A description of the Mei2-like protein family; structure, phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a “mei2-dot” in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 angstrom resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six beta-strands forming an anti-parallel beta-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVl

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta(1)) and 22 kDa (beta(2)), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta(1) and beta(2) subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between 1 and 2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the U. and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against UNA inhibited the aggregation response to stejnulxin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha(2)beta(1) as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antib

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent mole

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horsefly's salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin. (C) 2009 Elsevier Ltd. All rights reserved.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15,536 Da) and beta (14,873 Da). The mature amino acid sequences of the a (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers Of (alphabeta)(2) and (alphabeta)(4) with molecular weights of 63,680 and 128,518 Da, respectively. The (alphabeta)(2) complex is stabilized by an interchain disulfide bridge between the two alphabeta-heterodimers, whereas the stabilization of the (alphabeta)(4) complex seems to involve non-covalent interactions between the (alphabeta)(2) complexes. (C) 2002 Elsevier Science Ltd. All rights reserved.

DnaJ-like protein gene sll1384 is involved in phototaxis in Synechocystis sp PCC 6803

In Synechocystis sp. PCC 6803, gene sll1384 encodes a protein with a DnaJ domain at its N-terminal portion and a TPR domain at the C-terminal portion. An sll1384 mutant shows no difference from the wild type in adaptation to different temperatures, but almost completely loses its capability of phototactic movement. After complementation with sll1384, the mutant regains the phototaxis. As shown with electron microscopy, on the cell surface, mutant cells have pili that appear to be the same as that of the wild type. Also, the transformation efficiency remains unchanged in the mutant. It is postulated that Sll1384 regulates phototaxis of Synechocystis through protein-protein interaction. It is the first DnaJ-like protein gene identified in a cyanobacterium for a role in phototaxis.

Expression, purification, and characterization of recombinant Chinese shrimp crustin-like protein (CruFc) in Pichia pastoris

A crustin-like protein (CruFc) from Fenneropenaeus chinensis was expressed in Pichia pastoris and then purified to electrophoretic homogeneity on a Sephacryl S-100 column with a band corresponding to the expected one (13 kDa) shown by 15% SDS-PAGE. Western blot indicated that the rCruFc specifically reacted with polyclonal rabbit anti-Fenneropenaeus chinensis CruFc. Production in a 5 l bioreactor gave 237 mg rCruFc/l. Antimicrobial assay revealed that 4 mu M rCruFc inhibited growth of Staphylococcus aureus.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

An alpha 2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha sub(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha sub(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.