O-linked Glycosylation Sites Research Articles

On-Line LC−MS Approach Combining Collision-Induced Dissociation (CID), Electron-Transfer Dissociation (ETD), and CID of an Isolated Charge-Reduced Species for the Trace-Level Characterization of Proteins with Post-Translational Modifications

We have expanded our recent on-line LC-MS platform for large peptide analysis to combine collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10,000 Da) from complex proteins, such as beta-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approximately >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g., <or=2+ ions), which lack extensive ETD or CRCID fragmentation, can be characterized by the CID-MS2 step. A more targeted approach can then be followed in subsequent LC-MS runs to obtain additional information, if needed. Overall, the recently introduced ETD not only provides useful structural information, but also enhances the confidence of all assignments. The sensitivity of this new approach on the chromatographic time scale is similar to the previous Extended Range Proteomic Analysis (ERPA) using CID-MS2 and CID-MS3. The new LC-MS platform can be anticipated to be a useful approach for the comprehensive characterization of complex proteins.

Variants of the β1,3-Galactosyltransferase CgtB from the Bacterium Campylobacter Jejuni have Distinct Acceptor Specificities

The gene clusters encoding the lipooligosaccharide biosynthesis glycosyltransferases from Campylobacter jejuni have previously been divided in eight classes based on their genetic organization. Here, three variants of the beta1,3-galactosyltransferase CgtB from two classes were purified as fusions with the maltose-binding protein (MalE) from Escherichia coli and their acceptor preference was determined. The acceptor preference of each CgtB variant was directly related to the presence or absence of sialic acid in the acceptor, which correlated with the core oligosaccharide structure in vivo. The three variants were evaluated for their ability to use a derivitized monosaccharide, a GM2 ganglioside mimic, a GA2 ganglioside mimic as well as a peptide containing alpha-linked GalNAc. This characterization shows the flexibility of these galactosyltransferases for diverse acceptors. The CgtB variants were engineered via carboxy-terminal deletions and inversion of the gene fusion order. The combination of a 20 to 30 aa deletion in CgtB followed by MalE at its carboxy terminus significantly improved the glycosyltransferase activity (up to a 51.8-fold increase of activity compared to the full length enzyme) in all cases regardless of the acceptor tested. The improved enzyme CgtB(OH4384)DeltaC-MalE was used to galactosylate a glyco-peptide acceptor based on the interferon alpha2b protein O-linked glycosylation site as confirmed by the CE-MS analysis of the reaction products. This improved enzyme was also used successfully to galactosylate the human therapeutic protein IFNalpha2b[GalNAcalpha]. This constitutes the first report of the in vitro synthesis of the O-linked T-antigen glycan on a human protein by a bacterial glycosyltransferase and illustrates the potential of bacterial glycosyltransferases as tools for in vitro glycosylation of human proteins of therapeutic value.

Molecular cloning of pituitary glycoprotein α-subunit and follicle stimulating hormone and chorionic gonadotropin β-subunits from New World squirrel monkey and owl monkey

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey ( Saimiri sp.) and owl monkey ( Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone α-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature β subunits of follicle stimulating hormone (FSHβ) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHβ. New World primate glycoprotein hormone α-polypeptides and FSHβ subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the β-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the β-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGβ are 143 and 144 amino acids in length and 77% homologous with human CGβ. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGβ, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGβs. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

Characterization of Apin, a secreted protein highly expressed in tooth‐associated epithelia

We previously reported expression of a protein by enamel organ (EO) cells in rat incisors, originally isolated from the amyloid of Pindborg odontogenic tumors called Apin. The aim of the present study was to further characterize the Apin gene and its protein in various species, assess tissue specificity, and clarify its localization within the EO. Northern blotting and RT-PCR revealed that expression of Apin was highest in the EO and gingiva, moderate in nasal and salivary glands, and lowest in the epididymis. The protein sequences deduced from the cloned cDNA for rat, mouse, pig, and human were aligned together with those obtained from four other mammal genomes. Apin is highly conserved in mammals but is absent in fish, birds, and amphibians. Comparative SDS-PAGE analyses of the protein obtained from bacteria, transfected cells, and extracted from EOs all indicated that Apin is post-translationally modified, a finding consistent with the presence of predicted sites for phosphorylation and O-linked glycosylation. In rodent incisors, Apin was detected only in the ameloblast layer of the EO, starting at post-secretory transition and extending throughout the maturation stage. Intense labeling was visible over the Golgi region as well as on the apices of ameloblasts abutting the enamel matrix. Apin was also immunodetected in epithelial cells of the gingiva which bind it to the tooth surface (junctional epithelium). The presence of Apin at cell-tooth interfaces suggests involvement in adhesive mechanisms active at these sites, but its presence among other epithelial tissues indicates Apin likely possesses broader physiological roles.

Dermokine: An Extensively Differentially Spliced Gene Expressed in Epithelial Cells

Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.

O-Linked Glycosylation at Threonine 27 Protects the Copper Transporter hCTR1 from Proteolytic Cleavage in Mammalian Cells

The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wild-type hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.

Genomic organization and expression of CD8α and CD8β genes in fugu Takifugu rubripes

We have cloned cDNAs encoding the α and β chains of CD8 from the tiger pufferfish (fugu), Takifugu rubripes. The cDNA sequences encode a putative leader peptide, extracellular immunoglobulin variable region-like domain, stalk region, transmembrane region, and cytoplasmic tail. A protein tyrosine kinase p56 lck binding motif was not found in the putative fugu CD8α cytoplasmic tail. O-linked glycosylation sites were found in the stalk of both CD8 chains, suggesting possible stalk formation. Phylogenetic analysis showed that fugu CD8α and CD8β chains cluster with other vertebrate CD8α and CD8β chains, respectively. The fugu CD8 genes comprise six exons separated by five introns. The genes are tandemly aligned 3.6 kb apart and are in the same transcription orientation. Quantitative RT-PCR analysis demonstrated that fugu CD8 is expressed predominantly in lymphoid tissues. In situ hybridization showed that fugu CD8 genes are expressed in thymocytes and lymphocytes within lymphoid organs. Molecular characterization of CD8 in fish provides the basis for development of specific antibodies to identify T-cell subsets, as well as potentially important insights into the evolution of CD8 and the adaptive immunity.

Structural Evidence for a New IgG1 Antibody Sequence Allele of Cattle

Analysis of the heavy-chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV-1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1(a) (clone 2) and IgG1(b) (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216-230. The Thr224 and Thr226 of IgG1(a) were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1(b) were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three-dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro-Ala-Ser-Ser indicated a potential structure-enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O-linked glycosylation sites and increasing the susceptibility to degradation by protease-secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1(d). Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1(c) that is distinct in both sequence and structure from the known sequence variants.

O-Linked Glycosylation with Sialylated T-Antigen: A Novel Carbohydrate Determinant of von Willebrand Factor Antigen Levels.

The glycosylation-profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of the sialylated T-antigen (NeuAc( α 2–3)Gal( β 1–3)[NeuAc( α 2–6)]GalNAc). It is unknown yet if O-linked carbohydrates affect VWF levels. We therefore developed an immunosorbent-based assay using the lectin Peanut Agglutinin (PNA) to determine O-linked T-antigen on VWF. Control experiments showed that PNA-binding was: (i) specific to T-antigen that was de-sialylated via neuramidase-treatment; (ii) proportional to the number of O-linked glycosylation sites and (iii) independent of the extent of multimerisation. Using this assay, we found a strong correlation between PNA binding and VWF-antigen levels in a series of randomly selected plasma samples (252±125% for VWF<0.5 U/ml (n=15); 131±36% for VWF between 0.5 and 1.5 U/ml (n=32); 92±40% for VWF>1.5 U/ml (n=19); p<0.003). Reduced or increased PNA binding was also observed for patients characterized by increased (liver cirrhosis; n=58) or reduced (von Willebrand disease (VWD)-type 1; n=32) VWF-antigen levels, respectively. VWD-type 1 patients further displayed increased ratios of propeptide over mature VWF-antigen levels (0.38±0.18 versus 0.17±0.03 for patients (n=32) and controls (n=20), respectively; p<0.0001), which is indicative for reduced VWF survival in these patients. Interestingly, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank= 0.50; p=0.0039), suggesting a potential association between T-antigen glycosylation and VWF survival. Finally, we observed a marked decrease in PNA binding in post-DDAVP samples from various patients (131±45% versus 85±33% in pre- and post DDAVP, respectively; n=18; p=0.0013). Since these patients had distinct underlying pathologies (primary platelet function disorder, hemophilia A or VWD type 1, 2A or 2N), this seems to represent a general phenomenon that is not restricted to VWD. Moreover, it indicates that the O-linked glycosylation profile of VWF stored in endothelial storage-organelles may differ from that of constitutively secreted VWF.

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-than-predicted mass (approximately 3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.

Carbohydrates located on the top of the “cap” contribute to the adhesive and enzymatic functions of vascular adhesion protein‐1

Vascular adhesion protein 1 (VAP-1) is an endothelial adhesion molecule with an enzymatic activity. It deaminates biogenic amines, resulting in the formation of aldehydes and hydrogen peroxide. During the enzymatic reaction a transient Schiff base is formed between endothelial VAP-1 and its leukocytic ligand, and this interaction is important for lymphocyte adhesion. VAP-1 monomer has six potential N-linked, and three putative O-linked glycosylation sites and an SSSS sequence potentially forming an attachment site for an adjacent O-linked site. In this work we modeled the carbohydrate decorations on a structural model of VAP-1, and studied which of those potential glycosylation sites are utilized, and whether those decorations accessible to a lymphocyte ligand are important in lymphocyte adhesion and enzymatic activity of VAP-1. We show that, unlike the O-linked attachment sites, all six N-linked glycosylation sites are in use. Furthermore, mutation of the N-linked attachment sites strategically located on the top of the molecule reduces lymphocyte adhesion in non-static conditions, and enhances the catalytic activity of membrane-bound human VAP-1 in static conditions, suggesting that glycosylation regulates the functional properties of VAP-1.

GPR56 is a GPCR that is overexpressed in gliomas and functions in tumor cell adhesion

GPR56 (also known as TM7XN1) is a newly discovered orphan G-protein-coupled receptor (GPCR) of the secretin family that has a role in the development of neural progenitor cells and has been linked to developmental malformations of the human brain. GPR56 diverges from other secretin-like family members in that it has an extremely large N-terminal extracellular region (381 amino acids) and contains a novel feature among this new subclass, consisting of four cysteine residues that define a GPCR proteolytic site (GPS motif) located just before the first transmembrane spanning domain. The rest of the amino-terminal domain contains a large number of possible N- and O-linked glycosylation sites similar to mucin-like proteins. These features suggest a role in cell-cell, or cell-matrix interactions. Here, we demonstrate upregulation of GPR56 in glioblastoma multiforme tumors using functional genomics. Immunohistochemistry studies confirmed the expression of GPR56 protein in a majority of glioblastoma/astrocytoma tumor samples with undetectable levels of expression in normal adult brain tissue. Immunofluorescence analysis of human glioma cells using anti-GPR56 antibodies demonstrate that GPR56 is expressed on the leading edge of membrane filopodia and colocalizes with alpha-actinin. Purified recombinant GPR56 extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morphology and cell rounding. These results indicate that the extracellular domain may compete for unidentified ligand(s), and block the normal function of GPR56 in cell attachment. In reporter assays, overexpression of GPR56 activates the NF-kappaB, PAI-1 and TCF transcriptional response elements. These pathways have been implicated in cytoskeletal signaling, adhesion and tumor biology. The above results indicate that GPR56 serves as an adhesion GPCR and is involved in adhesion signaling.

Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris

The use of the methylotrophic yeast Pichia pastoris for large-scale recombinant production of proteins for therapeutic uses and/or biophysical characterisation has been gaining popularity. Here we describe the use of this organism for the production of a von Willebrand factor C domain from procollagen IIA for solution NMR studies. In this research, we specifically identified sites of O-linked glycosylation on the expressed protein, although the native protein is not glycosylated. We demonstrated that it was possible to remove the oligosaccharides by enzymatic digestion, however this approach proved to be prohibitively expensive for the scale of production required for high-resolution structural studies by NMR spectroscopy. After removal of the O-linked glycosylation sites by site-directed mutagenesis, we confirmed that the protein was no longer covalently glycosylated. However, analysis by 1H- and 13C-edited spectroscopy identified the presence of non-covalently associated glycans which were removed by lectin affinity chromatography. We have synthesised methods for the identification and removal of both covalently and non-covalently bound oligosaccharides from heterologous protein expressed in P. pastoris.

Complete gene sequence of a human Puumala hantavirus isolate, Puumala Umeå/hu: sequence comparison and characterisation of encoded gene products

Puumala virus is a member of the hantavirus genus in the Bunyaviridae family, and the major causative agent of haemorrhagic fever with renal syndrome in Europe. This study was conducted with a human Puumala virus isolate (PUUV Umeå/hu), and contains the determination of the first complete PUUV sequence from a human source. When the relationship to other Puumala viruses was analysed, a possible RNA segment exchange between two local strains of PUUV was noticed. Furthermore, the coding regions of PUUV Umeå/hu S- and M-segments were cloned, and a large set of gene products were expressed in mammalian cells. In addition, postulated N- and O-linked glycosylation sites in the two envelope proteins (G n and G c) were investigated individually by site-directed mutagenesis followed by gel-shift analysis. Our data demonstrate that N-linked glycosylation occurs at three sites in G n (N142, N357 and N409), and at one site in G c (N937). Also, one possible O-glycosylation site was identified in G c (T985). We conclude that the diversity between different Puumala virus isolates is high, and consequently characterization of local PUUV isolates is important for clinical diagnostic work. Finally, the obtained results concerning the encoded gene products are of great importance for the design of new vaccines.

Organization and expression of thirteen alternatively spliced exons in catfish CD45 homologs

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.

Organization and expression of thirteen alternatively spliced exons in catfish CD45 homologs

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa.

Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.

Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy.

Transferrin receptor (TfR) has been identified as a candidate IgA1 receptor expressed on human mesangial cells (HMC). TfR binds IgA1 but not IgA2, co-localizes with mesangial IgA1 deposits, and is overexpressed in patients with IgA nephropathy (IgAN). Here, structural requirements of IgA1 for its interaction with mesangial TfR were analyzed. Polymeric but not monomeric IgA1 interacted with TfR on cultured HMC and mediates internalization. IgA1 binding was significantly inhibited (>50%) by soluble forms of both TfR1 and TfR2, confirming that TfR serves as mesangial IgA1 receptor. Hypogalactosylated serum IgA1 from patients with IgAN bound TfR more efficiently than IgA1 from healthy individuals. Serum IgA immune complexes from patients with IgAN containing aberrantly glycosylated IgA1 bound more avidly to TfR than those from normal individuals. This binding was significantly inhibited by soluble TfR, highlighting the role of TfR in mesangial IgA1 deposition. For addressing the potential role of glycosylation sites in IgA1-TfR interaction, a variety of recombinant dimeric IgA1 molecules were used in binding studies on TfR with Daudi cells that express only TfR as IgA receptor. Deletion of either N- or O-linked glycosylation sites abrogated IgA1 binding to TfR, suggesting that sugars are essential for IgA1 binding. However, sialidase and beta-galactosidase treatment of IgA1 significantly enhanced IgA1/TfR interaction. These results indicate that aberrant glycosylation of IgA1 as well as immune complex formation constitute essential factors favoring mesangial TfR-IgA1 interaction as initial steps in IgAN pathogenesis.

N- and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colony-stimulating factor secreted by a Chinese hamster ovary cell line.

GM-CSF is one of several naturally occurring glycoproteins that regulate leukocyte production, migration and function. It has been produced in different cell types, with different properties that depend on the production process used. The purpose of this work was to characterize the recombinant human GM-CSF from an engineered Chinese hamster ovary cell line grown in suspension and as adherent culture for the identification of the glycosylation sites and the definition of the glycosidic moiety, including the degree of site occupancy. Both preparations exhibited size heterogeneity in SDS/PAGE with multiple bands containing glycoprotein forms with either two or one N-glycosylation sites occupied. Minor low molecular mass forms completely lacked N-linked oligosaccharides but contained 1-3 O-linked glycans. Twelve differently charged isoforms were detected in isoelectric focusing gels. At least 16 glycoforms, differing in the number of Hex-HexNAc units (Deltam 365 Da), were detected in MALDI-TOF MS spectra of the desialylated GM-CSFs. MALDI-TOF MS and HPAEC-PAD analysis indicated the presence of predominantly tri- and tetraantennary N-linked oligosaccharide chains with and without N-acetyllactosamine repeat units and some 10% of biantennary oligosaccharides, all containing more than 90% proximal alpha1-6-linked fucose. The oligosaccharide patterns of both GM-CSF preparations were found to be very similar. More than 90% of terminal galactose residues of the N-glycans were found alpha2-3 sialylated with NeuNAc (93%) or NeuNGc (7%). Site specific glycosylation was analysed by electrospray ionization MS and it was found that in the mono glycosylated GM-CSF form more than 90% of the Asn37 were occupied by N-glycans. O-glycosylation at the N-terminus of the polypeptide was detected at Ser7 and Ser9 or Thr10, in the predominantly doubly O-glycosylated glycoprotein form. In the triply modified GM-CSF molecules, Ser5 was additionally O-glycosylated. The major difference between both preparations was found in the MALDI spectra of the desialylated glycoproteins, revealing a higher proportion of forms with a single N-glycosylation site occupied in the preparation derived from suspension culture. ESI-MS and MALDI-MS analysis of endoproteolytically cleaved peptides as well as MALDI-TOF MS of the intact glycoprotein demonstrated the N- and C-termini integrity of the GM-CSF preparations.

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.