ABSTRACT
The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin-22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5 GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5 GlcNAc2 and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5 GlcNAc2 modified with a Glcα-1,2-Manß-1,2-Manß-1,3-Glcα-1,3-R tetrasaccharide. Expression of a Golgi-targeted GlcNAc transferase-I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5 GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in P. pastoris to produce particular human-type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.
Subject(s)
Glycoproteins , Polysaccharides , Protein Engineering/methods , Saccharomycetales , Synthetic Biology/methods , Animals , Glycoproteins/chemistry , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Humans , Mice , Polysaccharides/chemistry , Polysaccharides/genetics , Polysaccharides/metabolism , Saccharomycetales/genetics , Saccharomycetales/metabolismABSTRACT
TMEM165 is involved in a rare genetic human disease named TMEM165-CDG (congenital disorders of glycosylation). It is Golgi localized, highly conserved through evolution and belongs to the uncharacterized protein family 0016 (UPF0016). The use of isogenic TMEM165 KO HEK cells was crucial in deciphering the function of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor of many glycosylation enzymes. Severe Golgi glycosylation defects are observed in TMEM165 Knock Out Human Embryonic Kidney (KO HEK) cells and are rescued by exogenous manganese supplementation. Intriguingly, we demonstrate in this study that the observed Golgi glycosylation defect mainly depends on fetal bovine serum, particularly its manganese level. Our results also demonstrate that iron and/or galactose can modulate the observed glycosylation defects in TMEM165 KO HEK cells. While isogenic cultured cells are widely used to study the impact of gene defects on proteins' glycosylation patterns, these results emphasize the importance of the use of validated fetal bovine serum in glycomics studies.
Subject(s)
Antiporters/physiology , Cation Transport Proteins/physiology , Glycosylation/drug effects , Manganese/metabolism , Serum Albumin, Bovine/pharmacology , Antiporters/genetics , Calcium/metabolism , Cation Transport Proteins/genetics , Congenital Disorders of Glycosylation/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Ion TransportABSTRACT
Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis.
Subject(s)
Fungal Proteins/genetics , Golgi Apparatus/physiology , Manganese/pharmacology , Membrane Proteins/deficiency , Mutation , Antiporters , Cation Transport Proteins , Congenital Disorders of Glycosylation/genetics , Fungal Proteins/metabolism , Glycosylation/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Manganese/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolismABSTRACT
TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn2+-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+-induced degradation of TMEM165.
Subject(s)
Golgi Apparatus/drug effects , Lysosomes/drug effects , Manganese/pharmacology , Membrane Proteins/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Antiporters , Blotting, Western , Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Cation Transport Proteins , Dose-Response Relationship, Drug , Gene Knockdown Techniques , Glutamates/genetics , Glutamates/metabolism , Glycosylation/drug effects , Golgi Apparatus/metabolism , HEK293 Cells , HeLa Cells , Humans , Lysosomes/metabolism , Membrane Proteins/genetics , Microscopy, Confocal , Mutation , Proteolysis/drug effectsABSTRACT
BACKGROUND: Defects in TMEM165 gene cause a type-II Congenital Disorder of Glycosylation affecting Golgi glycosylation processes. TMEM165 patients exhibit psychomotor retardation, important osteoporosis, scoliosis, irregular epiphyses and thin bone cortex. TMEM165 protein is highly conserved in evolution and belongs to the family of UPF0016 membrane proteins which could be an unique group of Ca2+/H+ antiporters regulating Ca2+ and pH homeostasis and mainly localized in the Golgi apparatus. METHODS: RT-PCR from human brain tissues revealed TMEM165 splice-transcript variants. mRNA expression was analyzed by RT-Q-PCR. Expression plasmids allowed us to visualize isoform proteins and their subcellular localization. Their functions on glycosylation were achieved by looking at the gel mobility of highly glycosylated proteins in cells overexpressing isoforms. RESULTS: In this study, we highlight, as previously shown for other ion channels, the existence of TMEM165 splice-transcripts isoforms, in particular the Short-Form (SF) and the Long-Form (LF) transcripts, leading to a 129 aa and 259 aa protein isoform, respectively. These proteins both localize in the endoplasmic reticulum and have different effects on glycosylation compared to the wild-type protein (324 aa). We also point out that the SF is expressed at low levels in all human cells and tissues checked, excepted in brain, and forms homodimer. The LF was only expressed in the temporal lobe of human brain. GENERAL SIGNIFICANCE: The finding of numerous splice variants could lead to a family of TMEM165 isoforms. This family of TMEM165 splice transcripts could participate in the fine regulation of TMEM165 isoforms' functions and localizations.
Subject(s)
Alternative Splicing/genetics , Congenital Disorders of Glycosylation/genetics , Genetic Variation/genetics , Membrane Proteins/genetics , Amino Acid Sequence , Antiporters , Brain/metabolism , Calcium/metabolism , Cation Transport Proteins , Cell Line, Tumor , Endoplasmic Reticulum/genetics , Glycosylation , Golgi Apparatus/genetics , HeLa Cells , Humans , Protein Isoforms/genetics , RNA, Messenger/geneticsABSTRACT
IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.
Subject(s)
Glomerular Mesangium/metabolism , Immunoglobulin A/metabolism , Animals , Antibody Formation , Female , Immunoglobulin A/immunology , Male , Mice , Protein ConformationABSTRACT
Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Membrane Proteins/genetics , Mutation , Adolescent , Antiporters , Cation Transport Proteins , Cells, Cultured , Child , Child, Preschool , Dwarfism/genetics , Female , Fibroblasts , Golgi Apparatus/metabolism , Humans , Infant , Infant, Newborn , Male , Pedigree , Skin/cytologyABSTRACT
A paradigm regarding rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chain A of wild-type plants, up to 45% of the l-fucose is substituted by l-galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose-deficient mutant mur1, which provided insights into RGII chain A biosynthesis, and suggested that chain A truncation, rather than l-fucose to l-galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The ß-galacturonic acid of chain A exhibits up to two methyl etherifications in an organ-specific manner. Combined with variation in the length of side chain B, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition.
Subject(s)
Arabidopsis/chemistry , Galactose/chemistry , Pectins/chemistry , Plant Leaves/chemistry , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis/physiology , Cell Wall/metabolism , Chromatography, Liquid , Fucose/analysis , Fucose/metabolism , Galactose/analysis , Hexuronic Acids , Monosaccharides/chemistry , Mutation , Organ Specificity , Pectins/genetics , Pectins/metabolism , Plant Leaves/cytology , Plant Leaves/genetics , Plant Leaves/physiology , Spectrometry, Mass, Electrospray IonizationABSTRACT
Mechanisms underlying the disruption of self-tolerance in acquired autoimmunity remain unclear. Immunoglobulin A (IgA) nephropathy is an acquired autoimmune disease where deglycosylated IgA1 (IgA subclass 1) auto-antigens are recognized by IgG auto-antibodies, forming immune complexes that are deposited in the kidneys, leading to glomerulonephritis. In the intestinal microbiota of patients with IgA nephropathy, there was increased relative abundance of mucin-degrading bacteria, including Akkermansia muciniphila. IgA1 was deglycosylated by A. muciniphila both in vitro and in the intestinal lumen of mice. This generated neo-epitopes that were recognized by autoreactive IgG from the sera of patients with IgA nephropathy. Mice expressing human IgA1 and the human Fc α receptor I (α1KI-CD89tg) that underwent intestinal colonization by A. muciniphila developed an aggravated IgA nephropathy phenotype. After deglycosylation of IgA1 by A. muciniphila in the mouse gut lumen, IgA1 crossed the intestinal epithelium into the circulation by retrotranscytosis and became deposited in the glomeruli of mouse kidneys. Human α-defensins-a risk locus for IgA nephropathy-inhibited growth of A. muciniphila in vitro. A negative correlation observed between stool concentration of α-defensin 6 and quantity of A. muciniphila in the guts of control participants was lost in patients with IgA nephropathy. This study demonstrates that gut microbiota dysbiosis contributes to generation of auto-antigens in patients with IgA nephropathy and in a mouse model of this disease.
Subject(s)
Gastrointestinal Microbiome , Glomerulonephritis, IGA , Humans , Mice , Animals , Immunoglobulin A , Glomerulonephritis, IGA/genetics , Kidney , Immunoglobulin GABSTRACT
The C-type lectin receptor dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is an important player in the recognition of pathogens by dendritic cells. A plethora of pathogens including viruses, bacteria, parasites, and fungi are recognized by DC-SIGN through both mannose and fucose-containing glycans expressed on the pathogen surface. In this study, we identified semen clusterin as a novel DC-SIGN ligand. Semen clusterin, but not serum clusterin, expresses an extreme abundance of fucose-containing blood-type Ags such as Le(x) and Le(y), which are both excellent DC-SIGN ligands. These motifs enable semen clusterin to bind DC-SIGN with very high affinity (K(d) 76 nM) and abrogate the binding of HIV-1 to DC-SIGN. Depletion of clusterin from semen samples, however, did not completely prevent the ability of semen to inhibit the capture of HIV-1 by DC-SIGN, supporting that besides clusterin, semen contains other DC-SIGN ligands. Further studies are needed to characterize these ligands and define their contribution to the DC-SIGN-blocking activity mediated by semen. Clusterin is an enigmatic protein involved in a variety of physiologic and pathologic processes including inflammation, atherosclerosis, and cancer. Our results uncover an unexpected heterogeneity in the glycosylation pattern of clusterin and suggest that the expression of high concentrations of fucose-containing glycans enables semen clusterin to display a unique set of biological functions that might affect the early course of sexually transmitted infectious diseases.
Subject(s)
Cell Adhesion Molecules/metabolism , Clusterin/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Lectins, C-Type/metabolism , Receptors, Cell Surface/metabolism , Semen/immunology , Semen/metabolism , Adult , Antiviral Agents/blood , Antiviral Agents/metabolism , Cell Adhesion Molecules/blood , Clusterin/blood , Dendritic Cells/virology , Fucose/metabolism , Glycosylation , HIV-1/immunology , HIV-1/metabolism , Humans , Lectins, C-Type/blood , Ligands , Male , Mannose/metabolism , Middle Aged , Protein Binding/immunology , Receptors, Cell Surface/blood , Recombinant Proteins/blood , Recombinant Proteins/metabolism , Semen/virologyABSTRACT
Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.
Subject(s)
Cell Movement , Endoplasmic Reticulum/metabolism , Glycoproteins/chemistry , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Molecular Motor Proteins/metabolism , Protozoan Proteins/metabolism , Toxoplasma/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Movement/physiology , Fibroblasts/cytology , Fibroblasts/parasitology , Glycomics , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Host-Parasite Interactions/genetics , Humans , Mass Spectrometry , Membrane Proteins/genetics , Molecular Motor Proteins/genetics , Mutagenesis, Site-Directed , Plasmids , Protein Binding , Protein Transport/physiology , Proteomics , Protozoan Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Toxoplasma/genetics , TransfectionABSTRACT
OBJECTIVE: To investigate whether the glycosylation and sialylation levels of anti-proteinase 3 (anti-PR3) antibodies could affect their pathogenicity, and whether these levels could be correlated with the activity of granulomatosis with polyangiitis (Wegener's) (GPA). METHODS: Forty-two serum samples positive for anti-PR3 antibodies from 42 patients with active or weakly active/inactive GPA were included. Anti-PR3 antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and sialylation were assessed by enzyme-linked lectin assay. The glycosylation and sialylation levels of IgG purified from the serum of healthy donors and patients with active, remitted, or weakly active disease were assessed by permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil oxidative burst induced by purified IgG was assayed by spectrofluorimetry. RESULTS: The mean sialylation ratio of anti-PR3 antibodies was significantly lower in patients with active disease than in patients with weakly active or inactive disease, and this was inversely correlated with the Birmingham Vasculitis Activity Score (BVAS) (P < 0.0001). Similar results were obtained using the BVAS/GPA. The area under the receiver operating characteristic curve for the sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82 (P = 0.0006). The characterization of N-glycans showed a decrease in 2,6-linked sialylated N-glycans and an increase in dHex1 Hex3 HexNAc4 (mass/charge 1,836) agalactosylated structures in purified IgG from patients with active disease compared with controls. The anti-PR3 antibody-induced oxidative burst of neutrophils was inversely correlated with the sialylation levels of anti-PR3 IgG. CONCLUSION: The sialylation level of anti-PR3 antibodies contributes to the clinical activity of GPA, by modulating the oxidative burst of neutrophils induced by these autoantibodies.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/immunology , Granulomatosis with Polyangiitis/immunology , Myeloblastin/immunology , Adolescent , Adult , Aged , Antibodies, Antineutrophil Cytoplasmic/blood , Enzyme-Linked Immunosorbent Assay , Female , Glycosylation , Granulomatosis with Polyangiitis/blood , Humans , Male , Mass Spectrometry , Middle AgedABSTRACT
Glycosylation is a ubiquitous and universal cellular process in all domains of life. In eukaryotes, many glycosylation pathways occur simultaneously onto proteins and lipids for generating a complex diversity of glycan structures. In humans, severe genetic diseases called Congenital Disorders of Glycosylation (CDG), resulting from glycosylation defects, demonstrate the functional relevance of these processes. No real cure exists so far, but oral administration of specific monosaccharides to bypass the metabolic defects has been used in few CDG, then constituting the simplest and safest treatments. Oral D-Galactose (Gal) therapy was seen as a promising tailored treatment for specific CDG and peculiarly for TMEM165-CDG patients. TMEM165 deficiency not only affects the N-glycosylation process but all the other Golgi-related glycosylation types, then contributing to the singularity of this defect. Our previous results established a link between TMEM165 deficiency and altered Golgi manganese (Mn2+) homeostasis. Besides the fascinating power of MnCl2 supplementation to rescue N-glycosylation in TMEM165-deficient cells, D-Gal supplementation has also been shown to be promising in suppressing the observed N-glycosylation defects. Its effect on the other Golgi glycosylation types, most especially O-glycosylation and glycosaminoglycan (GAG) synthesis, was however unknown. In the present study, we demonstrate the differential impact of D-Gal or MnCl2 supplementation effects on the Golgi glycosylation defects caused by TMEM165 deficiency. Whereas MnCl2 supplementation unambiguously fully rescues the N- and O-linked as well as GAG glycosylations in TMEM165-deficient cells, D-Gal supplementation only rescues the N-linked glycosylation, without any effects on the other Golgi-related glycosylation types. According to these results, we would recommend the use of MnCl2 for TMEM165-CDG therapy.
ABSTRACT
We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-beta-d-galactopyranoside (GalNAcalpha-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N-glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic-recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N-glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.
Subject(s)
Cell Membrane/metabolism , Enterocytes/cytology , Galectin 4/metabolism , Glycoproteins/metabolism , Biomarkers/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Cell Polarity , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Enterocytes/metabolism , Glycoproteins/chemistry , Golgi Apparatus/metabolism , HT29 Cells , Humans , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.
Subject(s)
Evolution, Molecular , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Adaptor Proteins, Vesicular Transport/analysis , Adaptor Proteins, Vesicular Transport/metabolism , Animals , CHO Cells , Cells, Cultured , Cricetinae , Glycosylation , Glycosyltransferases/metabolism , Golgi Apparatus/enzymology , HeLa Cells , HumansABSTRACT
Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.
Subject(s)
Endoplasmic Reticulum/metabolism , Mannosidases/biosynthesis , Proteasome Endopeptidase Complex/metabolism , Up-Regulation , Glycosylation , HeLa Cells , Humans , Mannosephosphates/metabolism , Mannosidases/chemistry , Oligosaccharides/metabolism , Transfection , Uridine Diphosphate Glucose/metabolism , alpha-MannosidaseABSTRACT
Abnormalities of IgA arise in alcoholic cirrhosis, including mesangial IgA deposits with possible development of secondary IgA nephropathy (IgAN). Since little is known about circulating immune complexes in cases of secondary IgAN, we analyzed IgA-associated parameters in the serum of 32 patients with compensated or advanced alcoholic cirrhosis. Galactose deficiency and decreased sialylation of IgA1, as well as increased amounts of abnormally glycosylated polymeric IgA1, were detected in the serum of patients with advanced alcoholic cirrhosis. Moreover, aberrant IgA1 formed complexes with IgG and soluble CD89 in serum of patients with advanced alcoholic cirrhosis, similar to those found in primary IgAN. The IgA1 of alcoholic cirrhosis, however, had a modified N-glycosylation, not found in primary IgAN. In patients with alcoholic cirrhosis and IgAN, IgA deposits were associated with CD71 overexpression in mesangial areas, suggesting that CD71 might be involved in deposit formation. Although the IgA1 found in alcoholic cirrhosis bound more extensively to human mesangial cells than control IgA1, they differ from primary IgAN by not inducing mesangial cell proliferation. Thus, abnormally glycosylated IgA1 and soluble CD89-IgA and IgA-IgG complexes, features of primary IgAN, are also present in alcoholic cirrhosis. Hence, common mechanisms appear to be shared by diseases of distinct origins, indicating that common environmental factors may influence the development of IgAN.
Subject(s)
Antigen-Antibody Complex , Antigens, CD/blood , Glomerulonephritis, IGA/immunology , Immunoglobulin A/blood , Immunoglobulin G/blood , Kidney/immunology , Liver Cirrhosis, Alcoholic/immunology , Receptors, Fc/blood , Adult , Aged , Biopsy , Cell Proliferation , Cells, Cultured , Female , Galactose/blood , Galactose/deficiency , Glomerulonephritis, IGA/blood , Glomerulonephritis, IGA/pathology , Glycosylation , Humans , Kidney/pathology , Liver Cirrhosis, Alcoholic/blood , Liver Cirrhosis, Alcoholic/complications , Liver Cirrhosis, Alcoholic/pathology , Male , Mesangial Cells/immunology , Mesangial Cells/pathology , Middle Aged , ParisABSTRACT
The conserved oligomeric Golgi (COG) complex is a hetero-octameric complex essential for normal glycosylation and intra-Golgi transport. An increasing number of congenital disorder of glycosylation type II (CDG-II) mutations are found in COG subunits indicating its importance in glycosylation. We report a new CDG-II patient harbouring a p.R729W missense mutation in COG4 combined with a submicroscopical deletion. The resulting downregulation of COG4 expression additionally affects expression or stability of other lobe A subunits. Despite this, full complex formation was maintained albeit to a lower extent as shown by glycerol gradient centrifugation. Moreover, our data indicate that subunits are present in a cytosolic pool and full complex formation assists tethering preceding membrane fusion. By extending this study to four other known COG-deficient patients, we now present the first comparative analysis on defects in transport, glycosylation and Golgi ultrastructure in these patients. The observed structural and biochemical abnormalities correlate with the severity of the mutation, with the COG4 mutant being the mildest. All together our results indicate that intact COG complexes are required to maintain Golgi dynamics and its associated functions. According to the current CDG nomenclature, this newly identified deficiency is designated CDG-IIj.
Subject(s)
Carbohydrate Metabolism, Inborn Errors/metabolism , Carrier Proteins/metabolism , Golgi Apparatus/metabolism , Adult , Amino Acid Sequence , Carbohydrate Metabolism, Inborn Errors/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cells, Cultured , Child, Preschool , Female , Fibroblasts/metabolism , Glycosylation , Golgi Apparatus/chemistry , Golgi Apparatus/genetics , HeLa Cells , Humans , Infant , Male , Molecular Sequence Data , Protein Transport , Sequence Homology, Amino Acid , Vesicular Transport ProteinsABSTRACT
Mental retardation (MR) is the most frequent handicap among children and young adults. Although a large proportion of X-linked MR genes have been identified, only four genes responsible for autosomal-recessive nonsyndromic MR (AR-NSMR) have been described so far. Here, we report on two genes involved in autosomal-recessive and X-linked NSMR. First, autozygosity mapping in two sibs born to first-cousin French parents led to the identification of a region on 8p22-p23.1. This interval encompasses the gene N33/TUSC3 encoding one subunit of the oligosaccharyltransferase (OTase) complex, which catalyzes the transfer of an oligosaccharide chain on nascent proteins, the key step of N-glycosylation. Sequencing N33/TUSC3 identified a 1 bp insertion, c.787_788insC, resulting in a premature stop codon, p.N263fsX300, and leading to mRNA decay. Surprisingly, glycosylation analyses of patient fibroblasts showed normal N-glycan synthesis and transfer, suggesting that normal N-glycosylation observed in patient fibroblasts may be due to functional compensation. Subsequently, screening of the X-linked N33/TUSC3 paralog, the IAP gene, identified a missense mutation (c.932T-->G, p.V311G) in a family with X-linked NSMR. Recent studies of fucosylation and polysialic-acid modification of neuronal cell-adhesion glycoproteins have shown the critical role of glycosylation in synaptic plasticity. However, our data provide the first demonstration that a defect in N-glycosylation can result in NSMR. Together, our results demonstrate that fine regulation of OTase activity is essential for normal cognitive-function development, providing therefore further insights to understand the pathophysiological bases of MR.
Subject(s)
Genetic Predisposition to Disease , Hexosyltransferases/genetics , Intellectual Disability/genetics , Membrane Proteins/genetics , Tumor Suppressor Proteins/genetics , Adult , Amino Acid Sequence , Female , Genes, Recessive , Glycosylation , Humans , Male , Mental Retardation, X-Linked/genetics , Molecular Sequence Data , Mutation , Pedigree , Protein Subunits/genetics , SiblingsABSTRACT
BACKGROUND: Henoch-Schönlein purpura (HSP) and IgA nephropathy (IgAN) are characterized by mesangial deposition of polyclonal IgA eventually showing aberrant glycosylation, affinity for mesangial cells and/or co-precipitation with antigen, bacterial peptides, autoantibodies or soluble receptors. IgA were also suggested to be negatively charged and predominantly of λ type but rarely in a monoclonal form. METHODS: A gammopathy case with HSP provided us with a unique molecularly defined nephritogenic IgA1λ. Immunological analysis, biological activities, glycosylation analysis and finally IgA sequence were determined. RESULTS: Compared to IgA1 from healthy subjects or IgAN patients, IgA1 CAT showed hyposialylation but no hypogalactosylation, in agreement with underexpression of sialyltransferase genes by the plasma cell clone. IgA variable domains had low pIs with negatively charged complementarity-determining regions. Weak reactivity appeared against the cationic autoantigen lactoferrin, which was, however, absent from kidney deposits. Deposition also occurred in mice upon injection of only the polymeric form of IgA1 CAT, despite whether or not co-injected with lactoferrin. CONCLUSIONS: This monoclonal model of IgA nephritogenicity strongly suggests that beside hinge region glycosylation, V domains play a role in IgA stability and pathogenicity and supports the hypothesis that responses against cationic epitopes from pathogens or autoantigens may select negatively charged complementarity-determining regions prone either to bind charged structures of the mesangium or to promote by themselves IgA aggregation and deposition.