FNIII Domain Research Articles

Tenascin-C is expressed by human glioma in vivo and shows a strong association with tumor blood vessels

The extracellular matrix (ECM) protein tenascin-C (TN-C) is upregulated within glioma tissues and cultured glioma cell lines. TN-C possesses a multi-modular structure and a variety of functional properties have been reported for its domains. We describe five novel monoclonal antibodies identifying different domains of TN-C. The epitopes for these antibodies were investigated by using recombinantly expressed fibronectin type III domains of TN-C. The biological effects of TN-C fragments on glioma cell proliferation and adhesion were analyzed. The expression pattern of TN-C in human glioma tissue sections and in glioma cell lines was studied with the novel library of monoclonal antibodies. The immunocytochemical analyses of the established human glioma cell lines U-251-MG, U-373-MG and U-87-MG revealed distinct staining patterns for each antibody. Robust expression of TN-C was found within the tumor mass of surgery specimens from glioblastoma. In many cases, the expression of this ECM molecule was clearly associated with blood vessels, particularly with microvessels. Three of the new antibodies highlighted individual TN-C-expressing single cells in glioma tissues. The effect of TN-C domains on glioma cells was examined by a BrdU-proliferation assay and an adhesion assay. Short fragments of constitutively expressed TN-C-domains did not exert significant effects on the proliferation of glioma cells, whereas the intact molecule increased cell division rates. In contrast, the long fragment TNfnALL containing all of the FNIII domains of TN-C decreased proliferation. Additionally, we found strong differences between the adhesion-influencing properties of the recombinant fragments on glioma cells.

Molecular cloning and genomic characterization of novel Leptin-like genes in salmonids provide new insight into the evolution of the Leptin gene family

In the current study we describe the identification of novel leptin B homologous gene/s in the four salmonid species Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus). Homology modeling of Salmo salar (Ss) LepB1/B2 suggests that the protein satisfies parameters as long-chain four helical cytokine family and that the basic structural pattern of the protein follows that of human leptin (Zhang et al., 1997). Importantly, the docking studies suggested the SsLepB has binding affinity to the AA residues that identify the leptin binding and FNIII domains of the SsLep receptor (Rønnestad et al., 2010). Phylogenetic analyses support that LepB paralogs have most probably originated by 4R whole genome duplication (WGD) before speciation of the salmonid lineages. LepB1 and LepB2 genes are both present in the two closest relatives, the Atlantic salmon and the brown trout, while rainbow trout and charr have only preserved the long LepB1 variant in their genome. We have defined the sites of SsLepB mRNA expression at key life stages in Atlantic salmon and found that SsLepB1 and SsLepB2, although to different extent, were expressed in redundant and mostly complementary fashion in brain and gills throughout the lifecycle, suggesting that this pair of paralogs is likely undergoing early stages of subfunctionalization. Furthermore, we have quantified the expression profiles of SsLepB genes and of other two recently duplicated salmon leptins (SsLepA1, SsLepA2) during early development and show evidence that in fish, as in mammals and amphibians, leptin could play important roles in growth and development. This study provides an essential groundwork to further elucidate structural and functional evolution of this important hormone in salmonids as well as in other teleosts.

Antigenic differences between the EG95‐related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination

Cystic echinococcosis caused by Echinococcus granulosus remains an important and neglected issue in public health. The study of the likely efficacy of the currently available EG95 vaccine against other genotypes of the parasite is important to improve the vaccine as a potential tool to be used in control programmes. The recombinant vaccine EG95-1G1 was developed based on the G1 genotype of E. granulosus. Characterization of the eg95 gene family in the G6 genotype by genomic DNA cloning previously produced the first unequivocal information about the composition of the gene family in a different genotype. The information was used in this study to predict and express two EG95-related proteins from the G6 genotype as recombinants, for assessment of their capacity to bind antibodies raised in sheep vaccinated with the EG95-1G1 vaccine. The proteins (EG95-1G6 and EG95-5G6) from the G6 genotype of E. granulosus were unable to bind all the antibodies raised by sheep vaccinated with EG95-1G1. Differences in the amino acid sequence of EG95-related proteins from G6 and likely the differences in the encoded FnIII domain may be responsible for changes in the conformation of these epitopes.

Towards constructing extracellular matrix-mimetic hydrogels: An elastic hydrogel constructed from tandem modular proteins containing tenascin FnIII domains

Protein-based hydrogels have been developed for various biomedical applications where they provide artificial extracellular microenvironments that mimic the physical and biochemical characteristics of natural extracellular matrices (ECMs). In natural ECMs, a large number of proteins are tandem modular proteins consisting of many individually folded functional domains that confer structural and biological functionalities. Such tandem modular proteins are promising building blocks for constructing ECM-mimetic biomaterials. However, their use for such purposes has not been explored extensively. Tenascin-C (TNC) is an ECM tandem modular protein and plays an important role in mechanotransduction by regulating important cell–matrix interactions. The third FnIII domain of TNC (TNfn3) contains an RGD sequence and is known to bind integrins. Here we use the TNfn3 domain and resilin sequence-based tandem modular protein FRF4RF4R (F represents the TNfn3 domain and R represents the resilin sequence, respectively) as a building block to construct protein-based ECM-mimetic hydrogels. The tandem modular protein-based building block FRF4RF4R closely mimics the architecture of the naturally occurring tandem modular ECM protein TNC and incorporates intact RGD-containing FnIII domains. Our results demonstrate that tandem modular proteins containing TNfn3 can be readily photochemically crosslinked into elastic hydrogels, whose Young’s modulus can be tuned by the concentration of the tandem modular protein solution. In vitro studies demonstrate that none of the photochemical crosslinking reaction components are cytotoxic at the level tested, and the hydrogel supports the spread of human lung fibroblast cells. Our results demonstrate that FRF4RF4R-based hydrogel is a novel ECM-mimetic hydrogel.

Ligand-Induced Architecture of the Leptin Receptor Signaling Complex

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.

Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.

Studies on structural and average unfolding behaviours of FNIII domain of Contactin-1 protein by molecular dynamics simulation

Steered molecular dynamics (SMD) simulations are routinely used for unraveling folded domains of proteins to reveal intermediate structures and to compute associated forces and free energies. Using the example of a neuronal adhesion protein, Contactin, we discuss the effect of pulling velocities on unfolding forces and suggest a work–velocity scaling relation, which may give an estimate of the non-equilibrium nature of SMD simulations performed in complex environments. Implications of the existence of such a scaling relation and validity of Jarzynski's equality in the context of fast unfolding velocities are also discussed. Multiple unfolding pathways and protein intermediates along these pathways have been identified.

Abstract LB-149: The OPCML tumor suppressor binds and negatively regulates the Axl receptor tyrosine kinase, sensitizing cells to foretinib in epithelial ovarian cancer (EOC)

Abstract OPCML (opioid binding protein/cell adhesion like molecule) is a tumour suppressor gene that is silenced almost by somatic methylation in more than 80% of EOC cases, with loss of heterozygosity (LOH) occurring in 49% of cases [1,2]. OPCML binds a specific repertoire of receptor tyrosine kinases (RTKs) consisting of HER2, HER4 EphA2, FGFR1 and FGFR3. OPCML switches RTKs from clathrin to non clathrin dependent endocytosis, resulting in polyubiquitination of the RTK and targeting to the proteasome for destruction with associated signalling and growth inhibition [3]. We show that Axl, a TAM family RTK that is frequently activated in ovarian cancer [4], also interacts with and is downregulated by OPCML. Axl is downregulated following transient and stable OPCML expression in ovarian cancer lines SKOV3 and PEO1, with reciprocally increased Axl expression following stable knockdown of physiological OPCML in normal ovarian surface epithelial line OSE-C2. We performed co-IP and mammalian 2-hybrid (M 2-H) analysis between Axl and OPCML and determined that the Ig and FNIII domains are crucial to this interaction. We previously reported a complete loss of function proline 95 to arginine mutation [2]. Transfection of OPCMLWT protein in OPCML deficient cells promotes intercellular adhesion and adhesion to Collagen I and Fibronectin matrix, in association with Axl downregulation, In contrast OPCMLP95R transfection did not repress Axl and did not promote intercellular or cell to matrix adhesion. M 2-H data demonstrated that OPCMLP95R expression abolishes the interaction seen between OPCMLWT and Axl. We explored the impact of OPCML expression on cell signalling response to Foretinib (GSK1363089), a potent inhibitor of Axl [5]; OPCML led to a 3-fold sensitization to the tyrosine kinase inhibitor. The finding that OPCML, a frequently inactivated ovarian cancer tumor suppressor negatively regulates Axl, a frequently activated RTK in ovarian cancer and sensitises Axl positive OPCML deficient cells to Foretinib defines a promising predictive and therapeutic role for OPCML in ovarian cancer.

Molecular characterization and expression analysis of the putative interleukin 6 receptor (IL-6Rα and glycoprotein-130) in rainbow trout (Oncorhynchus mykiss): Salmonid IL-6Rα possesses a polymorphic N-terminal Ig domain with variable numbers of two repeats

Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this study, we have cloned both the IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa and is similar in size, extracellular domain structure (D1-D6) and presence of intracellular motifs important for signal transduction to tetrapod gp130s. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine-binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, a phylogenetic tree analysis confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts. The extra large D1 domain of the salmonid IL-6Rα molecules results partially from the insertions of two repetitive sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively. Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines, and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by LPS and Poly I:C in the cell lines studied. The expression patterns of the receptors suggest that high level expression of IL-6Rα is critical for IL-6 responsiveness.

Mechanical Unfolding of Cardiac Myosin Binding Protein-C by Atomic Force Microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.

Kinetic Partitioning Mechanism Governs the Folding of the Third FnIII Domain of Tenascin-C: Evidence at the Single-Molecule Level

Statistical mechanics and molecular dynamics simulations proposed that the folding of proteins can follow multiple parallel pathways on a rugged energy landscape from unfolded state en route to their folded native states. Kinetic partitioning mechanism is one of the possible mechanisms underlying such complex folding dynamics. Here, we use single-molecule atomic force microscopy technique to directly probe the multiplicity of the folding pathways of the third fibronectin type III domain from the extracellular matrix protein tenascin-C (TNfn3). By stretching individual (TNfn3) 8 molecules, we forced TNfn3 domains to undergo mechanical unfolding and refolding cycles, allowing us to directly observe the folding pathways of TNfn3. We found that, after being mechanically unraveled and then relaxed to zero force, TNfn3 follows multiple parallel pathways to fold into their native states. The majority of TNfn3 fold into the native state in a simple two-state fashion, while a small percentage of TNfn3 were found to be trapped into kinetically stable folding intermediate states with well-defined three-dimensional structures. Furthermore, the folding of TNfn3 was also influenced by its neighboring TNfn3 domains. Complex misfolded states of TNfn3 were observed, possibly due to the formation of domain-swapped dimeric structures. Our studies revealed the ruggedness of the folding energy landscape of TNfn3 and provided direct experimental evidence that the folding dynamics of TNfn3 are governed by the kinetic partitioning mechanism. Our results demonstrated the unique capability of single-molecule AFM to probe the folding dynamics of proteins at the single-molecule level.

Probing the Folded State of Fibronectin Type III Domains in Stretched Fibrils by Measuring Buried Cysteine Accessibility

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Nodes of Ranvier Act as Barriers to Restrict Invasion of Flanking Paranodal Domains in Myelinated Axons

Accumulation of voltage-gated sodium (Na(v)) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization, and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (Nfasc(NF¹⁸⁶)) in vivo results in nodal disorganization, including loss of Na(v) channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral nervous system (PNS) and central nervous system (CNS). Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in Nfasc(NF¹⁸⁶) mutant axons and occlude node formation. Our results suggest that Nfasc(NF¹⁸⁶)-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.

The First Type III Domain of Fibronectin is Associated with the Expression of Cytokines within the Lung Tumor Microenvironment

Recent studies have pointed to changes in tissue mechanics as a contributory element to the development of malignancies. Increased tissue rigidity is associated with the unfolding of the Type III domains of fibronectin within the extracellular matrix. The consequences of this unfolding on cellular functions within the lung are not well understood. In the present study, we evaluated the effect of a peptide representing a partially unfolded intermediate of the first Type III repeat of fibronectin (FnIII-1c) on inflammatory gene expression in adult human lung fibroblast cells. FnIII-1c induced expression of cytokines, CXCL1-3, IL-8 and TNF-α, by lung fibroblast cells. The increase in IL-8 expression was dependent on Toll-like receptor 2 and NFκB. Immunohistochemistry of tissue arrays representing squamous cell carcinoma of the lung revealed extensive stromal staining for IL-8 and fibronectin fibrils which were co-aligned with myofibroblasts. These data suggest a model in which unfolding of FnIII domains secondary to myofibroblast-generated tension may induce the release of cytokines by stromal fibroblasts present within the lung tumor.

Phenotypic effects of Ehlers–Danlos syndrome‐associated mutation on the FnIII domain of tenascin‐X

Tenascin-X (TNX) is an extracellular matrix (ECM) protein and interacts with a wide variety of molecules in the ECM as well as on the membrane. Deficiency of TNX causes a recessive form of Ehlers-Danlos syndrome (EDS) characterized by hyperelastic and fragile skin, easy bruising, and hypermobile joints. Three point mutations in TNX gene were found to be associated with hypermobility type EDS and one of such mutations is the V1195M mutation at the 7th fibronectin Type III domain (TNXfn7). To help elucidate the underlying molecular mechanism connecting this mutation to EDS, here we combined homology modeling, chemical denaturation, single molecule atomic force microscopy, and molecular dynamics (MD) simulation techniques to investigate the phenotypic effects of V1195M on TNXfn7. We found that the V1195M mutation does not alter the three-dimensional structure of TNXfn7 and had only mild destabilization effects on the thermodynamic and mechanical stability of TNXfn7. However, MD simulations revealed that the mutation V1195M significantly alters the flexibility of the C'E loop of TNXfn7. As loops play important roles in protein-protein and protein-ligand interactions, we hypothesize that the decreased loop flexibility by V1195M mutation may affect the binding of TNX to ECM molecules and thus adversely affect collagen deposition and fibrillogenesis. Our results may provide new insights in understanding the molecular basis for the pathogenesis of V1195M-resulted EDS.

Multidrug therapy in a patient with Rabson–Mendenhall syndrome

To the Editor: Insulin resistance is a common precursor to type 2 diabetes mellitus. It is characterised by impaired insulin signalling in several tissues, with compensatory hyperinsulinaemia. Extreme insulin resistance, on the other hand, is very rare. Although the molecular basis for extreme insulin resistance remains unknown in many patients, lipodystrophies and loss-of-function mutations in the insulin receptor gene are well established causes of this phenotype. In patients with lipodystrophy, type 2 diabetes is usually associated with several metabolic disturbances, including low HDL-cholesterol, hypertriacylglycerolaemia and non-alcoholic fatty liver disease, among others [1]. Mutations in the insulin receptor gene produce a spectrum of diseases including Donohue’s syndrome, Rabson–Mendenhall syndrome and type A insulin resistance; patients with these conditions have several different characteristics [2]. Severe cases are diagnosed at birth or in infancy due to the presence of signs of severe hyperinsulinaemia such as acanthosis nigricans, abnormal dentition, hyperandrogenism and pseudo-acromegaly. Type 2 diabetes develops early in life and its treatment is usually extremely challenging. Insulin is often used in very high doses, but microvascular complications develop early and aggressively in these patients [2, 3]. The use of other therapies (leptin, IGF-1, metformin and pioglitazone) has already been described in scattered reports, but usually as monotherapy and with limited efficacy [4]. A 17-year-old boy was brought to our attention for treatment of type 2 diabetes. His BMI was in the ageand sex-specific normal range (weight 44.8 kg, height 161.0 cm) and he had no lipodystrophy. He was the older son of non-consanguinous parents and his development was unremarkable except for abnormal dentition and short stature. He had had a history of severe acanthosis nigricans since 6 months of age affecting cervical, axillary and inguinal areas, and macrogenitalism without macro-orchidism had been diagnosed in early life. On examination he had signs of pseudoacromegaly and hyperandrogenism. Initial fasting glucose and insulin were 6.8 mmol/l and 2,102 pmol/l, respectively, with postprandial glucose (at 2 h) of 23.5 mmol/l. Postprandial insulin was above the laboratory limit of detection. Fasting triacylglycerol and HDL-cholesterol were normal at 0.56 mmol/l and 1.0 mmol/l, while HbA1c was 11.9%. On clinical and biochemical grounds type 2 diabetes secondary to Rabson–Mendehall syndrome was diagnosed. Sequencing of the insulin receptor gene revealed two mutations: (1) Asn851Ser, a novel mutation changing an exquisitely conserved residue in one of the extracellular FnIII domains of the beta subunit of the receptor; and (2) the previously described Ala1135Val in the highly conserved catalytic loop of the tyrosine kinase domain of the receptor [5]. R. O. Moreira (*) :R. L. Zagury : L. Zagury Instituto Estadual de Diabetes e Endocrinologia, Rua Moncorvo Filho 90, Rio de Janeiro, RJ, Brazil e-mail: rom_br@yahoo.com

Generation and Identification of Monoclonal Antibodies Against FNIII Domain D of Human Tenascin-C

Tenascin-C (TN-C), a key component of extracellular matrix (ECM), is strongly expressed in fetal and cancer tissues. Large-molecular-weight variants of TN-C, including different combinations of its alternative spliced FNIII repeats, are specifically expressed in tissues under certain pathological conditions. Here we report the production of monoclonal antibodies (MAbs) against FNIII domain D (FNIII D) of human TN-C. Complementary DNA encoding the FNIII D region was generated by RT-PCR from human osteosarcoma (OS) cell line, and the recombinant FNIII D-GST fusion protein was expressed and purified. Two hybridoma cell lines secreting monoclonal antibodies (MAbs) against FNIII D were obtained by routine murine hybridoma technique. The MAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). Both of them were applicable in Western blot and IHC. With our MAbs, we found TN-C was positive in OS and most of it was among the tumor stroma. To conclude, these MAbs to human FNIII D domain of TN-C may be useful for exploring OS pathogenesis and potential clinical application.

Non-Native Interactions Are Critical for Mechanical Strength in PKD Domains

SummaryExperimental observation has led to the commonly held view that native state protein topology is the principle determinant of mechanical strength. However, the PKD domains of polycystin-1 challenge this assumption: they are stronger than predicted from their native structure. Molecular dynamics simulations suggest that force induces rearrangement to an intermediate structure, with nonnative hydrogen bonds, that resists unfolding. Here we test this hypothesis directly by introducing mutations designed to prevent formation of these nonnative interactions. We find that these mutations, which only moderately destabilize the native state, reduce the mechanical stability dramatically. The results demonstrate that nonnative interactions impart significant mechanical stability, necessary for the mechanosensor function of polycystin-1. Remarkably, such nonnative interactions result from force-induced conformational change: the PKD domain is strengthened by the application of force.

A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four "site 1" and six "site 2" analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val(43), Phe(28), and Val(14) (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln(18) affects only IGF-1R and not IR binding. Alanine substitutions at Glu(12), Asp(15), Phe(19), Leu(53), and Glu(57) analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu(12), Phe(19), Leu(53), and Glu(57) that potentially engages the IGF-1R at one or more of the FnIII domains.