Epidermal Growth Factor-like Modules Research Articles

Molecular and Cellular Interactions of Scavenger Receptor SR-F1 With Complement C1q Provide Insights Into Its Role in the Clearance of Apoptotic Cells.

The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs via its collagen-like region. We identified two different binding regions on SR-F1: the N-terminal moiety interacts with C1q and CRT whereas the C-terminal moiety binds AcLDL. The role of SR-F1 N-linked glycans was also tested by mutating each of the three glycosylated asparagines. The three mutants retained binding activities for both AcLDL and C1q. A stable THP-1 cell line overexpressing SR-F1 was generated and C1q was shown to bind more strongly to the surface of SR-F1 overexpressing macrophages, with C1q/SR-F1 colocalization observed in some membrane areas. We also observed a higher level of CRT internalization for THP-1 SR-F1 cells. Increasing SR-F1 negatively modulated the uptake of apoptotic cells. Indeed, THP-1 cells overexpressing SR-F1 displayed a lower phagocytic capacity as compared with mock-transfected cells, which could be partially restored by addition of C1q in the extracellular milieu. Our data shed some light on the role of SR-F1 in efferocytosis, through its capacity to bind C1q and CRT, two proteins involved in this process.

Laminin G Domains of Protein S Bind and Inhibit Fixa

Background: Protein S (PS) is a vitamin K-dependent anticoagulant whose importance is distinguished by many hematological disorders in PS-deficient individuals. PS deficiency is associated with venous and arterial thrombosis, cerebral thrombophlebitis, myocardial infarction, ischemic stroke, and vascular calcification. The 75-kDa PS contains a γ-carboxyglutamic acid module, a thrombin sensitive region, four epidermal growth factor-like modules, and a sex hormone binding globulin-like region that consists of two laminin G domains (LG1 and LG2). PS has three different functions: (a) a cofactor for activated protein C; (b) a cofactor for tissue factor pathway inhibitor, and (c) a direct inhibitor of factor IXa (FIXa). Our laboratory discovered that PS binds FIXa with a dissociation constant (Kd) ~40 nM and inhibits FIXa's function. Currently, we are ascertaining the domain of PS responsible for binding FIXa. Result and Discussion : By using molecular modeling and biochemical assays, we demonstrated that the laminin G domains of PS are crucial for binding FIXa. However, we do not have any information regarding conformational or structural changes that occur when the LG domains and FIXa interact. To confirm our hypothesis, we used circular dichroism (CD), isothermal titration calorimetry (ITC) and biochemical assays to locate the region of the LG domains responsible for FIXa binding. CD spectroscopy gives details of protein secondary structure and conformational change. The conformational change was greatest for binding of LG1+2 to FIXa (LG1+2>LG1>LG2); thus, FIXa apparently has a greater affinity for LG1+2 than for either of the separate LG domains. ITC measures the stoichiometry and thermodynamics (binding affinity, enthalpy, entropy, and Gibbs free energy of protein-protein interaction. We observed that the binding affinity between LG1+2 and FIXa was higher than the affinity between either LG1 or LG2 and FIXa, with a trend of LG1+2>LG1>LG2. In addition, the differences in Gibbs free energy supported the same trend. The binding stoichiometry was 1:1 in all three cases, which implied that each LG construct binds one FIXa molecule. All three interactions were enthalpy driven, their binding was exothermic, and binding was favored by a negative enthalpy. In addition to the foregoing biophysical study, we performed biochemical assays to assess the activities of the LG domains. Thrombin generation assays were performed with PS-deficient plasma supplemented with each LG domain construct and with intact PS. The reactions were initiated with a low tissue factor concentration (0.4 pm) to selectively generate thrombin in the intrinsic pathway. With PS-deficient plasma, the amount of thrombin produced decreased from 255 nM to 155 nM on addition of LG1+2. This result was comparable to the effect of adding intact PS (148 nM thrombin produced). Overall, the structural studies and the plasma studies indicated that LG1+2 is required to mimic the function of PS in binding to and inhibiting FIXa. Similarly, we previously observed that the LG1+2 domain inhibited generation of FXa by FIXa (by ~55%), which was similar to the inhibition of FIXa by intact PS. We conclude that the LG1+2 domain mimics the function of PS in inhibiting FIXa. Future Direction: To exactly identify the region in the PS LG domains responsible for binding to FIXa, we have created multiple single and double amino acid substitutions in the LG domains. Currently, we are identifying particular PS mutants unable to bind FIXa to precisely map the FIXa interaction region in PS. Disclosures No relevant conflicts of interest to declare.

Recombinant mussel protein Pvfp-5β: A potential tissue bioadhesive

During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defense in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials, and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchoring the mussel to surfaces. Among these proteins, protein-5β (Pvfp-5β) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we established the first recombinant expression in Escherichia coli of Pvfp-5β. We characterized recombinant Pvfp-5β, finding that despite displaying a CD spectrum consistent with features of a random coil, the protein is correctly folded as indicated by MS and NMR analyses. Pvfp-5β folds as a β-sheet–rich protein as expected for an epidermal growth factor-like module. We examined the effects of Pvfp-5β on cell viability and adhesion capacity in NIH-3T3 and HeLa cell lines, revealing that Pvfp-5β has no cytotoxic effects at the protein concentrations used and provides good cell-adhesion strength on both glass and plastic plates. Our findings suggest that the adhesive properties of recombinant Pvfp-5β make it an efficient surface-coating material, potentially suitable for biomedical applications including regeneration of damaged tissues.

Molecular dynamics simulations on human fibulin-4 mutants D203A and E126K reveal conformational changes in EGF domains potentially responsible for enhanced protease lability and impaired extracellular matrix assembly

Fibulin-4 is a 50 kDa glycoprotein of elastic fibers and plays an important role in development and function of elastic tissues. Fibulin-4 consists of a tandem array of five calcium-binding epidermal growth factor-like modules flanked by N- and C-terminal domains. Mutations in the human fibulin-4 gene EFEMP2 have been identified in patients affected with various arteriopathies including aneurysm, arterial tortuosity, or stenosis, but the molecular basis of most genotype-phenotype correlations is unknown. Here we present biochemical and computer modelling approaches designed to gain further insight into changes in structure and function of two fibulin-4 mutations (E126K and D203A), which are potentially involved in Ca2+ binding in the EGF2 and EGF4 domain, respectively. Using recombinantly produced fibulin-4 mutant and wild type proteins we show that both mutations introduced additional protease cleavage sites, impaired extracellular assembly into fibers, and affected binding to to fibrillin-1, latent TGF-β-binding proteins, and the lysyl oxidase LOXL2. Molecular dynamics studies indicated that the E126K and D203A mutations do not necessarily result in a direct loss of the complexed Ca2+ ion after 500 ns simulation time, but in significantly enhanced fluctuations within the connecting loop between EGF3 and EGF4 domains and other conformational changes. In contrast, intentionally removing Ca2+ from EGF4 (D203A ΔCa) predicted dramatic changes in the protein structure. These results may explain the changes in protease cleavage sites, reduced secretion and impaired extracellular assembly of the E126K and D203A fibulin-4 mutants and provide further insight into understanding the molecular basis of the associated clinical phenotypes.

Extracellular Interactions between Fibulins and Transforming Growth Factor (TGF)-β in Physiological and Pathological Conditions.

Transforming growth factor (TGF)-β is a multifunctional peptide growth factor that has a vital role in the regulation of cell growth, differentiation, inflammation, and repair in a variety of tissues, and its dysregulation mediates a number of pathological conditions including fibrotic disorders, chronic inflammation, cardiovascular diseases, and cancer progression. Regulation of TGF-β signaling is multifold, but one critical site of regulation is via interaction with certain extracellular matrix (ECM) microenvironments, as TGF-β is primarily secreted as a biologically inactive form sequestrated into ECM. Several ECM proteins are known to modulate TGF-β signaling via cell–matrix interactions, including thrombospondins, SPARC (Secreted Protein Acidic and Rich in Cystein), tenascins, osteopontin, periostin, and fibulins. Fibulin family members consist of eight ECM glycoproteins characterized by a tandem array of calcium-binding epidermal growth factor-like modules and a common C-terminal domain. Fibulins not only participate in structural integrity of basement membrane and elastic fibers, but also serve as mediators for cellular processes and tissue remodeling as they are highly upregulated during embryonic development and certain disease processes, especially at the sites of epithelial–mesenchymal transition (EMT). Emerging studies have indicated a close relationship between fibulins and TGF-β signaling, but each fibulin plays a different role in a context-dependent manner. In this review, regulatory interactions between fibulins and TGF-β signaling are discussed. Understanding biological roles of fibulins in TGF-β regulation may introduce new insights into the pathogenesis of some human diseases.

Structure of astrotactin-2: a conserved vertebrate-specific and perforin-like membrane protein involved in neuronal development.

The vertebrate-specific proteins astrotactin-1 and 2 (ASTN-1 and ASTN-2) are integral membrane perforin-like proteins known to play critical roles in neurodevelopment, while ASTN-2 has been linked to the planar cell polarity pathway in hair cells. Genetic variations associated with them are linked to a variety of neurodevelopmental disorders and other neurological pathologies, including an advanced onset of Alzheimer's disease. Here we present the structure of the majority endosomal region of ASTN-2, showing it to consist of a unique combination of polypeptide folds: a perforin-like domain, a minimal epidermal growth factor-like module, a unique form of fibronectin type III domain and an annexin-like domain. The perforin-like domain differs from that of other members of the membrane attack complex-perforin (MACPF) protein family in ways that suggest ASTN-2 does not form pores. Structural and biophysical data show that ASTN-2 (but not ASTN-1) binds inositol triphosphates, suggesting a mechanism for membrane recognition or secondary messenger regulation of its activity. The annexin-like domain is closest in fold to repeat three of human annexin V and similarly binds calcium, and yet shares no sequence homology with it. Overall, our structure provides the first atomic-resolution description of a MACPF protein involved in development, while highlighting distinctive features of ASTN-2 responsible for its activity.

A novel FBN1 missense mutation (p.C102Y) associated with ectopia lentis syndrome in a Chinese family.

To characterize the disease-causing mutations in a Chinese family with ectopia lentis syndrome (ELS). Patients and their family members were given complete physical, ophthalmic, and cardiovascular examinations. Genomic DNA samples were extracted from the peripheral blood of the pedigree members and 100 healthy controls. Mutation screening was performed in the fibrillin-1 (FBN1) gene by bi-directional sequencing of the amplified products. The mutation was analyzed using two bioinformatics methods. A novel heterozygous c.305G>A mutation in exon 3 of FBN1 was detected. As a result of this change, a highly conserved cysteine residue was replaced by a tyrosine residue (p.C102Y). Another mutation was found in the same exon (c.303T>C), which did not change the amino acid sequence. Both mutations were discovered in each affected individual, but not in the unaffected family members, or in 100 ethnically matched controls. A bioinformatics analysis predicted that mutation p.C102Y would affect protein function. In the first epidermal growth factor-like module, we identified a novel FBN1 mutation (p.C102Y), which caused ELS in the family. Our study presented a unique phenotype, including some distinct ophthalmic findings, such as hypoplasia of the iris and anisometropia. Our results expanded the mutation spectrum of FBN1 and enriched the overall knowledge of genotype-phenotype correlations due to FBN1 mutations.

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.

The role of epidermal growth factor-like module containing mucin-like hormone receptor 2 in human cancers.

G-protein coupled receptors (GPCRs) are among the most diverse and ubiquitous proteins in all of biology. The epidermal growth factor-seven span transmembrane (EGF-TM7) subfamily of adhesion GPCRs is a small subset whose members are mainly expressed on the surface of leukocytes. The EGF domains on the N-terminus add significant size to these receptors and they are considered to be among the largest members of the TM7 family. Although not all of their ligands or downstream targets have been identified, there is evidence implicating the EGF-TM7 family diverse processes such as cell adhesion, migration, inflammation, and autoimmune disease. Recent studies have identified expression of EGF-TM7 family members on human neoplasms including those of the thyroid, stomach, colon, and brain. Their presence on these tissues is not surprising given the ubiquity of GPCRs, but because their functional significance and pathways are not completely understood, they are of tremendous clinical and scientific interest. Current evidence suggests that expression of certain EGF-TM7 receptors is correlated with tumor grade, confers a more invasive phenotype, and increases the likelihood of metastatic disease. In this review, we will discuss the structure, function, and regulation of these receptors. We also describe the expression of these receptors in human cancers and explore their potential mechanistic significance.

Basement Membrane Deposition of Nidogen 1 but Not Nidogen 2 Requires the Nidogen Binding Module of the Laminin γ1 Chain

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

A Molecular Analysis of Mutations at the Complex dumpy Locus in Drosophila melanogaster

The Drosophila dumpy gene consists of seventy eight coding exons and encodes a huge extracellular matrix protein containing large numbers of epidermal growth factor-like (EGF) modules and a novel module called dumpy (DPY). A molecular analysis of forty five mutations in the dumpy gene of Drosophila melanogaster was carried out. Mutations in this gene affect three phenotypes: wing shape, thoracic cuticular defects, and lethality. Most of the mutations were chemically induced in a single dumpy allele and were analyzed using a nuclease that cleaves single base pair mismatches in reannealed duplexes followed by dHPLC. Additionally, several spontaneous mutations were analyzed. Virtually all of the chemically induced mutations, except for several in a single exon, either generate nonsense codons or lesions that result in downstream stop codons in the reading frame. The remaining chemically induced mutations remove splice sites in the nascent dumpy message. We propose that the vast majority of nonsense mutations that affect all three basic dumpy phenotypes are in constitutive exons, whereas nonsense mutants that remove only one or two of the basic functions are in alternatively spliced exons. Evolutionary comparisons of the dumpy gene from seven Drosophila species show strong conservation of the 5′ ends of exons where mutants with partial dumpy function are found. In addition, reverse transcription PCR analyses reveal transcripts in which exons marked by nonsense mutations with partial dumpy function are absent.

Is There a Genetic Basis for Polycystic Ovary Syndrome?

Clinical evidence indicates that polycystic ovary syndrome (PCOS) has a heritable basis (1, 2), at least in part, which could result from a genetic etiology, epigenetic changes, or an admixture of the two causes. The apparent heritable nature of the syndrome has led investigators to search for the gene (or genes) that contribute to development of the PCOS phenotype. One impetus for genetic studies of PCOS was to unravel the etiology of the syndrome and provide greater precision to the diagnosis. Unfortunately, the phenotypic variability of the syndrome represents a major hindrance to that goal because the PCOS phenotype is based on criteria marked by a constellation of features, currently defined by the Rotterdam criteria (3). Thus, the PCOS phenotype is not a clearly delineated disease entity, and there is imprecision of diagnosis. Patients diagnosed with PCOS may satisfy the diagnostic criteria by having different features of the disease. Stated differently, PCOS can be conceptualized as a pathophysiological state of acyclic ovarian function rather than a single distinct disorder. This imprecision in diagnosis raises the strong possibility that PCOS with different root causes will be lumped into the same “diseased category” for comparison with normal controls. Genetic studies of PCOS are also confounded by variability between different investigators, especially if the study design or diagnostic criteria used across studies are not the same. The group of Ewens et al. (4) has used standard diagnostic criteria throughout their studies (oligomenorrhea and increased testosterone levels) to distinguish cases from controls. A potential problem is that any variability in diagnostic criteria between investigators may impact the ability of putative candidate genes identified in one population to be replicated in other populations if the initial population studied was phenotypically dissimilar, as a result of different diagnostic criteria. Investigators conducting genetic studies of PCOS have used several different approaches in different populations. Some have used a candidate gene approach. Other investigators have used linkage studies, either of case-control or family-based sib pairs. Association studies have also been conducted. Another approach is the transmission disequilibrium test—briefly, a family-based test of association designed to minimize error due to population stratification (5). Despite a decade of collaborative efforts, including analysis of 37 candidate genes (6), efforts to identify loci associated with PCOS have been plagued by a lack of replication between association and linkage studies (reviewed in Ref. 7). It seems clear that neither follistatin (8) nor lamin a/c (9) is a critical PCOS gene. So what genes are associated with PCOS? Surprisingly, in 2010 the best evidence of an association of a single gene (locus) with PCOS is the dinucleotide repeat microsatellite marker D19S884 (4, 6, 10–13). The D19S884 marker lies within intron 55 of the fibrillin-3 gene (FBN3, NM_032447). Allele 8 of the D19S884 marker contains 17 “GT” repeats and has been associated with PCOS. In contrast, allele 9 containing 18 GT repeats and allele 13 with 22 GT repeats show no association with PCOS. Fibrillin is a large extracellular matrix protein that forms microfibrils and contains epidermal growth factorlike modules, some of which have a calcium binding consensus sequence (calcium binding-epidermal growth factor-like modules). Related fibrillin family members regulate TGF action, but the reason as to why fibrillin-3 hasbeenassociatedwithPCOSremainsunclear, especially

The Relationship between Anti-merozoite Antibodies and Incidence of Plasmodium falciparum Malaria: A Systematic Review and Meta-analysis

One of the criteria to objectively prioritize merozoite antigens for malaria vaccine development is the demonstration that naturally acquired antibodies are associated with protection from malaria. However, published evidence of the protective effect of these antibodies is conflicting. We performed a systematic review with meta-analysis of prospective cohort studies examining the association between anti-merozoite immunoglobin (Ig) G responses and incidence of Plasmodium falciparum malaria. Two independent researchers searched six databases and identified 33 studies that met predefined inclusion and quality criteria, including a rigorous definition of symptomatic malaria. We found that only five studies were performed outside sub-Saharan Africa and that there was a deficiency in studies investigating antibodies to leading vaccine candidates merozoite surface protein (MSP)-1(42) and erythrocyte binding antigen (EBA)-175. Meta-analyses of most-studied antigens were conducted to obtain summary estimates of the association between antibodies and incidence of P. falciparum malaria. The largest effect was observed with IgG to MSP-3 C terminus and MSP-1(19) (responders versus nonresponders, 54%, 95% confidence interval [CI] [33%-68%] and 18% [4%-30%] relative reduction in risk, respectively) and there was evidence of a dose-response relationship. A tendency towards protective risk ratios (RR<1) was also observed for individual study estimates for apical membrane antigen (AMA)-1 and glutamate-rich protein (GLURP)-R0. Pooled estimates showed limited evidence of a protective effect for antibodies to MSP-1 N-terminal regions or MSP-1-EGF (epidermal growth factor-like modules). There was no significant evidence for the protective effect for MSP-2 (responders versus nonresponders pooled RR, MSP-2(FC27) 0.82, 95% CI 0.62-1.08, p = 0.16 and MSP-2(3D7) 0.92, 95% CI 0.75-1.13, p = 0.43). Heterogeneity, in terms of clinical and methodological diversity between studies, was an important issue in the meta-analysis of IgG responses to merozoite antigens. These findings are valuable for advancing vaccine development by providing evidence supporting merozoite antigens as targets of protective immunity in humans, and to help identify antigens that confer protection from malaria. Further prospective cohort studies that include a larger number of lead antigens and populations outside Africa are greatly needed to ensure generalizability of results. The reporting of results needs to be standardized to maximize comparability of studies. We therefore propose a set of guidelines to facilitate the uniform reporting of malaria immuno-epidemiology observational studies. Please see later in the article for the Editors' Summary.

Fibulins: multiple roles in matrix structures and tissue functions.

The fibulins are a family of secreted glycoproteins associated with basement membranes, elastic fibers, and other matrices. They are expressed in a variety of tissues. Association with these matrix structures is mediated by their ability to interact with many extracellular matrix constituents. The seven members of the family are defined by the presence of two structural modules, a tandem repeat of epidermal growth factor-like modules and a unique C-terminal fibulin-type module. They act not only as intermolecular bridges within the extracellular matrix to form supramolecular structures, but also as mediators for cellular processes and tissue remodeling. These important functions of fibulins in a wide range of biological processes have been shown in in vitro systems, gene knockout mice, and human genetic disorders. In this review, we describe the structure and function of these proteins and discuss the implication of fibulins in development and diseases.

Adipose tissue inflammation and liver fat in patients with highly active antiretroviral therapy-associated lipodystrophy

In this cross-sectional study, we sought to determine whether gene expression of macrophage markers and inflammatory chemokines in lipoatrophic subcutaneous abdominal adipose tissue and liver fat content are increased and interrelated in human immunodeficiency virus (HIV)-1-positive, highly active antiretroviral therapy (HAART)-treated patients with lipodystrophy (HAART+LD+; n = 27) compared with those without (HAART+LD-; n = 13). The study groups were comparable with respect to age, gender, and body mass index. The HAART+LD+ group had twofold more intra-abdominal (P = 0.01) and 1.5-fold less subcutaneous (P = 0.091) fat than the HAART+LD- group. As we have reported previously, liver fat was 10-fold higher in the HAART+LD+ compared with the HAART+LD- group (P = 0.00003). Inflammatory gene expression was increased in HAART-lipodystrophy: CD68 4.5-fold (P = 0.000013), tumor necrosis factor (TNF)-alpha 2-fold (P = 0.0094), chemokine (C-C motif) ligand (CCL) 2 2.5-fold (P = 0.0024), CCL3 7-fold (P = 0.0000017), integrin alphaM (ITGAM) 3-fold (P = 0.00067), epidermal growth factor-like module containing, mucin-like, hormone receptor-like (EMR)1 2.5-fold (P = 0.0038), and a disintegrin and metalloproteinase domain (ADAM)8 3.5-fold (P = 0.00057) higher in the HAART+LD+ compared with the HAART+LD- group. mRNA concentration of CD68 (r = 0.37, P = 0.019), ITGAM (r = 0.35, P = 0.025), CCL2 (r = 0.39, P = 0.012), and CCL3 (r = 0.54, P = 0.0003) correlated with liver fat content. In conclusion, gene expression of markers of macrophage infiltration and adipose tissue inflammation is increased in lipoatrophic subcutaneous abdominal adipose tissue of patients with HAART-associated lipodystrophy compared with those without. CD68, ITGAM, CCL2, and CCL3 expression is significantly associated with accumulation of liver fat.

Structure of the Extracellular Domain of Tie Receptor Tyrosine Kinases and Localization of the Angiopoietin-binding Epitope

Angiogenesis is essential for tissue repair and regeneration during wound healing but also plays important roles in many pathological processes including tumor growth and metastasis. The receptor protein tyrosine kinase Tie2 and its ligands, the angiopoietins, have important functions in the regulation of angiogenesis. Here, we report a detailed structural and functional characterization of the extracellular region of Tie2. Sequence analysis of the extracellular domain revealed an additional immunoglobulin-like domain resulting in a tandem repeat of immunoglobulin-like domains at the N terminus of the protein. The same domain organization was also found for the Tie1 receptor that shares a high degree of homology with Tie2. Based on structural similarities to other receptor tyrosine kinases and cell adhesion molecules, we demonstrate that the N-terminal two immunoglobulin-like domains of Tie2 harbor the angiopoietin-binding site. Using transmission electron microscopy we furthermore show that the extracellular domain of Tie receptors consists of a globular head domain and a short rod-like stalk that probably forms a spacer between the cell surface and the angiopoietin-binding site. Mutational analysis demonstrated that the head domain consists of the three immunoglobulin-like domains and the three epidermal growth factor-like modules and that the stalk is formed by the three fibronectin type III repeats. These findings might be of particular interest for drug development because Tie receptors are potential targets for treatment of angiogenesis-associated diseases.

Biophysical Characterization of the Signature Domains of Thrombospondin-4 and Thrombospondin-2

The signature domain of thrombospondins consists of tandem epidermal growth factor-like modules, 13 calcium-binding repeats, and a lectin-like module. Although very similar, the signature domains of thrombospondin-1 and -2 differ in several potentially important ways from the domains of thrombospondin-3, -4, and -5. We have compared matching recombinant segments representing the signature domains of thrombospondin-2 and -4. In the presence of 2 mM CaCl2, the far UV circular dichroism spectra of thrombospondin-2 and -4 constructs contain a strong negative band at 202 nm, but only the thrombospondin-2 construct has a band at 216 nm. Chelation of calcium shifted the negative bands to lower magnitudes. Titrations of the spectra demonstrated lower cooperativity and affinity for binding of calcium to thrombospondin-4 compared with thrombospondin-2. Atomic absorption spectroscopy demonstrated that the thrombospondin-4 constructs bind seven less calcium than the thrombospondin-2 construct at 0.6 mM CaCl2. In 2 mM CaCl2, the near UV circular dichroism spectra of thrombospondin-2, but not thrombospondin-4, contain a positive band at 292 nm that disappears upon calcium chelation. Intrinsic fluorescence spectra for both proteins were also sensitive to calcium, but the changes were simpler and more marked for thrombospondin-2 than for thrombospondin-4. In differential scanning calorimetry, the thrombospondin-2 construct melted in two distinct transitions at 53.5 and 81.8 degrees C, whereas the first transition for thrombospondin-4 constructs was observed at 63.5 degrees C. Thus, the studies revealed significant differences between the signature domains of thrombospondin-2 and thrombospondin-4 in calcium binding, fine structure, and inter-modular interactions.

Structure of the calcium-rich signature domain of human thrombospondin-2

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-A-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor-like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca(2+) ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

Laminin γ3 Chain Binds to Nidogen and Is Located in Murine Basement Membranes

Recently a novel laminin gamma3 chain was identified in mouse and human and shown to have the same modular structure as the laminin gamma1 chain. We expressed two fragments of the gamma3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The gamma3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the beta2 VI/V fragment. The gamma3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the gamma1 III3-5 fragment. These data suggested that laminins containing the gamma3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against gamma3 VI/V and gamma3 III3-5 showed no cross-reaction with homologous fragments from the gamma1 and gamma2 chains of laminin and allowed the establishment of gamma chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the gamma3 chain compared with the gamma1 chain in various tissue extracts of adult mice. The expression of gamma3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the gamma3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the gamma3 chain to basement membranes of these tissues.

Bovine model of Marfan syndrome results from an amino acid change (c.3598G&gt;A, p.E1200K) in a calcium-binding epidermal growth factor-like domain of fibrillin-1

Marfan Syndrome (MFS) is an autosomal dominant disorder caused by mutations in the fibrillin-1 gene (FBN1). Several calves, all sired by a phenotypically normal bull, were found to exhibit the major clinical and pathological characteristics of human MFS (aortic dissection, joint laxity, lens dislocation), and were recognized as potential models of the human disease. In this study, Fbn1 cDNA from affected animals was sequenced and a heterozygous c.3598G > A transition was detected in exon 29, which predicted the substitution of an evolutionarily conserved glutamic acid by lysine at position 1200 (p.E1200K). This residue is part of a calcium-binding epidermal growth factor-like (cbEGF-like) module, a domain that is frequently altered in human MFS. Analysis of genomic DNA from the original bull's sperm showed that less than 20% of the sperm harbored the mutation, consistent with the presence of germline mosaicism. This study validates the use of these animals as models of human MFS. These cows will be valuable for investigations into the molecular pathogenesis of MFS, and may lead to better therapeutic testing and evaluation of human Marfan patients.