N-glycosylation Inhibitor Research Articles

Abstract 444: ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted

Abstract GRP78 (a Mr 78kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr (i.e., N-linked glycosylation). This important biochemical event is essential for glycoprotein folding and function. When targeted in ER-/PR-/HER2+ breast tumor in athymic nude mice with the N-glycosylation inhibitor tunicamycin, the cancer growth was reduced ~55% in three weeks. Paraffin sections of the tumor exhibited reduced N-glycan level, and reduced angiogenesis but increased GRP78 expression in microvasculature as well as in the tumor tissue (J. Biol. Chem. 286, 29127–29138, 2011). This supported the presence of ER stress. We have now evaluated the effect of tunicamycin on the proliferation of metastatic ER-/PR-/HER2- (MDA-MB-231) and non-metastatic ER+ (MCF-7) human breast cancer cells. Tunicamycin inhibited proliferation of both cells types in a time and dose-dependent manner. Interestingly, GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 µg/ml) treated MDA-MB-231 and in MCF-7 cells and supported by both Western blotting and qPCR. Since GRP78 is an ER stress marker, so we have followed its intracellular localization by immunofluorescence microscopy after treating the cancer cells with tunicamycin as well as after subjecting them to various stress conditions. We have used unfixed cells and stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) or with anti-GRP antibody. There were expression of N-glycans but not GRP78. GRP78 however became detectable after permeabilization of cells with a brief exposure to ice-cold methanol. GRP78 was not detected in the conditioned media of the cancer cell but a high level of MMP-1 did. We have therefore, concluded that GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. This is obviously, contradicted the current dogma that GRP78 expression on the tumor cell surface interferes with the cancer therapeutic(s) and make them as tumor promoters and not tumor suppressors. Our study, thus, suggests strongly that anti-angiogenic and anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast tumor and all other sold tumors. Supported in part by the university funds and NIH/NIMHD G12MD007583 (KB). Citation Format: Jesus E. Serrano, Andrea Rivera-Ruiz, Krishna Baksi, Dipak K. Banerjee. ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 444.

The intrinsically disordered E-domains regulate the IGF-1 prohormones stability, subcellular localisation and secretion

Insulin-like growth factor-1 (IGF-1) is synthesised as a prohormone (proIGF-1) requiring enzymatic activity to yield the mature IGF-1. Three proIGF-1s are encoded by alternatively spliced IGF-1 mRNAs: proIGF-1Ea, proIGF-1Eb and proIGF-1Ec. These proIGF-1s have a common IGF-1 mature sequence but different E-domains. The structure of the E-domains has not been resolved, and their molecular functions are still unclear. Here, we show that E-domains are Intrinsically Disordered Regions that have distinct regulatory functions on proIGF-1s production. In particular, we identified a highly conserved N-glycosylation site in the Ea-domain, which regulated intracellular proIGF-1Ea level preventing its proteasome-mediated degradation. The inhibition of N-glycosylation by tunicamycin or glucose starvation markedly reduced proIGF-1Ea and mature IGF-1 production. Interestingly, 2-deoxyglucose, a glucose and mannose analogue, increased proIGF-1Ea and mature IGF-1 levels, probably leading to an accumulation of an under-glycosylated proIGF-1Ea that was still stable and efficiently secreted. The proIGF-1Eb and proIGF-1Ec were devoid of N-glycosylation sites, and hence their production was unaffected by N-glycosylation inhibitors. Moreover, we demonstrated that alternative Eb- and Ec-domains controlled the subcellular localisation of proIGF-1s, leading to the nuclear accumulation of both proIGF-1Eb and proIGF-1Ec. Our results demonstrated that E-domains are regulatory elements that control IGF-1 production and secretion.

N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

The sensitivity of cells to death receptor-induced apoptosis is commonly controlled by multiple checkpoints in order to limit induction of excessive or unnecessary death. Although cytotoxic in various cancer cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) does not trigger apoptosis in most non-transformed cells. The molecular nature of the checkpoints that normally protect the cells from TRAIL-induced death are not fully understood. Endoplasmic reticulum (ER) stress has been reported to switch the sensitivity of human cells to the cytotoxic effect of TRAIL, suggesting that this cellular state perturbs some of these protective mechanisms. We found that tunicamycin (TU), but no other ER stress inducers, sensitized mouse fibroblasts and hippocampal neuronal cells to TRAIL-induced apoptosis. Importantly, the sensitization was specific to TRAIL and not caused by differences in ER stress induction. Instead, it relied on the inhibition of N-glycosylation of the mouse TRAIL receptor (mTRAIL-R). Inhibition of N-glycosylation did not alter cell surface expression of mTRAIL-R but enhanced its ability to bind TRAIL, and facilitated mTRAIL-R oligomerization, which resulted in enhanced death-inducing signaling complex (DISC) formation and caspase-8 activation. Remarkably, reconstitution of mTRAIL-R-deficient cells with a version of mTRAIL-R mutated for the three N-glycosylation sites identified in its ectodomain confirmed higher sensitivity to TRAIL-induced apoptosis. Together, our results demonstrate that inhibition of N-glycosylation of mTRAIL-R, and not ER stress induction, sensitizes mouse cells to TRAIL-induced apoptosis. We therefore reveal a new mechanism restraining TRAIL cytotoxicity in mouse cells.

Abstract 476: The Role of N-glycosylation in the Myogenic Differentiation of Stem Cells to Vascular Smooth Muscle Cells

Cell fate decisions within the vasculature are crucial to the pathogenesis of vascular diseases, including, arteriosclerosis, atherosclerosis and restenosis after angioplasty. Notch signalling is involved in regulating cell fate in vasculature development during embryogenesis resulting in altered cell fate decisions leading to vascular disease. The Notch signalling pathway is highly regulated by a number of mechanisms including glycosylation, a post-translational modification. Our main objective was to define a putative role for N-glycosylation of the Notch1 receptor in controlling resident vascular stem cell fate in vitro . Utilising ligand Jagged-induced Notch signalling assay, qRT-PCR, immunocytochemistry, ectopic expression of Notch1 receptor, siRNA knockdown, pharmacological inhibition and enzyme linked lectin assay (ELLA), alterations in N-glycan decoration of the Notch1 receptor were assessed before evaluation of their effects on Notch signalling and Notch ligand promotion of myogenic differentiation. N-glycosylation of the Notch1 receptor was assessed using a combination of the HPLC and ELLA assays and confirmed the presence of N-glycans on the receptor, an effect that was abrogated following inhibition of glycosyltransferase activity with tunicamycin and lunatic fringe (Lfng) knockdown. Jagged1-induced Notch activation increased Notch target gene expression and promoted myogenic differentiation of bone-marrow derived mesenchymal stem cells and resident vascular stem cells. Selective knockdown of the Notch1 receptor in stem cells resulted in a significant decrease in Jagged1 stimulated Hey1 expression, a Notch1 target gene, concomitant with a reduction in myogenic differentiation due to decreased smooth muscle differentiation marker expression (CNN1 and MYH11 mRNA and protein levels). Inhibition of N-glycosylation with tunicamycin lead to a down regulation of smooth muscle differentiation markers, CNN1 and MYH11 independent of a reduction in Notch target gene expression. Lfng knockdown lead to a similar significant reduction in Jagged1 induced myogenic differentiation (reduced CNN1 expression). Collectively, these results suggest that N-glycosylation of Notch1 receptor is involved in Notch signalling leading to altered resident vascular stem cell fate.

GlcNAc-1-P-transferase-tunicamycin complex structure reveals basis for inhibition of N-glycosylation.

SummaryN-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase, GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity by inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. Our structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited for the design of MraY-specific inhibitors as potential antibiotics.

Multiplexed Digital mRNA Expression Analysis Profiles System-Wide Changes in mRNA Abundance and Responsiveness of UPR-Specific Gene Expression Changes During Batch Culture of Recombinant Chinese Hamster Ovary Cells.

The unfolded protein response (UPR) signaling pathway is viewed as critical for setting the effectiveness of recombinant protein expression in CHO cells. In this study, Nanostring nCounter technology is used to study expression of a group of genes associated with cellular processes linked to UPR activation under ER stress and the changing environment of a batch culture. Time course induction of ER stress, using tunicamycin (TM), shows a group of genes such as Chop, Trb3, Sqstm1, Grp78, and Herpud1 respond rapidly to TM inhibition of N-glycosylation, while others such as Atf5, Odz4, and Birc5 exhibits a delayed response. In batch culture, expression of "classical" UPR markers only increases when cells enter decline phase. In addition to providing a detailed analysis of the expression of process-relevant UPR markers during batch culture and in response to imposed chemical stress, we also highlighted six genes (Herpud1, Odz4, Sqstm1, Trb3, Syvn1, and Birc5) associated with the perception of ER stress responses in recombinant CHO cells. Herpud1 (involved in ER-associated degradation) exhibits a rapid (primary) response to stress and its relationship (and that of the other five genes) to the overall cellular UPR may identify novel targets to modulate recombinant protein production in CHO cells.

Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.

GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) is located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Nonmetastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/mL) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A) or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

Inhibition of N-Glycosylation towards Novel Anti-Cancer Chemotherapeutics

Inhibition of N-Glycosylation towards Novel Anti-Cancer Chemotherapeutics

Selective catalytic hydrogenation of the N-acyl and uridyl double bonds in the tunicamycin family of protein N-glycosylation inhibitors.

Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds. At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring opening under mild alkaline conditions.

Tunicamycin-induced endoplasmic reticulum stress suppresses plant immunity

Most secretory and membrane proteins are properly folded in the endoplasmic reticulum (ER) before being transferred to their functional destinations. Physiological and pathological stresses induce unfolded and misfolded protein accumulation in the ER, termed as ER stress. Under ER stress, cells initiate a protective response to maintain cellular homeostasis, which is referred as unfolded protein responses. Although protein processing in the ER has been known to regulate cell lifespan and disease, few evidences that prove the role of ER stress in plant immunity have been reported. We investigated the interaction between ER stress and pathogenicity in Arabidopsis by utilizing the N-glycosylation inhibitor, tunicamycin (TM) as an ER stress inducer. TM induced the accumulation of PR1 (pathogenesis-related protein 1) and callose in plant leaves, which are markers for PAMP-triggered immunity (PTI) activation. However, TM pre-treatment increased susceptibility of Arabidopsis to all bacterial pathogens tested. Moreover, TM resulted in cell death of plant leaves with an additive effect to hypersensitive response by bacterial effector proteins, suggesting TM-induced cell death is independent of the effector-triggered immunity. These results imply that TM-induced ER stress weakens overall immune system of plant not a specific immune pathway, probably via disruption of post-translational modification of immune-related proteins in the ER and subsequent cell death by apoptosis or autophagy. This study provides proves for the distinct suppressive effect of ER stress on the plant immune system.

Proteome-wide Identification of Glycosylation-dependent Interactors of Galectin-1 and Galectin-3 on Mesenchymal Retinal Pigment Epithelial (RPE) Cells

Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here, we aim to identify novel interacting binding partners for the β-galactoside-binding proteins galectin-1 (Gal-1) and galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion, and epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unraveling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pulldown experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor β PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced cross-linking with LRP1/PDGFRB and integrin-β1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with kifunensine, an inhibitor of complex-type N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.

Abstract 4509: GRP78 is neither expressed on ER-/PR-/Her2- human breast cancer cell surface nor secreted in the culture media

Abstract GRP78 (Mr78 kDa) is a glucose regulated protein. It is a member of the heat-shock protein (HSP) family but located in the lumen of the endoplasmic reticulum (ER). Its function as ER chaperone translocating protein across the ER membrane that needs to be glycosylated at the asparagine residue present in the sequeon Asn-X-Ser/Thr (N-linked glycosylation) is well recognized. This important biochemical event is essential for glycoprotein folding and function. For example, when the protein N-glycosylation is impaired with tunicamycin a protein N-glycosylation inhibitor, angiogenesis, a hallmark for tumor progression and metastasis is inhibited due to an induction of ER stress. Under such condition, GRP78 is overexpressed in microvasculature and in tumor tissue, concluding that GRP78 is a master regulator in ER stress induced unfolded protein response (upr)-mediated apoptosis in tumor microvasculature (J. Biol. Chem. 286, 29127-29138, 2011; Pure Appl. Chem. 84, 1907-1918, 2012). This contradicts the current dogma that supports GRP78 expression on the tumor cell surface interfering with the therapeutic(s) and making them as tumor promoters instead. To evaluate the GRP78 localization, we have used ER-/PR-/Her2- (i.e., triple negative) human breast cancer cell line MDA-MB-231 (Caucasian) cultured normally or without serum as well as before or after inducing the ER stress. After establishing the expression of GRP78 mRNA by qPCR and protein by western blotting, we focused on its cell surface expression. Unfixed cells were stained with Concanavalin A (Con A; Specificity = α-D-Mannose, α-D-Glucose, branched mannose), wheat germ agglutinin (WGA; Specificity = (GlcNAc-β-(1,4)-GlcNAc)1-4>β-GlcNAc-NeuAc) as well as with anti-GRP78 antibody followed by their detection by immunofluorescence microscopy using either Rhodamin or Alexa-conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining provided images of N-glycans on the cell surface and supported the intactness of the cell membrane. On the other hand, GRP78 fluorescence was absent from the surface of these cancer cells. Similar results were also obtained irrespective of cells cultured in the absence of serum and/or in the presence of 1µg/mL of tunicamycin. GRP78 fluorescence however was detected in cells after either fixing them with ice-cold methanol or after permeabilization with digitonin. In addition, western blotting also failed to detect GRP78 in the culture media of MDA-MB-231 cells. We, therefore, conclude that GRP78 is neither expressed on the outer-leaflet of the cell surface of ER-/PR-/Her2- human breast cancer cells MDA-MB-231 nor secreted in the culture media. Supported in part by Susan G. Komen for the Cure BCTR0600582 and NIH/NIMHD G12MD007583 (KB). Citation Format: Jesus E. Serrano, Eva C. Romero-Nutz, Neysharie Sanchez, Aditi Banerjee, Krishna Baksi, Dipak K. Banerjee. GRP78 is neither expressed on ER-/PR-/Her2- human breast cancer cell surface nor secreted in the culture media [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2017-4509

GRP78 is Neither Expressed on ER−/PR−/Her2− Human Breast Cancer Cell Surface nor Secreted

GRP78 (Mr78 kDa) is a glucose regulated protein. It is a member of the heat‐shock protein (HSP) family but located in the lumen of the endoplasmic reticulum (ER). Its function as ER chaperone translocating protein across the ER membrane that needs to be glycosylated at the asparagine residue present in the sequeon Asn‐X‐Ser/Thr (N‐linked glycosylation) is well recognized. This important biochemical event is essential for glycoprotein folding and function. For example, when the protein N‐glycosylation is impaired with tunicamycin a protein N‐glycosylation inhibitor, angiogenesis, a hallmark for tumor progression and metastasis is inhibited due to an induction of ER stress. Under such condition, GRP78 is overexpressed in microvasculature and in tumor tissue, concluding that GRP78 is a master regulator in ER stress induced unfolded protein response (upr)‐mediated apoptosis in tumor microvasculature (J. Biol. Chem. 286, 29127–29138, 2011; Pure Appl. Chem. 84, 1907–1918, 2012). This contradicts the current dogma that supports GRP78 expression on the tumor cell surface interfering with the therapeutic(s) and making them as tumor promoters instead. To evaluate the GRP78 localization, we have used ER−/PR−/Her2− (i.e., triple negative) human breast cancer cell line MDA‐MB‐231 (Caucasian) cultured normally or without serum as well as before or after inducing the ER stress. After establishing the expression of GRP78 mRNA by qPCR and protein by western blotting, we focused on its cell surface expression. Unfixed cells were stained with Concanavalin A (Con A; Specificity = α‐D‐Mannose, α‐D‐Glucose, branched mannose), wheat germ agglutinin (WGA; Specificity = (GlcNAc‐β‐(1,4)‐GlcNAc)1‐4>β‐GlcNAc‐NeuAc) as well as with anti‐GRP78 antibody followed by their detection by immunofluorescence microscopy using either Rhodamin or Alexa–conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining provided images of N‐glycans on the cell surface and supported the intactness of the cell membrane. On the other hand, GRP78 fluorescence was absent from the surface of these cancer cells. Similar results were also obtained irrespective of cells cultured in the absence of serum and/or in the presence of 1μg/mL of tunicamycin. GRP78 fluorescence however was detected in cells after either fixing them with ice‐cold methanol or after permeabilization with digitonin. In addition, western blotting also failed to detect GRP78 in the culture media of MDA‐MB‐231 cells. We, therefore, conclude that GRP78 is neither expressed on the outer‐leaflet of the cell surface of ER−/PR−/Her2− human breast cancer cells MDA‐MB‐231 nor secreted in the culture media.Support or Funding InformationSupported in part by NSF EPS‐1002410 (DKB) and NIH/NIMHD G12MD007583 (KB).

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

Interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1): Implication of N-glycosylation in FAM5C secretion

N-glycosylation of proteins is important for protein folding and function. We have recently reported that FAM5C/BRINP3 contributes to the tumor necrosis factor-α-induced expression of leukocyte adhesion molecules in vascular endothelial cells (ECs). However, regulatory mechanism of the FAM5C biosynthesis is poorly understood. Co-immunoprecipitation assay revealed the interaction of FAM5C with UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), a glycoprotein folding-sensor enzyme. FAM5C ectopically expressed in HEK293 cells was localized to the endoplasmic reticulum and co-localized with endogenously expressed UGGT1. Molecular size of FAM5C was reduced by treatment with N-glycosidase F and in FAM5C-expressing cells cultured in the presence of the N-glycosylation inhibitor tunicamycin. FAM5C was secreted by the cells and the secretion of FAM5C was blocked by tunicamycin. Among six potential N-glycosylation sites, the potential site at Asn168 was not N-glycosylated, and Asn337, Asn456, Asn562, Asn609, and Asn641 mutants were poorly secreted by the cells. These results demonstrated that FAM5C is an N-glycosylated protein and N-glycosylation is necessary for the secretion of FAM5C.

N-glycosylation of human sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is essential for stability, secretion and activity.

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted N-linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its function in vivo, remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of its N-glycans and secretion. Glycosylation of Asn356 in SMPDL3A, an N-linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specific N-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A.

Synergistic killing of FLT3ITD-positive AML cells by combined inhibition of tyrosine-kinase activity and N-glycosylation.

Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein (CHOP). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML.

High glucose induces N-linked glycosylation-mediated functional upregulation and overexpression of Cav3.2 T-type calcium channels in neuroendocrine-like differentiated human prostate cancer cells

Given that Cav3.2 T-type Ca2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Cav3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Cav3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Cav3.2 in neuroendocrine-like differentiated prostate cancer cells.

Global Analysis of Secreted Proteins and Glycoproteins in Saccharomyces cerevisiae.

Protein secretion is essential for numerous cellular activities, and secreted proteins in bodily fluids are a promising and noninvasive source of biomarkers for disease detection. Systematic analysis of secreted proteins and glycoproteins will provide insight into protein function and cellular activities. Yeast (Saccharomyces cerevisiae) is an excellent model system for eukaryotic cells, but global analysis of secreted proteins and glycoproteins in yeast is challenging due to the low abundances of secreted proteins and contamination from high-abundance intracellular proteins. Here, by using mild separation of secreted proteins from cells, we comprehensively identified and quantified secreted proteins and glycoproteins through inhibition of glycosylation and mass spectrometry-based proteomics. In biological triplicate experiments, 245 secreted proteins were identified, and comparison with previous experimental and computational results demonstrated that many identified proteins were located in the extracellular space. Most quantified secreted proteins were down-regulated from cells treated with an N-glycosylation inhibitor (tunicamycin). The quantitative results strongly suggest that the secretion of these down-regulated proteins was regulated by glycosylation, while the secretion of proteins with minimal abundance changes was contrarily irrelevant to protein glycosylation, likely being secreted through nonclassical pathways. Glycoproteins in the yeast secretome were globally analyzed for the first time. A total of 27 proteins were quantified in at least two protein and glycosylation triplicate experiments, and all except one were down-regulated under N-glycosylation inhibition, which is solid experimental evidence to further demonstrate that the secretion of these proteins is regulated by their glycosylation. These results provide valuable insight into protein secretion, which will further advance protein secretion and disease studies.

A Rare UGT2B7 Variant Creates a Novel N-Glycosylation Site at Codon 121 with Impaired Enzyme Activity.

The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism.