Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.     

Identification and characterization of a novel Golgi protein, golgin-67

In the course of screening a lambdagt11 human leukemic T-cell cDNA expression library with an antibody specific to the mitotic target of Src, Sam68, we identified and cloned a cDNA encoding a novel protein with a predicted molecular mass of 51.4 kDa. Polyclonal antibodies raised to a His(6)-tagged construct of this protein, detected a approximately 67-kDa protein in immunoprecipitation experiments, and cytological studies showed that this protein localized to the Golgi complex, through colocalization experiments with specific Golgi markers. Therefore, we designated this protein golgin-67. Sequence analysis revealed that golgin-67 is a highly coiled-coil protein, with potential Cdc2 and Src kinase phosphorylation motifs. It has sequence homologies to other Golgi proteins, including the coatamer complex I vesicle docking protein, GM130. Structurally, golgin-67 resembles, golgin-84, an integral membrane Golgi protein with an N-terminal coiled-coil domain and a single C-terminal transmembrane domain. The C-terminal region of golgin-67, which contains a predicted transmembrane domain, was demonstrated to be essential for its Golgi localization.     

Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.     

Lbc proto-oncogene product binds to and could be negatively regulated by metastasis suppressor nm23-H2

Lbc was identified as transforming gene from human leukemic cells and encodes Rho type guanine nucleotide exchange factor with 47kDa molecular weight. We isolated overlapping cDNAs of Lbc from human lung tissue. Full-length Lbc cDNA encodes 309kDa huge protein with Ht31 PKA anchoring motif, Dof domain, C1 domain, and coiled-coil structure. In order to analyze the regulatory mechanism of its activity, we searched for binding proteins. By yeast two-hybrid screening, we identified metastasis suppressor nm23-H2 as binding protein, which interacts with amino-terminal region of Lbc containing Dof domain. nm23 gene family encodes nucleoside diphosphate kinase, however, the binding of nm23-H2 to Lbc was independent of kinase activity. nm23-H1, which binds to Rac-specific GEF Tiam1, could not bind to Lbc suggesting nm23-H2 would be specific regulator for Lbc. Expression of nm23-H2 in cells leads to decrease the amount of GTP-bound Rho and suppress stress fiber formation stimulated by expression of Lbc. Our data suggest that metastasis suppressor nm23-H2 could regulate Lbc negatively by binding to amino-terminal region of Lbc proto-oncogene product.     

Isolation and characterization of EMILIN-2, a new component of the growing EMILINs family and a member of the EMI domain-containing superfamily

EMILIN (elastin microfibril interfase located Protein) is an elastic fiber-associated glycoprotein consisting of a self-interacting globular C1q domain at the C terminus, a short collagenous stalk, an extended region of potential coiled-coil structure, and an N-terminal cysteine-rich domain (EMI domain). Using the globular C1q domain as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a novel protein. Determination of the entire primary structure demonstrated that this EMILIN-binding polypeptide is highly homologous to EMILIN. The domain organization is superimposable, one important difference being a proline-rich (41%) segment of 56 residues between the potential coiled-coil region and the collagenous domain absent in EMILIN. The entire gene (localized on chromosome 18p11.3) was isolated from a BAC clone, and it is structurally almost identical to that of EMILIN (8 exons, 7 introns with identical phases at the exon/intron boundaries) but much larger (about 40 versus 8 kilobases) than that of EMILIN. Given these findings we propose to name the novel protein EMILIN-2 and the prototype member of this family EMILIN-1 (formerly EMILIN). The mRNA expression of EMILIN-2 is more restricted compared with that of EMILIN-1; highest levels are present in fetal heart and adult lung, whereas, differently from EMILIN-1, adult aorta, small intestine, and appendix show very low expression, and adult uterus and fetal kidney are negative. Finally, the EMILIN-2 protein is secreted extracellularly by in vitro-grown cells, and in accordance with the partial coexpression in fetal and adult tissues, the two proteins shown extensive but not absolute immunocolocalization in vitro.     

JEAP, a novel component of tight junctions in exocrine cells.

Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells.     

Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera of healthy individuals. Full-length tNOX mRNA is present in both normal and tumor cells but appears not to be expressed in either. Our research suggests alternative splicing as the basis for the cancer specificity of tNOX expression at the cell surface. Four splice variants were found. Of these, the exon 4 minus and exon 5 minus forms present in cancer cell lines were absent in noncancer cell lines. In contrast to full-length tNOX cDNA, transfection of COS cells with tNOX exon 4 minus cDNA resulted in overexpression of mature 34 kDa tNOX protein at the plasma membrane. The exon 4 minus form resulted in initiation of translation at a downstream M231 initiation site distinct from that of full-length mRNA. With replacement of M231 by site-directed mutagenesis, no translation of exon 4 minus cDNA or cell surface expression of 34 kDa mature tNOX was observed. The unprocessed molecular mass of 47 kDa of the exon 4 minus cDNA translated from methionine 231 corresponded to that of the principal native tNOX form of the endoplasmic reticulum. Taken together, the molecular basis of cancer-cell-specific expression of 34 kDa tNOX appears to reside in the cancer-specific expression of exon 4 minus splice variant mRNA.     

Ferroportin is expressed on the mucous granule membrane of a subpopulation of goblet cells in the duodenum of the rat

Ferroportin is a basolateral transporter involved in the release of iron from cells. In addition to expression on the basolateral membrane of enterocytes, ferroportin is also seen on the microvillus membrane. This led us to consider that ferroportin might be expressed by other cells of the intestine where it contributes to iron metabolism. Ferroportin gene and protein expression in rat duodenum was studied by in situ hybridisation and immunohistochemistry, respectively in rats with different efficiencies of iron absorption. Ferroportin mRNA localised to enterocytes of the villus only. Ferroportin was demonstrated in enterocytes and in 30% of goblet cells. In goblet cells it localised to the mucous granule membrane. In iron-loaded intestine some goblet cells contained iron suggesting that ferroportin may transport iron into the mucous granule where it would be lost during discharge of mucous. The finding of ferroportin in iron deficient goblet cells also suggests an additional role to iron excretion.     

Isolation and functional characterization of a dynamin-like gene from Plasmodium falciparum

A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain. Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain. Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region. Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage. Morphological studies indicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60-100 nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes. The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P. falciparum parasite at the erythrocyte stage. Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite.     

GRIP domain-mediated targeting of two new coiled-coil proteins,GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.     

Immumochemical and immunocytochemial characterization of a novel monoclonal antibody recognizing a 140 kDa protein in cerebral pericytes of the rat

Summary A monoclonal antibody that recognizes a 140 kDa peripheral plasma membrane protein in pericytes of nervous tissues of the rat is described. Microvessels of brain cortex and perineurium of peripheral nerves are shown to react positively to this antibody. The antigen is absent in brain regions that lack a blood-brain barrier, i.e., choroid plexuses and area postrema. Antigen expression starts as early as day 18 of embryonic development. By means of immuno-electron microscopy the 140 kDa antigen was detected as clusters along the entire circumference of cerebral pericytes. The same antigenic determinant is also expressed in apical domains of plasma membranes of a variety of transporting epithelia, such as hepatocytes, enterocytes of the small intestine, and epithelial cells of proximal tubules of the kidney. We postulate the 140 kDa protein as being a constituent of the pericytes involved in regulative functions of the blood-brain barrier.     

A dynamic, cytoplasmic triacylglycerol pool in enterocytes revealed by ex vivo and in vivo coherent anti-Stokes Raman scattering imaging

The absorptive cells of the small intestine, enterocytes, are not generally thought of as a cell type that stores triacylglycerols (TGs) in cytoplasmic lipid droplets (LDs). We revisit TG metabolism in enterocytes by ex vivo and in vivo coherent anti-Stokes Raman scattering (CARS) imaging of small intestine of mice during dietary fat absorption (DFA). We directly visualized the presence of LDs in enterocytes. We determined lipid amount and quantified LD number and size as a function of intestinal location and time post-lipid challenge via gavage feeding. The LDs were confirmed to be primarily TG by biochemical analysis. Combined CARS and fluorescence imaging indicated that the large LDs were located in the cytoplasm, associated with the tail-interacting protein of 47 kDa. Furthermore, in vivo CARS imaging showed real-time variation in the amount of TG stored in LDs through the process of DFA. Our results highlight a dynamic, cytoplasmic TG pool in enterocytes that may play previously unexpected roles in processes, such as regulating postprandial blood TG concentrations.     

Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.     

The expression and regulation of the iron transport molecules hephaestin and IREG1: implications for the control of iron export from the small intestine

The amount of iron in the body is controlled at the point of absorption in the proximal small intestine. Dietary iron enters the intestinal epithelium via the brush-border transporter DMT1 and exits through the basolateral membranes. The basolateral transfer of iron requires two components: a copper-containing iron oxidase known as hephaestin and a membrane transport protein IREG1. The amount of iron traversing the enterocytes is directly related to body iron requirements and inversely related to the iron content of the intestinal epithelium. We propose that body signals control iron absorption by first acting on crypt enterocytes to determine the expression of basolateral transport components. This, in turn, modulates the intracellular iron content of mature epithelial cells, which ultimately determines the activity of the brush-border transporter DMT1.     

Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.

In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, we report the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain.