Type-1 Repeats Research Articles

Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8β (C8β) in Nile tilapia (Oreochromis niloticus)

Nile tilapia (Oreochromis niloticus), one of the most important groups of food fishes in the world, has frequently suffered from serious challenge from pathogens in recent years. Immune responses of Nile tilapia should be understood to protect the aquaculture industry of this fish. The complement system has an important function in recognizing bacteria, opsonizing these pathogens by phagocytes, or killing them by direct lysis. In this study, two Nile tilapia complement component genes, complement component 1 inhibitor (C1INH) and complement component 8β subunit (C8β), were cloned and their expression characteristics were analyzed. C1INH cDNA was found containing a 1791 bp open reading frame (ORF) encoding a putative protein with 597 amino acids, a 101 bp 5′-untranslated region (UTR) and a 236 bp 3′-UTR. The predicted protein structure for this gene consisted of two Ig-like domains and glycosyl hydrolase family-9 active site signature 2. The C8β cDNA consisted of a 1761 bp ORF encoding 587 amino acids, a 15 bp 5′-UTR and a 170 bp 3′-UTR. The predicted protein of C8β contained three motifs, thrombospondin type-1 repeat, membrane attack complex/perforin domain, and LDL-receptor class A. Expression analysis revealed that these two complement genes were highly expressed in the liver, however, were weakly expressed in the gill, heart, brain, kidney, intestine, spleen and dorsal muscle tissues. The present study provided insights into the complement system and immune functions of Nile tilapia.

Connective tissue growth factor (CCN2) in blood vessels

The CCN family comprise the products of six immediate-early response genes (Cyr61, Ctgf, Nov and Wisp1-3) and are multi-functional proteins, characterised by four discrete protein modules in which reside functional domains: an insulin-like growth factor binding protein-like module (IGFBP) but has low affinity for IGFBPs, a von Willebrand factor type C repeat module (VWC) which mediates integrin and growth factor binding, a thrombospondin type-1 repeat module (TSP-1), and a cysteine-knot-containing module (CT). These modules mediate a host of interactions such as growth factor binding, integrin recognition, and interaction(s) with heparin and proteoglycans (reviewed in Holbourn et al., 2008; Chen and Lau, 2009).The CCN family are involved in many normal and pathological cellular processes and have a plethora of functions including cell proliferation, angiogenesis, wound healing, and fibrogenesis, tumourigenesis. In addition, many roles have been described for CCN family members in the cardiovascular system (Table 1). The focus of this review is the role of connective tissue growth factor (CCN2, CTGF) in blood vessels and in vascular pathology.

6-Alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin Type-1 repeats by protein O-fucosyltransferases 1 and 2

Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

TROP2 is epigenetically inactivated and modulates IGF‐1R signalling in lung adenocarcinoma

Trop-2, a cell surface glycoprotein, contains both extracellular epidermal growth factor-like and thyroglobulin type-1 repeat domains. Low TROP2 expression was observed in lung adenocarcinoma tissues as compared with their normal counterparts. The lack of expression could be due to either the loss of heterozygosity (LOH) or hypermethylation of the CpG island DNA of TROP2 upstream promoter region as confirmed by bisulphite sequencing and methylation-specific (MS) polymerase chain reaction (PCR). 5-Aza-2′-deoxycytidine treatment on lung cancer cell (CL) lines, CL1-5 and A549, reversed the hypermethylation status and elevated both TROP2 mRNA and protein expression levels. Enforced expression of TROP2 in the lung CL line H1299 reduced AKT as well as ERK activation and suppressed cell proliferation and colony formation. Conversely, silencing TROP2 with shRNA transfection in the less efficiently tumour-forming cell line H322M enhanced AKT activation and increased tumour growth. Trop-2 could attenuate IGF-1R signalling-mediated AKT/β-catenin and ERK activation through a direct binding of IGF1. In conclusion, inactivation of TROP2 due to LOH or by DNA methylation may play an important role in lung cancer tumourigenicity through losing its suppressive effect on IGF-1R signalling and tumour growth.

Maturation of Thyroglobulin Protein Region I

In vertebrates, the thyroglobulin (Tg) gene product must be exported to the lumen of thyroid follicles for thyroid hormone synthesis. In toto, Tg is composed of multiple type-1 repeats connected by linker and hinge (altogether considered as "region I," nearly 1,200 residues); regions II-III (~720 residues); and cholinesterase-like (ChEL) domain (~570 residues). Regions II-III and ChEL rapidly acquire competence for secretion, yet regions I-II-III require 20 min to become a partially mature disulfide isomer; stabilization of a fully oxidized form requires ChEL. Transition from partially mature to mature Tg occurs as a discrete "jump" in mobility by nonreducing SDS-PAGE, suggesting formation of at most a few final pairings of Cys residues that may be separated by significant intervening primary sequence. Using two independent approaches, we have investigated which portion of Tg is engaged in this late stage of its maturation. First, we demonstrate that this event is linked to oxidation involving region I. Introduction of the Tg-C1245R mutation in the hinge (identical to that causing human goitrous hypothyroidism) inhibits this maturation, although the Cys-1245 partner remains unidentified. Second, we find that Tg truncated after its fourth type-1 repeat is a fully independent secretory protein. Together, the data indicate that final acquisition of secretory competence includes conformational maturation in the interval between linker and hinge segments of region I.

Vertebral defects in patients with Peters plus syndrome and mutations in B3GALTL

Peters plus syndrome (PPS-MIM 261540) is a rare genetic disorder characterized by anterior chamber eye anomalies, disproportionate short stature, mild to severe psychomotor delay, dysmorphic facial features, cleft lip/palate sometimes associated with malformations of the internal organs. 1 It is inherited as an autosomal recessive pattern and caused by mutation of the B3GALTL gene (MIM 610308), a beta-1,3-glucosyltransferase involved in the synthesis of the unusual O-linked disaccharide glucosylbeta-1,3-fucose-O- found on the thrombospondin type-1 repeats (TSRs) of many biologically important proteins. 2 We report a case of a 41/2 year old Moroccan

Role of NOV and CTGF in PDGF and TGF-β signaling in hepatic fibrogenesis

Aims: CYR61 (CCN1), CTGF (CCN2) and NOV (CCN3) belong to the CCN protein family. They share a modular structure with a secretary signal peptide, an insulin-like growth factor binding protein-like domain, a von Willebrand factor type C and a Thrombospondin type-1 repeat module and a cysteine knot containing module. These cysteine-rich proteins are secreted and play an essential role in fibrogenesis [1]. Recently it was demonstrated that NOV and CTGF appear to act in a Yin/Yang manner to regulate extracellular matrix (ECM) metabolism [2]. Here we aimed to analyse the synergy of CTGF and TGF-β in the regulation of ECM synthesis in the hepatic stellate cell line GRX and cell proliferation in EA·hy 926 endothelial cells.

Genome-Wide Association Study of Stroke in Sickle Cell Anemia.

Abstract 1528Poster Board I-551Stroke is a potentially lethal complication of sickle cell anemia (SCA) and one marker of sickle vasculopathy. Candidate gene studies conducted have demonstrated that stroke is associated with polymorphisms (SNPs) in several genes whose interactions can be used to build risk prediction models. For an unbiased discovery of the complex genetic basis of this complication, we conducted a genome-wide association study in 1387 SCA patients from the Cooperative Study of Sickle Cell Disease to identify single nucleotide polymorphisms (SNPs) associated with stroke. The data included 145 patients with at least one stroke event (cases), and 1242 stroke free patients (controls). Cases and controls had approximately the same median age (18 years) and similar gender composition (cases: 56% males; controls: 52% males). DNA was genotyped with the IIlumina Human610-Quad that includes approximately 600,000 SNPs and we removed samples with a call rate < 93%, and samples with a mismatch between gender reported in the database and heterozygosity of more than 5% SNPs in chromosome X. Error rate was estimated to be less than 5% based on agreement between known repeated samples and identical samples matched using genome-wide identity by descent using the software PLINK. We examined general, allelic, dominant and recessive associations of each individual SNP using Bayesian tests and scored the evidence of association of each model by its posterior probability. We assumed uniform probability on competitive models, so that the posterior odds of each model of association relative to the model of no association is equivalent to the Bayes factor (BF) and conducted extensive simulations to compute the expected number of false positive associations for different thresholds of the BF. The simulations showed that the false positive rate of the Bayesian decision rule changes with the allele frequency and suggested using a BF > 10,000 to reduce the expected number of false positive associations to less than 1 in 100,000 independent tests. Twenty-six SNPs passed this threshold, 15 SNPs were in intragenic regions and 10 SNPs were in known genes, including one SNP in the brain specific angiogenesis inhibitor BAI1 (rs11167147, odds for stroke in carriers of the AC or CC genotype = 0.25 relative to carriers of the AA genotype, BF>22,000) and one SNP in the regulator of angiogenes AIMP1 (rs7654865: odds for stroke in carriers of the AC or CC genotype = 0.10 relative relative to carriers of he AA genotype, BF>10,000). SNPs in other genes involved in angiogenesis (ANGPT1, ANGPT4 and TEK) were also associated with stroke, although none of the associations reached genome-wide significance. The regulation of angiogenesis is controlled by a balance between stimulators and inhibitors. BAI1 is a p53 target gene specifically expressed in the brain that is a transmembrane protein containing an extracellular domain with thrombospondin type-1 repeats that can exhibit anti-angiogenic activity. BAI1 is a mediator in the p53-signaling pathway; p53 has been shown to result in the decreased expression of VEGF and increased expression of BAI1. The VEGF system is integrated into the p53 transcriptional network and both pathways can be abnormal in SCA vessels. AIMP1 encodes a cytokine induced by apoptosis that is involved in the control of angiogenesis, inflammation and wound healing. It induces the expression of TNFRSF1A in endothelial cells and has anti-angiogenic functions via inhibition of HIF1α activities. HIF1α is involved in mediating angiogenic growth of endothelial cells. None of the SNPs in genes that we found associated with stroke in earlier studies reached genome-wide significance, although several SNPs in BMP6, ADCY9, EDN1, ERG, MET, SELP, TEK and TGFBR3 reached lesser statistical significance. We also looked for replication of SNPs that have been associated with stroke in the general population; rs12229103 (NINJ2) was significantly associated with stroke (BF>10). This SNP is within 20kb from rs12425791 that was found to be associated with stroke in the general population. Also SNPs in IMPA2 and AIM1 were significantly associated. Although confirmation of our genetic studies in an independent sample of individuals is needed and functional studies are warranted, our findings provide suggestive evidence for a major role of genes involved in angiogenesis in the modulation of stroke risk, a finding is in agreement with previous work suggesting that angiogenesis is dysregulated in SCA. DisclosuresNo relevant conflicts of interest to declare.

Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan

Protein C-mannosylation is the attachment of alpha-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the (36)WDAWGPWSECSRTC(49) sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp(39) and Trp(42). C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-(3)H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.

Vasculostatin Inhibits Intracranial Glioma Growth and Negatively RegulatesIn vivoAngiogenesis through a CD36-Dependent Mechanism

Angiogenesis is a critical physiologic process that is appropriated during tumorigenesis. Little is known about how this process is specifically regulated in the brain. Brain angiogenesis inhibitor-1 (BAI1) is a brain-predominant seven-transmembrane protein that contains five antiangiogenic thrombospondin type-1 repeats (TSR). We recently showed that BAI1 is cleaved at a conserved proteolytic cleavage site releasing a soluble, 120 kDa antiangiogenic factor called vasculostatin (Vstat120). Vstat120 has been shown to inhibit in vitro angiogenesis and suppress subcutaneous tumor growth. Here, we examine its effect on the intracranial growth of malignant gliomas and further study its antitumor mechanism. First, we show that expression of Vstat120 strongly suppresses the intracranial growth of malignant gliomas, even in the presence of the strong proangiogenic stimulus mediated by the oncoprotein epidermal growth factor receptor variant III (EGFRvIII). This tumor-suppressive effect is accompanied by a decrease in tumor vascular density, suggesting a potent antiangiogenic effect in the brain. Second, and consistent with this interpretation, we find that treatment with Vstat120 reduces the migration of cultured microvascular endothelial cells in vitro and inhibits corneal angiogenesis in vivo. Third, we show that these antivascular effects critically depend on the presence of the cell surface receptor CD36 on endothelial cells in vitro and in vivo, supporting the role of Vstat120 TSRs in mediating these effects. These results advance the understanding of brain-specific angiogenic regulation, and suggest that Vstat120 has therapeutic potential in the treatment of brain tumors and other intracerebral vasculopathies.

The effect of thrombospondin-1 on breast cancer metastasis

Thrombospondin-1 (TSP-1) has been proposed to have both pro-metastatic and anti-metastatic properties. To elucidate its role in breast cancer metastasis, we compared tumor progression in the polyomavirus middle T antigen (Pyt) transgenic mouse and the TSP-1-null Pyt transgenic mouse. We characterized the tumors in these mice at 45, 60 and 90 days of age. Tumor size, areas of necrosis, macrophage infiltration, levels of active and total TGF-beta, vessel morphology, and lung and blood metastasis were measured in these mice. Mammary tumors were larger in the TSP-1-null mouse, and vessels were larger, but fewer in number in these tumors. The level of total TGF-beta was significantly higher in the Pyt tumors at 90 days of age. Importantly, significantly fewer metastases were observed in the lungs of the TSP-1-null/Pyt mouse. Primary Pyt tumor cells were more migratory than TSP-1-null Pyt tumor cells on collagen. Treatment of Pyt mice with recombinant proteins that contain the type-1 repeats of TSP-1 resulted in decreased primary tumor growth and metastasis. Sequences that are involved in CD36 binding and those required for TGF-beta activation mediated the inhibition of primary tumor growth. Thus, TSP-1 in the mammary tumor microenvironment inhibits angiogenesis and tumor growth, but promotes metastasis to the lung in the Pyt transgenic mouse. The ability of TSP-1 to support metastasis correlates with its ability to promote tumor cell migration.

Two decades of thyroglobulin type-1 domain research

Thyroglobulin type-1 repeats are primarily found in thyroglobulin and several other functionally unrelated proteins. Because a few of them exhibit inhibitory activity against cysteine proteases they were named thyropins (thyroglobulin type-1 domain protease inhibitors). In contrast to cystatins, the best-characterized group of papain-like protease inhibitors, they exhibit greater selectivity in their interactions with target proteases. Interestingly, a few members inhibit aspartic protease cathepsin D and metalloproteases. In contrast to the inhibitory fragment of the major histocompatibility complex class II-associated p41 form of invariant chain, whose structural integrity appears mandatory for its inhibitory properties, short polypeptides derived from insulin-like growth factor-binding proteins exhibit the same activity as the structure of the whole fragment. Taken together, the results indicate that the thyroglobulin type-1 repeat is a structural motif occasionally employed as an inhibitor of proteases.

O-Fucosylation of Thrombospondin Type 1 Repeats in ADAMTS-like-1/Punctin-1 Regulates Secretion: IMPLICATIONS FOR THE ADAMTS SUPERFAMILY

The ADAMTS superfamily contains several metalloproteases (ADAMTS proteases) as well as ADAMTS-like molecules that lack proteolytic activity. Their common feature is the presence of one or more thrombospondin type-1 repeats (TSRs) within a characteristic modular organization. ADAMTS like-1/punctin-1 has four TSRs. Previously, O-fucosylation on Ser or Thr mediated by the endoplasmic reticulum-localized enzyme protein-O-fucosyltransferase 2 (POFUT2) was described for TSRs of thrombospondin-1, properdin, and F-spondin within the sequence Cys-Xaa(1)-Xaa(2)-(Ser/Thr)-Cys-Xaa-Xaa-Gly (where the fucosylated residue is underlined). On mass spectrometric analysis of tryptic peptides from recombinant secreted human punctin-1, the appropriate peptides from TSR2, TSR3, and TSR4 were found to bear either a fucose monosaccharide (TSR3, TSR4) or a fucose-glucose disaccharide (TSR2, TSR3, TSR4). Although mass spectral analysis did not unambiguously identify the relevant peptide from TSR1, metabolic labeling of cells expressing TSR1 and the cysteine-rich module led to incorporation of [(3)H]fucose into this construct. Mutation of the putative modified Ser/Thr residues in TSR2, TSR3, and TSR4 led to significantly decreased levels of secreted punctin-1. Similarly, expression of punctin-1 in Lec-13 cells that are deficient in conversion of GDP-mannose to GDP-fucose substantially decreased the levels of secreted protein, which were restored upon culture in the presence of exogenous l-fucose. In addition, mutation of the single N-linked oligosaccharide in punctin-1 led to decreased levels of secreted punctin-1. Taken together, the data define a critical role for N-glycosylation and O-fucosylation in the biosynthesis of punctin-1. From a broad perspective, these data suggest that O-fucosylation may be a widespread post-translational modification in members of the ADAMTS superfamily with possible regulatory consequences.

ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis

ADAMTS-like 2 (ADAMTSL2), is a secreted protein resembling the ancillary domains of the ADAMTS proteases, but with distinct structural features. It has 7 thrombospondin type-1 repeats (TSRs), but an unusually long spacer module, which in both humans and mice, contains a novel insertion bearing six N-glycosylation sites. The ADAMTSL2 protein expressed in HEK293F and COS-1 cells, is a cell-surface and extracellular matrix binding glycoprotein, with N-linked carbohydrate constituting ∼ 20% by mass. The 4.0 kb Adamtsl2 mRNA is found most abundantly in adult mouse liver, lung and spleen by northern blotting. During mouse embryogenesis, Adamtsl2 was expressed most strongly in the third week of gestation. Adamtsl2 mRNA was detected by in situ hybridization in developing skeletal muscle, liver, bronchial and arterial smooth muscle, skin, intervertebral disc, perichondrium, pancreas and spinal cord. Immunohistochemical localization of ADAMTSL2 protein was similar to mRNA expression. Detection of Adamtsl2 mRNA and protein in developing skeletal myotubes, but not undifferentiated myogenic precursors led us to investigate its regulation during in vitro myogenic differentiation. In C2C12 and 23A2 myogenic cells, but not in 23A2 cells rendered non-myogenic by expression of G12V:H-Ras (9A2 cells), differentiation induced by serum starvation triggered expression of Adamtsl2 mRNA, coordinately with Myog, a marker of muscle differentiation. Furthermore, activation of the key myogenic determinant MyoD in 10T1/2 fibroblasts also triggered expression of Adamtsl2 mRNA. Collectively, the data suggest that induction of Adamtsl2 mRNA is an integral feature of myogenesis.

Angiogenic Response Caused by Oncolytic Herpes Simplex Virus–Induced Reduced Thrombospondin Expression Can Be Prevented by Specific Viral Mutations or by Administering a Thrombospondin-Derived Peptide

Wild-type (WT) herpes simplex virus (HSV) causes some pathology, such as ocular keratitis, by increasing infected tissue vascularity, possibly reflecting altered angiogenic factor expression in infected cells. Oncolytic HSVs possess specific mutations enabling selective replication in tumor cells. We investigated whether this ability to enhance infected tissue vascularity is retained in oncolytic HSV, which could be an undesirable effect of oncolytic HSVs that may need to be addressed when treating tumors with oncolytic HSVs. s.c. tumors derived from U87 human glioma cells in athymic mice were treated with oncolytic HSVs G207 or G47Delta in the presence or absence of a recombinant protein composed of the three type-1 repeats (3TSR) of thrombospondin-1 (TSP-1). Real-time reverse transcription-PCR and Western blot of infected cultured cells measured angiogenic factor expression. Microvessel density was assessed using immunofluorescence. G207-treated U87 s.c. tumors had elevated microvessel densities compared with saline- and G47Delta-treated tumors, and G207 treatment caused delayed tumor growth resumption. G207-infected U87 and U373 cells exhibited reduced protein, not mRNA, expression of angiogenesis inhibitors TSP-1 and thrombospondin-2 (TSP-2). 3TSR restored the G207-treated tumor microvessel density to the low level of G47Delta-treated tumors and prevented delayed growth resumption. Oncolytic HSV G207 thus retains the ability of WT HSV to increase infected tissue vascularity. In infected tumors, this increased vascularity is mediated by reduced TSP-1 and TSP-2 levels and causes delayed tumor growth resumption. Incorporating viral mutations, such as those seen in G47Delta or administering thrombospondin-derived peptides, counteracts the angiogenic effect of oncolytic HSV and should be considered when designing oncolytic HSV therapies.

Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura.

The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.

Correction: Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by β1 integrins

Correction: Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by β1 integrins

Thrombospondin-1 Mimetic Peptide Inhibitors of Angiogenesis and Tumor Growth: Design, Synthesis, and Optimization of Pharmacokinetics and Biological Activities

The heptapeptide 1, NAc-Gly-Val-DIle-Thr-Arg-Ile-ArgNHEt, a structurally modified fragment derived from the second type-1 repeat of thrombospondin-1 (TSP-1), is known to possess antiangiogenic activity. However, therapeutic utility could not be demonstrated because this peptide has a very short half-life in rodents. To optimize the PD/PK profile of 1, we initiated a systematic SAR study. The initial structural modifications were performed at positions 5 and 7 of peptide 1 and at the N- and C-termini. Out of several hundred peptides synthesized, the nonapeptide 5 (ABT-526) emerged as a promising lead. ABT-526 inhibited VEGF-induced HMVEC cell migration and tube formation in the nanomolar range and increased apoptosis of HUAEC cells. ABT-526 showed acceptable PK in rodents, dog, and monkey. ABT-526, when incorporated in an angiogenic pellet implanted in the rat cornea at 10 microM, reduced neovascularization by 92%. Substitution of DalloIle in place of DIle in ABT-526 provided nonapeptide 6 (ABT-510), which was 30-fold less active than ABT-526 in the EC migration but 20-fold more active in the tube formation assay. In comparison to ABT-526, ABT-510 has increased water solubility and slower clearance in dog and monkey. Radiolabeled ABT-510 demonstrated saturable binding to HMVEC cells at 0.02-20 nM concentrations and was displaceable by TSP-1. ABT-510 and ABT-526 were shown to significantly increase apoptosis of HUAEC cells. ABT-510 was effective in blocking neovascularization in the mouse Matrigel plug model and inhibited tumor growth in the mouse Lewis lung carcinoma model. Previous studies had shown that ABT-510 was effective in inhibiting the outgrowth of murine melanoma metastases in syngeneic mice and in blocking the growth of human bladder carcinoma implanted in nude mice. It had been also shown that ABT-510 could regress tumor lesions in pet dogs or cause unexpected stabilization of the disease in advanced canine cancer. ABT-526 and ABT-510 are the first compounds in the class of potent inhibitors of angiogenesis that mimic the antiangiogenic function of TSP-1. ABT-510 is currently in phase II clinical studies.

β1 Integrins Are Receptors for Thrombospondin 1

Thrombospondin 1 (TSP1) is an endogenous antiangiogenic protein, and it is known that some of these effects are mediated by binding of the type-1 repeat (TSR) domains to the cell surface receptor CD36. However, Short et al. show that TSP1 and a TSR-containing peptide inhibited migration of human umbilical vein endothelial cells (HUVEC), which do not express CD36, in response to vascular endothelial growth factor (VEGF). Fluorescence-activated cell sorting (FACS) indicated that β1 integrins were abundant on HUVEC, and antibodies against β1 integrins that activated β1 integrins signaling inhibited cell migration in a dose-dependent manner. Concentrations of activating antibodies against β1 integrins that were too low to inhibit migration directly interfered with the inhibitory activity of the TSR peptide, but antibodies against β3 integrins and nonactivating antibodies against β1 integrins did not. Small interfering RNAs (siRNAs) were used to decrease β1 or β3 integrins, and individually these did not block VEGF-induced migration; however, siRNA treatment to reduce β1 integrin did block the inhibitory effect of the TSR peptide. Immunoblotting of HUVEC proteins bound to beads coated with TSR peptide indicated a direct interaction between TSR and α5 β1 integrins. Antibodies against α5 or α3 blocked the TSR peptide inhibition of VEGF-induced migration. Both antagonists of phospholipase C-γ (PLC-γ) and of phosphoinositide 3-kinase (PI3K) inhibited migration. TSR and the PLC-γ antagonist had additive effects, whereas TSR and the PI3K inhibitor did not. The p85 subunit of PI3K was found in a complex with β1 integrin; thus, PI3K appears to be involved in the inhibitory effects of β1 signaling on migration. Finally, the involvement of the β1 integrins in mediating TSP1 inhibitory effects was present in cells that do express the CD36 receptor for TSR. In cells that do express CD36, antibodies to CD36 blocked inhibition of migration by the TSR peptide; however, migration in response to full-length TSP1 was not blocked by the antibody to CD36 but was blocked by the β1 integrin antibody. Thus, inhibition of migration by TSP1 appears to involve both CD36 and β1 integrins and can be mediated solely by β1 integrins in cells lacking CD36. Understanding the mechanisms of inhibitors of angiogenesis may aid in designing such inhibitors for cancer therapy. S. M. Short, A. Derrien, R. P. Narsimhan, J. Lawler, D. E. Inger, B. R. Zetter, Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by β1 integrins. J. Cell Biol. 168 , 643-653 (2005). [Abstract] [Full Text]

Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by β1 integrins

The anti-angiogenic effect of thrombospondin-1 has been shown to be mediated through binding of the type-1 repeat (TSR) domain to the CD36 transmembrane receptor. We now report that the TSR domain can inhibit VEGF-induced migration in human umbilical vein endothelial cells (HUVEC), cells that lack CD36. Moreover, we identified β1 integrins as a critical receptor in TSR-mediated inhibition of migration in HUVEC. Using pharmacological inhibitors of downstream VEGF receptor effectors, we found that phosphoinositide 3-kinase (PI3k) was essential for TSR-mediated inhibition of HUVEC migration, but that neither PLCγ nor Akt was necessary for this response. Furthermore, β1 integrins were critical for TSR-mediated inhibition of microvascular endothelial cells, cells that express CD36. Together, our results indicate that β1 integrins mediate the anti-migratory effects of TSR through a PI3k-dependent mechanism.