Integral Membrane Glycoprotein Research Articles

Voltage-Gated Sodium Channels: Biophysics, Pharmacology, and Related Channelopathies

Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named “channel partners” or “channel interacting proteins” (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium “channelopathies”. This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field.

Secreted amyloid precursor proteins promote proliferation and glial differentiation of adult hippocampal neural progenitor cells

Amyloid precursor protein (APP) is an integral membrane glycoprotein present at high levels in nerve cells. Two soluble secreted forms, sAPPα and sAPPβ, are processed from APP by two mutually exclusive proteolytic pathways. sAPPα shows a range of neuroprotective and growth factor properties, including reduction of neuronal injury and improvement in memory performance, in contrast to the generally less potent sAPPβ. In addition, sAPPα has been shown to increase the proliferation of both embryonic neural stem cells and neural progenitor cells (NPCs) derived from the subventricular zone (SVZ) of the adult brain. However, an effect of sAPPα (or sAPPβ) on adult hippocampal progenitor cell proliferation and differentiation has not previously been observed. In this study, we examined the effect of both the α- and β-cleaved ectodomains of sAPP on adult NPCs isolated from the subgranular zone (SGZ) of the rat hippocampus in the presence or absence of depolarizing conditions. Assays were performed to examine the effect of sAPPα and sAPPβ on SGZ-derived adult NPC proliferation in parallel with SVZ-derived cells and on differentiation with SGZ-derived cells. We observed both sAPPα and sAPPβ increased the proliferation of SGZ-derived NPCs in vitro. Further, treatment of SGZ-derived NPCs with either sAPPα or sAPPβ increased the number of cells expressing the astrocytic marker GFAP and promoted cell survival. The effect on differential fate was observed in both the presence and absence of depolarizing conditions. Thus, both sAPPα and sAPPβ exert a complex range of effects on SGZ-derived adult NPCs, including increasing NPC proliferation, maintaining cell viability, yet promoting glial over neuronal differentiation. These findings provide the first direct support for the secreted forms of APP regulating SGZ-derived NPCs, and raise the possibility some or all of the effects may have therapeutic benefit in models of neurological disease.

Structural insights into human GPCR protein OA1: a computational perspective

Human ocular albinism type 1 protein (OA1)-a member of the G-protein coupled receptor (GPCR) superfamily-is an integral membrane glycoprotein expressed exclusively by intracellular organelles known as melanocytes, and is responsible for the proper biogenesis of melanosomes. Mutations in the Oa1 gene are responsible for the disease ocular albinism. Despite its clinical importance, there is a lack of in-depth understanding of its structure and mechanism of activation due to the absence of a crystal structure. In the present study, homology modeling was applied to predicting OA1 structure following thorough sequence analysis and secondary structure predictions. The predicted model had the signature residues and motifs expected of GPCRs, and was used for carrying out molecular docking studies with an endogenous ligand, L-DOPA and an antagonist, dopamine; the results agreed quite well with the available experimental data. Finally, three sets of explicit molecular dynamics simulations were carried out in lipid bilayer, the results of which not only confirmed the stability of the predicted model, but also helped witness some differences in structural features such as rotamer toggle switch, helical tilts and hydrogen bonding pattern that helped distinguish between the agonist- and antagonist-bound receptor forms. In place of the typical "D/ERY"-motif-mediated "ionic lock", a hydrogen bond mediated by the "DAY" motif was observed that could be used to distinguish the agonist and antagonist bound forms of OA1. In the absence of a crystal structure, this study helped to shed some light on the structural features of OA1, and its behavior in the presence of an agonist and an antagonist, which might be helpful in the future drug discovery process for ocular albinism.

Involvement of Human Natural Killer-1 (HNK-1) Sulfotransferase in the Biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl Tetrasaccharide Found in α-Thrombomodulin from Human Urine

Thrombomodulin (TM) is an integral membrane glycoprotein, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (β-TM) and a non-PG form without a CS chain (α-TM) and hence is a part-time PG. An α-TM preparation isolated from human urine contained the glycosaminoglycan linkage region tetrasaccharide GlcUAβ1-3Galβ1-3Galβ1-4xylose, and the nonreducing terminal GlcUA residue is 3-O-sulfated. Because the human natural killer-1 sulfotransferase (HNK-1ST) transfers a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue in the HNK-1 antigen precursor trisaccharide, GlcUAβ1-3Galβ1-4GlcNAc, the sulfotransferase activity toward the linkage region was investigated. In fact, the activity of HNK-1ST toward the linkage region was much higher than that toward the glucuronylneolactotetraosylceramide, the precursor of the HNK-1 epitope. HNK-1ST may be responsible for regulating the sorting of α- and β-TM. Furthermore, HNK-1ST also transferred a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue of a chondroitin chain. Intriguingly, the HNK-1 antibody recognized CS chains and the linkage region if they contained GlcUA(3-O-sulfate), suggesting that HNK-1ST not only synthesizes the HNK-1 epitope but may also be involved in the generation of part-time PGs.

Lipid Droplet Formation Is Dispensable for Endoplasmic Reticulum-associated Degradation

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are destroyed by cytoplasmic proteasomes through a process known as ER-associated degradation. Substrates of this pathway are initially sequestered within the ER lumen and must therefore be dislocated across the ER membrane to be degraded. It has been proposed that generation of bicellar structures during lipid droplet formation may provide an "escape hatch" through which misfolded proteins, toxins, and viruses can exit the ER. We have directly tested this hypothesis by exploiting yeast strains defective in lipid droplet formation. Our data demonstrate that lipid droplet formation is dispensable for the dislocation of a plant toxin and the degradation of both soluble and integral membrane glycoproteins.

Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in four groups of nine worm-free calves challenged with either 8000 H. contortus or Haemonchus placei infective larvae. Vaccinates received 50 μg of the antigen and 1 mg QuilA adjuvant three times 21 days apart, while the controls got adjuvant alone. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Immunization resulted in high titre antibodies against the vaccine antigens and significant reduction in egg output and worm numbers of both challenge species, compared with control calves. It was concluded that vaccination of calves with native parasite gut membrane glycoproteins obtained from H. contortus conferred protection against both H. placei and H. contortus.

Synaptophysin Regulates the Kinetics of Synaptic Vesicle Endocytosis in Central Neurons

Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity.

Design, synthesis, and evaluation of 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate (8MDP-fluor), as a novel equilibrative nucleoside transporter probe.

Nucleoside transporters are integral membrane glycoproteins that play critical roles in physiological nucleoside and nucleobase fluxes, and influence the efficacy of many nucleoside chemotherapy drugs. Fluorescent reporter ligands/substrates have been shown to be useful in the analysis of nucleoside transporter (NT) protein expression and discovery of new NT inhibitors. In this study, we have developed a novel dipyridamole (DP)-based equilibrative nucleoside transporter 1 (ENT1) fluorescent probe. The potent ENT1 and ENT2 inhibitor analogue of dipyridamole, 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (4, 8MDP), was modified to replace one β-hydroxyethyl group of the amino substituent at the 2-position with a β-aminoethyl group and then conjugated through the amino group to 6-(fluorescein-5-carboxamido)hexanoyl moiety to obtain a new fluorescent molecule, 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate, designated 8MDP-fluorescein (8MDP-fluor, 6). The binding affinities of 8MDP-fluor at ENT1 and ENT2 are reflected by the uridine uptake inhibitory K(i) values of 52.1 nM and 285 nM, respectively. 8MDP-fluor was successfully demonstrated to be a flow cytometric probe for ENT1 comparable to the nitrobenzylmercaptopurine riboside (NBMPR) analogue ENT1 fluorescent probe SAENTA-X8-fluorescein (SAENTA-fluor, 1). This is the first reported dipyridamole-based ENT1 fluorescent probe, which adds a novel tool for probing ENT1, and possibly ENT2.

Neuronal glycoprotein M6a induces filopodia formation via association with cholesterol‐rich lipid rafts

A neuronal integral membrane glycoprotein M6a has been suggested to be involved in a number of biological processes, including neuronal remodeling and differentiation, trafficking of mu-opioid receptors, and Ca(2+) transportation. Moreover, pathological situations such as chronic stress in animals and depression in humans have been associated with alterations in M6a sequence and expression. The mechanism of action of M6a is essentially unknown. In this work, we analyze the relevance of M6a distribution in plasma membrane, namely its lipid microdomain targeting, for its biological function in filopodia formation. We demonstrate that M6a is localized in membrane microdomains compatible with lipid rafts in cultured rat hippocampal neurons. Removal of cholesterol from neuronal membranes with methyl-β-cyclodextrin decreases M6a-induced filopodia formation, an effect that is reversed by the addition of cholesterol. Inhibition of Src kinases and MAPK prevents filopodia formation in M6a-over-expressing neurons. Src-deficient SYF cells over-expressing M6a fail to promote filopodia formation. Taken together, our findings reveal that the association of M6a with lipid rafts is important for its role in filopodia formation and Src and MAPK kinases participate in M6a signal propagation.

Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells

N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in human embryonic kidney 293 (HEK293) cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.

Alternatively spliced Tissue Factor: discovery, insights, clinical implications

Following the molecular cloning of human Tissue Factor (TF) in mid-1980's, great strides have been made in the understanding of TF biology, TF's crucial roles in the initiation of blood coagulation and embryonic development, and TF's contribution to the pathobiology of various disease states. The 21st century brought about a rather unexpected turn in the "TF journey"--a few years back it was reported that the TF gene produces not one, but two proteins with distinct structural and functional characteristics. The so-called "full-length TF" (flTF) - a much-studied integral membrane glycoprotein long presumed to be, and experimentally handled as "the TF" in hundreds of laboratories around the world - is now known to be one of the two TF forms naturally occurring in humans as well as mice. The other, recently discovered form is termed alternatively spliced TF (asTF) which, unlike flTF, lacks a transmembrane domain and can thus be secreted. In this review, we summarize the literature on asTF by discussing asTF's biologic roles as they are currently understood, tackling a number of questions pertaining to asTF's evident and proposed biologic properties, and briefly covering the emerging field of regulated TF pre-mRNA processing.

Cell surface targeting of myelin oligodendrocyte glycoprotein (MOG) in the absence of endoplasmic reticulum molecular chaperones

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.

Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor

BackgroundTissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371–3382). MethodsIn this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein. Results and conclusionsFractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form. ConclusionThese observations may attribute to the difference in the ability of TF–FVIIa complex to activate factor X (FX). General significanceStructural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF.

Scavenger receptor BI: a multi-purpose player in cholesterol and steroid metabolism.

Scavenger receptor class B type I (SR-BI) is an important member of the scavenger receptor family of integral membrane glycoproteins. This review highlights studies in SR-BI knockout mice, which concern the role of SR-BI in cholesterol and steroid metabolism. SR-BI in hepatocytes is the sole molecule involved in selective uptake of cholesteryl esters from high-density lipoprotein (HDL). SR-BI plays a physiological role in binding and uptake of native apolipoprotein B (apoB)-containing lipoproteins by hepatocytes, which identifies SR-BI as a multi-purpose player in lipid uptake from the blood circulation into hepatocytes in mice. In adrenocortical cells, SR-BI mediates the selective uptake of HDL-cholesteryl esters, which is efficiently coupled to the synthesis of glucocorticoids (i.e. corticosterone). SR-BI knockout mice suffer from adrenal glucocorticoid insufficiency, which suggests that functional SR-BI protein is necessary for optimal adrenal steroidogenesis in mice. SR-BI in macrophages plays a dual role in cholesterol metabolism as it is able to take up cholesterol associated with HDL and apoB-containing lipoproteins and can possibly facilitate cholesterol efflux to HDL. Absence of SR-BI is associated with thrombocytopenia and altered thrombosis susceptibility, which suggests a novel role for SR-BI in regulating platelet number and function in mice. Transgenic expression of cholesteryl ester transfer protein in humanized SR-BI knockout mice normalizes hepatic delivery of HDL-cholesteryl esters. However, other pathologies associated with SR-BI deficiency, i.e. increased atherosclerosis susceptibility, adrenal glucocorticoid insufficiency, and impaired platelet function are not normalized, which suggests an important role for SR-BI in cholesterol and steroid metabolism in man. In conclusion, generation of SR-BI knockout mice has significantly contributed to our knowledge of the physiological role of SR-BI. Studies using these mice have identified SR-BI as a multi-purpose player in cholesterol and steroid metabolism because it has distinct roles in reverse cholesterol transport, adrenal steroidogenesis, and platelet function.

Evaluation of Non-Proteolytic Functions of Murine Alternatively Spliced Tissue Factor.

AbstractAbstract 1135 Background.Aside from hemostatic maintenance, Tissue Factor (TF) also plays a major role in such pathophysiological processes as thrombogenesis and cancer progression. TF protein occurs naturally in two forms: full length TF (flTF), a well–studied integral membrane glycoprotein that serves as an obligatory enzymatic co-factor of the serine protease FVIIa, and alternatively spliced TF (asTF), which lacks a transmembrane domain and can be secreted. asTF has a unique 40 amino acid C-terminus and can be detected, alongside flTF, in organized arterial thrombi (Bogdanov et al, Nat Med 2003 Apr; 9(4):458-62). Following the discovery of human asTF, the murine form of asTF (masTF) was identified and characterized (Bogdanov et al, J Thromb Haemost. 2006 Jan;4(1):158-67). Like human asTF, masTF lacks a transmembrane domain due to the exclusion of exon 5 from the primary transcript during its splicing, possesses a unique 93 amino acid C-terminus, and exhibits minimal coagulant potential. Most recently, hasTF was discovered to induce cell adhesion and angiogenesis via integrin ligation, independent of FVIIa, PAR-2 cleavage and/or any other proteolytic events (van den Berg et al, Proc Natl Acad Sci U.S.A. 2009 Nov 17;106(46):19497-502). It has yet to be investigated whether masTF exhibits non-proteolytic biologic activity analogous to that of hasTF. As murine models comprise the preferred in vivo platform in preclinical cardiovascular and cancer research, it is highly warranted to ascertain these possible functional properties of masTF. In this study, we performed an initial set of experiments to address this issue. Results.N-terminally His-tagged recombinant masTF mature protein was generated in E. Coli, purified, and assessed by Coomassie staining; masTF's identity was successfully verified by western blotting. Analogously to hasTF, masTF induced adhesion of murine endothelial cells (bEnd.3) in a time-dependent fashion. A 15-fold increase over BSA (n = 3; p < 0.0001) was observed as early as 1 hour after the experiment's onset; at 4 hours, bEnd.3 cells exposed to masTF displayed a 37-fold increase over BSA (p < 0.0005). We noted that masTF also induced bEnd.3 cells to display characteristic endothelial morphology, whereas BSA did not. We subsequently used a wound healing assay to ascertain whether masTF promotes directional migration of bEnd.3 cells, and used VEGF (100 ng/mL) as a positive control to assess the degree of potency. Per wound, the area of complete closure generated by masTF (50 nM) was ∼4.9-fold greater than that of the vehicle (n = 3; p < 0.0001), an effect similar to that elicited by VEGF (area of closure ∼7-fold greater than that of the vehicle; p < 0.0001). Microarray analysis of masTF-treated bEnd.3 cells (Affymetrix Gene 1.0 ST platform) revealed upregulation of the genes encoding multiple CXC chemokines, with CXCL2, CXCL10, and CXCL1 topping the list (R = 1.42, 1.37, and 1.32, respectively). Notably, expression of the major adhesion molecule VCAM-1 was also upregulated (R = 1.3), suggesting that masTF may promote interactions between murine endothelial cells and leukocytes. Indeed, we found that stimulating bEnd.3 cells with 50 nM masTF caused J774A.1 cells (murine monocytes/macrophages) to adhere with a 75% increased affinity (n = 3; p = 0.0001 vs. vehicle) when exposed to orbital shear conditions. Depletion of masTF from the medium by Ni-charged beads and denaturation by heat eliminated increased monocyte adhesion, while addition of polymyxin B and non-charged beads had no effect, confirming that the observed biologic phenomena were elicited by the masTF protein. Conclusions.We report, for the first time, that murine asTF appears to possess non-proteolytic biologic properties analogous to those of human asTF, which indicates that the alternatively spliced TF may be a general cell agonist eliciting changes in gene expression via integrin ligation. We are currently investigating whether the endothelial surface molecules interacting with masTF, as well as the intracellular signaling pathways activated by masTF in endothelial cells, are analogous to those engaged by hasTF. Disclosures:No relevant conflicts of interest to declare.

Proteolysis-induced N-terminal Ectodomain Shedding of the Integral Membrane Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Is Accompanied by Tyrosine Phosphorylation of Its C-terminal Domain and Recruitment of Src and PKCδ

CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCdelta to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.

The physiological role of endoglin in the cardiovascular system

Endoglin (CD105) is an integral membrane glycoprotein that serves as a coreceptor for members of the transforming growth factor-β superfamily of proteins. A major role for endoglin in regulating transforming growth factor-β-dependent vascular remodeling and angiogenesis has been postulated based on the following: 1) endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1, a disease characterized by vascular malformations; 2) endoglin knockout mice die at midgestation because of defective angiogenesis; 3) endoglin is overexpressed in neoangiogenic vessels, during inflammation, and in solid tumors; and 4) endoglin regulates the expression and activity of endothelial nitric oxide synthase, which is involved in angiogenesis and vascular tone. Besides the predominant form of the endoglin receptor (long endoglin isoform), two additional forms of endoglin have been recently reported to play a role in the vascular pathology and homeostasis: the alternatively spliced short endoglin isoform and a soluble endoglin form that is proteolytically cleaved from membrane-bound endoglin. The purpose of this review is to underline the role that the different forms of endoglin play in regulating angiogenesis, vascular remodeling, and vascular tone, as well as to analyze the molecular and cellular mechanisms supporting these effects.

Study of potential inhibitors of thyroid iodide uptake by using CHO cells stably expressing the human sodium/iodide symporter (hNIS) protein

Thyroid gland is highly dependent on dietary intake of iodine for normal function, so it is particularly subjected to "endocrine disruptor" action. The human sodium/iodide symporter (hNIS) is an integral plasma membrane glycoprotein mediating the active transport of iodide into thyroid follicular cells, a crucial step for thyroid hormone biosynthesis. Beyond to perchlorate and thyocianate ions a few other inhibitors of iodide uptake have been described. The aim of this study was to investigate if 10 substances usually used as drugs in clinical practice were able to inhibit NIS-mediated iodide uptake in vitro. A CHO cell line stably expressing hNIS was used to test any inhibition of NIS-mediated iodide uptake exerted by drugs. Perchlorate and thyocianate ions were used as positive controls. None of the analyzed substances was able to significantly inhibit iodide uptake in our system. As we expected, perchlorate and thyocianate ions were able to inhibit iodide uptake in a dose-dependent manner. In conclusion, we carried out an in vitro assay to evaluate the potential inhibitory effect of common drugs on NISmediated iodide uptake by using CHO-hNIS cells. None of the analyzed substances was able to inhibit iodide uptake; only perchlorate and thyocianate were able to inhibit iodide uptake in a dose-dependent manner.

The gene structure and promoter region of the vaccine target aminopeptidase H11 from the blood-sucking nematode parasite of ruminants, Haemonchus contortus

Aminopeptidase H11, an integral membrane glycoprotein present only in the gut of Haemonchus contortus, could provide substantial protection as shown by 90% reduction in fecal egg counts, while its recombinant version expressed in E. coli induced little. To investigate the characteristics further, we amplified mRNA of H11 gene via reverse transcriptase polymerase chain reaction, followed by isolation of its 1,517-bp 5'-flanking region and determination of its genomic organization. The H11 gene contained 25 exons separated by 24 introns and spans 14,959 bp of genomic DNA. Analysis of the 1,517 bp 5'-flanking region of the H11 gene revealed a putative "TATA-less" promoter. Partial sequences of the last exon and its 3'-UTR of H11 isoform H11-4 were also identified upstream to the H11 gene with the same transcription orientation. The 1,517-bp 5'-flanking region and part of the first exon of the H11 gene were subcloned into the vector upstream of green fluorescence protein reporter gene and microinjected into the gonads of Caenorhabditis elegans. The transformed animals exhibited fluorescence in the distal intestine in the L4 larvae stage and adult worms. This study characterized gene structure of aminopeptidase H11, demonstrated different transcriptional pattern of its promoter region between free-living and blood-sucking nematode species, and highlights the utility of C. elegans as a heterologous system to study the biology roles of H11 isoforms.

Abstract 2244: Internalization of basigin-2, a poor prognosis marker, by a monoclonal antibody

Abstract Basigin-2 (EMMPRIN, CD147) is widely expressed on the surface of numerous tumor types and is associated with poor prognosis. Basigin is an integral membrane glycoprotein also expressed on numerous normal cell types. Several functions have been described for basigin-2 within both normal and malignant tissues. The best characterized is its ability to induce the expression of matrix metalloproteinases in stromal cells. As such, it is suggested to play a role in tumor metastasis. Because of its association with poor prognosis and possible role in metastasis, we examined the ability of basigin-2 to be targeted on the cell surface and internalized as a possible gateway for entry into tumor cells, or for down-regulation for inhibition of metastasis. Belton et al. have documented internalization of basigin-2 by a recombinant soluble protein corresponding to the extracellular domain (ECD) of basigin-2 (J Biol Chem, 2008, 283:17805). We have extended this investigation to examine antibody-mediated endocytosis. A monoclonal antibody to the basigin ECD (also reported in Belton et al.) has been used in an internalization assay using biotinylated antibody mixed with streptavidinylated-saporin (the ribosome-inactivating protein). This complex will eliminate cells only if there is antibody-mediated endocytosis, since saporin has no mechanism of cell entry. HeLa cells, which are known to express basigin-2, were challenged and a potent cytotoxicity was observed: the ED50 of the biotinylated antibody-streptavidin-saporin complex is 370 pM. This is evidence that the antibody internalizes in HeLa cells. Similar data were observed with ovarian carcinoma cells (OVCAR3, SKOV3). Non-tumor cells with low expression have low sensitivity to the complex and there was a correlation between basigin-2 expression on the cell surface and cytotoxicity. We conclude that basigin-2 internalizes in response to antibody binding. Further work is necessary to determine if this can be used for introduction of molecules into basigin-2-expressing cells or if down-regulation would have an effect on metastasis promoted by this poor prognosis molecule. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2244.