The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4

BackgroundC-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation.MethodsWe established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope.ResultsWe found that MFAP4 is C-mannosylated at Trp²³⁵ in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion.ConclusionsOur results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion.General significanceThese results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.     

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)

BackgroundC-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear.MethodsWe established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan.ResultsWe identified that ADAMTS4 is C-mannosylated at Trp⁴⁰⁴ in the metalloprotease domain and at Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ in the thrombospondin type 1 repeat (TSR). The replacement of Trp⁴⁰⁴ with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity.ConclusionsOur results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity.General significanceBecause C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C²-Man-)Trp.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan

Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the ³⁶WDAWGPWSECSRTC⁴⁹ sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp³⁹ and Trp⁴². C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-³H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.The ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type-1 repeats) superfamily (1) consists of 19 secreted metalloproteases (ADAMTS proteases) and six ADAMTS-like proteins in humans. ADAMTS-like proteins closely resemble the ancillary domains of ADAMTS proteases and like them have a conserved modular organization containing one or more thrombospondin type-1 repeats (TSRs)² (2–5). TSRs are modules of ∼50 amino acids having a characteristic six-cysteine signature. The prototypic ADAMTSL, ADAMTSL1, also referred to as punctin-1 because of its punctate distribution in the substratum of transfected cells, is a 525-residue glycoprotein containing four TSRs (4). A longer punctin-1 variant arising from alternative splicing, containing 13 TSRs and homologous to ADAMTSL3, is predicted by the human genome sequencing project ({"type":"entrez-nucleotide","attrs":{"text":"NM_001040272","term_id":"334688819","term_text":"NM_001040272"}}NM_001040272) but has not yet been physically cloned and expressed. The function of ADAMTSL1/punctin-1 is unknown. Recently, ADAMTSL2 and ADAMTSL4 mutations were identified in the genetic disorders geleophysic dysplasia (6) and recessive isolated ectopia lentis, respectively (2). In genome-wide analysis, the ADAMTSL3 locus has been associated with variation in human height (7). Thus, in addition to known genetic disorders caused by ADAMTS mutations (8, 9), ADAMTSL family members are now also implicated in human disease. Among the ADAMTS proteases, ADAMTS5 and ADAMTS4 are strongly associated with cartilage destruction in arthritis (10–12).Like most secreted proteins, the ADAMTS superfamily members undergo post-translational modification and are predicted to contain N-linked oligosaccharides. In addition, TSRs of ADAMTS superfamily members, by virtue of high sequence similarity to the corresponding motifs in thrombospondin 1 and properdin, are predicted to contain two uncommon types of glycosylation. Specifically, TSRs often contain the sequence motifs W⁰XXW⁺³ and C¹X_2–3(S/T)C²XXG, which are consensus sites for protein C-mannosylation of the W⁰ residue and O-fucosylation (of Ser/Thr) respectively, in close proximity to each other (13, 14). In recently published work, it was shown that ADAMTSL1 and ADAMTS13 are modified by O-fucosylation, the covalent attachment to Ser or Thr residues of fucose or a fucose-glucose disaccharide (15, 16). Punctin-1 contains consensus sequences for O-fucosylation in all four of its TSRs, but the presence of the glycans was previously only confirmed on TSR2, -3, and -4 (16). Addition of O-fucose is mediated by protein O-fucosyltransferase 2 (POFUT2), which is a distinct transferase from that which catalyzes addition of O-linked fucose to epidermal growth factor-like repeats (POFUT1) (17, 18). A β3-glucosyltransferase subsequently adds glucose to the 3′-OH of the fucose (19, 20). It was further demonstrated that O-fucosylation, which occurs after completion of TSR folding, was rate-limiting for secretion of punctin-1 and ADAMTS13 (15, 16). This role was inferred from the following two experimental observations. 1) Expression of wild-type punctin-1 and ADAMTS13 in Lec13 cells, which do not fucosylate proteins, led to their decreased secretion (15, 16). 2) Mutation of the modified Ser or Thr residues greatly reduced secretion of punctin-1 and ADAMTS13 (15, 16).Protein C-mannosylation is the attachment of an α-mannopyranosyl residue to the indole C-2 of tryptophan via a C-C linkage (14, 21). Unlike O-fucosylation, it can utilize protein primary structure rather than tertiary structure as the determinant, i.e. mannose is added to unfolded polypeptides or unstructured synthetic peptides (22). C-Mannosylation uses dolichyl-phosphate mannose (Dol-P-Man) as the precursor and appears to be enzyme-catalyzed within the endoplasmic reticulum (23), but the responsible mannosyltransferase has not yet been identified. A variety of mammalian cell lines can perform this modification (24). Proteins known to be C-mannosylated include human RNase 2, interleukin 12, the mucins MUC5AC and MUC5B, and several proteins containing TSRs, such as thrombospondin-1, F-spondin, and components of complement (C6 and C7) and properdin (13, 21, 25–27).Krieg et al. (22) proposed a model in which the C-mannosyltransferase bound directly to the WXXW⁺³ motif, analogous to the Asn-X-(Thr/Ser) motif for N-glycosylation, and later analysis showed that both the Trp residues in the W⁰XXW⁺³XXX motif and the sole Trp residue in a (F/Y¹)XXW⁺³ motif could be modified (13). Based on meta-analysis of the C-mannosylation literature, Julenius (28) used a neural network approach to develop a prediction algorithm for protein C-mannosylation, termed NetCGlyc. This analysis suggested that Cys was an acceptable substitute for Trp at the +3 position (i.e. permitting C-mannosylation of W⁰ in a W⁰SSC motif). Julenius (28) reported a clear preference for small and/or polar residues (Ser, Ala, Gly, and Thr) at the +1 position, whereas Phe and Leu were not allowed. The NetCGlyc algorithm provides a useful guide for prediction of C-mannosylation sites, especially in the ADAMTS superfamily, which has a large number of TSRs (27). Here we specifically inquired whether the short form of punctin-1, the prototypic ADAMTSL, is modified by C-mannosylation, analyzed the role of Trp residues in the punctin TSRs, and investigated its possible functional significance in punctin-1 biosynthesis. By demonstrating that TSR1 of ADAMTS5 is also C-mannosylated, we extended the analysis to identify this unusual modification in an ADAMTS protease.

TABLE 1

Predicted C-mannosylation sites^a in the ADAMTS superfamilyOpen in a separate window^aThe full-length human reference ADAMTS sequences from GenBank™ were analyzed at the NetCGly 1.0 server for prediction of C-mannosylation sites. For prediction of O-fucosylation sites in the same peptide, the consensus sequence C¹X_2–3(S/T)C² XXG was utilized.^bThe sequence context in which the predicted modified Trp residue occurs is provided, and the residue with predicted modification is numbered. Ser/Thr residues predicted to be O-fucosylated based on the consensus sequence CXX(S/T)C are underlined.^cSequences containing predicted C-mannosylation sites that are not within TSRs are shown in italics.     

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.     

Altered N-glycan profile of IgG-depleted serum proteins in Hashimoto's thyroiditis

BackgroundHashimoto's thyroiditis (HT) is an autoimmune disease characterized by chronic inflammation of thyroid gland. Although HT is the most common cause of hypothyroidism, the pathogenesis of this disease is not fully understood. Glycosylation of serum proteins was examined in HT only to a limited extent. The study was designed to determine the glycosylation pattern of IgG-depleted sera from HT patients.MethodsSerum N-glycans released by N-glycosidase F (PNGase F) digestion were analyzed by normal-phase high-performance liquid chromatography (NP-HPLC). N-glycan structures in each collected HPLC fraction were determined by liquid chromatography-mass spectrometry (LC-MS) and exoglycosidase digestion. Fucosylation and sialylation was also analyzed by lectin blotting.ResultsThe results showed an increase of monosialylated tri-antennary structure (A3G3S1) and disialylated diantennary N-glycan with antennary fucose (FA2G2S2). Subsequently, we analyzed the serum N-glycan profile by lectin blotting using lectins specific for fucose and sialic acid. We found a significant decrease of Lens culinaris agglutinin (LCA) staining in HT samples, which resulted from the reduction of α1,6-linked core fucose in HT serum. We also observed an increase of Maackia amurensis II lectin (MAL-II) reaction in HT due to the elevated level of α2,3-sialylation in HT sera.ConclusionsThe detected alterations of serum protein sialylation might be caused by chronic inflammation in HT. The obtained results complete our previous IgG N-glycosylation analysis in autoimmune thyroid patients and show that the altered N-glycosylation of serum proteins is characteristic for autoimmunity process in HT.General SignificanceThyroid autoimmunity is accompanied by changes of serum protein sialylation.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

A role for N-glycosylation in active adenosine deaminase 2 production

BackgroundAdenosine deaminase 2 (ADA2) regulates extracellular levels of adenosine and the optimal expression of ADA2 is essential for modulating the immune system. However, the mechanisms regulating the production of active ADA2 enzyme are not fully understood. In this study, we examined the role of N-glycosylation in the formation of functional structures and the secretory pathway of ADA2.MethodsWe investigated the roles of N-glycosylation in the activity, homodimerization, and secretion of ADA2 via site-directed mutagenesis and the application of N-glycosylation inhibitors. Subcellular localization of ADA2 along with the endoplasmic reticulum (ER) glucosidase inhibitor was observed under confocal fluorescence microscope.ResultsInhibiting the initial N-glycosylation of ADA2 in the ER via site-directed mutagenesis or treatment with N-glycosylation inhibitors reduced the intracellular ADA2 activity and secretion. At this time, decreases in the ADA2 homodimers and ADA2 aggregation were observed in the cells. Treating the cells with castanospermine, an inhibitor of N-glycan editing in the ER, resulted in a reduction of the localization rate to the Golgi and markedly suppressed the ADA2 secretion.ConclusionsThese data suggest that the initial N-glycosylation and N-glycan editing in the ER are essential for the production of an active ADA2 enzyme and proper trafficking to the extracellular space.General significanceWith sufficient N-glycosylation in the ER, ADA2 exerts its function and is secreted extracellularly.     

Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs

Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.     

N-Glycosylation of Asparagine 8 Regulates Surface Expression of Major Histocompatibility Complex Class I Chain-related Protein A (MICA) Alleles Dependent on Threonine 24

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn⁸) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr²⁴) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.     

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.     

Distinct Roles of N-Glycosylation at Different Sites of Corin in Cell Membrane Targeting and Ectodomain Shedding

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.     

Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1)

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn²¹¹ residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved ²¹¹NDS N-glycosylation motif, but not other N-glycosylation sites (Asn²⁶⁰, Asn³⁷¹, and Asn³⁹⁴), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr⁴⁸⁴, Tyr⁵²⁰, Tyr⁷⁹², and Tyr⁷⁹⁷. The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved ²¹¹NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.     

Modification of N-glycosylation modulates the secretion and lipolytic function of apoptosis inhibitor of macrophage (AIM)

The mouse macrophage-derived apoptosis inhibitor of macrophage (AIM), which is incorporated into adipocytes and induces lipolysis by suppressing fatty acid synthase (FAS) activity, possesses three potential N-glycosylation sites. Inactivation of N-glycosylation sites revealed that mouse AIM contains two N-glycans in the first and second scavenger receptor cysteine-rich domains, and that depletion of N-glycans decreased AIM secretion from producing cells. Interestingly, the lack of N-glycans increased AIM lipolytic activity through enhancing AIM incorporation into adipocytes. Although human AIM contains no N-glycan, attachment of N-glycans increased AIM secretion. Thus, the N-glycosylation plays important roles in the secretion and lipolytic function of AIM.

Structured summary of protein interactions

AIMphysically interacts with FAS by anti tag coimmunoprecipitation (View interaction)     

Enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide

N-Glycosylation is a common form of protein post-translational modification in Pichia pastoris and greatly affects folding and secretion. The propeptide of the Pseudomonas aeruginosa elastase (PAE) is indispensable for proper folding and secretion of the enzyme. We have studied the effect of introducing N-glycosylation sites to the propeptide of the recombinant elastase (rPAE) on its expression levels in P. pastoris. Addition of N-glycosylation sites to the propeptide at N51 or N93 enhanced rPAE production levels by 104 or 57 %, respectively, while addition at N11 or N127 led to a 25 or 50 % decrease, respectively. The introduced N-glycosylation sites in the propeptide at these four sites exerted a null effect on the N-glycosylation degree of mature rPAE.     

N-glycosylation-mutated HCV envelope glycoprotein complex enhances antigen-presenting activity and cellular and neutralizing antibody responses

BackgroundThe development of an efficient vaccine and broadly cross-neutralizing antibodies of hepatitis C virus (HCV) remains a priority. The heavily glycosylated viral envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Bacteria-derived unmethylated CpG DNA, a potent stimulator of immune cells, is important for vaccine research.MethodsHere, the immunogenicities of wild type (WT) E1E2, five N-glycosylation site mutated E1E2 glycoproteins, and five CpG-coupled E1E2 N-glycosylation mutated glycoproteins were analyzed in BALB/c mice by DNA vaccination using in vivo electroporation.ResultsThe E1E2 protein expression levels were examined and shown to be unaffected by these N-glycosylation mutations. We found that a CpG-coupled E1-N209D-E2-N430D DNA vaccine (named CpG-E1E2-M4) induced the highest cellular immune response compared to the WT E1E2, CpG-E1E2, and other mutants. Furthermore, the CpG-E1E2-M4 anti-serum effectively neutralized the infection of cell-cultured HCV (HCVcc, genotype 2a)- and HCV pseudo particles (HCVpp, genotypes 1 to 7) to Huh-7.5.1 hepatocytes. Additionally, CpG-E1E2-M4 enhanced the Interleukin-12 (IL-12) production and antigen-presenting activity of CD11c⁺ dendritic cells (DCs) by inducing CD4⁺ Th1 polarization and the production of perforin and granzyme B (GrB) in CD8⁺ T cells.ConclusionsAs our knowledge this is the first study revealing that the naturally poor immunogenicity of E1E2 can be enhanced by the deletion of N-glycans combined with the addition of immune activator CpG by DNA vaccination.General significanceDeletion of N-glycans can enhance viral immunogenicity. The selected CpG-E1E2-M4 mutant is a novel potential HCV DNA vaccine that elicits enhanced CD4⁺ Th1 and CD8⁺ T cell responses and neutralizing antibody production against HCV infection. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.