Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL
Klaus W. WagnerElizabeth A. PunnooseThomas JanuarioDavid A. LawrenceRobert PittiKate LancasterDori LeeMelissa von GoetzSharon YeeKlára TótpálLing HuwViswanatham KattaGuy CavetS.G. HymowitzLukas C. AmlerAvi Ashkenazi
590
Citation
38
Reference
10
Related Paper
Citation Trend
Keywords:
Ectodomain
N-linked glycosylation
Cite
N-linked glycosylation
Fucosyltransferase
Cite
Citations (21)
N-glycosylation is an important post-translational modification necessary to maintain the structural and functional properties of proteins. Impaired N-glycosylation has been observed in several diseases. It is significantly modified by the state of cells and is used as a diagnostic or prognostic indicator for multiple human diseases, including cancer and osteoarthritis (OA). Aim of the study was to explore the N-glycosylation levels of subchondral bone proteins in patients with primary knee OA (KOA) and screen for potential biological markers for the diagnosis and treatment of primary KOA. A comparative analysis of total protein N-glycosylation under the cartilage was performed in medial subchondral bone (MSB, N = 5) and lateral subchondral bone (LSB, N = 5) specimens from female patients with primary KOA. To analyse the N-glycosylation sites of the proteins, non-labelled quantitative proteomic and N-glycoproteomic analyses were performed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) data. Parallel reaction monitoring (PRM) validation experiments were carried out on differential N-glycosylation sites of proteins in selected specimens, including MSB (N = 5) and LSB (N = 5), from patients with primary KOA. In total, 1149 proteins with 1369 unique N-chain glycopeptides were detected, and 1215 N-glycosylation sites were found, in which ptmRS scores for 1163 N-glycosylation sites were ≥ 0.9. In addition, N-glycosylation of the total protein in MSB compared to that in LSB was identified, in which 295 N-glycosylation sites were significantly different, including 75 upregulated and 220 downregulated N-glycosylation sites in MSB samples. Importantly, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses of proteins with differential N-glycosylation sites showed that they were primarily associated with metabolic pathways including ECM-receptor interactions, focal adhesion, protein digestion and absorption, amoebiasis, and complement and coagulation cascades. Finally, PRM experiments confirmed the N-glycosylation sites of collagen type VI, alpha 3 (COL6A3, VAVVQHAPSESVDN[+3]ASMPPVK), aggrecan core protein (ACAN, FTFQEAAN[+3]EC[+57]R, TVYVHAN[+3]QTGYPDPSSR), laminin subunit gamma-1 (LAMC1, IPAIN[+3]QTITEANEK), matrix-remodelling-associated protein 5 (MXRA5, ITLHEN[+3]R), cDNA, FLJ92775, highly similar to Homo sapiens melanoma cell adhesion molecule (MCAM), mRNA(B2R642, C[+57]VASVPSIPGLN[+3]R), and aminopeptidase fragment (Q59E93, AEFN[+3]ITLIHPK) in the array data of the top 20 N-glycosylation sites. These abnormal N-glycosylation patterns provide reliable insights for the development of diagnostic and therapeutic methods for primary KOA.
N-linked glycosylation
Cite
Citations (4)
N-linked glycosylation
Membrane glycoproteins
Cite
Citations (26)
N-linked glycosylation
Heterologous
Heterologous expression
Cite
Citations (10)
B7-H6 is a novel immune checkpoint molecule that triggers NK cell cytotoxicity, but the role of N-glycosylation in B7-H6 is poorly understood. We here identified the existence of N-glycosylation of B7-H6 in different cell lines and exogenous expression cells by PNGase F digestion and tunicamycin blockage. Subsequently, we demonstrated that B7-H6 contains 6 functional N-linked glycosylation sites by single site mutation and electrophoresis. Phylogenetical and structural analysis revealed that N43 and N208 glycan are conserved in jawed vertebrates and may thus contribute more to the biological functions. We further demonstrated that N43 and N208 glycosylation are essential for B7-H6 to trigger NK cell activation. Mechanistically, we found that N43 and N208 glycan contributed to the stability and membrane expression of B7-H6 protein. Lack of N208 glycosylation led to membrane B7-H6 shedding, while N43 mutation resulted in impaired B7-H6/NKp30 binding affinity. Together, our findings highlight the significance of N-linked glycosylation in B7-H6 biological functions and suggest potential targets for modulating NK cell-mediated immunity.
Tunicamycin
N-linked glycosylation
Cite
Citations (2)
Organic anion transporting polypeptides (OATPs) have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins. Putative N-glycosylation sites located in the extracellular loops 2 and 5 is considered a common feature of all OATPs and some members have been demonstrated to be glycosylated proteins. However, experimental evidence is still lacking on how such a post-translational modification affect the transport activity of OATPs and which of the putative glycosylation sites are utilized in these transporter proteins. In the present study, we substituted asparagine residues that are possibly involved in N-glycosylation with glutamine residues and identified three glycosylation sites (Asn134, Asn503 and Asn516) within the structure of OATP1B1, an OATP member that is mainly expressed in the human liver. Our results showed that Asn134 and Asn516 are used for glycosylation under normal conditions; however, when Asn134 was mutagenized, an additional asparagine at position 503 is involved in the glycosylation process. Simultaneously replacement of all three asparagines with glutamines led to significantly reduced protein level as well as loss of transport activity. Further studies revealed that glycosylation affected stability of the transporter protein and the unglycosylated mutant was retained within endoplasmic reticulum.
N-linked glycosylation
Cite
Citations (59)
N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.
N-linked glycosylation
Cite
Citations (56)
Melanin‐concentrating hormone (MCH) is known to act through two G‐protein‐coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn 13 , Asn 16 and Asn 23 ) for N ‐linked glycosylation in its extracellular amino‐terminus which may modulate its reactivity. Site‐directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N ‐linked glycosylation sites in MCHR1 were glycosylated, and that N ‐linked glycosylation of Asn 23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N ‐linked glycosylation of the MCHR1.
N-linked glycosylation
Site-directed mutagenesis
Melanin-concentrating hormone
Cite
Citations (36)
The amino-terminal ectodomain of human thyrotropin receptor (TSHR) contains six potential N-linked glycosylation sites (N-Xaa-S/T). This study was designed to evaluate the functional role of TSHR carbohydrates in detail. Because our previous mutagenesis study by Asn to Gln substitutions suggested the critical role of the first and third glycosylation sites (amino acids 77 and 113) for expression of the functional TSHR, we first constructed TSHR mutants having these two glycosylation sites to elucidate whether these two sites are sufficient for TSHR function and expression; this mutant however proved to be nonfunctional. Also the expression levels and function of TSHR mutants with a Ser/Thr to Ala substitution at the first or third glycosylation site were found to be intact. These data indicate that our previous data appear to result from amino acid substitution itself, not from disruption of glycosylation. The next series of the mutants was therefore constructed to identify at least how many glycosylation sites are necessary. Neither TSH binding nor cAMP response was detected in TSHR mutants with three glycosylation sites. However, the mutants with four glycosylation sites were fully functional in terms of TSH binding and cAMP production, although the expression levels were 30 to 40% of that in wild-type TSHR. Finally, Western blot revealed that all six glycosylation sites are actually glycosylated. These data indicate that 1) TSHR ectodomain contains six N-linked carbohydrates, and 2) glycosylation of at least four sites appears necessary for expression of the functional TSHR.
Ectodomain
Thyrotropin receptor
N-linked glycosylation
Cite
Citations (39)