脊椎动物趋化因子受体CXCR基因分化研究

为了探讨脊椎动物CXCR(CXC chemokine receptor)在基因组上进化和分化的规律,采用生物信息学软件绘制了脊椎动物7个CXCR的基因结构图,分析了它们的系统进化关系,研究这些受体在不同物种基因同源性.结果表明,脊椎动物CXCR 7个成员在进化上发生了不同程度地分化.人、鼠、蜥蜴CXCR1和CXCR2在同一条染色体上,蛋白相似率很高,在进化树上混杂聚集在一起,形成CXCR1/2;而硬骨鱼类CXCR1和CXCR2发生了分化,形成了单独的CXCR1和CXCR2.鱼类CXCR3分化为3个基因,其中CXCR3b1、CXCR3b2与其他脊椎动物CXCR3同源性较高,聚集在一起,而CXCR3a分化较大.脊椎动物CXCR5和CXCR6基因较保守,基因同源性高.CXCR4和CXCR7在哺乳类、鸟类、爬行类和两栖类均仅一个基因,但在硬骨鱼类中它们各自分化为2个基因.CXCR4和CXCR7位于同一条染色体上.鱼类CXCR4a和CXCR7a与其他脊椎动物CXCR4和CXCR7基因同源性较高.而CXCR4b和CXCR7b这两个基因无论是从基因结构还是基因同源性上都发生了一定程度的分化.     

Chemokine Receptor CXCR7 Is a Functional Receptor for CXCL12 in Brain Endothelial Cells

The chemokine CXCL12 regulates multiple cell functions through its receptor, CXCR4. However, recent studies have shown that CXCL12 also binds a second receptor, CXCR7, to potentiate signal transduction and cell activity. In contrast to CXCL12/CXCR4, few studies have focused on the role of CXCR7 in vascular biology and its role in human brain microvascular endothelial cells (HBMECs) remains unclear. In this report, we used complementary methods, including immunocytofluorescence, Western blot, and flow cytometry analyses, to demonstrate that CXCR7 was expressed on HBMECs. We then employed short hairpin RNA (shRNA) technology to knockdown CXCR7 in HBMECs. Knockdown of CXCR7 in HBMECs resulted in significantly reduced HBMEC proliferation, tube formation, and migration, as well as adhesion to matrigel and tumor cells. Blocking CXCR7 with a specific antibody or small molecule antagonist similarly disrupted HBMEC binding to matrigel or tumor cells. We found that tumor necrosis factor (TNF)-α induced CXCR7 in a time and dose-response manner and that this increase preceded an increase in vascular cell adhesion molecule-1 (VCAM-1). Knockdown of CXCR7 resulted in suppression of VCAM-1, suggesting that the reduced binding of CXCR7-knockdown HBMECs may result from suppression of VCAM-1. Collectively, CXCR7 acted as a functional receptor for CXCL12 in brain endothelial cells. Targeting CXCR7 in tumor vasculature may provide novel opportunities for improving brain tumor therapy.     

The Cell Surface Receptor Tartan Is a Potential In Vivo Substrate for the Receptor Tyrosine Phosphatase Ptp52F

Receptor-linked protein-tyrosine phosphatases (RPTPs) are essential regulators of axon guidance and synaptogenesis in Drosophila, but the signaling pathways in which they function are poorly defined. We identified the cell surface receptor Tartan (Trn) as a candidate substrate for the neuronal RPTP Ptp52F by using a modified two-hybrid screen with a substrate-trapping mutant of Ptp52F as “bait.” Trn can bind to the Ptp52F substrate-trapping mutant in transfected Drosophila S2 cells if v-Src kinase, which phosphorylates Trn, is also expressed. Coexpression of wild-type Ptp52F causes dephosphorylation of v-Src-phosphorylated Trn. To examine the specificity of the interaction in vitro, we incubated Ptp52F-glutathione S-transferase (GST) fusion proteins with pervanadate-treated S2 cell lysates. Wild-type Ptp52F dephosphorylated Trn, as well as most other bands in the lysate. GST “pulldown” experiments demonstrated that the Ptp52F substrate-trapping mutant binds exclusively to phospho-Trn. Wild-type Ptp52F pulled down dephosphorylated Trn, suggesting that it forms a stable Ptp52F-Trn complex that persists after substrate dephosphorylation. To evaluate whether Trn and Ptp52F are part of the same pathway in vivo, we examined motor axon guidance in mutant embryos. trn and Ptp52F mutations produce identical phenotypes affecting the SNa motor nerve. The genes also display dosage-dependent interactions, suggesting that Ptp52F regulates Trn signaling in SNa motor neurons.Receptor-linked protein-tyrosine phosphatases (RPTPs) are enzymes with extracellular (XC) domains, a single transmembrane domain, and one or two cytoplasmic protein tyrosine phosphatase (PTP) homology domains. Many RPTPs have XC sequences that resemble those of cell adhesion molecules (for a review, see reference 33). This sequence organization suggests that RPTPs can couple cell-cell recognition events to dephosphorylation of cytoplasmic substrates. Interestingly, while phosphotyrosine (PY) pathways involved in cell growth and differentiation typically involve receptor tyrosine kinases that bind to growth factors and are regulated by nontransmembrane PTPs, those that control axon guidance often use RPTPs and nontransmembrane TKs. This implies that the cues that affect PY signaling in axonal growth cones may interact with RPTPs rather than with receptor tyrosine kinases (reviewed in reference 14).There are 17 active RPTPs encoded in the human genome, while Drosophila has six. Most of the mammalian RPTPs are expressed in nonneural tissues, but four of the six fly RPTPs are expressed only by central nervous system (CNS) neurons in late embryos. All published zygotic phenotypes produced by Rptp mutations are alterations in axon guidance or synaptogenesis. These results suggest that the major functions of the Drosophila RPTPs are in neural development (for a review, see reference 16). Analysis of axon guidance phenotypes in embryos bearing single or multiple Rptp mutations is consistent with the idea that RPTP interactions with ligands at growth cone choice points convey “information,” in the form of changes in substrate phosphorylation within growth cones, that is used to determine pathway decisions.In the Drosophila neuromuscular system, 36 motor axons grow out within six nerve bundles in each abdominal hemisegment, and each axonal growth cone makes a series of genetically determined guidance decisions that direct it to the appropriate muscle fiber (for a review, see reference 27). Our work on Rptp mutant combinations suggests that each pathway decision uses a specific subset of the six RPTPs. RPTPs can exhibit functional redundancy, so that the loss of one does not produce a defect unless another RPTP is also absent, or competition, in which loss of one RPTP suppresses the phenotype produced by loss of another (5, 6, 31). Examination of RPTP expression patterns suggests that the RPTPs are expressed by most (or possibly all) CNS neurons, including motor neurons. If so, the requirements for individual RPTPs for execution of particular guidance decisions cannot be due to selective expression of these RPTPs on specific motor axons. These requirements might instead be determined by the expression patterns of RPTP ligands, so that only RPTPs whose ligands were localized to the vicinity of a growth cone choice point would participate in that pathway decision. Alternatively (or in addition), the necessity of a particular RPTP for a pathway decision might arise from selective expression of RPTP substrates, so that an RPTP would be important for guidance decisions made by a growth cone of a specific motor neuron only if that neuron expressed the relevant substrate(s).Evaluation of such models requires identification of specific XC ligands and intracellular substrates for the Drosophila RPTPs. Only one set of ligands has been identified thus far. These are the heparan sulfate proteoglycans Syndecan (Sdc) and Dallylike (Dlp), which bind to the Lar RPTP with nanomolar affinity and contribute to its functions in axon guidance and synapse growth (9, 15). Similarly, little is known about substrate specificity in vivo. Lar can dephosphorylate the Enabled (Ena) protein, which regulates the growth cone cytoskeleton, and genetic interaction studies suggest that Ena may be an in vivo substrate for Lar (35). The transmembrane protein gp150 can be dephosphorylated by Ptp10D in cell culture and intact fly larvae, but genetics has not provided evidence that Ptp10D and gp150 are in the same signaling pathway in vivo (7).The identification of in vivo substrates for RPTPs has been hampered by the fact that purified RPTP cytoplasmic domains often do not exhibit high selectivity in vitro when tested for dephosphorylation activity on peptides or proteins. The most fruitful method for finding substrates for both RPTPs and cytoplasmic PTPs has been the use of “substrate-trapping” mutants. The most effective substrate traps were devised by Tonks and coworkers, and are created by changing an invariant Asp (D) residue within the PTP active site to Ala (A) (8). The D residue has an abnormal pK and is thus able to donate a proton to the phosphorus-oxygen bond, facilitating displacement of the tyrosine (Y) OH by the invariant Cys (C) nucleophile of the enzyme. This creates a phosphoenzyme intermediate. The dephosphorylated substrate then dissociates, and water attacks the Cys-phosphate bond, releasing the phosphate and reconstituting the enzyme. In D→A mutants, the polarization of the phosphorus-oxygen bond by protonation cannot take place, and the PY substrate remains bound to the enzyme. Substrate-trapping mutants expressed in cells often bind to only a few phosphoproteins, suggesting that PTPs exhibit high specificity in vivo (see, for example, reference 11).We conducted a modified yeast two-hybrid screen to find Drosophila phosphoproteins that bind selectively to RPTP substrate-trapping mutants. We identified the cell surface receptor Tartan (Trn) in this screen and showed that it is a substrate for the Ptp52F RPTP in Drosophila Schneider 2 (S2) cells. Axon guidance phenotypes in trn mutants are identical to those seen in Ptp52F mutants, and trn and Ptp52F exhibit dosage-dependent genetic interactions. These results suggest that Ptp52F is a regulator of Trn signaling in motor neurons in vivo.     

The E3 Ubiquitin Ligase Atrophin Interacting Protein 4 Binds Directly To The Chemokine Receptor CXCR4 Via a Novel WW Domain-mediated Interaction

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.     

Upregulated Expression of C-X-C Chemokine Receptor 4 Is an Independent Prognostic Predictor for Patients with Gastric Cancer

Aberrant chemokine (C-X-C motif) receptor CXCR4 expressions in malignant tissues have been reported, but its role in gastric cancer prognosis remains unknown. Our studies were designed to investigate the expression and prognostic significance of CXCR4 in patients with gastric cancer. CXCR4 expression was retrospectively analyzed by immunohistochemistry in 97 patients with gastric adenocarcinoma from China. Results were assessed for association with clinical features and overall survival by using Kaplan-Meier analysis. Prognostic values of CXCR4 expression and clinical outcomes were evaluated by Cox regression analysis. A molecular prognostic stratification scheme incorporating CXCR4 expression was determined by using receiver operating characteristic (ROC) analysis. The results show that CXCR4 predominantly localized in the cell membranes and cytoplasm. The protein level of CXCR4 was upregulation in gastric cancer tissues and upregulated expression of CXCR4 was only significantly associated with Lauren classification (P<0.001). Increased CXCR4 expression in gastric cancer tissues was positively correlated with poor overall survival of gastric cancer patients (P<0.001). Further multivariate Cox regression analysis suggested that intratumoral CXCR4 expression was an independent prognostic indicator for the disease. Applying the prognostic value of intratumoral CXCR4 density to TNM stage system showed a better prognostic value in patients with gastric cancer. In conclusion, intratumoral CXCR4 expression was recognized as an independent prognostic marker for the overall survival of patients with gastric cancer. On the basis of TNM stage, detection of CXCR4 expression will be helpful for predicting prognosis for patients with gastric cancer.     

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

Background

The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/Principal Findings

We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping.

Conclusions/Significance

We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.     

A Putative Cell Surface Receptor for Anemia-Inducing Feline Leukemia Virus Subgroup C Is a Member of a Transporter Superfamily

Domestic cats infected with the horizontally transmitted feline leukemia virus subgroup A (FeLV-A) often produce mutants (termed FeLV-C) that bind to a distinct cell surface receptor and cause severe aplastic anemia in vivo and erythroblast destruction in bone marrow cultures. The major determinant for FeLV-C-induced anemia has been mapped to a small region of the surface envelope glycoprotein that is responsible for its receptor binding specificity. Thus, erythroblast destruction may directly or indirectly result from FeLV-C binding to its receptor. To address these issues, we functionally cloned a putative cell surface receptor for FeLV-C (FLVCR) by using a human T-lymphocyte cDNA library in a retroviral vector. Expression of the 2.0-kbp FLVCR cDNA in naturally resistant Swiss mouse fibroblasts and Chinese hamster ovary cells caused substantial susceptibility to FeLV-C but no change in susceptibilities to FeLV-B and other retroviruses. The predicted FLVCR protein contains 555 amino acids and 12 hydrophobic potential membrane-spanning sequences. Database searches indicated that FLVCR is a member of the major-facilitator superfamily of transporters and implied that it may transport an organic anion. RNA blot analyses showed that FLVCR mRNA is expressed in multiple hematopoietic lineages rather than specifically in erythroblasts. These results suggest that the targeted destruction of erythroblasts by FeLV-C may derive from their greater sensitivity to this virus rather than from a preferential susceptibility to infection.     

Cell Surface Estrogen Receptor Alpha Is Upregulated during Subchronic Metabolic Stress and Inhibits Neuronal Cell Degeneration

In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER) has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP), a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2) was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation) compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a powerful cell-survival signal. These results shed new light on the pathogenetic mechanisms leading to neuronal cell degeneration.     

Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface,Promoting Oligomerization,and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.     

Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8^{- /-}) and wild-type (WT) mice. We found that CCR8^-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca²⁺ flux and CCL1-driven PMφ aggregation. Similar to CCR8^-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8^-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8^-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8^-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.     

Identification of a Receptor for Extracellular Renalase

Background

An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI) and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI) in wild type (WT) mice. Therefore, we sought to identity the receptor for extracellular renalase.

Methods and Results

RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.

Conclusions

PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.     

Ganglioside GT1b Is a Putative Host Cell Receptor for the Merkel Cell Polyomavirus

The Merkel cell polyomavirus (MCPyV) was identified recently in human Merkel cell carcinomas, an aggressive neuroendocrine skin cancer. Here, we identify a putative host cell receptor for MCPyV. We found that recombinant MCPyV VP1 pentameric capsomeres both hemagglutinated sheep red blood cells and interacted with ganglioside GT1b in a sucrose gradient flotation assay. Structural differences between the analyzed gangliosides suggest that MCPyV VP1 likely interacts with sialic acids on both branches of the GT1b carbohydrate chain. Identification of a potential host cell receptor for MCPyV will aid in the elucidation of its entry mechanism and pathophysiology.Members of the polyomavirus (PyV) family, including simian virus 40 (SV40), murine PyV (mPyV), and BK virus (BKV), bind cell surface gangliosides to initiate infection (2, 8, 11, 15). PyV capsids are assembled from 72 pentamers (capsomeres) of the major coat protein VP1, with the internal proteins VP2 and VP3 buried within the capsids (7, 12). The VP1 pentamer makes direct contact with the carbohydrate portion of the ganglioside (10, 12, 13) and dictates the specificity of virus interaction with the cell. Gangliosides are glycolipids that contain a ceramide domain inserted into the plasma membrane and a carbohydrate domain that directly binds the virus. Specifically, SV40 binds to ganglioside GM1 (2, 10, 15), mPyV binds to gangliosides GD1a and GT1b (11, 15), and BKV binds to gangliosides GD1b and GT1b (8).Recently, a new human PyV designated Merkel cell PyV (MCPyV) was identified in Merkel cell carcinomas, a rare but aggressive skin cancer of neuroendocrine origin (3). It is as yet unclear whether MCPyV is the causative agent of Merkel cell carcinomas (17). A key to understanding the infectious and transforming properties of MCPyV is the elucidation of its cellular entry pathway. In this study, we identify a putative host cell receptor for MCPyV.Because an intact infectious MCPyV has not yet been isolated, we generated recombinant MCPyV VP1 pentamers in order to characterize cellular factors that bind to MCPyV. VP1 capsomeres have been previously shown to be equivalent to virus with respect to hemagglutination properties (4, 16), and the atomic structure of VP1 bound to sialyllactose has demonstrated that the capsomere is sufficient for this interaction (12, 13). The MCPyV VP1 protein (strain w162) was expressed and purified as described previously (1, 6). Briefly, a glutathione S-transferase-MCPyV VP1 fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose affinity chromatography. The fusion protein was eluted using glutathione and cleaved in solution with thrombin. The thrombin-cleaved sample was then rechromatographed on a second glutathione-Sepharose column to remove glutathione transferase and any uncleaved protein. The unbound VP1 was then chromatographed on a P-11 phosphocellulose column, and peak fractions eluting between 400 and 450 mM NaCl were collected. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie blue staining (Fig. ​(Fig.1A,1A, left) and immunoblotting using an antibody (I58) that generally recognizes PyV VP1 proteins (Fig. ​(Fig.1A,1A, right) (9). Transmission electron microscopy (Philips CM10) analysis confirmed that the purified recombinant MCPyV VP1 formed pentamers (capsomeres), which did not assemble further into virus-like particles (Fig. ​(Fig.1B).1B). In an initial screening of its cell binding properties, we tested whether the MCPyV VP1 pentamers hemagglutinated red blood cells (RBCs). The MCPyV VP1 pentamers were incubated with sheep RBCs and assayed as previously described (5). SV40 and mPyV recombinant VP1 pentamers served as negative and positive controls, respectively. We found that MCPyV VP1 hemagglutinated the RBCs with the same efficiency as mPyV VP1 (protein concentration/hemagglutination unit) (Fig. ​(Fig.1C,1C, compare rows B and C from wells 1 to 11), suggesting that MCPyV VP1 engages a plasma membrane receptor on the RBCs. The recombinant murine VP1 protein used for comparison was from the RA strain, a small plaque virus (4). Thus, MCPyV VP1 has the hemagglutination characteristics of a small plaque mPyV (12, 13).Open in a separate windowFIG. 1.Characterization of MCPyV VP1. Recombinant MCPyV VP1 forms pentamers and hemagglutinates sheep RBCs. (A) Coomassie blue-stained SDS-PAGE and an immunoblot of the purified recombinant MCPyV VP1 protein are shown. Molecular mass markers are indicated. (B) Electron micrograph of the purified MCPyV VP1. MCPyV VP1 (shown in panel A) was diluted to 100 μg/ml and absorbed onto Formvar/carbon-coated copper grids. Samples were washed with phosphate-buffered saline, stained with 1% uranyl acetate, and visualized by transmission electron microscopy at 80 kV. Bar = 20 nm. (C) Sheep RBCs (0.5%) were incubated with decreasing concentrations of purified recombinant SV40 VP1 (row A), mPyV VP1 (row B), and MCPyV VP1 (row C). Wells 1 to 11 contain twofold serial dilutions of protein, starting at 2 μg/ml (well 1). Well 12 contains buffer only and serves as a negative control. Well 7 (rows B and C) corresponds to 128 hemagglutination units per 2 μg/ml VP1 protein.To characterize the chemical nature of the putative receptor for MCPyV, total membranes from RBCs were purified as described previously (15). The plasma membranes (30 μg) were incubated with MCPyV VP1 (0.5 μg) and floated on a discontinuous sucrose gradient (15). After fractionation, the samples were analyzed by SDS-PAGE, followed by immunoblotting with I58. VP1 was found in the bottom of the gradient in the absence of the plasma membranes (Fig. ​(Fig.2A,2A, first panel). In the presence of plasma membranes, a fraction of the VP1 floated to the middle of the gradient (Fig. ​(Fig.2A,2A, second panel), supporting the hemagglutination results that suggested that MCPyV VP1 binds to a receptor on the plasma membrane.Open in a separate windowFIG. 2.MCPyV VP1 binds to a protease-resistant, sialic acid-containing receptor on the plasma membrane. (A) Purified recombinant MCPyV VP1 was incubated with or without the indicated plasma membranes. The samples were floated in a discontinuous sucrose gradient, and the fractions were collected from the top of the gradient, subjected to SDS-PAGE, and immunoblotted with the anti-VP1 antibody I58. (B) Control and proteinase K-treated plasma membranes were subjected to SDS-PAGE, followed by Coomassie blue staining. (C) HeLa cells treated with proteinase K (4 μg/ml) were incubated with MCPyV at 4°C, and the resulting cell lysate was probed for the presence of MCPyV VP1. (D) As described in the legend to panel C, except 293T cells were used. (E) Purified MCPyV VP1 was incubated with plasma membranes pretreated with or without α2-3,6,8 neuraminidase and analyzed as described in the legend to panel A.To determine whether the receptor is a protein or a lipid, plasma membrane preparations (30 μg) were incubated with proteinase K (Sigma), followed by analysis with SDS-PAGE and Coomassie blue staining. Under these conditions, the majority of the proteins in the plasma membranes were degraded by the protease (Fig. ​(Fig.2B,2B, compare lanes 1 and 2). Despite the lack of proteins, the proteinase K-treated plasma membranes bound MCPyV VP1 as efficiently as control plasma membranes (Fig. ​(Fig.2A,2A, compare the second and third panels), demonstrating that MCPyV VP1 interacts with a protease-resistant receptor. The absence of VP1 in the bottom fraction in Fig. ​Fig.2A2A (third panel) is consistent with the fact that the buoyant density of the membranes is lowered by proteolysis. Of note, a similar result was seen with binding of the mPyV to the plasma membrane (15). Binding of MCPyV to the cell surface of two human tissue culture cells (i.e., HeLa and 293T) was also largely unaffected by pretreatment of the cells with proteinase K (Fig. 2C and D, compare lanes 1 and 2), further indicating that a nonproteinaceous molecule on the plasma membrane engages the virus.We next determined whether the protease-resistant receptor contains a sialic acid modification. Plasma membranes (10 μg) were incubated with a neuraminidase (α2-3,6,8 neuraminidase; Calbiochem) to remove the sialic acid groups. In contrast to the control plasma membranes, the neuraminidase-treated membranes did not bind MCPyV VP1 (Fig. ​(Fig.2E,2E, compare first and second panels), indicating that the MCPyV receptor includes a sialic acid modification.Gangliosides are lipids that contain sialic acid modifications. We asked if MCPyV VP1 binds to gangliosides similar to other PyV family members. The structures of the gangliosides used in this analysis (gangliosides GM1, GD1a, GD1b, and GT1b) are depicted in Fig. ​Fig.3A.3A. To assess a possible ganglioside-VP1 interaction, we employed a liposome flotation assay established previously (15). When liposomes (consisting of phosphatidyl-choline [19 μl of 10 mg/ml], -ethanolamine [5 μl of 10 mg/ml], -serine [1 μl of 10 mg/ml], and -inositol [3 μl of 10 mg/ml]) were incubated with MCPyV VP1 and subjected to the sucrose flotation assay, the VP1 remained in the bottom fraction (Fig. ​(Fig.3B,3B, first panel), indicating that VP1 does not interact with these phospholipids. However, when liposomes containing GT1b (1 μl of 1 mM), but not GM1 (1 μl of 1 mM) or GD1a (1 μl of 1 mM), were incubated with MCPyV VP1, the vesicles bound this VP1 (Fig. ​(Fig.3B).3B). A low level of virus floated partially when incubated with liposomes containing GD1b (Fig. ​(Fig.3B),3B), perhaps reflecting a weak affinity between MCPyV and GD1b. Importantly, MCPyV binds less efficiently to neuraminidase-treated GT1b-containing liposomes than to GT1b-containing liposomes (Fig. ​(Fig.3B,3B, sixth panel), suggesting that the GT1b sialic acids are involved in virus binding. This finding is consistent with the ability of neuraminidase to block MCPyV binding to the plasma membrane (Fig. ​(Fig.2E).2E). The level of virus flotation observed in the neuraminidase-treated GT1b-containing liposomes is likely due to the inefficiency of the neuraminidase reaction with a high concentration of GT1b used to prepare the vesicles.Open in a separate windowFIG. 3.MCPyV VP1 binds to ganglioside GT1b. (A) Structures of gangliosides GM1, GD1a, GD1b, and GT1b. The nature of the glycosidic linkages is indicated. (B) Purified MCPyV VP1 protein was incubated with liposomes only or with liposomes containing the indicated gangliosides. The samples were analyzed as described in the legend to Fig. ​Fig.2A.2A. Where indicated, GT1b-containing liposomes were pretreated with α2-3,6,8 neuraminidase and analyzed subsequently for virus binding. (C to E) The indicated viruses were incubated with liposomes and analyzed as described in the legend to panel B.As controls, GM1-containing liposomes bound SV40 (Fig. ​(Fig.3C),3C), GD1a-containing liposomes bound mPyV (Fig. ​(Fig.3D),3D), and GD1b-containing liposomes bound BKV (Fig. ​(Fig.3E),3E), demonstrating that the liposomes were functionally intact. We note that, while all of the MCPyV VP1 floated when incubated with liposomes containing GT1b (Fig. ​(Fig.3B,3B, sixth panel), a significant fraction of SV40, mPyV, and BKV VP1 remained in the bottom fraction despite being incubated with liposomes containing their respective ganglioside receptors (Fig. 3C to E, second panels). This result is likely due to the fact that in contrast to MCPyV, which are assembled as pentamers (Fig. ​(Fig.1B),1B), the SV40, mPyV, and BKV used in these experiments are fully assembled particles: their larger and denser nature prevents efficient flotation. Nonetheless, we conclude that MCPyV VP1 binds to ganglioside GT1b efficiently.The observation that GD1a does not bind to MCPyV VP1 suggests that the monosialic acid modification on the right branch of GT1b (Fig. ​(Fig.3A)3A) is insufficient for binding. Similarly, the failure of GD1b to bind MCPyV VP1 suggests that the sialic acid on the left arm of GT1b is necessary for binding. Together, these observations suggest that MCPyV VP1 interacts with sialic acids on both branches of GT1b (Fig. ​(Fig.4).4). A recent structure of SV40 VP1 in complex with the sugar portion of GM1 (10) demonstrated that although SV40 VP1 binds both branches of GM1 (Fig. ​(Fig.4),4), only a single sialic acid in GM1 is involved in this interaction. In the case of mPyV, structures of mPyV VP1 in complex with different carbohydrates (12, 13) revealed that the sialic acid-galactose moiety on the left branch of GD1a (and GT1b) is sufficient for mPyV VP1 binding (Fig. ​(Fig.4).4). Although no structure of BKV in complex with the sugar portion of GD1b (or GT1b) is available, in vitro binding studies (8) have suggested that the disialic acid modification on the right branch of GD1b (and GT1b) is responsible for binding BKV VP1 (Fig. ​(Fig.4).4). Thus, it appears that the unique feature of the MCPyV VP1-GT1b interaction is that the sialic acids on both branches of this ganglioside are likely involved in capsid binding.Open in a separate windowFIG. 4.A potential model of the different VP1-ganglioside interactions (see the text for discussion).The identification of a potential cellular receptor for MCPyV will facilitate the study of its entry mechanism. An important issue for further study is to determine whether MCPyV targets Merkel cells preferentially, and if so, whether GT1b is found in higher levels in these cells to increase susceptibility.     

Cell Surface Heparan Sulfate Is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8.1

An immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposi's sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1DeltaTMFc). K8.1DeltaTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1DeltaTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.     

A Model for Migratory B Cell Oscillations from Receptor Down-Regulation Induced by External Chemokine Fields

A long-standing paradigm in B cell immunology is that effective somatic hypermutation and affinity maturation require cycling between the dark zone and light zone of the germinal center. The cyclic re-entry hypothesis was first proposed based on considerations of the efficiency of affinity maturation using an ordinary differential equations model for B cell population dynamics. More recently, two-photon microscopy studies of B cell motility within lymph nodes in situ have revealed the complex migration patterns of B lymphocytes both in the preactivation follicle and post-activation germinal center. There is strong evidence that chemokines secreted by stromal cells and the regulation of cognate G-protein coupled receptors by these chemokines are necessary for the observed spatial cell distributions. For example, the distribution of B cells within the light and dark zones of the germinal center appears to be determined by the reciprocal interaction between the level of the CXCR4 and CXCR5 receptors and the spatial distribution of their respective chemokines CXCL12 and CXCL13. Computer simulations of individual-based models have been used to study the complex biophysical and mechanistic processes at the individual cell level, but such simulations can be challenging to parameterize and analyze. In contrast, ordinary differential equations are more tractable, but traditional compartment model formalizations ignore the spatial chemokine distribution that drives B cell redistribution. Motivated by the desire to understand the motility patterns observed in an individual-based simulation of B cell migration in the lymph node, we propose and analyze the dynamics of an ordinary differential equation model incorporating explicit chemokine spatial distributions. While there is experimental evidence that B cell migration patterns in the germinal center are driven by extrinsically regulated differentiation programs, the model shows, perhaps surprisingly, that feedback from receptor down-regulation induced by external chemokine fields can give rise to spontaneous interzonal and intrazonal oscillations in the absence of any extrinsic regulation. While the extent to which such simple feedback mechanisms contributes to B cell migration patterns in the germinal center is unknown, the model provides an alternative hypothesis for how complex B cell migration patterns might arise from very simple mechanisms.