Listeria monocytogenes Triggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

Martins, Mariana; Custódio, Rafael; Camejo, Ana; Almeida, Maria Teresa; Cabanes, Didier; Sousa, Sandra

doi:10.1074/jbc.m112.422568

Cited by 26 publications

(26 citation statements)

References 45 publications

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“…Recently, Vip, an important virulent factor in Listeria monocytogenes was shown to bind surface exposed gp96, which was identified as a 390 amino acid sequence in the N-terminal domain. This information is in contrary to the results obtained from transmembrane prediction server, TMPred, which predicted a 172 amino acid extracellular domain in gp96 [21]. Since the presence of an extracellular 390 amino acid sequence was experimentally verified, we analyzed the sequences downstream of this region for a putative transmembrane sequence.…”

Section: Resultsmentioning

confidence: 77%

“…Our studies have shown that the non-fimbrial adhesin, OmpA is a major virulence component of E. coli K1 and that it interacts with Ecgp96 on the surface of HBMEC to bind to and invade [3]. A recent study also showed that Vip, a major virulence factor in Listeria monocytogenes binds to the N-terminal extracellular domain of gp96 (a homologue of Ecgp96) in epithelial cells [21]. Gp96 also acts as a receptor for a wide variety of pathogens, including Clostridium difficile , adherent-invasive E. coli , Neisseria gonorrheae and Staphylococcus aureus and for bovine adeno-associated virus (BAAV) [17–20, 36, 37].…”

Section: Discussionmentioning

confidence: 99%

“…This is supported by (i) Mutation of N-glycosylated asparagines blocks the invasion by ~75%, (ii) Suppression of Ecgp96 using specific siRNA blocks invasion in CHO cells, implying that gp96 is the primary receptor for OmpA in these cells (iii) Confirmation that these putative sites are indeed glycosylated by Western blotting, (iv) Enzymatic removal of GlcNAc1-4GlcNAc, but not mannose in Ecgp96, inhibits bacterial binding and invasion, and (v) Pre-incubation of bacteria with either chitin hydrolysate or anti-OmpA antibody significantly attenuates bacterial adhesion and invasion, which corroborates our previous observation that OmpA binds GlcNAc1-4GlcNAc moieties. Based on experimental evidence provided by Martins et al, Ecgp96 seems to have a larger extracellular domain (390 amino acids) than a 172 amino acid extracellular domain predicted using transmembrane prediction server, TMPred [21]. A highly correlative statistical analysis of helix-helix interactions in membrane proteins reveal a common GXXXG motif, which could represent a transmembrane region [27], and we found the sequence ‘GVVDS’ downstream of extracellular domain in Ecgp96 that concurred with the GXXXG motif rule [27].…”

Section: Discussionmentioning

confidence: 99%

“…Ecgp96 was implicated for the first time as a bacterial receptor for OmpA of E. coli K1 to invade HBMEC [16]. Several studies have now identified gp96, the non-endothelial homologue of Ecgp96, as a receptor for a number of bacteria [17–21]. Our previous studies showed that TLR2 stabilizes Ecgp96 on the membrane of HBMEC to facilitate OmpA binding.…”

Section: Introductionmentioning

confidence: 99%

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Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Krishnan

Prasadarao²

2014

Microbes and Infection

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Bacterial meningitis is a serious central nervous system infection and Escherichia coli K1 (E. coli K1) is one of the leading etiological agents that cause meningitis in neonates. Outer membrane protein A (OmpA) of E. coli K1 is a major virulence factor in the pathogenesis of meningitis, and interacts with human brain microvascular endothelial cells (HBMEC) to cross the blood-brain barrier. Using site-directed mutagenesis, we demonstrate that two N-glycosylation sites (NG1 and NG2) in the extracellular domain of OmpA receptor, Ecgp96 are critical for bacterial binding to HBMEC. E. coli invasion assays using CHO-Lec1 cells that express truncated N-glycans, and sequential digestion of HBMEC surface N-glycans using specific glycosidases showed that GlcNAc1-4GlcNAc epitopes are sufficient for OmpA interaction with HBMEC. Lack of NG1 and NG2 sites in Ecgp96 inhibits E. coli OmpA induced F-actin polymerization, phosphorylation of protein kinase C-α, and disruption of transendothelial electrical resistance required for efficient invasion of E. coli in HBMEC. Furthermore, the microvessels of cortex and hippocampus of the brain sections of E. coli K1 infected mice showed increased expression of glycosylated Ecgp96. Therefore, the interface of OmpA and GlcNAc1-4GlcNAc epitope interaction would be a target for preventative strategies against E. coli K1 meningitis.

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Section: Resultsmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Krishnan

Prasadarao²

2014

Microbes and Infection

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“…Concurrently, we have shown that Lm infection redistributes the ER chaperone Gp96 to the PM through an uncharacterized mechanism [12]. Gp96 is an ER-resident HSP90 paralogue, which controls the expression and folding of proteins assigned to the secretory pathway and has crucial roles in cellular homoeostasis, host development and immunity [13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore‐forming toxins

et al. 2016

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During infection, plasma membrane (PM) blebs protect host cells against bacterial pore-forming toxins (PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT-induced blebbing. Gp96 interacts with non-muscle myosin heavy chain IIA (NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA-Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod-like structures from polarized migrating cells, a process that also promotes NMHCIIA-Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O (LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO-dependent killing.

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Cell membrane gp96 facilitates HER2 dimerization and serves as a novel target in breast cancer

Sun

Hou

et al. 2015

Int. J. Cancer

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HER2 receptor dimerization is a critical step in the HER2 activation process. Here, we demonstrated that heat shock protein gp96 on cell membrane interacts with HER2, facilitates HER2 dimerization and promotes cell proliferation. Cell membrane gp96 levels were observed to correlate with HER2 phosphorylation in primary breast tumors. Finally, we provide evidence that targeting gp96 with a specific monoclonal antibody led to decreased cell growth and increased apoptosis in vitro, and suppression of tumor growth in vivo. Our work represents a new therapeutic strategy for inhibiting HER2 signaling in cancer.The heat shock protein 90 (Hsp90) family contains four members in humans, HSP90a, HSP90b, gp96 (grp94) and Trap-1. As one of the most abundant chaperones in the endoplasmic reticulum (ER), gp96 associates with client proteins and guide their maturation and assembly. In contrast to the other members of HSP90 family that may bind and activate hundreds of client proteins, gp96 has a relatively strict binding selectivity and is known to associate with only a handful of confirmed clients, including certain Toll-like receptors, 1,2 integrins, 1 insulin-like growth factors 3 and immunoglobulin. 4 A recent study by proteomic plasma membrane profiling has revealed that besides TLRs and integrins, gp96 is required for the cell surface expression of four members of LDL receptor family. To date, these gp96-regulated proteins are structurally characterized by the presence of bpropeller and leucine rich repeats (LLR), suggesting an important role of gp96 in recognizing these motifs. 5The normally ER-resident gp96 may translocate to cell membrane under bacterial infection, 6,7 or in certain tumor cells. 8,9Cell membrane-expressed gp96 is shown to correlate with malignancy in breast cancer cells. 10 However, until now few cell membrane gp96 client proteins have been identified in regulation of various cell physiological processes, including HER2 11,12 and the metalloprotease pro-ADAMTS9 (ADAMTS9). 13 The biological role of cell membrane gp96 in tumor cells deserves further investigation.Signaling through HER2 receptors is critically involved in multiple human cancers. HER2 functions as the preferred heterodimeric partner within the HER family, and HER2-containing heterodimers are characterized by slow rates of ligand dissociation and increased recycling back to the plasma membrane for reactivation, allowing potent and prolonged activation of downstream signaling pathways.14 HER2 overexpression occurs in approximately 25% of breast cancers and is related to adverse tumor characteristics and a poor prognosis in patients. 15 Due to its central roles in the regulation of HER-mediated signaling and tumorigenesis, HER2 is an ideal target for therapeutic development, including monoclonal antibodies to prevent homo or heterodimerization. [16][17][18][19] In this study, we use several approaches to examine the interaction of cell surface gp96 and HER2, and define the regulation of gp96 in HER2 dimerization and signaling. We then use an a...

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Listeria monocytogenes Triggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

Cited by 26 publications

References 45 publications

Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore‐forming toxins

Cell membrane gp96 facilitates HER2 dimerization and serves as a novel target in breast cancer

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