The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the ... more The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesic...
The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antherae... more The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man 0.2:4.0:1.4: 13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normalphase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannosetype glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man 9-GlcNAc 2 , which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi a-mannosidase digestion, and 1 H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc 1 Man 9 GlcNAc 2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc 1 Man 9 GlcNAc 2 glycan. There were no processed glucosylated oligosaccharides such as Glc 1 Man 5±8 GlcNAc 2. Furthermore, Glc 1 Man 9 GlcNAc 2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc 1 Man 9 GlcNAc 2 may play a role in the folding of arylphorin or in the assembly of hexamers.
Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potential... more Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man (9Ϫ5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)branched triantennary glycans (1 %) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm A(1,3)-fucosylation (forming the Lewis x determinant), A(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man A(1,3)-arm. This isomeric form is indicative of the action of a novel β-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.
Glycosylation of proteins has been shown to play a role in a variety of cellular events. Thanks t... more Glycosylation of proteins has been shown to play a role in a variety of cellular events. Thanks to recent advances in obtaining conformational constraints across glycosidic linkages, structural characterisation of glycoproteins has improved considerably. It is now becoming apparent that N-glycosylation of a folded protein can have a significant stabilising effect on large regions of the backbone structure.
Proceedings of the National Academy of Sciences, 2005
Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular ... more Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck ( Marmota monax ), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core α-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The co...
Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), ... more Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquitoborne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.
ABSTRACTHepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and li... more ABSTRACTHepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and liver cancer. Using comparative glycoproteomics, we identified a glycoprotein that is altered both in amount and in glycosylation as a function of liver fibrosis and cirrhosis. Specifically, this altered glycoprotein is an immunoglobulin G (IgG) molecule reactive to the heterophilic alpha-Gal epitope [Galα-1-3Galβ1-(3)4GlcNAc-R]. While similar changes in glycosylation have been observed in several autoimmune diseases, the specific immunoglobulins and their antigen recognition profiles were not determined. Thus, we provide the first report identifying the specific antigenic recognition profile of an immunoglobulin molecule containing altered glycosylation as a function of liver disease. This change in glycosylation allowed increased reactivity with several fucose binding lectins and permitted the development of a plate-based assay to measure this change. Increased lectin reactivity was obse...
The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was ... more The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of R1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex 7 HexNAc 4 dHex 1, Negative ion ESI-MS/MS confirmed the presence of the R1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex 6 HexNAc 4 dHex 1 Neu5Ac 1 , with the sialic acid linked to the terminal 1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.
Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures a... more Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn354 are inaccessible because the complex glycans at Asn445 block access to the site. On IgE, the additional CH2 hinge domain blocks access to the oligomannose glycans at Asn394 on one H chain by adopting an asymmet...
The fundamental importance of correct protein glycosylation is abundantly clear in a group of dis... more The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type–specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve ...
MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-l... more MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionizationmass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di-or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galβ1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid α2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material.
The monoglucosylated oligomannose N-linked oligosaccharide (Glc 1 Man 9 GlcNAc 2) is a retention ... more The monoglucosylated oligomannose N-linked oligosaccharide (Glc 1 Man 9 GlcNAc 2) is a retention signal for the calnexin-calreticulin quality control pathway in the endoplasmic reticulum. We report here the presence of such monoglucosylated N-glycans on the human complement serum glycoprotein C3. This finding represents the first report of monoglucosylated glycans on a human serum glycoprotein from non-diseased individuals. The presence of the glucose moiety in 5% of the human C3 glycoprotein suggests that this glycosylation site is sequestered within the protein and is consistent with previous studies identifying a cryptic conglutinin binding site on C3 that becomes exposed upon its conversion to iC3b.
Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the o... more Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the only biomarker that is used routinely and there is a need for further complementary biomarkers both in terms of sensitivity and specificity.N-glycosylation changes in ovarian cancer serum glycoproteins include a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLex) on haptoglobinβ-chain,α1-acid glycoprotein andα1-antichymotrypsin. These changes are also present in chronic inflammation but not in malignant melanoma, where there are low levels of inflammatory processes. Acute phase proteins carrying increased amounts of SLexhave an increased half-life. Sialylation of acute phase proteins also decreases apoptosis favouring survival of cancer cells. Cancer cells produce inflammatory cytokines which influence glycosylation processing in liver parenchymal cells. Altered glycosylation of the acute phase protein transferrin plays an important role in iron homeostasis. Glycos...
Critical Reviews in Biochemistry and Molecular Biology, 1998
The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational ev... more The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucintype O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosaccharides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.
The conditions required for mammalian‐type complex N‐linked glycosylation of human proteins produ... more The conditions required for mammalian‐type complex N‐linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N‐linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn‐4h) cells cultured in serum‐free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 ± 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn‐4h cells cultured in serum‐containing medium, only 24 ± 3%...
The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (G... more The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP Sc from the brains of Syrian hamsters contains the same set of glycans as PrP C , but a higher proportion of tri-and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.
This article was published in an Elsevier journal. The attached copy is furnished to the author f... more This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author's institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the ... more The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesic...
The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antherae... more The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man 0.2:4.0:1.4: 13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normalphase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannosetype glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man 9-GlcNAc 2 , which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi a-mannosidase digestion, and 1 H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc 1 Man 9 GlcNAc 2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc 1 Man 9 GlcNAc 2 glycan. There were no processed glucosylated oligosaccharides such as Glc 1 Man 5±8 GlcNAc 2. Furthermore, Glc 1 Man 9 GlcNAc 2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc 1 Man 9 GlcNAc 2 may play a role in the folding of arylphorin or in the assembly of hexamers.
Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potential... more Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man (9Ϫ5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)branched triantennary glycans (1 %) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm A(1,3)-fucosylation (forming the Lewis x determinant), A(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man A(1,3)-arm. This isomeric form is indicative of the action of a novel β-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.
Glycosylation of proteins has been shown to play a role in a variety of cellular events. Thanks t... more Glycosylation of proteins has been shown to play a role in a variety of cellular events. Thanks to recent advances in obtaining conformational constraints across glycosidic linkages, structural characterisation of glycoproteins has improved considerably. It is now becoming apparent that N-glycosylation of a folded protein can have a significant stabilising effect on large regions of the backbone structure.
Proceedings of the National Academy of Sciences, 2005
Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular ... more Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck ( Marmota monax ), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core α-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The co...
Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), ... more Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquitoborne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.
ABSTRACTHepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and li... more ABSTRACTHepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and liver cancer. Using comparative glycoproteomics, we identified a glycoprotein that is altered both in amount and in glycosylation as a function of liver fibrosis and cirrhosis. Specifically, this altered glycoprotein is an immunoglobulin G (IgG) molecule reactive to the heterophilic alpha-Gal epitope [Galα-1-3Galβ1-(3)4GlcNAc-R]. While similar changes in glycosylation have been observed in several autoimmune diseases, the specific immunoglobulins and their antigen recognition profiles were not determined. Thus, we provide the first report identifying the specific antigenic recognition profile of an immunoglobulin molecule containing altered glycosylation as a function of liver disease. This change in glycosylation allowed increased reactivity with several fucose binding lectins and permitted the development of a plate-based assay to measure this change. Increased lectin reactivity was obse...
The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was ... more The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of R1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex 7 HexNAc 4 dHex 1, Negative ion ESI-MS/MS confirmed the presence of the R1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex 6 HexNAc 4 dHex 1 Neu5Ac 1 , with the sialic acid linked to the terminal 1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.
Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures a... more Analysis of the glycosylation of human serum IgD and IgE indicated that oligomannose structures are present on both Igs. The relative proportion of the oligomannose glycans is consistent with the occupation of one N-linked site on each heavy chain. We evaluated the accessibility of the oligomannose glycans on serum IgD and IgE to mannan-binding lectin (MBL). MBL is a member of the collectin family of proteins, which binds to oligomannose sugars. It has already been established that MBL binds to other members of the Ig family, such as agalactosylated glycoforms of IgG and polymeric IgA. Despite the presence of potential ligands, MBL does not bind to immobilized IgD and IgE. Molecular modeling of glycosylated human IgD Fc suggests that the oligomannose glycans located at Asn354 are inaccessible because the complex glycans at Asn445 block access to the site. On IgE, the additional CH2 hinge domain blocks access to the oligomannose glycans at Asn394 on one H chain by adopting an asymmet...
The fundamental importance of correct protein glycosylation is abundantly clear in a group of dis... more The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type–specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve ...
MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-l... more MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionizationmass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di-or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galβ1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid α2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material.
The monoglucosylated oligomannose N-linked oligosaccharide (Glc 1 Man 9 GlcNAc 2) is a retention ... more The monoglucosylated oligomannose N-linked oligosaccharide (Glc 1 Man 9 GlcNAc 2) is a retention signal for the calnexin-calreticulin quality control pathway in the endoplasmic reticulum. We report here the presence of such monoglucosylated N-glycans on the human complement serum glycoprotein C3. This finding represents the first report of monoglucosylated glycans on a human serum glycoprotein from non-diseased individuals. The presence of the glucose moiety in 5% of the human C3 glycoprotein suggests that this glycosylation site is sequestered within the protein and is consistent with previous studies identifying a cryptic conglutinin binding site on C3 that becomes exposed upon its conversion to iC3b.
Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the o... more Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the only biomarker that is used routinely and there is a need for further complementary biomarkers both in terms of sensitivity and specificity.N-glycosylation changes in ovarian cancer serum glycoproteins include a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLex) on haptoglobinβ-chain,α1-acid glycoprotein andα1-antichymotrypsin. These changes are also present in chronic inflammation but not in malignant melanoma, where there are low levels of inflammatory processes. Acute phase proteins carrying increased amounts of SLexhave an increased half-life. Sialylation of acute phase proteins also decreases apoptosis favouring survival of cancer cells. Cancer cells produce inflammatory cytokines which influence glycosylation processing in liver parenchymal cells. Altered glycosylation of the acute phase protein transferrin plays an important role in iron homeostasis. Glycos...
Critical Reviews in Biochemistry and Molecular Biology, 1998
The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational ev... more The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucintype O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosaccharides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.
The conditions required for mammalian‐type complex N‐linked glycosylation of human proteins produ... more The conditions required for mammalian‐type complex N‐linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N‐linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn‐4h) cells cultured in serum‐free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 ± 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn‐4h cells cultured in serum‐containing medium, only 24 ± 3%...
The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (G... more The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP Sc from the brains of Syrian hamsters contains the same set of glycans as PrP C , but a higher proportion of tri-and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.
This article was published in an Elsevier journal. The attached copy is furnished to the author f... more This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author's institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
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