ABSTRACT
Sphingosylphosphorylcholine (SPC) is a lysosphingolipid that exerts multiple functions, including acting as a spasmogen, as a mitogenic factor for various types of cells, and sometimes as an inflammatory mediator. Currently, liquid chromatography/tandem mass spectrometry (LC/MS/MS) is used for the quantitation of SPC. However, because of the complicated procedures required it may not be cost effective, hampering its regular usage in a routine practical SPC monitoring. In this report, we have generated RNA aptamers that bind to SPC with high affinity using an in vitro selection procedure and developed an enzyme-linked aptamer assay system using the minimized SPC aptamer that can successfully distinguish SPC from the structurally related sphingosine 1-phosphate (S1P). This is the first case of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process being performed with a lysosphingolipid. The SPC aptamers would be valuable tools for the development of aptamer-based medical diagnosis and for elucidating the biological role of SPC.
Subject(s)
Aptamers, Nucleotide/chemistry , Phosphorylcholine/analogs & derivatives , SELEX Aptamer Technique/methods , Sphingosine/analogs & derivatives , Aptamers, Nucleotide/genetics , Base Sequence , Biological Assay , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Phosphorylcholine/analysis , Phosphorylcholine/chemistry , Reproducibility of Results , Sphingosine/analysis , Sphingosine/chemistry , Surface Plasmon ResonanceABSTRACT
Proopiomelanocortin (POMC) is the precursor of melanocyte-stimulating hormone (MSH) and beta-endorphin, and is suggested to have evolved by the insertion and deletion of ancestral MSH segments. Here, the primary structure of POMC was determined with cDNA cloning of brown tree snakes of Squamata and American alligators of Crocodylia to show an overview of the molecular evolution of POMC in reptiles. Snake and alligator POMCs are composed of alpha-, beta-, and gamma-MSH segments and a single beta-END segment as in other tetrapods; however, the gamma-MSH segment in snake POMC has a mutation in the essential sequence from His-Phe-Arg-Trp to His-(d)-(d)-Arg, in which (d) means deletion. It is conceivable that the ancestry of snake gamma-MSH had weak functional constraint and lacked biological significance during evolution. Phylogenetic analyses using the neighbor-joining method show that snake prePOMC is most diverged, and alligator prePOMC is most conserved in reptilian POMCs while it shows the highest sequence identity with ostrich prePOMC. These relationships are comparable to those observed in mitochondrial DNA. On the other hand, analyses of the pituitary with mass spectrometry revealed several peptides by post-translational processing as predicted by the locations of processing sites consisting of basic amino acid residues in snake and alligator POMCs. Remarkably, the monobasic site at the N-terminal side of the snake beta-MSH is suggested to act as a processing site. Thus, the study shows the divergence of snake POMC such as the critical mutation of gamma-MSH and high conservation of hormone organization of alligator POMC.
Subject(s)
Alligators and Crocodiles/genetics , Colubridae/genetics , Peptides/analysis , Peptides/chemistry , Pituitary Gland/chemistry , Pro-Opiomelanocortin/chemistry , Pro-Opiomelanocortin/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression Regulation , Mass Spectrometry , Molecular Sequence Data , PhylogenyABSTRACT
Telomerase is an enzyme that catalyzes addition of telomeric repeat sequences to the 3'-termini of eukaryotic chromosome DNA. The catalytic core of telomerase consists of a protein component, telomerase reverse transcriptase (TERT), for the catalysis and an RNA component, telomerase RNA (TR), containing the template for the sequence. Human telomerase RNA (hTR) consists of 451 nucleotides (nt) and contains consecutive G-stretches in the 5'-terminal region. We examined the effects of the 5'-terminal sequence (nt 1-17) in hTR, which is assumed to be a single-stranded region (region 1), on interaction and telomerase activity in vitro. Mutation and binding experiments for hTR and its variants suggest that region 1 has repressive effects on telomerase activity by interaction with the region(s) in the 3'-half part. We prepared various hTR variants with mutations in region 1 and two possible target regions (region 2: nt 229-244; region 3: nt 284-297). Studies on these variants showed that region 1 can interact with regions 2 and 3 and the interactions between regions 1 and 3 may contribute to the repressive effects of region 1. We found that a mutation in region 2 markedly enhances telomerase activity. We also found that some deletion and sequence mutations in region 1 enhance the activity.
Subject(s)
RNA/chemistry , Telomerase/genetics , Base Sequence , Circular Dichroism , DNA/chemistry , DNA, Antisense/pharmacology , Electrophoretic Mobility Shift Assay , G-Quadruplexes , Guanine/chemistry , Humans , Mutation , RNA/genetics , Telomerase/metabolismABSTRACT
Human telomerase RNA (hTR) is assumed to contain a 17-nt segment in a single-stranded state at the 5'-terminus. There are four stretches of consecutive G residues, which are apt to form a quadruplex structure, in the segment. It is reported that deletion of some part of this region enhances telomerase activity when the core telomerase enzyme is reconstituted by addition of telomerase reverse transcriptase. To elucidate the reason for such effects, we constructed hTR mutants with deletion of the segment (hTRdelta20) and with the segment containing four G-to-A displacement to interrupt the G-stretch sequences (hTR17A) and their activity was compared with that of the wild-type hTR (hTRW). hTRdelta20 showed much higher activity than the other while hTR17A showed activity similar to that of hTRW. This result suggests that the lower activity for full-length hTR is not due to putative quadruplex formation.
