Fibronectin Type III-like Domains Research Articles

The Listeria innocua chitinase LinChi78 has a unique region that is necessary for hydrolytic activity.

Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase from Listeria innocua has a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.

Study of Fibronectin Type III-Like Domains Role in Activation of gp130 Receptor.

Chimeric gp130 receptors were produced to study the role of three fibronectin type III-like domains in activation of gp130 receptor machinery. The ligand-induced dimerization of gp130 was sufficient to trigger STAT3 signaling pathway. These findings can be used as the basis in designing novel therapeutic gp130 inhibitors.

Non-productive adsorption of bacterial β-glucosidases on lignins is electrostatically modulated and depends on the presence of fibronection type III-like domain

Non-productive adsorption of cellulases onto lignins is an important mechanism that negatively affects the enzymatic hydrolysis of lignocellulose biomass. Here, we examined the non-productive adsorption of two bacterial β-glucosidases (GH1 and GH3) on lignins. The results showed that β-glucosidases can adsorb to lignins through different mechanisms. GH1 β-glucosidase adsorption onto lignins was found to be strongly pH-dependent, suggesting that the adsorption is electrostatically modulated. For GH3 β-glucosidase, the results suggested that the fibronectin type III-like domain interacts with lignins through electrostatic and hydrophobic interactions that can partially, or completely, overcome repulsive electrostatic forces between the catalytic domain and lignins. Finally, the increase of temperature did not result in the increase of β-glucosidases adsorption, probably because there is no significant increase in hydrophobic regions in the β-glucosidases structures. The data provided here can be useful for biotechnological applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts.

Analysis of the xynB5 gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in Caulobacter crescentus.

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-L-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (Km 0.24 ± 0.0005 mM, Vmax 0.041 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.27 mM(-1) s(-1)), followed by β-xylosidase (Km 0.64 ± 0.032 mM, Vmax 0.055 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.14 mM(-1)s(-1)) and finally α-L-arabinosidase (Km 1.45 ± 0.05 mM, Vmax 0.091 ± 0.0004 µmol min(-1) mg(-1) and Kcat/Km 0.1 mM(-1) s(-1)). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.

Interactions by the Fungal Flo11 Adhesin Depend on a Fibronectin Type III-like Adhesin Domain Girdled by Aromatic Bands

Saccharomyces cerevisiae harbors a family of GPI-anchored cell wall proteins for interaction with its environment. The flocculin Flo11, a major representative of these fungal adhesins, confers formation of different types of multicellular structures such as biofilms, flors, or filaments. To understand these environment-dependent growth phenotypes on a molecular level, we solved the crystal structure of the N-terminal Flo11A domain at 0.89-Å resolution. Besides a hydrophobic apical region, the Flo11A domain consists of a β sandwich of the fibronectin type III domain (FN3). We further show that homophilic Flo11-Flo11 interactions and heterophilic Flo11-plastic interactions solely depend on the Flo11A domain and are strongly pH dependent. These functions of Flo11A involve an apical region with its surface-exposed aromatic band, which is accompanied by acidic stretches. Together with electron microscopic reconstructions of yeast cell-cell contact sites, our data suggest that Flo11 acts as a spacer-like, pH-sensitive adhesin that resembles a membrane-tethered hydrophobin.

The chitinase C gene PsChiC from Pseudomonas sp. and its synergistic effects on larvicidal activity.

Pseudomonas sp. strain TXG6-1, a chitinolytic gram-negative bacterium, was isolated from a vegetable field in Taixing city, Jiangsu Province, China. In this study, a Pseudomonas chitinase C gene (PsChiC) was isolated from the chromosomal DNA of this bacterium using a pair of specific primers. The PsChiC gene consisted of an open reading frame of 1443 nucleotides and encoded 480 amino acid residues with a calculated molecular mass of 51.66 kDa. The deduced PsChiC amino acid sequence lacked a signal sequence and consisted of a glycoside hydrolase family 18 catalytic domain responsible for chitinase activity, a fibronectin type III-like domain (FLD) and a C-terminal chitin-binding domain (ChBD). The amino acid sequence of PsChiCshowed high sequence homology (> 95%) with chitinase C from Serratia marcescens. SDS-PAGE showed that the molecular mass of chitinase PsChiC was 52 kDa. Chitinase assays revealed that the chitobiosidase and endochitinase activities of PsChiCwere 51.6- and 84.1-fold higher than those of pET30a, respectively. Although PsChiC showed little insecticidal activity towards Spodoptera litura larvae, an insecticidal assay indicated that PsChiC increased the insecticidal toxicity of SpltNPV by 1.78-fold at 192 h and hastened death. These results suggest that PsChiC from Pseudomonas sp. could be useful in improving the pathogenicity of baculoviruses.

Shear Stress-induced Redistribution of Vascular Endothelial-Protein-tyrosine Phosphatase (VE-PTP) in Endothelial Cells and Its Role in Cell Elongation

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.

1H, 15N and 13C backbone chemical shift assignment of titin domains A59–A60 and A60 alone

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone 15N, 13C and 1H chemical shift and 13Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.

Tenascin C Promiscuously Binds Growth Factors via Its Fifth Fibronectin Type III-Like Domain

Tenascin C (TNC) is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII) domains. The fifth TNCIII domain (TNCIII5) has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII), specifically FNIII12–14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1–5), as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF) family, the fibroblast growth factor (FGF) family, the transforming growth factor beta (TGF-β) superfamily, the insulin-like growth factor binding proteins (IGF-BPs), and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1–5 with TGF-β1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.

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.

Fibronectin Type III-like Domains of Neurofascin-186 Protein Mediate Gliomedin Binding and Its Clustering at the Developing Nodes of Ranvier

The cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Ig-CAMs) play a crucial role in the organization of the node of Ranvier in myelinated axons. In the peripheral nervous system, Gliomedin (Gldn) secreted by Schwann cell microvilli binds NgCAM-related CAM (NrCAM) and Neurofascin-186 (NF186) and direct the nodal clustering of voltage-gated sodium channels (Nav). NF186 is the single axonal Gldn partner to ensure Nav clustering at nodes, whereas NrCAM is only required in glial cells (Feinberg, K., Eshed-Eisenbach, Y., Frechter, S., Amor, V., Salomon, D., Sabanay, H., Dupree, J. L., Grumet, M., Brophy, P. J., Shrager, P., and Peles, E. (2010) Neuron 65, 490-502). The olfactomedin domain of Gldn is implicated in the interaction with nodal Ig-CAMs. However, the interacting modules of NrCAM or NF186 involved in Gldn association are unknown. Here, we report that fibronectin type III-like (FnIII) domains of both Ig-CAMs mediate their interaction with Gldn in pulldown and cell binding assays. Using surface plasmon resonance assays, we determined that NrCAM and NF186 display similar affinity constant for their association with Gldn (K(D) of 0.9 and 5.7 nm, respectively). We characterized the FnIII domains 1 and 2 of NF186 as interacting modules that ensure association with Gldn. We found that the soluble FnIII domains of NF186 (FnIII-Fc) bind on Schwann cells and inhibit Gldn and Nav clustering at heminodes, the precursors of mature nodes in myelinating cultures. Our study reveals the unexpected importance of FnIII domains of Ig-CAMs in the organization of nodes of Ranvier in peripheral axons. Thus, NF186 utilizes distinct modules to organize the multimeric nodal complex.

Promotion of cell spreading and migration by vascular endothelial‐protein tyrosine phosphatase (VE‐PTP) in cooperation with integrins

Vascular endothelial-protein tyrosine phosphatase (VE-PTP) is a receptor-type protein tyrosine phosphatase with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III-like domains in its extracellular region. VE-PTP is expressed specifically in endothelial cells and is implicated in regulation of angiogenesis. The molecular basis for such regulation by VE-PTP has remained largely unknown, however. We now show that forced expression of VE-PTP promoted cell spreading as well as formation of lamellipodia and filopodia in cultured fibroblasts plated on fibronectin. These effects of VE-PTP on cell morphology required its catalytic activity as well as activation of integrins and Ras. In addition, VE-PTP-induced cell spreading and lamellipodium formation were prevented by inhibition of Src family kinases or of Rac or Cdc42. Indeed, forced expression of VE-PTP increased the level of c-Src phosphorylation at tyrosine-416. Moreover, the VE-PTP-induced changes in cell morphology were suppressed by expression of dominant negative forms of FRG or Vav2, both of which are guanine nucleotide exchange factors for Rho family proteins and are activated by tyrosine phosphorylation. Forced expression of VE-PTP also enhanced fibronectin-dependent migration of cultured fibroblasts. Conversely, depletion of VE-PTP by RNA interference in human umbilical vein endothelial cells or mouse endothelioma cells inhibited cell spreading on fibronectin. These results suggest that VE-PTP, in cooperation with integrins, regulates the spreading and migration of endothelial cells during angiogenesis.

Tertiary and Secondary Structure Elasticity of a Six-Ig Titin Chain

The protein titin functions as a mechanical spring conferring passive elasticity to muscle. Force spectroscopy studies have shown that titin exhibits several regimes of elasticity. Disordered segments bring about a soft, entropic spring-type elasticity; secondary structures of titin's immunoglobulin-like (Ig-) and fibronectin type III-like (FN-III) domains provide a stiff elasticity. In this study, we demonstrate a third type of elasticity due to tertiary structure and involving domain-domain interaction and reorganization along the titin chain. Through 870 ns of molecular dynamics simulations involving 29,000–635,000 atom systems, the mechanical properties of a six-Ig domain segment of titin (I65-I70), for which a crystallographic structure is available, are probed. The results reveal a soft tertiary structure elasticity. A remarkably accurate statistical mechanical description for this elasticity is derived and applied. Simulations also studied the stiff, secondary structure elasticity of the I65-I70 chain due to the unraveling of its domains and revealed how force propagates along the chain during the secondary structure elasticity response.

Expression of PTPRO in the interneurons of adult mouse olfactory bulb

PTPRO is a receptor-type protein tyrosine phosphatase (PTP) with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III-like domains in its extracellular region. In the chick, PTPRO mRNA has been shown to be particularly abundant in embryonic brain, and PTPRO is implicated in axon growth and guidance during embryonic development. However, the temporal and spatial expression of PTPRO protein in the mammalian CNS, particularly in the juvenile and adult mammalian brain, has not been evaluated in any detail. By immunohistofluorescence analysis with a monoclonal antibody to PTPRO, we show that PTPRO is widely expressed throughout the mouse brain from embryonic day 16 to postnatal day 1, while expression is largely confined to the olfactory bulb (OB) and olfactory tubercle in the adult brain. In the OB, PTPRO protein is expressed predominantly in the external plexiform layer, the granule cell layer, and the glomerular layer (GL). In these regions, expression of PTPRO is predominant in interneurons such as gamma-aminobutyric acid (GABA)-ergic or calretinin (CR)-positive granule cells. In addition, PTPRO is expressed in GABAergic, CR-positive, tyrosine hydroxylase-positive, or neurocalcin-positive periglomerular cells in the GL. Costaining of PTPRO with other neuronal markers suggests that PTPRO is likely to be localized to the dendrites or dendritic spines of these olfactory interneurons. Thus, PTPRO might participate in regulation of dendritic morphology or synapse formation of interneurons in the adult mouse OB.

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Analysis of the involvement of chitin-binding domain of ChiCW in antifungal activity, and engineering a novel chimeric chitinase with high enzyme and antifungal activities.

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, ChiCWdeltaFC (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since ChiCWdeltaAC (lacking the chitin-binding domain) could not be expressed in Escherichia coli as ChiCWdeltaFC did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW (ChBD(ChiCW)) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with ChBD(ChiCW). When the chitin-binding domain of ChiA1 was replaced with ChBD(ChiCW), the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that ChBD(ChiCW) may play an important role in the antifungual activity of ChiCW.

Role of exposed aromatic residues in substrate-binding of CBM family 5 chitin-binding domain of alkaline chitinase

Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S-transferase fusion protein. This fusion protein showed binding abilities to insoluble chitin and chitosan. Two surface-exposed aromatic residues (Trp541 and Trp542) were found in the tertiary-structure model of ChBD and targeted for mutational analysis. Single and double mutations of the two aromatic residues decreased the chitin- and chitosan-binding abilities. It was revealed that these residues would be important for substrate-binding of the ChBD.

Novel IL31RA gene mutation and ancestral OSMR mutant allele in familial primary cutaneous amyloidosis

Primary cutaneous amyloidosis (PCA) is an itchy skin disorder associated with amyloid deposits in the superficial dermis. The disease is relatively common in Southeast Asia and South America. Autosomal dominant PCA has been mapped earlier to 5p13.1-q11.2 and two pathogenic missense mutations in the OSMR gene, which encodes the interleukin-6 family cytokine receptor oncostatin M receptor beta (OSMRbeta), were reported. Here, we investigated 29 Taiwanese pedigrees with PCA and found that 10 had heterozygous missense mutations in OSMR: p.D647V (one family), p.P694L (six families), and p.K697T (three families). The mutation p.P694L was associated with the same haplotype in five of six families and also detected in two sporadic cases of PCA. Of the other 19 pedigrees that lacked OSMR pathology, 8 mapped to the same locus on chromosome 5, which also contains the genes for 3 other interleukin-6 family cytokine receptors, including interleukin-31 receptor A (IL31RA), which can form a heterodimeric receptor with OSMRbeta through interleukin-31 signaling. In one family, we identified a point mutation in the IL31RA gene, c.1562C>T that results in a missense mutation, p.S521F, which is also sited within a fibronectin type III-like repeat domain as observed in the OSMR mutations. PCA is a genetically heterogeneous disorder but our study shows that it can be caused by mutations in two biologically associated cytokine receptor genes located on chromosome 5. The identification of OSMR and IL31RA gene pathology provides an explanation of the high prevalence of PCA in Taiwan as well as new insight into disease pathophysiology.

SAP‐1 is a microvillus‐specific protein tyrosine phosphatase that modulates intestinal tumorigenesis

SAP-1 (PTPRH) is a receptor-type protein tyrosine phosphatase (RPTP) with a single catalytic domain in its cytoplasmic region and fibronectin type III-like domains in its extracellular region. The cellular localization and biological functions of this RPTP have remained unknown, however. We now show that mouse SAP-1 mRNA is largely restricted to the gastrointestinal tract and that SAP-1 protein localizes to the microvilli of the brush border in gastrointestinal epithelial cells. The expression of SAP-1 in mouse intestine is minimal during embryonic development but increases markedly after birth. SAP-1-deficient mice manifested no marked changes in morphology of the intestinal epithelium. In contrast, SAP-1 ablation inhibited tumorigenesis in mice with a heterozygous mutation of the adenomatous polyposis coli gene. These results thus suggest that SAP-1 is a microvillus-specific RPTP that regulates intestinal tumorigenesis.

Neogenin Interacts with Hemojuvelin through Its Two Membrane-Proximal Fibronectin Type III Domains

Hemojuvelin is a recently identified iron-regulatory protein that plays an important role in affecting the expression of hepcidin, a key iron regulatory hormone. Although the underlying mechanism of this process is not clear, several hemojuvelin-binding proteins, including the cell surface receptor neogenin and bone morphogenetic protein (BMP) cytokines, have been identified. The ectodomain of neogenin is composed of four immunoglobulin-like (Ig) domains followed by six fibronectin type III-like (FNIII) domains. Here we report expression of soluble versions of hemojuvelin and neogenin for biochemical characterization of their interaction and the interaction of HJV with BMP-2. Hemojuvelin normally undergoes an autocatalytic cleavage, and as in vivo, recombinant hemojuvelin exists as a mixture of cleaved and uncleaved forms. Neogenin binds to cleaved and noncleaved hemojuvelin, as verified by its binding to an uncleaved mutant hemojuvelin. We localized the hemojuvelin binding site on neogenin to the membrane-proximal fifth and sixth FNIII domains and the juxtamembrane linker and showed that a fragment containing only this region binds 2-3 orders of magnitude more tightly than the entire neogenin ectodomain. Binding to the most membrane-proximal region of neogenin may play a role in regulating the levels of soluble and membrane-bound forms of hemojuvelin, which in turn would influence the amount of free BMP-2 available for binding to its receptors and triggering transcription of the hepcidin gene. Our finding that BMP-2 and neogenin bind simultaneously to hemojuvelin raises the possibility that neogenin is part of a multiprotein complex at the hepatocyte membrane involving BMP, its receptors, and hemojuvelin.