EGF-like Module Research Articles

Investigating the role of non-synonymous variant D67N of ADGRE2 in chronic myeloid leukemia

BackgroundChronic myeloid leukaemia (CML) is a type of blood cancer that begins in the hematopoietic stem cells. It is primarily characterized by a specific chromosomal aberration, the Philadelphia chromosome. While the fusion gene is a major contributor to CML, several other genes including ADGRE2, that are reported as highly expressed in hematopoietic stem cells and could be utilized as a therapeutic marker in leukemic patients are implicated in the disease’s progression. Until recently, little research had been conducted to identify single nucleotide polymorphisms (SNPs) associated with CML. Therefore, this study aims to investigate the influence of non-synonymous variants on the structure and function of the gene encoding adhesion G protein-coupled receptor E2, ADGRE2, and to evaluate their association with CML and its clinical and pathological characteristics.MethodsNon-synonymous SNPs of ADGRE2 were retrieved from the ENSEMBL, COSMIC, and gnomAD genome browsers, and the pathogenicity of deleterious variants was assessed using several established computational tools, including SIFT, CADD, REVEL, PolyPhen, and MetaLR.ResultsVarious in silico analyses explored the impact of damaging SNP on the function, stability, and structure of EGF-like modules containing mucin-like hormone receptor-like2 (EMR2) protein encoded by the ADGRE2 gene. Genotype analysis was performed on collected blood samples, revealing that altered genotype TT of variant rs765071211 (C/T) was associated significantly with CML patients compared to the control. Further in vitro and in vivo analyses suggest that this SNP holds potential for clinical translation.

Spatiotemporal EP4–fibulin-1 expression is associated with vascular intimal hyperplasia

Abstract Aims Cyclooxygenase-2–derived prostaglandin E2 (PGE2) is thought to promote vascular intimal hyperplasia (IH). It has been reported that the PGE2 receptor EP4 is upregulated in injured vessels and that EP4 signalling in vascular smooth muscle cells (VSMCs) promotes IH. In contrast, EP4 in endothelial cells has been demonstrated to restrain IH. We aimed to investigate spatiotemporal expression of EP4 and whether modulating EP4 signalling could be a viable therapeutic strategy. Methods and results We generated EP4 reporter mice (Ptger4-IRES-nlsLacZ) and found temporary but prominent EP4 expression in VSMCs of the proliferative neointima 2 weeks after femoral artery wire injury. Injury-induced IH was diminished in VSMC-targeted EP4 heterozygous deficient mice (Ptger4fl/+;SM22-Cre) 2 and 4 weeks after vascular injury compared to that in SM22-Cre, whereas injury-induced IH was exacerbated in VSMC-targeted EP4-overexpressing mice (Ptger4-Tg) compared to controls (non-Tg). We then investigated the downstream signalling of EP4 in VSMCs. Stimulation of EP4 increased mRNA and protein levels of the glycoprotein fibulin-1 in Ptger4-Tg VSMCs. Fibulin-1C recombinant proteins increased VSMC proliferation and migration through transforming growth factor (TGF)- β/Smad3, and EP4-mediated proliferation and migration were attenuated in Fbln1fl/fl;SM22-Cre VSMCs and in CRISPR/Cas9-mediated Fbln1 knockdown in Ptger4-Tg VSMCs. We generated multiple deletion mutants of fibulin-1C and found that EGF-like modules 6–8 appear to be involved in fibulin-1-mediated proliferation. Among binding partners of fibulin-1, extracellular matrix protein 1 (ECM1) was also upregulated by EP4 stimulation, and fibulin-1C and ECM1 proteins additively enhanced VSMC proliferation and migration. Injury-induced IH was attenuated in VSMC-targeted fibulin-1 deletion mice (Fbln1fl/fl;SM22-Cre) compared to Fbln1fl/fl. Furthermore, systemic EP4 antagonist administration reduced injury-induced IH in wild-type mice. Conclusion EP4 was upregulated in VSMCs of proliferative IH, and EP4 signalling promoted IH, at least in part through fibulin-1. An EP4 antagonist might be considered as a therapeutic strategy for IH.

Impact of naturally forming human α/β-tryptase heterotetramers in the pathogenesis of hereditary α-tryptasemia.

Both α-tryptase and β-tryptase are preferentially expressed by human mast cells, but the purpose of α-tryptase is enigmatic, because its tetramers lack protease activity, whereas β-tryptase tetramers are active proteases. The monogenic disorder called hereditary α-tryptasemia, due to increased α-tryptase gene copies and protein expression, presents with clinical features such as vibratory urticaria and dysautonomia. We show that heterotetramers composed of 2α- and 2β-tryptase protomers (α/β-tryptase) form naturally in individuals who express α-tryptase. α/β-Tryptase, but not homotetramer, activates protease-activated receptor-2 (PAR2), which is expressed on cell types such as smooth muscle, neurons, and endothelium. Also, only α/β-tryptase makes mast cells susceptible to vibration-triggered degranulation by cleaving the α subunit of the EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) mechanosensory receptor. Allosteric effects of α-tryptase protomers on neighboring β-tryptase protomers likely result in the novel substrate repertoire of α/β-tryptase tetramers that in turn cause some of the clinical features of hereditary α-tryptasemia and of other disorders involving mast cells.

Human EGF-derived direct and reverse short linear motifs: conformational dynamics insight into the receptor-binding residues

Short linear motifs (SLiMs) have been recognized to perform diverse functions in a variety of regulatory proteins through the involvement in protein–protein interactions, signal transduction, cell cycle regulation, protein secretion, etc. However, detailed molecular mechanisms underlying their functions including roles of definite amino acid residues remain obscure. In our previous studies, we demonstrated that conformational dynamics of amino acid residues in oligopeptides derived from regulatory proteins such as alpha-fetoprotein (AFP), carcino-embryonic antigen (CEA), and pregnancy specific β1-glycoproteins (PSGs) contributes greatly to their biological activities. In the present work, we revealed the 22-member linear modules composed of direct and reverse AFP14–20-like heptapeptide motifs linked by CxxGY/FxGx consensus motif within epidermal growth factor (EGF), growth factors of EGF family and numerous regulatory proteins containing EGF-like modules. We showed, first, the existence of similarity in amino acid signatures of both direct and reverse motifs in terms of their physicochemical properties. Second, molecular dynamics (MD) simulation study demonstrated that key receptor-binding residues in human EGF in the aligned positions of the direct and reverse motifs may have similar distribution of conformational probability densities and dynamic behavior despite their distinct physicochemical properties. Third, we found that the length of a polypeptide chain (from 7 to 53 residues) has no effect, while disulfide bridging and backbone direction significantly influence the conformational distribution and dynamics of the residues. Our data may contribute to the atomic level structure–function analysis and protein structure decoding; additionally, they may provide a basis for novel protein/peptide engineering and peptide-mimetic drug design.

A novel mutation of laminin \u03b22 (LAMB2) in two siblings with renal failure

This report describes a novel mutation of LAMB2, the gene associated with Pierson syndrome (microcoria-congenital nephrosis syndrome), in two female siblings. The c.970T>C p.(Cys324Arg) mutation in the LAMB2 gene affects one of the eight highly conserved cysteine residues within the first EGF-like module of the laminin β2 protein. These residues form disulfide bonds in order to achieve a correct 3D structure of the protein. The reported phenotype is considered a relatively mild variant of Pierson syndrome and is associated with later-onset (18 months) therapy-resistant nephrotic syndrome leading to renal failure, and ocular abnormalities consisting of high myopia, microcoria, diverse retinal abnormalities, hence a low level of visual acuity. Importantly, the reported LAMB2 mutation was associated with normal neurological development in both siblings.Conclusion: this report presents the variability of the renal, ocular and neurological phenotypes associated with LAMB2 mutations and underscores the importance of ophthalmologic examination in all children with unexplained renal insufficiency or nephrotic syndrome.What is known• LAMB2 mutations are associated with Pierson syndrome• Pierson syndrome is associated with congenital nephrotic syndrome, microcoria and neurological deficitsWhat is new• A novel mutation in the LAMB2 gene in two female siblings• Genotype and clinical phenotype description of a novel LAMB2 mutation

Comparative evaluation of eosinophils in normal mucosa, dysplastic mucosa and oral squamous cell carcinoma with hematoxylin-eosin, Congo red, and EMR1 immunohistochemical staining techniques.

Background:Oral squamous cell carcinoma is the most common malignant lesion of the oral cavity, and it involves various molecular mechanisms. The development of oral squamous cell carcinoma is influenced by the host immune cells, such as eosinophils. The present study was conducted to compare the presence of eosinophils in normal mucosa, dysplastic mucosa, and oral squamous cell carcinoma by -hematoxylin- eosin staining, Congo red staining, and epidermal growth factor-like (EGF-like) module containing a mucin–like hormone receptor1 (EMR1) immunohistochemical marker.Methods:In this cross-sectional study, 60 paraffinized samples were selected, consisting of 20 normal mucosae, 20 dysplastic mucosae, and 20 squamous cell carcinoma samples. After confirmation of the diagnosis, the mean number of eosinophils was evaluated by hematoxylin-eosin, Congo red, and immunohystochemical staining techniques. The data were analyzed by SPSS-10 software using the Kruskal-Wallis and Friedman tests.Results:The results showed that the number of eosinophils in dysplastic mucosa was significantly higher than the number in normal mucosa, and the number of eosinophils in squamous cell carcinoma was significantly higher than the number in dysplastic mucosa in all staining techniques (p<0.001). Moreover, the comparison of staining techniques showed a significantly higher number of eosinophils in EMR1immunohistochemicalmarker than were observed when Congo red and hematoxylin - eosin (H&E) staining techniques were used (p<0.001).Conclusion:It can be argued that eosinophil contributes to the identification of lesions that have a higher potential of malignant transformation. Moreover, eosinophil can be suggested as an indicator in the differentiation of oral lesions in cases with borderline diagnosis and in targeted molecular therapy.

Dietary rapeseed/canola-oil supplementation reduces serum lipids and liver enzymes and alters postprandial inflammatory responses in adipose tissue compared to olive-oil supplementation in obese men.

Obesity is associated with hyperlipidemia, hepatic steatosis, and low-grade inflammation. Studies have shown that MUFA as well as PUFA have beneficial effects on blood lipids and the inflammatory state. This study investigates the effects of a daily supplementation of either 50 g of rapeseed/canola (RA) or olive (OL) oil over 4 wk on serum lipids, serum liver enzymes, and inflammatory gene expression in subcutaneous (s. c.) adipose tissue in obese men. Consuming RA resulted in increased serum n-3 fatty acids and a reduction in total cholesterol, LDL cholesterol, and serum aspartate aminotransferase compared to OL. In s. c. adipose tissue, gene expression of the pro-inflammatory cytokine IL6 was reduced in RA compared to OL. However, after 4 h after a test meal, containing the appropriate oil, white bread, and 400 mL of liquid diet drink (835 kcal in total), gene expression of IL6, IL1B, and EMR1 (egf-like module containing Mucin-like hormone receptor-like 1) was increased in RA and of monocyte chemoattractant protein-1 (CCL2) in both RA and OL. This demonstrates that consuming RA for 4 wk improves serum lipids, liver enzymes, and basal inflammation in s. c. adipose tissue, but it mediates an acute pro-inflammatory response in adipose tissue upon consuming a meal.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Modification of EGF-Like Module 1 of Thrombospondin-1, an Animal Extracellular Protein, by O-Linked N-Acetylglucosamine

Thrombospondin-1 (TSP-1) is known to be subject to three unusual carbohydrate modifications: C-mannosylation, O-fucosylation, and O-glucosylation. We now describe a fourth: O-β-N-acetylglucosaminylation. Previously, O-β-N-acetylglucosamine (O-β-GlcNAc) was found on a threonine in the loop between the fifth and sixth cysteines of the 20th epidermal growth factor (EGF)-like module of Drosophila Notch. A BLAST search based on the Drosophila Notch loop sequence identified a number of human EGF-like modules that contain a similar sequence, including EGF-like module 1 of TSP-1 and its homolog, TSP-2. TSP-1, which has a potentially modifiable serine in the loop, reacted in immuno-blots with the CTD110.6 anti-O-GlcNAc antibody. Antibody reactivity was diminished by treatment of TSP-1 with β-N-acetylhexosaminidase. TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6. Analysis of tandem modules of TSP-1 localized reactivity of CTD110.6 to EGF-like module 1. Top-down mass spectrometric analysis of EGF-like module 1 demonstrated the expected modifications with glucose (+162 Da) and xylose (+132 Da) separately from modification with N-acetyl hexosamine (+203 Da). Mass spectrometric sequence analysis localized the +203-Da modification to Ser580 in the sequence 575CPPGYSGNGIQC586. These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.

Deletion of the SPARC acidic domain or EGF-like module reduces SPARC-induced migration and signaling through p38 MAPK/HSP27 in glioma

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.

Comparative domain modeling of human EGF-like module EMR2 and study of interaction of the fourth domain of EGF with chondroitin 4-sulphate

EMR2 is an EGF-like module containing mucin-like hormone receptor-2 precursor, a G-protein coupled receptor (G-PCR). Mutation in EMR2 causes complicated disorders like polycystic kidney disease (PKD). The structure of EMR2 shows that the fifth domain is comprised of EGF-TM7 helices. Functional assignment of EMR2 by support vector machine (SVM) revealed that along with transporter activity, several novel functions are predicted. A twenty amino acid sequence “MGGRVFLVFLAFCVWLTLPG” acts as the signal peptide responsible for posttranslational transport. Eight amino acids are involved in N-glycosylation sites and two cleavage sites are Leu517 and Ser518 in EMR2. The residue Arg241 is responsible for interaction with glycosaminoglycan and chondroitin sulfate. On the basis of structure, function and ligand binding sites, competitive EMR2 inhibitors designed may decrease the rate of human diseases like Usher's syndrome, bilateral frontoparietal polymicrogyria and PKD.

Leukocyte adhesion-GPCR EMR2 is aberrantly expressed in human breast carcinomas and is associated with patient survival

EGF-like module containing mucin-like hormone receptor 2 (EMR2) is a leukocyte-restricted adhesion G protein-coupled receptor. Aberrant expression of EMR2 and its highly homologous molecule CD97 have been reported in various human cancers. Herein, we investigate the expression of EMR2 in neoplastic breast human tissue and its relationship with patient survival. EMR2 expression in normal and neoplastic breast tissue was assessed by immunohistochemistry in sections from 10 normal controls and micro-arrayed tissue cores from 69 cases of ductal carcinoma in situ (DCIS) and 272 invasive carcinomas. The pattern and intensity of staining was correlated with the clinicopathological characteristics of each case and the disease outcome. While absent in normal breast epithelium, EMR2 was significantly up-regulated in the cytoplasmic and nuclear compartments of both DCIS and invasive carcinoma, with invasive samples displaying significantly higher expression levels compared with in situ disease. In invasive disease, EMR2 cytoplasmic expression was significantly associated with higher tumour grade but not with patient age, nodal status, tumour size, estrogen receptor expression, relapse-free or overall survival. In contrast, EMR2 nuclear expression correlated negatively with higher tumour grade. Of note, EMR2 nuclear expression was associated with longer relapse-free survival as well as overall survival. This study indicates that EMR2 is expressed in neoplastic breast epithelium and suggests that expression patterns of EMR2 are relevant in breast cancer progression. The association of improved patient survival with higher nuclear expression levels identifies EMR2 as a potential biomarker in patients with invasive breast cancer.

Systemic human Netrin-1 gene delivery by adeno-associated virus type 8 alters leukocyte accumulation and atherogenesis in vivo

Atherosclerosis is an inflammatory disorder of arteries. Atherosclerotic plaque, in its early to intermediate stages, is composed largely of lipid-engorged foam cells. These foam cells are derived from the trafficking of monocytes (Mo) into the arterial intima, attracted to the site by chemoattractants. Given that foam cells are derived from the trafficking of Mo, the use of Netrin-1, an Mo chemorepellent, may be useful in limiting Mo accumulation and subsequent plaque formation. To investigate the potential of Netrin-1 for limiting atherosclerosis, we systemically delivered its human (h) cDNA by adeno-associated virus type 8 (AAV8, single-stranded structure) delivery into low-density lipoprotein receptor knockout (LDLR-/-) mice and placed the animals on a high cholesterol diet (HCD). Compared with control neomycin resistance (Neo) gene delivery/HCD, hNetrin-1 delivery resulted in a significant reduction in plaque formation, as determined by larger aortic lumen size, thinner intima-media thickness and lower blood velocity than the Neo/HCD control (all statistically significant). Indices of monocyte/macrophage (Mo/MΦ) accumulation, CD68, integrin, alpha M (ITGAM) and egf-like module containing, mucin-like, hormone receptor-like 1 (EMR-1), were reduced in hNetrin-1/HCD-treated animal's aortas and spleens compared with Neo/HCD-treated animals. Unexpectedly, CD25 and foxp3 (regulatory T cells (Tregs)) in the aorta were strongly upregulated. This is the first time the Mo/MΦ chemorepellent approach, and specific Netrin-1 gene delivery, has been performed for the reduction of Mo/MΦ burden and atherosclerosis. In addition, Netrin-1 has never before been linked to altered Treg levels. These data strongly suggest that hNetrin-1 gene delivery can reduce Mo/MΦ accumulation, inflammation and subsequent plaque formation.

Interactions among Stalk Modules of Thrombospondin-1

Thrombospondin-1 is a trimeric, modular calcium-binding glycoprotein. The subunit is composed of an N-terminal module; oligomerization domain; stalk modules including a von Willebrand factor type C module, three properdin or thrombospondin type 1 repeat (TSR) modules, and two thrombospondin-type EGF-like modules; and a C-terminal signature domain comprising single copies of the epidermal growth factor (EGF)-like, wire, and lectin-like modules. Conformational changes in the signature domain influence ligand binding to the N-terminal modules. Interactions have been demonstrated among the modules of the signature domain and the thrombospondin-type EGF-like modules. We have extended this analysis to the rest of the stalk modules. Differential scanning calorimetry revealed interactions between the most C-terminal TSR module and the EGF-like modules. Calorimetry and differences in expression levels of single versus tandem modules indicated that the three TSRs interact with each other as well. No evidence of interactions between the von Willebrand factor type C and TSR modules were detected by differential scanning calorimetry, circular dichroism, or intrinsic fluorescence. These results indicate that the TSR and thrombospondin-type EGF-like stalk modules act as a unit that may relay conformational information between the N-terminal and C-terminal parts of the protein.

Interactions among the Epidermal Growth Factor-like Modules of Thrombospondin-1

Epidermal growth factor (EGF)-like modules are defined in part by six cysteines joined by disulfides in a 1–3, 2–4, and 5–6 pattern. Thrombospondin-1 (TSP-1) is a multimodular glycoprotein with three EGF-like modules, E1, E2, and E3, arranged in tandem. These modules likely propagate conformational changes between surrounding C-terminal and N-terminal elements of TSP-1 and interact with other extracellular molecules. E1, E2, and their homologs in other TSPs are unique among EGF-like modules in having two residues rather than one between Cys-4 and Cys-5. In addition, E2 has a calcium-binding site and an unusually long loop between Cys-5 and Cys-6. The structure of E1, E2, or E3 expressed alone changed little upon heating as monitored by far-UV CD, whereas more marked changes occurred in E12, E23, and E123 tandem constructs. The individual modules denatured in differential scanning calorimetry experiments only at >85 °C. E12, E23, or E123 tandem constructs, however, had a transition in the range of 44–70 °C. The temperature of the transition was higher when calcium was present and higher with E123 than with E12 or E23. Isothermal titration calorimetry demonstrated KD values of binding of calcium to E2, E12, E23, or E123 at 25 °C of 11.5, 2.9, 2.2, or 0.3 μm, respectively. Monoclonal antibodies HB8432 and C6.7, which recognize epitopes in E2, bound to E12, E23, or E123 with greater affinity than to E2 alone. These results indicate that interactions among the modules of E123 influence the tertiary structure and calcium binding of E2.

Structural basis for EGFR ligand sequestration by Argos

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signaling, called Argos, was identified in Drosophila. Argos functions by directly binding to (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6Å‐resolution crystal structure of Argos bound to Spitz, an EGFR ligand. Contrary to expectations and previous reports, Argos itself does not contain an EGF‐like domain. Instead, a trio of closely related domains (resembling a three‐finger toxin fold) form a clamp‐like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF domain of Spitz. The three‐domain clamp of Argos resembles the human urokinase‐type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF‐like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues.

Interaction Among The Stalk Modules Of Thrombospondin-1

Thrombospondin-1 is a trimeric multi-module calcium-binding glycoprotein that is involved in important biological processes, such as angiogenesis, signal pathway, tumor formation and synaptogenesis. The subunit is composed of an N-terminal module, oligomerization module, the stalk modules, which including von Willebrand Factor type-C (vWF-C) module, three properdin or thrombospondin type 1 (TSR) modules, and three EGF-like modules, and finally the C-terminal calcium-binding wire module and the lectin-like module. Recent research indicates that conformational changes in the C-terminal modules influence ligand binding to the N-terminal modules. The second and the third EGF-like modules have been shown to interact closely with both the wire and the lectin-like modules. My work focuses on the interaction among the stalk modules and if they can propagate conformational changes between C-terminal and N-terminal elements of TSP-1 and interact with other extracellular molecules.Series of recombinant proteins have been generated based on the sequence of stalk modules. We have applied biophysical methods including far UV circular dichroism, intrinsic fluorescence, differential scanning calorimetry, isothermal titration calorimetry, and competitional ELISA. We have found there are strong interactions among the three EGF-like modules. There is one calcium binding site with in the second EGF-like module, the calcium concentration influences the stability of all the three modules from the ITC results. Based on DSC, far UV CD and fluorescence results, there are also strong interaction among the three TSRs and between TSRs and EGF-like modules. We did not find evidence for the interaction between vWF-C and TSR modules.Overall, we have demonstrated that there are strong interactions among the stalk modules of TSP-1, which help relay conformational information between the N-terminal and C-terminal parts of the protein.

Mutations Targeting Intermodular Interfaces or Calcium Binding Destabilize the Thrombospondin-2 Signature Domain

Thrombospondins (THBSs) are a family of secreted calcium-binding glycoproteins with roles in angiogenesis, cell motility, apoptosis, cytoskeletal organization, and extracellular matrix organization. The THBS-2 signature domain (three epidermal growth factor (EGF)-like modules, a wire module with 13 calcium-binding repeats, and a lectin-like module) binds 30 calcium ions and forms extensive interactions among its parts. We explored the significance of these structural elements by examining the impact of 10 different mutations known to result in pseudoachondrodysplasia or multiple epiphyseal dysplasia when found in the homologous wire and lectin-like modules of thrombospondin-5 (THBS-5). A variety of observations indicate that the mutations result in unstable THBS-5 proteins that aggregate in the endoplasmic reticulum. We introduced the mutations into homologous sites of a THBS-2 construct, for which the crystal structure is known, and determined the effects of the mutations on structure as assayed by differential scanning calorimetry and expression of the epitope for the 4B6.13 conformation-sensitive antibody. Abnormalities were found in one or more of several readouts: stability of interactions between the wire and lectin-like modules, stabilities of the EGF-like and wire modules, expression of the 4B6.13 epitope in soluble protein, and expression of the 4B6.13 epitope in substrate-adsorbed protein at different calcium concentrations. The patterns of abnormalities support the idea that the EGF-like, wire, and lectin-like modules constitute a dynamic and interactive calcium-sensitive structure in which a distortion at one site is transmitted to distal sites, leading to global changes in the protein.

Influences of the N700S Thrombospondin-1 Polymorphism on Protein Structure and Stability

Thrombospondins (THBSs) are multimodular, secreted proteins characterized by a signature domain comprising a unique set of 13 calcium-binding repeats flanked by epidermal growth factor (EGF)-like and lectin-like modules. A polymorphism that changes a conserved Asn to Ser at residue 700 in the most N-terminal calcium-binding repeat of THBS-1 (repeat 1C) is found in 8-10% of European populations and has been linked to increased risk of premature coronary artery disease. The Ser substitution leads to altered stability in the EGF-like and wire modules of the THBS-1 signature domain as assessed by differential scanning calorimetry carried out in 2 mm or 200 mum calcium. Studies of the melting profiles of the THBS-2 signature domain proteins with Asn or Ser at position 702 (homologous to 700 in THBS-1) revealed that the impact of the Ser allele is similar in both THBS-1 and THBS-2. Structure determination of the Ser(702) THBS-2 variant in 2 mm calcium showed that repeat 1C contains two bound calcium ions as in the crystal of the Asn(702) protein, including the ion that is coordinated by Asn(702), and is associated with changes in conformation of repeat 1C and the adjacent EGF-like modules. The Ser substitution leads to the decreased ability of soluble THBS-2 signature domain protein to bind 4B6.13, a conformation-sensitive monoclonal antibody that recognizes an epitope in repeat 1C. These results indicate that although THBS harboring the Ser allele binds a full complement of calcium ions, repeat 1C is altered, leading to destabilization of surrounding structures.

Structural basis for EGFR ligand sequestration by Argos

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-beta family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.