A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124.

(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 µM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.

Hypoxia increases cellular levels of phosphatidic acid and lysophospholipids in undifferentiated Caco-2 cells.

Cancer cells are known to survive in a hypoxic microenvironment by altering their lipid metabolism as well as their energy metabolism. In this study, Caco-2 cells derived from human colon cancer, were found to have elevated intracellular levels of phosphatidic acid and its lysoform, lysophosphatidic acid (LPA), under hypoxic conditions. Our results suggested that the elevation of LPA in Caco-2 cells was mainly due to the combined increases in cellular levels of lysophosphatidylcholine and lysophosphatidylethanolamine by phospholipase A2 and subsequent hydrolysis to LPA by lysophospholipase D. We detected the Ca2+ -stimulated choline-producing activities toward exogenous lysophosphatidylcholines in whole Caco-2 cell homogenates, indicating their involvement in the LPA production in intact Caco-2 cells.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

Selectin-Binding Assay by Flow Cytometry.

Cell-cell interactions mediated by selectins and their ligand glycans play pivotal roles in a variety of biological processes represented by leukocyte recruitment to inflammatory sites, lymphocyte homing, and extravasation of cancer cells. The interactions are enhanced at least partly through the upregulation of the selectin-ligand glycan expression, which is observed, for instance, during the activation of leukocytes or epithelial-mesenchymal transition of cancer cells. Selectin-binding assays such as cell adhesion assay or rolling assay have long been used to directly evaluate the activity of these cells in the selectin-mediated processes. In this chapter, we introduce a highly quantitative assay by flow cytometry using recombinant selectin-Ig(Fc) chimera proteins, showing our procedure and tips for E-selectin-binding assay of colon cancer cells undergoing epithelial-mesenchymal transition.

BGN/TLR4/NF-B Mediates Epigenetic Silencing of Immunosuppressive Siglec Ligands in Colon Cancer Cells.

Human Toll-like receptor (TLR) signaling plays a vital role in intestinal inflammation by activating the NF-B pathway. By querying GENT2 datasets, we identified the gene expression level of TLR2 and TLR4 as being substantially increased in colorectal cancer. Introduction of shRNAs for TLR4 but not TLR2 dramatically recovered disialyl Lewisa and sialyl 6-sulfo Lewisx glycans, which are preferentially expressed in non-malignant colonic epithelial cells and could serve as ligands for the immunosuppressive molecule Siglec-7. We screened several TLR4 ligands and found that among them BGN is highly expressed in cancers and is involved in the epigenetic silencing of Siglec-7 ligands. Suppression of BGN expression substantially downregulated NF-B activity and the marker H3K27me3 in the promoter regions of the SLC26A2 and ST6GalNAc6 genes, which are involved in the synthesis of those glycans, and restored expression of normal glycans as well as Siglec-7 binding activities. We show that in the presence of TLR4, inflammatory stimuli initiate a positive loop involving NF-B that activates BGN and further enhances TLR4 activity. Present findings indicate a putative mechanism for the promotion of carcinogenesis by loss of immunosuppressive ligands by the BGN/TLR4/ NF-B pathway.

SSEA3 and Sialyl Lewis a Glycan Expression Is Controlled by B3GALT5 LTR through Lamin A-NFYA and SIRT1-STAT3 Signaling in Human ES Cells.

B3GALT5 is involved in the synthesis of embryonic stem (ES) cell marker glycan, stage-specific embryonic antigen-3 (SSEA3). This gene has three native promoters and an integrated retroviral long terminal repeat (LTR) promoter. We found that B3GALT5-LTR is expressed at high levels in human ES cells. B3GALT5-LTR is also involved in the synthesis of the cancer-associated glycan, sialyl Lewis a. Sialyl Lewis a is expressed in ES cells and its expression decreases upon differentiation. Retinoic acid induced differentiation of ES cells, decreased the short form of NFYA (NFYAs), increased phosphorylation of STAT3, and decreased B3GALT5-LTR expression. NFYAs activated, and constitutively-active STAT3 (STAT3C) repressed B3GALT5-LTR promoter. The NFYAs and STAT3C effects were eliminated when their binding sites were deleted. Retinoic acid decreased the binding of NFYA to B3GALT5-LTR promoter and increased phospho-STAT3 binding. Lamin A repressed NFYAs and SSEA3 expression. SSEA3 repression mediated by a SIRT1 inhibitor was reversed by a STAT3 inhibitor. Repression of SSEA3 and sialyl Lewis a synthesis mediated by retinoic acid was partially reversed by lamin A short interfering RNA (siRNA) and a STAT3 inhibitor. In conclusion, B3GALT5-LTR is regulated by lamin A-NFYA and SIRT1-STAT3 signaling that regulates SSEA3 and sialyl Lewis a synthesis in ES cells, and sialyl Lewis a is also a ES cell marker.

Roles of p53 Family Structure and Function in Non-Canonical Response Element Binding and Activation.

The p53 canonical consensus sequence is a 10-bp repeat of PuPuPuC(A/T)(A/T)GPyPyPy, separated by a spacer with up to 13 bases. C(A/T)(A/T)G is the core sequence and purine (Pu) and pyrimidine (Py) bases comprise the flanking sequence. However, in the p53 noncanonical sequences, there are many variations, such as length of consensus sequence, variance of core sequence or flanking sequence, and variance in number of bases making up the spacer or AT gap composition. In comparison to p53, the p53 family members p63 and p73 have been found to have more tolerance to bind and activate several of these noncanonical sequences. The p53 protein forms monomers, dimers, and tetramers, and its nonspecific binding domain is well-defined; however, those for p63 or p73 are still not fully understood. Study of p63 and p73 structure to determine the monomers, dimers or tetramers to bind and regulate noncanonical sequence is a new challenge which is crucial to obtaining a complete picture of structure and function in order to understand how p63 and p73 regulate genes differently from p53. In this review, we will summarize the rules of p53 family non-canonical sequences, especially focusing on the structure of p53 family members in the regulation of specific target genes. In addition, we will compare different software programs for prediction of p53 family responsive elements containing parameters with canonical or non-canonical sequences.

Sialyl Glycan Expression on T Cell Subsets in Asthma: a correlation with disease severity and blood parameters.

Memory T helper (Th) and regulatory T (Treg) cells play key roles in asthma. Certain sialyl carbohydrate determinants for selectins profoundly affect the migratory properties of memory Th cells, and the suppressive function of Treg cells. Previous studies have shown that the proportion of CCR4+ memory Th cells expressing sialyl 6-sulfo Lewis X (LeX) is elevated in asthma patients. We aim to investigate the roles of different sialyl glycans on T cell subsets in asthma. Using flow cytometry, we assessed the expression of three sialyl glycans, sialyl 6-sulfo LeX, cyclic sialyl 6-sulfo LeX, and sialyl LeX on memory Th and Treg cells, in the peripheral blood of asthmatic children. We also assessed the relationships between glycan-expressing cell percentages and asthma clinical parameters. Compared with controls, asthmatic children showed higher proportions of memory Th cells expressing sialyl LeX and sialyl 6-sulfo LeX. The proportions of memory Th cells with sialyl 6-sulfo LeX and cyclic sialyl 6-sulfo LeX expression in asthmatic children correlated with absolute eosinophil count and IgE level, respectively. Children with moderate-to-severe asthma had lower numbers of sialyl LeX positive Treg cells. Our study suggests that sialyl glycans on T cells may play important roles in the pathogenesis of asthma.

Synergistic activation of the NEU4 promoter by p73 and AP2 in colon cancer cells.

More than 50% of colon cancers bear mutations in p53, one of the most important tumor suppressors, and its family members p63 or p73 are expected to contribute to inhibiting the progression of colon cancers. The AP2 family also acts as a tumor suppressor. Here we found that p73 and AP2 are able to activate NEU4, a neuraminidase gene, which removes the terminal sialic acid residues from cancer-associated glycans. Under serum starvation, NEU4 was up-regulated and one of the NEU4 target glycans, sialyl Lewis X, was decreased, whereas p73 and AP2 were up-regulated. Sialyl Lewis X levels were not, however, decreased under starvation conditions in p73- or AP2-knockdown cells. p53 and AP2 underwent protein-protein interactions, exerting synergistic effects to activate p21, and interaction of p53 with AP2 was lost in cells expressing the L350P mutation of p53. The homologous residues in p63 and p73 are L423 and L377, respectively. The synergistic effect of p53/p63 with AP2 to activate genes was lost with the L350P/L423P mutation in p53/p63, but p73 bearing the L377P mutation was able to interact with AP2 and exerted its normal synergistic effects. We propose that p73 and AP2 synergistically activate the NEU4 promoter in colon cancer cells.

Epigenetic silencing of the synthesis of immunosuppressive Siglec ligand glycans by NF-κB/EZH2/YY1 axis in early-stage colon cancers.

Normal colonic epithelial cells express sialyl 6-sulfo Lewisx and disialyl Lewisa on their cell surface, which are ligands for the immunosuppressive molecule Siglec-7. Expression of these normal glycans is frequently lost upon malignant transformation by silencing DTDST and ST6GalNAc6 at the early stage of colorectal carcinogenesis, and leads to production of inflammatory mediators that facilitate carcinogenesis. Indeed, by querying The Cancer Genome Atlas datasets, we confirmed that the level of DTDST or ST6GalNAc6 mRNA is substantially decreased at the early stage of colorectal carcinogenesis. Cultured colon cancer cell lines were used in this study including DLD-1, HT-29, LS174T and SW620. Their promoter regions were strongly marked by repressive mark H3K27me3, catalyzed by EZH2 that was markedly upregulated in early stage of colorectal carcinogenesis. Suppression of EZH2 substantially downregulated H3K27me3 mark and upregulated DTDST and ST6GalNAc6 as well as expression of normal glycans and Siglec-binding activities. Transcription factor YY1 was vital for the recruitment of PRC2-containing EZH2 to both promoters. Inhibition of NF-κB substantially reduced EZH2 transcription and restored their mRNAs as well as the production of normal Siglec ligand glycans in the results obtained from in vitro studies on cultured colon cancer cell lines. These findings provide a putative mechanism for promotion of carcinogenesis by loss of immunosuppressive molecules by epigenetic silencing through NF-κB-mediated EZH2/YY1 axis.

BLIMP1 transcriptionally induced by EGFR activation and post-translationally regulated by proteasome and lysosome is involved in keratinocyte differentiation, migration and inflammation.

BACKGROUND: B lymphocyte-induced maturation protein-1 (BLIMP1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of BLIMP1 expression in keratinocytes and crosstalk between EGFR and BLIMP1 in skin homeostasis. OBJECTIVE: The aim of the study was to investigate the regulation and functional link between EGFR and BLIMP1 in human epidermal keratinocytes. METHODS: Immunoblotting and Q-PCR were used to determine the molecular mechanism of BLIMP1 expression induced by EGFR in primary human epidermal keratinocytes (NHEK) and HaCaT cells. In functional assay, effects of BLIMP1 knockdown on EGFR-mediated cytokine production, differentiation, and migration in NHEK were evaluated by Q-PCR, ELISA, immunoblotting, and/or wound-healing assay. RESULTS: EGFR activation by EGFR ligands could upregulate the protein and mRNA levels of BLIMP1 in NHEK and HaCaT cells. This effect was dependent on PKC, p38, and ERK activation. Additionally, the stability of BLIMP1 protein was under the control of the proteasome and lysosome degradation systems. EGF could also upregulate BLIMP1 expression in skin squamous cell carcinomas. In addition, BLIMP1 knockdown enhanced the EGFR-mediated IL8, CXCL5 and IL6 gene expression and keratinocyte migration, but reduced the EGFR-mediated suppression of differentiation marker K10. CONCLUSIONS: Our findings shed new insights into the regulation of BLIMP1 expression by EGFR-mediated gene transcription and proteasome/lysosome-mediated degradation in keratinocytes. Functionally, BLIMP1 is a negative regulator of EGF-induced inflammation and migration in keratinocytes, and exerts a gene-specific regulation on keratinocyte differentiation.

Distinct substrate specificities of human GlcNAc-6-sulfotransferases revealed by mass spectrometry-based sulfoglycomic analysis.

Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O-sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer-specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2-based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2-based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1- and core 2-based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3-)ß1-3Galß1-4GlcNAc(6-SO3-)ß1-3Galß1-3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.

Gangliosides and Tumors.

Tumor-associated gangliosides play important roles in regulation of signal transduction induced by growth-factor receptors including EGFR, FGFR, HGF and PDGFR in a specific microdomain called glycosynapse in the cancer cell membranes, and in interaction with glycan recognition molecules involved in cell adhesion and immune regulation including selectins and siglecs. As the genes involved in the synthesis and degradation of tumor-associated gangliosides were identified, biological functions became clearer from the experimental results employing forced overexpression and/or knockdown/knockout of the genes. Studies on the regulatory mechanisms for their expression also achieved great advancements. Epigenetic silencing of glycan-related genes is a dominant mechanism in glycan alteration at early stages of carcinogenesis. Development of hypoxia resistance involving activation of a transcription factor HIF, and acquisition of cancer stem cell-like characteristics through epithelial-mesenchymal transition are important mechanisms for glycan modulations in the later stages of cancer progression. In the initial stages of studies, the gangliosides which specifically appear in cancers attracted attention under the name of tumor-associated gangliosides. However, it became apparent that not only the cancer-associated gangliosides but also the normal gangliosides present in nonmalignant cells and tissues perform important biological functions, and some of them tend to disappear in cancer cells resulting in the loss of the physiological functions, and this sometimes facilitates progression of cancers.

Inhibition of Endothelial SCUBE2 (Signal Peptide-CUB-EGF Domain-Containing Protein 2), a Novel VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) Coreceptor, Suppresses Tumor Angiogenesis.

OBJECTIVE: SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), expressed on the endothelial cell surface, functions as a novel coreceptor for VEGFR2 (vascular endothelial growth factor receptor 2) and enhances VEGF-induced signaling in adult angiogenesis. However, whether SCUBE2 plays a role in pathological angiogenesis and whether anti-SCUBE2 antibody is an effective strategy for blocking tumor angiogenesis remain unknown. The aim of this study was to investigate the pathological role and targeting therapy of SCUBE2 in tumor vasculature. APPROACH AND RESULTS: Immunohistochemistry revealed that SCUBE2 is highly expressed in endothelial cells of numerous carcinomas. Genetic endothelial cell knockout of SCUBE2 and pharmacological inhibition with the anti-SCUBE2 monoclonal antibody SP.B1 significantly reduced xenograft tumor growth, decreased tumor vascular density, increased apoptosis, and decreased the proliferation of tumor cells. Mechanistic studies revealed that SP.B1 binds to SCUBE2 and induces its internalization for lysosomal degradation, thereby reducing its cell surface level and inhibiting the binding of and downstream signaling of VEGF, including VEGFR2 phosphorylation and AKT/MAPK (mitogen-activated protein kinase) activation. Importantly, dual combination therapy with anti-SCUBE2 monoclonal antibody and anti-VEGF antibody or chemotherapy was more effective than single-agent therapy. CONCLUSIONS: Endothelial cell surface SCUBE2 is a VEGFR2 coreceptor essential for pathological tumor angiogenesis, and anti-SCUBE2 monoclonal antibody acting as an internalization inducer may provide a potent combination therapy for tumor angiogenesis.

Concerted mass spectrometry-based glycomic approach for precision mapping of sulfo sialylated N-glycans on human peripheral blood mononuclear cells and lymphocytes.

Despite well-recognized biological importance, mass spectrometry (MS)-based glycomic identification of sulfo-, sialylated terminal glyco-epitopes on the N-glycans of various immune cell types remains technically challenging and rarely reported. Previous studies with monoclonal antibody have implicated a regulated expression of 6-sulfo-α2-6-sialyl LacNAc on B cells in peripheral lymph nodes and the circulating peripheral blood lymphocytes but its occurrence on leukemia cells or lymphomas have not been critically addressed. In this study, we have extended our previously developed MS-based sulfoglycomic platform by incorporating additional complementary analytical approaches in order to achieve a high sensitivity mapping and relative quantification of the detected sulfated glycotopes down to the level of defining their sialyl linkages. We showed that discovery mode sulfoglycomics and precise location of sulfate were best achieved by multimode MS analyses of fractionated, permethylated sulfated N-glycans. On the other hand, the relative degree of sulfation on individual N-glycans could be more efficiently inferred from the respective extracted ion chromatograms of native, non-sulfated and sulfated target N-glycans in single LC-MS/MS runs. The GlcNAc-6-O-sulfated α2-6-sialyl LacNAc, which constitutes the higher affinity ligand for the human inhibitory co-receptor of B cells, CD22, was found to be commonly carried on a range of complex type N-glycans from human CD19+ and CD4+ lymphocytes. We further showed that its occurrence on the most abundant α2-6-disialylated biantennary structure from the peripheral blood mononuclear cells of patients diagnosed as B-cell chronic lymphocytic leukemia varied within ±2-fold abundance from the mean value determined for isolated CD19+ lymphocytes and cultured B-CLL cells.

FUT8 promotes breast cancer cell invasiveness by remodeling TGF-ß receptor core fucosylation.

BACKGROUND: Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-ß signaling during breast cancer progression remains largely unknown. METHOD: In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-ß-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-ß1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model. RESULTS: Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-ß-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-ß receptor complexes might be core fucosylated by FUT8 to facilitate TGF-ß binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis. CONCLUSIONS: Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-ß signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.

Downregulation of miR-199a/b-5p is associated with GCNT2 induction upon epithelial-mesenchymal transition in colon cancer.

ß-1,6-N-acetylglucosaminyltransferase 2 (GCNT2), which encodes a key glycosyltransferase for blood group I antigen synthesis, is induced upon epithelial-mesenchymal transition (EMT). Our results indicate that GCNT2 is upregulated upon EMT induced with epidermal growth factor and basic FGF in cultured human colon cancer cells. GCNT2 knockdown or overexpression decreases or increases, respectively, malignancy-related characteristics of colon cancer cells and I antigen levels. MiR-199a/b-5p is markedly downregulated upon EMT in colon cancer cells. Here, we find that miR-199a/b-5p consistently regulates GCNT2 expression in reporter assays and that it binds directly to the GCNT2 3' untranslated region intracellularly in RNA-induced silencing complex-trap assays. Overexpression of miR-199a/b-5p decreases GCNT2 expression and suppresses I antigen production. Based on these findings, we propose that miR-199a/b-5p regulates GCNT2 and I antigen expression in colon cancer cells undergoing EMT.

Endothelial SCUBE2 Interacts With VEGFR2 and Regulates VEGF-Induced Angiogenesis.

OBJECTIVE: Vascular endothelial growth factor (VEGF), a major mediator of angiogenesis, exerts its proangiogenic action by binding to VEGFR2 (VEGF receptor 2), the activity of which is further modulated by VEGFR2 coreceptors such as neuropilins. However, whether VEGFR2 is regulated by additional coreceptors is not clear. To investigate whether SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), a peripheral membrane protein expressed in vascular endothelial cells (ECs) known to bind other signaling receptors, functions as a VEGFR2 coreceptor and to verify the role of SCUBE2 in the VEGF-induced angiogenesis. APPROACH AND RESULTS: SCUBE2 lentiviral overexpression in human ECs increased and short hairpin RNA knockdown inhibited VEGF-induced EC growth and capillary-like network formation on Matrigel. Like VEGF, endothelial SCUBE2 was upregulated by hypoxia-inducible factor-1α at both mRNA and protein levels. EC-specific Scube2 knockout mice were not defective in vascular development but showed impaired VEGF-induced neovascularization in implanted Matrigel plugs and recovery of blood flow after hind-limb ischemia. Coimmunoprecipitation and ligand-binding assays showed that SCUBE2 forms a complex with VEGF and VEGFR2, thus acting as a coreceptor to facilitate VEGF binding and augment VEGFR2 signal activity. SCUBE2 knockdown or genetic knockout suppressed and its overexpression promoted the VEGF-induced activation of downstream proangiogenic and proliferating signals, including VEGFR2 phosphorylation and mitogen-activated protein kinase or AKT activation. CONCLUSIONS: Endothelial SCUBE2 may be a novel coreceptor for VEGFR2 and potentiate VEGF-induced signaling in adult angiogenesis.

Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain.

Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with α2,3-sialylated and α1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLex). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of α2,3-sialic acid and α1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLex fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more α2,3-sialylated and α1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.