ions and showed alkaline pH optima (pH 8.4).

N-linked glycosylation of platelet P2Y12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression

P2Y12 receptor is a Gi-coupled adenosine diphosphate (ADP) receptor with a critical role in platelet aggregation. It contains two potential N-linked glycosylation sites at its extra cellular amino-terminus, which may modulate its activity. Studies of both tunicamycin treatment and site-directed mutagenesis have revealed a dispensable role of the N-linked glycosylation in the receptor’s surface expression and ligand binding activity. However, the non-glycosylated P2Y12 receptor is defective in the P2Y12-mediated inhibition of the adenylyl cyclase activity. Thus the study uncovers an unexpected vital role of N-linked glycans in receptor’s signal transducing step but not in surface expression or ligand binding.

Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1–283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS–PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.

N-linked glycosylation of the human bradykinin B2 receptor is required for optimal cell-surface expression and coupling.

To investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHO-K1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cell-surface to a much greater extent than the non-glycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function.

Phylogenetic Analysis of the Hemagglutinin (H) Gene of Canine Distemper Viruses Isolated from Wild Masked Palm Civets (Paguma larvata)

Hemagglutinin (H) gene of two CDV isolates, the Haku93 and Haku00 strains, from masked palm civets was molecularly analyzed. H genes of both two CDVs contained one open reading frame encoding 607 amino acids. Nucleotide and predicted amino acid sequences of H gene of the CDV Haku93 and Haku00 revealed high similarity to those of recent field isolates such as the Yanaka and Tanu96, while they showed limited identity to those of old vaccine strains. Potential N-linked glycosylation sites in both Haku93 and Haku00 were identical to other recent CDV isolates. Phylogenetic analysis revealed that the CDV strains derived from masked palm civets were classified into the group of recent Japanese CDV isolates.

HCV E2 glycoprotein: mutagenesis of N-linked glycosylation sites and its effects on E2 expression and processing

An expression system for analysis of the synthesis and processing of the E2 glycoprotein of a hepatitis C virus (HCV) genotype 1a strain was developed in transiently transfected cells. E2 proteins representing the entire length of the protein, including the transmembrane segment (E2) as well as two truncated versions (E2 660 and E2 715), were characterized for acquisition of N-linked glycans and transport to the media of transfected cells. To investigate the utilization of the 10 potential N-linked glycosylation sites on this E2 protein, a series of mutations consisting of single or multiple (two, three, four or eight) ablations of asparagine residues in the background of the E2 660 construct were analyzed. E2 660 proteins harboring single or multiple site mutations were produced at levels similar to that of wild-type protein, but secretion of the single mutants was mildly diminished, and elimination of two or more sites dramatically reduced delivery of the protein to the media. Similar results were obtained in Huh-7 cells with respect to intracellular synthesis and secretion of the mutant proteins. Analysis of oligosaccharide composition using endoglycosidase digestion revealed that all of the glycan residues on the intracellular forms of E2 660, E2 715, and E2 contained N-linked glycans modified into high-mannose carbohydrates, in contrast to the secreted forms, which were endo H resistant. The parental E2 660 protein could be readily detected in Huh-7 cells using anti-polyhistidine or antibody to recombinant E2. In contrast, E2 660 lacking the eight N-linked glycans was expressed but not detectable with anti-E2 antibody, and proteins lacking four glycans exhibited reduced reactivity. These experiments provide direct evidence that the presence of multiple N-linked glycans is required for the proper folding of the E2 protein in the ER and secretory pathway as well as for formation of its antigenic structure.

Identification and partial characterization of Taastrup virus: a newly identified member species of the Mononegavirales

We present a 8904-nt sequence of the central part of the RNA genome of a novel virus with a filovirus-like, nonidentical morphology named Taastrup virus (TV) detected in the leafhopper Psammotettix alienus. Sequence analysis identified five potential open reading frames (ORFs) and a complex pattern of homologies to various members of the Mononegavirales suggests a genome organization with the following order of genes: 3′-N-P-M-G-L-5′. Sequence analyses reveal an unusually large glycoprotein (G) containing both potential O-linked (14) and N-linked (9) glycosylation sites—a feature shared with the glycoproteins of Filoviridae and Pneumovirinae, and a nucleoprotein (N) with homology to the nucleoprotein of viral hemorrhagic septicemia virus (VHSV), a member of the Rhabdoviridae. Highly conserved domains were identified in the RNA-dependent RNA polymerase (L) between TV and other viruses within the order of Mononegavirales, and homology was found in particular with members of the Rhabdoviridae. The sequence similarities and the unique filovirus-like but nonidentical morphology unambiguously refer this newly identified virus to the order of Mononegavirales but to no family more than any, to other within the order.

Characterization of a tissue-specific and developmentally regulated β-1,3-glucanase gene family in Prunus persica

We report here on the characterization of a peach β-1,3-glucanase (βGlu) gene family including two newly isolated basic βGlu genes, PpGns2 and PpGns3, linked in a tandem array. The deduced mature proteins PpGNS2 and PpGNS3 share 80% amino acid sequence identity, while PpGNS2 is nearly identical to PpGNS1 (99% identity). The deduced PpGNS3 protein contains a potential N-linked glycosylation site and a tetrapeptide sequence for sorting function, whereas these sites are absent in both PpGNS1 and PpGNS2. βGlu gene expression during flowering and early fruit development is both temporally and spatially regulated. The βGlu transcript reaches a high level in opening flower buds, and then decreases to very low levels at full-bloom. The transcript levels then increase once again at petal-fall (young fruitlets). Gene-specific expression studies in peach revealed that alternate members of βGlu genes were expressed at different stages of development. The PpGns1 transcript was specifically localized in calyx and carpels of opening flower buds. The PpGns3 transcript was mainly expressed at a high level during early fruit set. The PpGns2 transcript was not detected in all tissues analyzed. Transgenic tobacco plants carrying the 5′-promoter region of each of the βGlu genes fused to the uidA reporter gene revealed that PpGns2 was transcriptionally active, and was exclusively expressed in both cotyledons and first leaves of transgenic seedlings. In spite of their highly conserved 5′-flanking regions, the regulation of PpGns1 and PpGns2 was quite distinct. Upon closer investigation of these 5′-flanking regions, significant variations associated with ACGT core-containing motifs and their flanking sequences were observed.

N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A.

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower ( Brassica oleracea var. botrytis ) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc2Man6. LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40% decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.