A mammalian homolog of the zebrafish transmembrane protein 2 (TMEM2) is the long-sought-after cell-surface hyaluronidase

Hyaluronan (HA) is an extremely large polysaccharide (glycosaminoglycan) involved in many cellular functions. HA catabolism is thought to involve the initial cleavage of extracellular high-molecular-weight (HMW) HA into intermediate-size HA by an extracellular or cell-surface hyaluronidase, internalization of intermediate-size HA, and complete degradation into monosaccharides in lysosomes. Despite considerable research, the identity of the hyaluronidase responsible for the initial HA cleavage in the extracellular space remains elusive. HYAL1 and HYAL2 have properties more consistent with lysosomal hyaluronidases, whereas CEMIP/KIAA1199, a recently identified HA-binding molecule that has HA-degrading activity, requires the participation of the clathrin-coated pit pathway of live cells for HA degradation. Here we show that transmembrane protein 2 (TMEM2), a mammalian homolog of a protein playing a role in zebrafish endocardial cushion development, is a cell-surface hyaluronidase. Live immunostaining and surface biotinylation assays confirmed that mouse TMEM2 is expressed on the cell surface in a type II transmembrane topology. TMEM2 degraded HMW-HA into ∼5-kDa fragments but did not cleave chondroitin sulfate or dermatan sulfate, indicating its specificity to HA. The hyaluronidase activity of TMEM2 was Ca2+-dependent; the enzyme's pH optimum is around 6–7, and unlike CEMIP/KIAA1199, TMEM2 does not require the participation of live cells for its hyaluronidase activity. Moreover, TMEM2-expressing cells could eliminate HA immobilized on a glass surface in a contact-dependent manner. Together, these data suggest that TMEM2 is the long-sought-after hyaluronidase that cleaves extracellular HMW-HA into intermediate-size fragments before internalization and degradation in the lysosome.

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Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00374.2012 ◽

2013 ◽

Vol 304 (1) ◽

pp. H94-H103 ◽

Cited By ~ 20

Author(s):

Aude Belliard ◽

Yoann Sottejeau ◽

Qiming Duan ◽

Jessa L. Karabin ◽

Sandrine V. Pierre

Keyword(s):

Cell Surface ◽

Atpase Activity ◽

Ion Transport ◽

Cell Viability ◽

Ischemia Reperfusion ◽

Resistant Mutant ◽

Surface Expression ◽

Live Cells ◽

Mutant Form ◽

Dependent Manner

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase α1- and α3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase α1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport.

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frizzled regulates mirror-symmetric pattern formation in the Drosophila eye

Development ◽

10.1242/dev.121.9.3045 ◽

1995 ◽

Vol 121 (9) ◽

pp. 3045-3055 ◽

Cited By ~ 38

Author(s):

L. Zheng ◽

J. Zhang ◽

R.W. Carthew

Keyword(s):

Pattern Formation ◽

Cell Surface ◽

Transmembrane Protein ◽

Photoreceptor Cells ◽

Mirror Image ◽

Morphogenetic Furrow ◽

Symmetric Pattern ◽

Mosaic Analysis ◽

Drosophila Eye ◽

A Cell

Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. We have investigated the mechanisms by which this pattern formation occurs and our results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. We propose that frizzled mediates a bidirectional signal emanating from the equator.

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Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

10.1101/2021.12.14.472513 ◽

2021 ◽

Author(s):

Mizuki Yamamoto ◽

Jin Gohda ◽

Ayako Kobayashi ◽

Keiko Tomita ◽

Youko Hirayama ◽

...

Keyword(s):

Cell Surface ◽

Spike Protein ◽

Dependent Manner ◽

Furin Cleavage ◽

Entry Pathway ◽

Furin Cleavage Site ◽

Metalloproteinase Inhibitors ◽

A Cell ◽

A Disintegrin And Metalloproteinase ◽

Human Coronaviruses

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future.

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Effects of Herpes Simplex Virus on Structure and Function of Nectin-1/HveC

Journal of Virology ◽

10.1128/jvi.76.5.2424-2433.2002 ◽

2002 ◽

Vol 76 (5) ◽

pp. 2424-2433 ◽

Cited By ~ 38

Author(s):

Claude Krummenacher ◽

Isabelle Baribaud ◽

James F. Sanzo ◽

Gary H. Cohen ◽

Roselyn J. Eisenberg

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cell Surface ◽

Cell Aggregation ◽

Viral Envelope ◽

Actin Binding ◽

Dependent Manner ◽

A Cell ◽

Adhesive Site ◽

Simplex Virus

ABSTRACT Herpes simplex virus (HSV) entry requires the interaction between the envelope glycoprotein D (gD) and a cellular receptor such as nectin-1 (also named herpesvirus entry mediator C [HveC]) or HveA/HVEM. Nectin-1 is a cell adhesion molecule found at adherens junctions associated with the cytoplasmic actin-binding protein afadin. Nectin-1 can act as its own ligand in a homotypic interaction to bridge cells together. We used a cell aggregation assay to map an adhesive functional site on nectin-1 and identify the effects of gD binding and HSV early infection on nectin-1 function. Soluble forms of nectin-1 and anti-nectin-1 monoclonal antibodies were used to map a functional adhesive site within the first immunoglobulin-like domain (V domain) of nectin-1. This domain also contains the gD-binding site, which appeared to overlap the adhesive site. Thus, soluble forms of gD were able to prevent nectin-1-mediated cell aggregation and to disrupt cell clumps in an affinity-dependent manner. HSV also prevented nectin-1-mediated cell aggregation by occupying the receptor. Early in infection, nectin-1 was not downregulated from the cell surface. Rather, detection of nectin-1 changed gradually over a 30-min period of infection, as reflected by a decrease in the CK41 epitope and an increase in the CK35 epitope. The level of detection of virion gD on the cell surface increased within 5 min of infection in a receptor-dependent manner. These observations suggest that cell surface nectin-1 and gD may undergo conformational changes during HSV entry as part of an evolving interaction between the viral envelope and the cell plasma membrane.

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Dopamine reduces cell surface Na+/H+ exchanger-3 protein by decreasing NHE3 exocytosis and cell membrane recycling

AJP Renal Physiology ◽

10.1152/ajprenal.00251.2017 ◽

2017 ◽

Vol 313 (4) ◽

pp. F1018-F1025 ◽

Cited By ~ 1

Author(s):

Ming Chang Hu ◽

I. Alexandru Bobulescu ◽

Henry Quiñones ◽

Serge M. Gisler ◽

Orson W. Moe

Keyword(s):

Cell Membrane ◽

Cell Surface ◽

Proximal Tubule ◽

Apical Cell ◽

Brefeldin A ◽

Dependent Manner ◽

Culture Model ◽

A Cell ◽

The Many ◽

Dose Dependent

The intrarenal autocrine-paracrine dopamine (DA) system mediates a significant fraction of the natriuresis in response to a salt load. DA inhibits a number of Na+ transporters to effect sodium excretion, including the proximal tubule Na+/H+ exchanger-3 (NHE3). DA represent a single hormone that regulates NHE3 at multiple levels, including translation, degradation, endocytosis, and protein phosphorylation. Because cell surface NHE3 protein is determined by the balance between exocytotic insertion and endocytotic retrieval, we examined whether DA acutely affects the rate of NHE3 exocytosis in a cell culture model. DA inhibited NHE3 exocytosis at a dose-dependent manner with a half maximal around 10−6 M. The DA effect on NHE3 exocytosis was blocked by inhibition of protein kinase A and by brefeldin A, which inhibits endoplasmic reticulum-to-Golgi transport. NHE3 directly interacts with the ε-subunit of coatomer protein based on yeast-two-hybrid and coimmunoprecipitation. Because NHE3 has been shown to be recycled back to the cell membrane after endocytosis, we measured NHE3 recycling using a biochemical reinsertion assay and showed that reinsertion of NHE3 back to the membrane is also inhibited by DA. In conclusion, among the many mechanisms by which DA reduces apical membrane NHE3 and induces proximal tubule natriuresis, one additional mechanism is inhibition of exocytotic insertion and reinsertion of NHE3 in the apical cell surface.

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Thrombin adhesive properties: induction by plasmin and heparan sulfate.

The Journal of Cell Biology ◽

10.1083/jcb.123.5.1279 ◽

1993 ◽

Vol 123 (5) ◽

pp. 1279-1287 ◽

Cited By ~ 17

Author(s):

R Bar-Shavit ◽

Y Eskohjido ◽

J W Fenton ◽

J D Esko ◽

I Vlodavsky

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Chondroitin Sulfate ◽

Dermatan Sulfate ◽

Basement Membranes ◽

Dependent Manner ◽

Heparin Binding ◽

Adhesive Properties ◽

Adhesive Protein ◽

Adhesive Molecule

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.

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Identification of the atypical cadherin FAT1 as a novel glypican-3 interacting protein in liver cancer cells

Scientific Reports ◽

10.1038/s41598-020-79524-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Panpan Meng ◽

Yi-Fan Zhang ◽

Wangli Zhang ◽

Xin Chen ◽

Tong Xu ◽

...

Keyword(s):

Cell Migration ◽

Cell Surface ◽

Transmembrane Protein ◽

Tyrosine Phosphatase ◽

Interacting Protein ◽

Extracellular Signaling ◽

Putative Receptor ◽

Elevated Expression ◽

A Cell ◽

Receptor Tyrosine Phosphatase

AbstractGlypican-3 (GPC3) is a cell surface heparan sulfate proteoglycan that is being evaluated as an emerging therapeutic target in hepatocellular carcinoma (HCC). GPC3 has been shown to interact with several extracellular signaling molecules, including Wnt, HGF, and Hedgehog. Here, we reported a cell surface transmembrane protein (FAT1) as a new GPC3 interacting protein. The GPC3 binding region on FAT1 was initially mapped to the C-terminal region (Q14517, residues 3662-4181), which covered a putative receptor tyrosine phosphatase (RTP)-like domain, a Laminin G-like domain, and five EGF-like domains. Fine mapping by ELISA and flow cytometry showed that the last four EGF-like domains (residues 4013-4181) contained a specific GPC3 binding site, whereas the RTP domain (residues 3662-3788) and the downstream Laminin G-2nd EGF-like region (residues 3829-4050) had non-specific GPC3 binding. In support of their interaction, GPC3 and FAT1 behaved concomitantly or at a similar pattern, e.g. having elevated expression in HCC cells, being up-regulated under hypoxia conditions, and being able to regulate the expression of EMT-related genes Snail, Vimentin, and E-Cadherin and promoting HCC cell migration. Taken together, our study provides the initial evidence for the novel mechanism of GPC3 and FAT1 in promoting HCC cell migration.

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Quantitative Imaging of Single Live Cells Reveals Spatiotemporal Dynamics of Multistep Signaling Events of Chemoattractant Gradient Sensing in Dictyostelium

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0544 ◽

2005 ◽

Vol 16 (2) ◽

pp. 676-688 ◽

Cited By ~ 90

Author(s):

Xuehua Xu ◽

Martin Meier-Schellersheim ◽

Xuanmao Jiao ◽

Lauren E. Nelson ◽

Tian Jin

Keyword(s):

Cell Surface ◽

G Protein ◽

G Proteins ◽

Receptor Occupancy ◽

Live Cells ◽

Second Phase ◽

Gradient Sensing ◽

Protein Activation ◽

G Protein Activation ◽

A Cell

Activation of G-protein-coupled chemoattractant receptors triggers dissociation of Gα and Gβγ subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PHCrac-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naïve cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PHCrac-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PHCrac-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity.

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Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

Infection and Immunity ◽

10.1128/iai.66.12.5703-5710.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 5703-5710 ◽

Cited By ~ 110

Author(s):

Ashu Sharma ◽

Hakimuddin T. Sojar ◽

Ingrid Glurich ◽

Kiyonobu Honma ◽

Howard K. Kuramitsu ◽

...

Keyword(s):

Immune Response ◽

Recombinant Protein ◽

Cell Surface ◽

Host Immune Response ◽

Cloning And Expression ◽

Dependent Manner ◽

Leucine Rich Repeat ◽

Clotting Factors ◽

Binding Studies ◽

A Cell

ABSTRACT Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed inEscherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.

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Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor

Blood ◽

10.1182/blood.v95.12.3951.012k21_3951_3958 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3951-3958 ◽

Cited By ~ 5

Author(s):

George B. Stefano ◽

Patrick Cadet ◽

Christophe Breton ◽

Yannick Goumon ◽

Vincent Prevot ◽

...

Keyword(s):

Nitric Oxide ◽

Estrogen Receptor ◽

Cell Surface ◽

Intracellular Calcium ◽

Cell Surface Receptor ◽

Dependent Manner ◽

No Release ◽

Human Granulocytes ◽

A Cell ◽

17Β Estradiol

We tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17β-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17β-estradiol conjugated to bovine serum albumin (E2-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17β-estradiol and E2-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17β-estradiol–stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca2+]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N′,N′-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca2+]i chelator, reduced [Ca2+]i in response to E2-BSA, and depleting [Ca2+]i stores abolished the effect of 17β-estradiol on NO release. Confocal photomicrographs using E2-BSA–FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase–polymerase chain reaction, human neutrophil granulocytes expressed ER but not ERβ, suggesting that ER may be the membrane receptor for 17β-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca2+]i.

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