Sequence and Properties of Cagein, a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus.

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as "basal-body cages." The major protein comprising Euplotes cages has been previously identified and termed "cagein." Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear "nanochromosome" of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.

Induction of Duffy gene (FY) in human endothelial cells and in mouse.

Duffy Blood Group protein is a glycoprotein with seven transmembrane domains that binds to C-X-C and C-C chemokines. The antigen is constitutively expressed in endothelial and epithelial cells of several nonerythroid tissues and in Purkinje cells of the cerebellum. We studied the effect of proinflammatory cytokines on Duffy gene expression in endothelial cells from human umbilical vein (HUVEC) and human pulmonary arteries (HPAEC). Also, we studied the effect of inflammatory agents like bacterial lipopolysaccharide (LPS) on Duffy gene induction in mouse. Reverse transcription-PCR and mRNA blot analyses showed that Duffy mRNA was present in these cells in negligible amounts. However, treatment with tumor necrosis factor-alpha for 6-24h resulted in a 5 to 8-fold increase in Duffy mRNA. On the other hand, treatment with interleukin-1 (IL-1), IL-6 or LPS did not have any effect. Fluorescence microscopy and fluorescence activated cell sorting showed greater expression of Duffy protein in treated cells correlating the increase in mRNA synthesis with an increase in antigen production. In mice, Duffy gene was induced in lungs and brain with LPS treatment indicating that the induction is a physiological event. Vascular endothelial cells may induce Duffy protein to regulate leukocytes and/or chemokine trafficking.

Plateins: a novel family of signal peptide-containing articulins in euplotid ciliates.

In euplotid ciliates, the cortex is reinforced by alveolar plates--proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed alpha-, beta-, and gamma-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related beta- or gamma-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic alpha-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two alpha-platein genes from E. aediculatus. Even more acidic proteins, the derived alpha1- and alpha2-plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valine- and proline-rich 12-mers) are present in all three plateins. In beta/gamma-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the alpha-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of beta/gamma-platein, but near the N-terminus of alpha-plateins. All three plateins contain canonical N-terminal signal sequences, unique among known cytoskeletal proteins. The presence of start-transfer sequences correlates well with the final intra-alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin-related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre-fission morphogenesis are underway.

Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliate Euplotes define a new family of articulins.

Protistan cells employ a wide variety of strategies to reinforce and give pattern to their outermost cortical layers. Whereas some use common cytoskeletal elements such as microtubules, others are based on novel cytoskeletal proteins that are as-yet-unknown in higher eukaryotes. The hypotrich ciliate Euplotes possesses a continuous monolayer of scales or plates, located within flattened membranous sacs ('alveoli') just below the plasma membrane, and this provides rigidity and form to the cell. Using immunological techniques, the major proteins comprising these 'alveolar plates' have been identified and termed alpha-, beta-, and gamma-plateins. The present report describes work leading to the molecular characterization of three plateins, alpha 1 and alpha 2 (predicted M(r)s of 61 and 56 kDa) and a beta/gamma form (M(r)=73 kDa). All three proteins have features that are hallmarks of articulins, a class of cytoskeletal proteins that has been identified in the cortex of a wide variety of protistan cells, including certain flagellates, ciliates, dinoflagellates and PLASMODIUM: Chief among these common features are a prominent primary domain of tandem 12-amino acid repeats, rich in valine and proline, and a secondary domain of fewer, shorter repeating units. However, variations in amino acid use within both primary and secondary repetitive domains, and a much more acidic character (predicted pIs of 4.7-4.9), indicate that the plateins represent the first proteins in a new subclass or family of articulins. This conclusion is supported by another novel feature of the plateins, the presence of a canonical hydrophobic signal peptide at the N-terminus of each derived platein sequence. This correlates well with the final cellular location of the plateins, which are assembled into plates within the membrane-limited alveolar sacs. To our knowledge, this is the first report in any eukaryote of cytoskeletal proteins with such start-transfer sequences. Confocal immunofluorescence microscopy, using antibodies to the plateins as probes, reveals that new alveolar plates (enlarging in cortical zones undergoing morphogenesis) label more faintly than mature parental plates. During plate assembly (or polymerization), the plateins thus appear to exist in a more soluble form.