Putative N-linked Glycosylation Sites Research Articles

Deletion of the single putative N-glycosylation site of the porcine circovirus type 2 Cap protein enhances specific immune responses by DNA immunisation in mice

The immunogenicity of a putative N-linked glycosylation site located at amino acids 143-145 (N143YS) of the porcine circovirus 2 (PCV2) Cap protein was investigated. Eukaryotic vectors expressing wild-type PCV2 Cap (pCap) and N-linked glycosylation site mutant Cap (pCap-m) were constructed and the immunogenicity of these proteins was determined following DNA vaccination in BALB/c mice. pCap-m elicited significantly higher Cap-specific T lymphocyte proliferative activity, percentage of CD8(+) T cells, ratio of immunoglobulin (Ig) G2a:IgG1 and levels of interferon-γ compared to pCap (P<0.05). These results indicate that deletion of the N-glycosylation site in the PCV2 Cap protein enhances specific immune responses and may have a role in Cap-based DNA vaccines with enhanced immunogenicity.

Interaction of Human, Rat, and Mouse Immunoglobulin A (IgA) with Staphylococcal Superantigen-like 7 (SSL7) Decoy Protein and Leukocyte IgA Receptor

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.

The Genetic Bottleneck in Vertical Transmission of Subtype C HIV-1 Is Not Driven by Selection of Especially Neutralization-Resistant Virus from the Maternal Viral Population

Subtype C human immunodeficiency virus type 1 (HIV-1C) continues to cause the majority of new cases of mother-to-child transmission (MTCT), and yet there are limited data on HIV-1C transmission. We amplified env from plasma RNA for 19 HIV-1C MTCT pairs, 10 transmitting in utero (IU) and 9 transmitting intrapartum (IP). There was a strong genetic bottleneck between all mother-infant pairs, with a majority of transmission events involving the transmission of a single virus. env genes of viruses transmitted to infants IP, but not IU, encoded Env proteins that were shorter and had fewer putative N-linked glycosylation sites in the V1-V5 region than matched maternal sequences. Viruses pseudotyped with env clones representative of each maternal and infant population were tested for neutralization sensitivity. The 50% inhibitory concentration of autologous serum was similar against both transmitted (infant) and nontransmitted (maternal) viruses in a paired analysis. Mother and infant Env proteins were also similar in sensitivity to soluble CD4, to a panel of monoclonal antibodies, and to heterologous HIV-1C sera. In addition, there was no difference in the breadth or potency of neutralizing antibodies between sera from 50 nontransmitting and 23 IU and 23 IP transmitting HIV-1C-infected women against four Env proteins from heterologous viruses. Thus, while a strong genetic bottleneck was detected during MCTC, with viruses of shorter and fewer glycosylation sites in env present in IP transmission, our data do not support this bottleneck being driven by selective resistance to antibodies.

Cloning and Characterization of the Antiviral Activity of Feline Tetherin/BST-2

Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection.

Mutation of putative N-Linked Glycosylation Sites in Japanese encephalitis Virus Premembrane and Envelope proteins enhances humoral immunity in BALB/C mice after DNA vaccination

Swine are an important host of Japanese encephalitis virus (JEV). The two membrane glycoproteins of JEV, prM and E, each contain a potential N-linked glycosylation site, at positions N15 and N154, respectively. We constructed plasmids that contain the genes encoding wild-type prME (contain the signal of the prM, the prM, and the E coding regions) and three mutant prME proteins, in which the putative N-linked glycosylation sites are mutated individually or in combination, by site-directed mutagenesis. The recombinant plasmids were used as DNA vaccines in mice. Our results indicate that immunizing mice with DNA vaccines that contain the N154A mutation results in elevated levels of interleukin-4 secretion, induces the IgG1 antibody isotype, generates greater titers of anti-JEV antibodies, and shows complete protection against JEV challenge. We conclude that mutation of the putative N-glycosylation site N154 in the E protein of JEV significantly enhances the induced humoral immune response and suggest that this mutant should be further investigated as a potential DNA vaccine against JEV.

Molecular cloning and characterization of Izumo1 gene from sheep and cashmere goat reveal alternative splicing

We cloned the cDNA and genomic DNA encoding for Izumo1 of cashmere goat (Capra hircus) and sheep (Ovis aries). Analysis of 4.6 kb Izumo1 genomic sequences in sheep and goat revealed a canonical open reading frame (ORF) of 963 bp spliced by eight exons. Sheep and goat Izumo1 genes share >99% identity at both DNA and protein levels and are also highly homologous to the orthologues in cattle, mouse, rat and human. Extensive cloning and analysis of Izumo1 cDNA revealed three (del 69, del 182 and del 217) and two (del 69 and ins 30) alternative splicing isoforms in goat and sheep, respectively. All of the isoforms are derived from splicing at typical GT-AG sites leading to partial or complete truncation of the immunoglobulin (Ig)-like domain. Bioinformatics analysis showed that caprine and ovine Izumo1 proteins share similar structure with their murine orthologue. There are a signal peptide at the N-terminus (1-22 aa), a transmembrane domain at the C-terminus (302-319 aa), and an extracellular Ig-like region in the middle (161-252 aa) with a putative N-linked glycosylation site (N(205)-N-S). Alignment of Izumo1 protein sequences among 15 mammalian species displayed several highly conserved regions, including LDC and YRC motifs with cysteine residues for potential disulfide bridge formation, CPNKCG motif upstream of the Ig-like domain, GLTDYSFYRVW motif upstream of the putative N-linked glycosylation site, and a number of scattered cysteine residues. These distinctive features are very informative to pinpoint the important gene motifs and functions. The C-terminal regions, however, are more variable across species. Izumo1 cDNA sequences of goat, sheep, and cow were found to be largely homologous, and the molecular phylogenetic analysis is consistent with their morphological taxonomy. This implies the Izumo1 gene evolves from the same ancestor, and the mechanism of sperm-egg fusion in mammals may be under the same principle in which Izumo1 plays an important role.

A delta-class glutathione transferase from the Chinese mitten crab Eriocheir sinensis: cDNA cloning, characterization and mRNA expression

Glutathione-S-transferases (GSTs), a major superfamily of antioxidative enzymes, play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The delta-class GST cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) from the Chinese mitten crab, Eriocheir sinensis. The 938 bp E. sinensis GST gene, encoding a polypeptide of 216 amino acids, showed significant similarity to homologous genes in insects. The deduced amino acid sequence of the crab GST contains conserved features of this gene family including the G-site (1–82 aa, tripeptide glutathione binding site) in the N-terminal region and an H-site (88–213 aa, substrate binding site) in the C-terminus. Additionally, a kinase C phosphorylation site (ITI) and one putative N-linked glycosylation site (DNIT) for N-linked carbohydrate chains were also identified. Quantitative real-time RT-PCR (qRT-PCR) was employed to investigate the distribution of GST mRNA in different tissues and its temporal expression in haemocytes of crabs challenged with Aeromonas hydrophila. Different levels of GST mRNA expression were detected in haemocytes, hepatopancreas and muscle, while it was not detected in the gills, intestines and stomach. GST transcription was significantly induced in haemocytes at 6 h post-bacterial challenge ( P < 0.05) and dropped to basal levels at 12 h, presumably down-regulated by increased bacteremia by that time. These findings suggest that GST could play a critical role in immunity, and this positive feedback mechanism can allow for efficient activation of the early innate immune response to infection.

Genetic characterization and susceptibility on poultry and mammal of H7N6 subtype avian influenza virus isolated in Japan in 2009

From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the “Manual of Standards for Diagnostic Tests and Vaccines” by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals.

Mutation of putative N-linked glycosylation sites on the human nucleotide receptor P2X7 reveals a key residue important for receptor function.

The nucleotide receptor P2X(7) is an immunomodulatory cation channel and a potential therapeutic target. P2X(7) is expressed in immune cells such as monocytes and macrophages and is activated by extracellular ATP following tissue injury or infection. Ligand binding to P2X(7) can stimulate ERK1/2, the transcription factor CREB, enzymes linked to the production of reactive oxygen species and interleukin-1 isoforms, and the formation of a nonspecific pore. However, little is known about the biochemistry of P2X(7), including whether the receptor is N-linked glycosylated and if this modification affects receptor function. Here we provide evidence that P2X(7) is sensitive to the glycosidases EndoH and PNGase F and that the human receptor appears glycosylated at N187, N202, N213, N241, and N284. Mutation of N187 results in weakened P2X(7) agonist-induced phosphorylation of ERK1/2, CREB, and p90 ribosomal S6 kinase, as well as a decreased level of pore formation. In further support of a role for glycosylation in receptor function, treatment of RAW 264.7 macrophages with the N-linked glycosylation synthesis inhibitor tunicamycin attenuates P2X(7) agonist-induced, but not phorbol ester-induced, ERK1/2 phosphorylation. Interestingly, residue N187 belongs to an N-linked glycosylation consensus sequence found in six of the seven P2X family members, suggesting this site is fundamentally important to P2X receptor function. To address the mechanism whereby N187 mutation attenuates receptor activity, we developed a live cell proteinase K digestion assay that demonstrated altered cell surface expression of P2X(7) N187A. This is the first report to map human P2X(7) glycosylation sites and reveal residue N187 is critical for receptor trafficking and function.

Targeting of neutral cholesterol ester hydrolase to the endoplasmic reticulum via its N-terminal sequence

Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn(270), Asn(367), and Asn(389)) of NCEH are glycosylated. Glycosylation at Asn(270), which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells.

A comparative study of HIV-1 clade C env evolution in a Zambian infant with an infected rhesus macaque during disease progression

To evaluate whether HIV-1 clade C (HIV-C) envelope variations that arise during disease progression in rhesus macaque model reflect changes that occur naturally in human infection. An infant macaque was infected with SHIV-1157i, an R5 tropic clade C SHIV, that expresses a primary HIV-C envelope derived from an infected human infant and monitored over a 5-year period. Genetic variation of the V1-V5 envelope region, which is the main target for humoral immune responses, derived from the infected macaque and infant was examined. The V1-V5 envelope region was cloned and sequenced from longitudinal peripheral blood mononuclear cell samples collected from the infected macaque and infant. Phylogenetic analysis [phylogenetic tree, diversity, divergence, ratio of nonsynonymous (dN) and synonymous substitution (dS) and dN distribution] was performed. Plasma RNA viral load, CD4(+) T-cell count, changes in the length of V1-V5 region, putative N-linked glycosylation site number and distribution were also measured. Phylogenetic analysis revealed that changes in the macaque closely reflected those of the infant during disease progression. Similar distribution patterns of dN and hot spots were observed between the macaque and infant. Analysis of putative N-linked glycosylation sites revealed several common variations between the virus populations in the two host species. These variations correlate with decline of CD4 T-cell count in the macaque and might be linked with disease progression. SHIV-C infection of macaque is a relevant animal model for studying variation of primary HIV-C envelope during disease progression and could be used to analyze the selection pressures that are associated with those changes.

Genetic variation analysis of porcine reproductive and respiratory syndrome virus isolated in China from 2002 to 2007 based on ORF5

The complete open reading frame 5 (ORF5) sequences of 34 field porcine reproductive and respiratory syndrome virus (PRRSV) isolates from China in 2002–2007 were detected and compared with the different variable Chinese isolates S1, CH-1a, HB-1, HB-2 and JXA1. The results showed that all isolates were of type 2 PRRSV and could be assigned to two clusters. The isolates in cluster sg1 was high similar with the highly pathogenic PRRSV strain JXA1, while sg2 clustered with type 2 PRRSV isolate VR2332. It was interesting that the isolate SH02 which was isolated from Shanghai in 2002 has 98.8% identity with JXA1 emerged in 2006. And the ZJJ07 isolate was found to be a natural recombinant between a Chinese highly pathogenic SY0608 isolate and a VR-2332 derivative NH04 isolate. Analysis of the potential glycosylation sites indicated that they were frequently mutated and formed five putative N-linked glycosylation (NGS) sites patterns based on N30, 33–35, 44 and 51 in those isolates. It indicated that the highly variable PRRSV strain with different NGS patterns spread widely in China. The great genetic diversity could be taken into consideration for the control and prevention of this disease.

Stanniocalcin 2 is positively and negatively controlled by 1,25(OH)2D3 and PTH in renal proximal tubular cells

We have previously identified a second mammalian stanniocalcin (STC2) in humans and demonstrated that STC2 inhibits phosphate uptake in an opossum renal proximal tubular cell line (opossum kidney (OK) cells). However, the regulation of Stc2 gene expression in OK cells is not well understood. In this study, we identified the opossum Stc2 cDNA sequence. The opossum STC2 amino acid sequence had 78.8% homology with human STC2, and has a conserved putative N-linked glycosylation site. Next, we investigated the regulation of Stc2 gene expression by the classical calcium and phosphate-regulating factors 1,25(OH)(2)D(3) and PTH in OK cells. In western blot analysis using affinity-purified anti-STC2 antibody, the secretion of STC2 protein was stimulated by 1,25(OH)(2)D(3) in a dose-dependent manner. By contrast, PTH suppressed the induction of STC2 protein secretion by 1,25(OH)(2)D(3). Real-time PCR analysis revealed that Stc2 mRNA expression was increased by 1,25(OH)(2)D(3) in a dose- and time-dependent manner. In addition, actinomycin D, an RNA synthesis inhibitor, prevented the effects of 1,25(OH)(2)D(3) on Stc2 gene expression. On the other hand, PTH and phorbol 12,13-myristic acetate, a specific PKC activator, but not 8-bromo-cyclic AMP, a specific PKA activator, reduced the mRNA levels of Stc2. In addition, Gö6976, a specific PKC inhibitor, abolished the downregulation of Stc2 mRNA expression by PTH. Furthermore, we demonstrated that the renal Stc2 mRNA expression was increased by 1,25(OH)(2)D(3) and decreased by PTH in vivo. These results suggest that STC2 is positively and negatively controlled by 1,25(OH)(2)D(3) and PTH in renal proximal tubular cells.

The Campylobacter jejuni/coli cjaA (cj0982c) Gene Encodes an N-Glycosylated Lipoprotein Localized in the Inner Membrane

The Campylobacter coli 72Dz/92 cjaA gene (orthologue of cj0982c of C. jejuni NCTC 11168) product is a highly immunogenic, amino acid-binding protein. CjaA was palmitic acid-modified when processed in E. coli. In addition, site-directed mutagenesis of the Cys residue of the LAAC motif of its signal sequence confirmed that CjaA is a lipoprotein when processed in Campylobacter. Localization of the protein appeared to be host dependent. In Campylobacter, CjaA was recovered mainly as an inner-membrane protein, whereas in E. coli most of the protein was present in the periplasmic space. Interestingly, antiserum raised against Campylobacter glycine-extracted material also recognized CjaA produced by Campylobacter and Escherichia coli, indicating that at least part of the protein may be surface exposed. Site-directed mutagenesis of the Asn residues of two putative N-linked glycosylation sites (NIS and NFT) showed that CjaA is glycosylated and that only the first N-X-S/T sequeon serves as a glycan acceptor.

Analysis of Putative N-Linked Glycosylation Sites and Variable Region of Envelope HIV-1 CRF07_BC Recombinant in Intravenous Drug Users in Xinjiang Autonomous Region, China

To investigate more closely the determinants of transmission and escape in HIV-1 internal proteins, we analyzed the characterization of putative N-linked glycosylation sites (PNGSs) and the variable loop of CRF07_BC recombinant human immunodeficiency virus type 1 (HIV-1), isolated from intravenous drug users (IDUs). We studied the characterization of PNGSs and the variable loop in the C1-C5 and V1-V4 regions of the HIV-1 env gene in 12 intravenous drug users (IDUs) who were divided into two groups according to the length of infection time. In addition, two IDUs were longitudinally monitored from the time of seroconversion for 1.5 and 2.5 years. The longitudinal characterization within the individuals on PNGSs and the variable loop in the C1-C5 and V1-V4 region were also observed. Based on the above analysis, we found that PNGSs and the variable loop appeared to increase over time within IDU transmission of CRF07_BC recombinant HIV-1. We argue that limited PNGSs and the length of variable loops may be involved in selective transmission and escape.

Identification and phylogenetic analysis on lipopolysaccharide and β-1,3-glucan binding protein (LGBP) of kuruma shrimp Marsupenaeus japonicus

A lipopolysaccharide (LPS) and beta-1,3-glucan binding protein (LGBP) gene was cloned from hemocytes of kuruma shrimp Marsupenaeus japonicus by reverse-transcription polymerase chain reaction (RT-PCR), cloning and sequencing of overlapping PCR, and rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of M. japonicus LGBP is 1062 bp and encodes a 354 amino acid (aa) sequence with a 23 aa signal peptide. The calculated molecular mass of the mature protein (331 aa) is 40.15 kDa with an estimated pI of 4.78. The M. japonicus LGBP sequence contains (1) two putative N-linked glycosylation sites, (2) two putative integrin-binding motifs, (3) a kinase C phosphorylation site (KCPS), (4) a glucanase motif (GM), and (5) two potential polysaccharide recognition motifs (polysaccharide binding motif (PsBM) and beta-glucan recognition motif (GRM)), and with features of tryptophan-rich, slight homology to lysozyme, and slight homology to lectin. A sequence comparison showed that the deduced amino acids of M. japonicus LGBP has an overall high similarity to penaeid LGBP and betaGBP (85.6-89.9%), lobster Homarus gammarus betaGBP (77.0%), and crayfish Pacifastacius leniusculus LGBP (67.8%). The phylogenetic analysis revealed that M. japonicus LGBP grouped together with other crustacean LGBP and betaGBP, and was close to termite GNBP, but was far way from moth betaGBP, betaGRP, fly GNBP, and mosquito betaGRP. The LGBP of M. japonicus was strongly expressed in hemocytes. The LGBP mRNA transcript in hemocytes of M. japonicus was significantly upregulated 12-48 h after a LPS injection, indicating activation of the innate immune system through the binding of the LGBP and LPS complex.

Up-regulation of Ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT–PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.

Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli

Type I (α/β) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon ( Salmo salar) IFN-α (SasaIFN-α1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-α1 (rSasaIFN-α1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-α subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-α, the mature form of the SasaIFN-α2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-α2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-α2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 × 10 6 U mg −1 against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-α2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-α2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity.

Immunocytochemical and Phylogenetic Analyses of an Arginine Vasotocin-Dependent Aquaporin, AQP-h2K, Specifically Expressed in the Kidney of the Tree Frog, Hyla japonica

Water movement occurs across the plasma membrane of various cells of animals, plants, and microorganisms through specialized water-channel proteins called aquaporins (AQPs). We have identified a new member of the amphibian AQP family, AQP-h2K, from the kidneys of Hyla japonica. This protein consists of 280 amino acid residues with two NPA (Asn-Pro-Ala) sequence motifs and a mercury-sensitive cysteine residue just upstream from the second NPA motif. There are two putative N-linked glycosylation sites at Asn-120 and Asn-128 and one protein kinase A phosphorylation site at Ser-262. The AQP-h2K protein was specifically expressed in the apical membrane and/or cytoplasm of principal cells in the kidney collecting ducts. After stimulation with arginine vasotocin, it was translocated from the cytoplasmic pool to the apical membrane. Phylogenetic analysis of AQP proteins from anurans and mammals identified six clusters of anuran AQPs: types 1, 2, 3, and 5 and two anuran-specific types, designated a1 and a2. The cluster AQPa2 contains Hyla AQP-h2 and AQP-h3, which are expressed in the anuran urinary bladder and ventral pelvic skin. AQP-h2K belongs to the type 2, together with mammalian (human and mouse) AQP2, suggesting that AQP-h2K is an anuran ortholog of the neurohypophysial hormone-regulated mammalian AQP2 and that the AQP2 molecule is already present in the anuran mesonephros.

Molecular Cloning of cDNAs and Genes for Three α-Glucosidases from European Honeybees,Apis melliferaL., and Heterologous Production of Recombinant Enzymes inPichia pastoris

cDNAs encoding three alpha-glucosidases (HBGases I, II, and III) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 bp in length, and included ORFs of 1,767, 1,743, and 1,704 bp encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases I, II, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (38-44% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.