Consensus Sequence For N-linked Glycosylation Research Articles

Dimerization is required for the glycosylation of S1-S2 linker of sea urchin voltage-gated proton channel Hv1

Multimerization of ion channels is essential for establishing the ion-selective pathway and tuning the gating regulated by membrane potential, second messengers, and temperature. Voltage-gated proton channel, Hv1, consists of voltage-sensor domain and coiled-coil domain. Hv1 forms dimer, whereas voltage-dependent channel activity is self-contained in monomer unlike many ion channels, which assemble to form ion-conductive pathways among multiple subunits. Dimerization of Hv1 is necessary for cooperative gating, but other roles of dimerization in physiological aspects are still largely unclear. In this study, we show that dimerization of Hv1 takes place in ER. Sea urchin Hv1 (Strongylocentrotus purpuratus Hv1: SpHv1) was glycosylated in the consensus sequence for N-linked glycosylation within the S1-S2 extracellular loop. However, glycosylation was not observed in the monomeric SpHv1 that lacks the coiled-coil domain. A version of mHv1 in which the S1-S2 loop was replaced by that of SpHv1 showed glycosylation and its monomeric form was not glycosylated. Tandem dimer of monomeric SpHv1 underwent glycosylation, suggesting that dimerization of Hv1 is required for glycosylation. Moreover, when monomeric Hv1 has a dilysine motif in the C-terminal end, which is known to act as a retrieval signal from Golgi to ER, prolonging the time of residency in ER, it was glycosylated. Overall, our results suggest that monomeric SpHv1 does not stay long in ER, thereby escaping glycosylation, while the dimerization causes the proteins to stay longer in ER. Thus, the findings highlight the novel significance of dimerization of Hv1: regulation of biogenesis and maturation of the proteins in intracellular compartments.

N-linked glycosylation of thermostable lipase from Bacillus thermocatenulatus to improve organic solvent stability

A thermostable lipase from Bacillus thermocatenulatus was glycosylated by forming the consensus sequence (–NXS/T–) for N-linked glycosylation by site-directed mutagenesis. Among the eight BTL2 mutants including the consensus sequence, six BTL2 mutants, A277 N, A290 N, Y200 N, T236 N, T238 N, and P261 N, were glycosylated. Among the six mutants, glycosylated A277 N and T236 N showed higher stability in the presence of 25% (v/v) DMSO (74.3 and 72.8% of initial activity was remained after incubation at 45 °C for 20 h, respectively) than deglycosylated A277 N and T236 N (57.2 and 45.1% of initial activity was remained, respectively). These glycosylated mutants also showed higher remaining activity than wild-type BTL2 (56.0% of the initial activity were remained). Furthermore, the glycosylated mutant T236 N showed longer half-lives in the presence of 25% (v/v) ethylene glycol, DMSO, and DMF (161, 133, and 56.7 h at 45 °C, respectively) than deglycosylated mutant T236 N (107, 91.9, and 42.8 h, respectively). N-linked glycosylation may be a promising approach for preparing enzymes to retain their activity in the presence of organic solvents.

Enhancing FVIII Expression By Modifying B-Domain Sequence for Hemophilia A Gene Therapy

Background: We have previously shown that conserving six N-linked glycosylation sites of the human factor VIII (FVIII) B-domain sequences in a synthetic linker, V3 (31aa/N6), increased FVIII expression by 3-fold over B-domain deleted FVIII (BDD-FVIII) following AAV mediated gene transfer in mice (McIntosh et. al. Blood 2013). These N-linked glycosylation consensus sequences (N-X-T/S) are highly conserved across different species but their role in the context of transgenic FVIII expression following gene transfer remains unknown. We, therefore, undertook systemic B-domain linker engineering studies to elucidate the mechanism by which the V3-linker improves FVIII expression following AAV mediated gene transfer.Methods: A series of FVIII variants were made by modifying the B-domain linker size and amino acid sequence and then cloned into AAV or lentiviral vectors. The latter were used for making stable FVIII producer cell lines to facilitate biochemical characterization including glycome profile using MALDI-TOF mass spectrometry. The AAV vectors, in which expression of the FVIII gene was directed by a small synthetic liver specific promoter (HLP) were used to assess transgene expression in-vitro and in murine models.Results: Purified recombinant FVIII-V3 had the same biological activity as that of full-length and BDD FVIII protein concentrates when assessed by thrombin generation and vWF/FVIII binding affinity assays (Dissociation constant (KD) <1nM) suggesting that V3 linker did not interfere with the potency of FVIII. Replacement of the asparagine residues with alanine (FVIII-V5) within the six N-linked glycosylation motifs resulted in a >20 reduction in glycan structures but the expression profile and biological properties of FVIII-V5 were comparable to those of FVIII-V3. In contrast, FVIII-V1 (44aa), in which the N-linked glycosylation triplets in FVIII-V3 were flanked by two additional amino acids that are normally adjacent to these moieties in the native FVIII B-domain, was expressed at 3-fold low levels when compared to FVIII-V3 and V5 in mice. Similarly, randomly scrambling the amino acid sequence of the V3-linker (FVIII-V2) without modifying size resulted in a 7-fold decrease in FVIII expression levels in mice. Notably, FVIII-V3 and V5 mRNA levels were 4-fold higher than the levels observed with BDD-FVIII and FVIII-V1 variants. Deletion analysis of the V3 B-domain linker identified a shorter form (FVIII-N2 19aa/N2) that exhibited similar levels of FVIII secretion to those observed with FVIII-V3.Conclusion: We have critically evaluated the role of the FVIII B domain V3 linker on human FVIII expression in human cell lines and mice following AAV mediated gene transfer. In all cases, the FVIII-V3 variant performed better than the BDD-FVIII, in large part due to an increase in FVIII-V3 mRNA levels. Although the FVIII-V3 molecule is fully glycosylated, our studies show that glycan content did not significantly influence FVIII expression but increasing size and modifying amino-acid content of the linker, markedly reduced FVIII expression following AAV mediated gene transfer in mice. Therefore, the results presented here support the use of FVIII-V3 for gene therapy for hemophilia A in our on-going investigator lead Phase 1 trial. DisclosuresMcIntosh:Freeline: Consultancy, Equity Ownership; BioMarin: Patents & Royalties. Dane:Freeeline: Employment, Equity Ownership. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties.

Mice with an N-Ethyl-N-Nitrosourea (ENU) Induced Tyr209Asn Mutation in Natriuretic Peptide Receptor 3 (NPR3) Provide a Model for Kyphosis Associated with Activation of the MAPK Signaling Pathway.

Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.

A Novel CSF3R Mutation Uncovers the Importance of Membrane-Proximal N-Glycosylation for Receptor Regulation

Mutations in CSF3R (aka GCSFR) occur in the majority of patients with Chronic Neutrophilic Leukemia (CNL) and also are rarely found in adult and pediatric Acute Myeloid Leukemia. The most common CSF3R mutation in CNL is T618I (aka T595I), a point mutation in the membrane-proximal extracellular domain that causes ligand independence. Mutations that lead to a premature stop in the cytoplasmic domain are also found in CNL and result in increased expression of CSF3R on the cell surface. Understanding the biology of other novel mutations in CSF3R may lead to insight into both receptor biology and oncogenesis.The CSF3R N610H mutation was identified in a patient with a myeloproliferative neoplasm, which was most consistent with a JAK2, CALR, MPL mutation negative primary myelofibrosis. This patient had a history of mild leukocytosis for several years with most recent white blood cell counts between 13.3 and 15.3 x 103/uL. A bone marrow biopsy revealed 90% cellularity with mildly increased reticulin fibrosis, increased myeloid to erythroid ratio, no overt dysplasia, and less than 5% blasts. The cells were karotypically normal with a micro deletion of the 3' end of PDGFRB (5q) identified by FISH at 59%. The patient has had minimal symptoms with no anemia or thrombocytopenia and is currently being monitored but not receiving any intervention. The patient's bone marrow was next-generation sequenced using a 42-gene myeloid malignancy targeted mutation hotspot panel which revealed a mutation at N610H in CSF3R at a 50% mutant allele frequency.Given its proximity to the most common CSF3R mutation found in Chronic Neutrophilic Leukemia (T618I, aka T595I), we were interested in understanding whether the N610H mutation might contribute to disease biology. N610 (also known as N586 in the traditional numbering system that does not include the signal peptide) is part of an N-X-T motif, which is a consensus sequence for N-linked glycosylation. Haniu et al (Biochemistry 1996) demonstrated that N610 is one of 8 sites that are N-glycosylated on CSF3R. We confirm by mass spectrometry (MS) based analysis that N610 is occupied with a bisecting complex N-glycan (in vitro). We further found that the N610H mutation and a more conservative N610Q substitution are highly activating in CSF3R, leading to cytokine-independent growth in the murine Ba/F3 cell line. Furthermore, like the T618I mutation, these mutations render the receptor ligand-independent. N610H and N610Q lead to a robust increase in downstream signaling through the JAK/STAT pathway as demonstrated by an increase in the levels of phospho-STAT3. The loss of N-glycosylation in the membrane-proximal region of CSF3R may therefore increase ligand-independent receptor activation and promote oncogenesis. This study highlights the insight that rare human mutations can provide into the relationship between receptor structure and function. DisclosuresRadich:Ariad: Consultancy; Novartis: Consultancy, Research Funding; Gilliad: Consultancy; Incyte: Consultancy. Bertozzi:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Catalent Biologics: Membership on an entity's Board of Directors or advisory committees; Verily: Membership on an entity's Board of Directors or advisory committees; Enable Bioscience: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Oh:CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Characterization of Missense Mutations in Factor VIII That Lead to Abnormal N-Linked Glycosylation

Most secreted proteins are glycosylated on the asparagine (N) residue with the consensus sequence N-X-S/T(X≠Proline).Coagulation factor VIII (FVIII) is heavily N-linked glycosylated with 5 consensus sites outside the B domain. However, the roles of these glycans are not well understood. Meanwhile, missense mutations which could create additional N-linked glycosylation sites have largely not been characterized in hemophilia A patients.In this study we first expressed individual domains of FVIII and determined that the A2, Cand C2 domains are efficiently secreted. The A1(N42,N239), A3 (N1810)and C1 (N2118)domains are glycosylated, whereas N582 in the A2 domain is not glycosylated. Only one hemophilia A missense mutation, S241C in the A1 domain, was found to abolish the consensus sequence for N-linked glycosylation at N239. We confirmed that the S241C mutant lost one glycan and became unstable inside cells. We also tested the other three glycosylation sites and found that elimination of the N-linked glycan at N2118 (N2118Q mutation) impaired the secretion of the C domain. This defect could not be rescued by adding another N-linked glycan (at N2062) in the C1 domain, indicating that the N2118 glycan is specifically required for the secretion of the C domain.We next searched the CHAMP F8 Mutation Database and the FVIII Variant Database and identified 19 missense mutations that potentially create an ectopic glycosylation site.These mutations are located in A1, A2, A3 and C1 domains, but none in the C2 domain. Only two mutations (I566T and M1772T) have previously been characterized.We found that all but one (I2071T) of these mutations gained an additional N-linked glycan. We further studied missense mutations in the A2 (A469T, A469S, I566T, M614T and G701S) and the C domain (W2062S, I2071T and D2131N) because these domains are secreted in cell culture. Whereas secretion of I566T, W2062S and D2131N mutants was comparable to their wild-type counterparts, secretion of other mutants decreased to 5%-30% of WT (P<0.05). Mutants that secreted into culture media nevertheless have low FVIII activity (<2%), indicating that these mutations cause cross reactive material positive hemophilia A.The consequences of additional N-linked glycan were further investigated using the A2 domain mutants, since this domain is normally unglycosylated. After treating with tunicamycin to block the N-linked glycosylation process in the endoplasmic reticulum (ER),the secretion of A2 domain with I566T andG701Smutants, which had relatively high secretion levels, decreased significantly. On the other hand, removing the additional glycan boosted the secretion of A469S and A469T, two low-secretion mutants.Tunicamycin treatment had no effect on another low secretion mutant,M614T.These results suggest that amino acid substitution in I566T andG701Smutationsis detrimental to the proper folding of the protein and the additional N-glycan plays a stabilization role. On the other hand, additional N-glycan plays a destabilization role in A469S and A469T mutations, contributing to disruption of folding in these mutants. For theM614Tmutation,the amino acid substitution alone is likely sufficient todestroy the protein folding.We also studied interactions of abnormally glycosylated mutants with ER chaperones.All the mutants with low secretion levels significantly induced expression of GRP78 to 1.5-2.0 folds(P<0.05), while mutants that maintain higher secretion levels did not affect GRP78 expression. The low secretion mutants also had increased binding to GRP78 and calreticulin, but not to calnexin.Therefore ER chaperones play a key role in the ER quality control of FVIII mutants. In conclusion, our results indicate that the effects of abnormal N-linked glycosylation on FVIII folding and secretionvary widely, from detrimental to beneficial. The impact of a particular glycan is likely determined by the location and the underlying amino acid change caused by the mutation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rational design of hyper-glycosylated interferon beta analogs: A computational strategy for glycoengineering

Glycoengineering has been successfully used to improve the physicochemical and pharmaceutical properties of therapeutics. One aspect of glycoengineering is to introduce new N-linked glycosylation consensus sequences (Asn, X, Thr/Ser) into desirable positions in the peptide backbone by mutational insertion to generate proteins with increased sialic acid content. In the current work, human interferon beta (huIFN-β) was used as a model to identify the potential positions for the addition of new N-glycosylation sites. A computational strategy was employed to predict the structural distortions and functional alterations that might be caused by the change in amino acid sequence. Accordingly, three-dimensional (3D) structures of the designed huIFN-β analogs were generated by comparative modeling. Molecular dynamics (MD) simulation was carried out to assess the molecular stability and flexibility profile of the structures. Subsequently, for the purpose of glycoengineering huIFN-β, analogs with 3D structures more similar to the wild-type huIFN-β and exposed Asn residue in the new N-glycosylation site were identified. These modeling procedures indicated that the addition of the new N-glycosylation site in the loop regions had lower constraining effects on the tertiary structure of the protein. This computational strategy can be applied to avoid alterations in the 3D structure of proteins caused by changes in the amino acid sequences, when designing novel hyper-glycosylated therapeutics. This in turn reduces labor-intensive experimental analyses of each analog.

N-Linked Glycosylation Is Required for Transferrin-Induced Stabilization of Transferrin Receptor 2, but Not for Transferrin Binding or Trafficking to the Cell Surface

Transferrin receptor 2 (TfR2) is a member of the transferrin receptor-like family of proteins. Mutations in TfR2 can lead to a rare form of the iron overload disease, hereditary hemochromatosis. TfR2 is proposed to sense body iron levels and increase the level of expression of the iron regulatory hormone, hepcidin. Human TfR2 (hTfR2) contains four potential Asn-linked (N-linked) glycosylation sites on its ectodomain. The importance of glycosylation in TfR2 function has not been elucidated. In this study, by employing site-directed mutagenesis to remove glycosylation sites of hTfR2 individually or in combination, we found that hTfR2 was glycosylated at Asn 240, 339, and 754, while the consensus sequence for N-linked glycosylation at Asn 540 was not utilized. Cell surface protein biotinylation and biotin-labeled Tf indicated that in the absence of N-linked oligosaccharides, hTfR2 still moved to the plasma membrane and bound its ligand, holo-Tf. However, without N-linked glycosylation, hTfR2 did not form the intersubunit disulfide bonds as efficiently as the wild type (WT). Moreover, the unglycosylated form of hTfR2 could not be stabilized by holo-Tf. We further provide evidence that the unglycosylated hTfR2 behaved in manner different from that of the WT in response to holo-Tf treatment. Thus, the putative iron-sensing function of TfR2 could not be achieved in the absence of N-linked oligosaccharides. On the basis of our analyses, we conclude that unlike TfR1, N-linked glycosylation is dispensable for the cell surface expression and holo-Tf binding, but it is required for efficient intersubunit disulfide bond formation and holo-Tf-induced stabilization of TfR2.

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.

Migration of the Swine Influenza Virus δ-Cluster Hemagglutinin N-Linked Glycosylation Site from N142 to N144 Results in Loss of Antibody Cross-Reactivity

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a >8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization.

Abstract 163: Functional Analysis of Novel Mutations in Human SR-BI

Scavenger receptor class B, type I (SR-BI) is an 82 kDa glycosylated high density lipoprotein (HDL) receptor that is imperative for mediating HDL-cholesterol (HDL-C) uptake into the liver for whole body cholesterol excretion. Recently, two novel point mutations in human SR-BI, S112F or T175A, were discovered in patients with high HDL-C levels. We hypothesize that the high HDL-cholesterol levels are a direct result of each mutant SR-BI’s inability to bind and mediate the selective uptake of cholesteryl esters (CE) from HDL particles. To test this hypothesis, we created SR-BI cDNA encoding the S112F- or T175A point mutations. Cell surface biotinylation and flow cytometry analysis confirmed the plasma membrane localization of both mutant receptors, although T175A-SR-BI expressed at slightly lower levels than wild-type SR-BI. Following transient transfection into COS-7 cells, both S112F- and T175A-SR-BI mutant receptors displayed a greater than 75% decrease in [125I]HDL binding and a greater than 90% decrease in selective uptake of [3H]HDL-COE, as compared to wild-type SR-BI. Both mutant SR-BI receptors also displayed an impaired ability to mediate the release of free cholesterol from cells to HDL. PFO-PAGE revealed that the mutant receptors maintained their ability to form dimers and higher order oligomers, suggesting that the lack of function is not due to a change in the oligomeric status of SR-BI. Interestingly, introduction of alanine at residue 175 disrupts the consensus sequence for N-linked glycosylation at Asn173. Immunoblot analysis confirmed that the T175A-SR-BI migrated slightly faster than wild-type SR-BI, and we hypothesize that receptor function may be compromised due to changes in the glycosylation pattern of this receptor. It is also important to note that S112 and T175 are located in a highly conserved hydrophobic region of the extracellular domain that has previously been shown to be important for receptor function. As such, we speculate that mutations at one or both of these residues may also have a significant impact on HDL-cholesterol transport due to changes in hydrophobicity of this region of SR-BI.

Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.

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.

The Role of Rhodopsin Glycosylation in Protein Folding, Trafficking, and Light-Sensitive Retinal Degeneration

Several mutations in the N terminus of the G-protein-coupled receptor rhodopsin disrupt NXS/T consensus sequences for N-linked glycosylation (located at N2 and N15) and cause sector retinitis pigmentosa in which the inferior retina preferentially degenerates. Here we examined the role of rhodopsin glycosylation in biosynthesis, trafficking, and retinal degeneration (RD) using transgenic Xenopus laevis expressing glycosylation-defective human rhodopsin mutants. Although mutations T4K and T4N caused RD, N2S and T4V did not, demonstrating that glycosylation at N2 was not required for photoreceptor viability. In contrast, similar mutations eliminating glycosylation at N15 (N15S and T17M) caused rod death. Expression of T17M was more toxic than T4K to transgenic photoreceptors, further suggesting that glycosylation at N15 plays a more important physiological role than glycosylation at N2. Together, these results indicate that the structure of the rhodopsin N terminus must be maintained by an appropriate amino acid sequence surrounding N2 and may require a carbohydrate moiety at N15. The mutant rhodopsins were rendered less toxic in their dark inactive states, because RD was abolished or significantly reduced when transgenic tadpoles expressing T4K, T17M, and N2S/N15S were protected from light exposure. Regardless of their effect on rod viability, all of the mutants primarily localized to the outer segment and Golgi and showed little or no endoplasmic reticulum accumulation. Thus, glycosylation was not crucial for rhodopsin biosynthesis or trafficking. Interestingly, expression of similar bovine rhodopsin mutants did not cause rod cell death, possibly attributable to greater stability of bovine rhodopsin.

Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b

ASICs (acid-sensing ion channels) are H(+)-gated Na(+) channels with a widespread expression pattern in the central and the peripheral nervous system. ASICs have a simple topology with two transmembrane domains, cytoplasmic termini and a large ectodomain between the transmembrane domains; this topology has been confirmed by the crystal structure of chicken ASIC1. ASIC1a and ASIC1b are two variants encoded by the asic1 gene. The variable part of the protein includes the cytoplasmic N-terminus, the first transmembrane domain and approximately the first third of the ectodomain. Both variants contain two consensus sequences for N-linked glycosylation in the common, distal part of the ectodomain. In contrast with ASIC1a, ASIC1b contains two additional consensus sequences in the variable, proximal part of the ectodomain. Here we show that all the extracellular asparagine residues within the putative consensus sequences for N-glycosylation carry glycans. The two common distal glycans increase surface expression of the channels, but are no absolute requirement for channel activity. In sharp contrast, the presence of at least one of the two proximal glycans, which are specific to ASIC1b, is an absolute requirement for surface expression of ASIC1b. This result suggests substantial differences in the structure of the proximal ectodomain between the two ASIC1 variants.

Analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) from the lycopodiophyte Selaginella kraussiana suggests that XTH sequence characteristics and function are highly conserved during the evolution of vascular plants

A tissue print followed by a xyloglucan endotransglycosylase assay revealed that XET activity is present at sites of cell elongation in both roots and shoots of the lycopodiophyte Selaginella kraussiana. This paper provides the first report and analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) cDNA sequence, isolated from a club moss. In silico analysis of the deduced amino acid sequence revealed a strong conservation of the XET-domain described in higher plants. The catalytic site (DEIDLEFLG) varies in only one amino acid compared with the consensus sequence and was shown to be functional after recombinant expression of Sk-XTH1 in Pichia pastoris. Sk-XTH1 displays xyloglucan endotransglycosylase activity over a broad pH (4.5-7.5) and temperature range (4-30 degrees C), but it shows no hydrolase activity. The catalytic site is followed by a consensus sequence for N-linked glycosylation. Four terminal cysteines were shown to stabilize a putative XET-C terminal extension region, which includes conserved amino acids, involved in the recognition and binding of the substrates. The N-linked sugar interactions as well as the disulphide bridges were shown to be necessary to perform XET activity. The presence of a highly conserved XTH sequence and function in a microphyllophyte suggests that XTHs were present before the divergence of lycopodiophytes and euphyllophytes. It also points to a possible key role for XTHs in the production of a cell wall that allowed the further evolution of land plants.

Evolution of the uniquely adaptable lentiviral envelope in a natural reservoir host

BackgroundThe ability of emerging pathogens to infect new species is likely related to the diversity of pathogen variants present in existing reservoirs and their degree of genomic plasticity, which determines their ability to adapt to new environments. Certain simian immunodeficiency viruses (SIVcpz, SIVsm) have demonstrated tremendous success in infecting new species, including humans, resulting in the HIV-1 and HIV-2 epidemics. Although SIV diversification has been studied on a population level, the essential substrates for cross-species transmission, namely SIV sequence diversity and the types and extent of viral diversification present in individual reservoir animals have not been elucidated. To characterize this intra-host SIV diversity, we performed sequence analyses of clonal viral envelope (env) V1V2 and gag p27 variants present in individual SIVsm-infected sooty mangabeys over time.ResultsSIVsm demonstrated extensive intra-animal V1V2 length variation and amino acid diversity (le38%), and continual variation in V1V2 N-linked glycosylation consensus sequence frequency and location. Positive selection was the predominant evolutionary force. Temporal sequence shifts suggested continual selection, likely due to evolving antibody responses. In contrast, gag p27 was predominantly under purifying selection. SIVsm V1V2 sequence diversification is at least as great as that in HIV-1 infected humans, indicating that extensive viral diversification in and of itself does not inevitably lead to AIDS.ConclusionPositive diversifying selection in this natural reservoir host is the engine that has driven the evolution of the uniquely adaptable SIV/HIV envelope protein. These studies emphasize the importance of retroviral diversification within individual host reservoir animals as a critical substrate in facilitating cross-species transmission.

Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site‐specific glycosylation in yeast

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.

Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system

To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system. A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells. RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot. An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut. Northern blot showed a single 3.0 kb transcript in selected digestive organs. pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Immunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins. pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.

Membrane Topology of the STT3 Subunit of the Oligosaccharyl Transferase Complex

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.