Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

A new Arabidopsis nucleic-acid-binding protein gene is highly expressed in dividing cells during development

An Arabidopsis thaliana cDNA encoding a new RNA-binding protein (RBP37) was cloned from a silique cDNA library. The predicted amino acid sequence corresponds to a RBP containing two RNA recognition motifs (RRM) and a basic domain. An affinity for nucleic acids was confirmed in binding assays using in vitro synthesised AtRBP37 protein. In situ hybridisation experiments on sections of flowers and siliques showed expression only in growing organs: gynoecium, petals, filaments and during early-embryogenesis expression is located in the embryo proper and the suspensor up to late heart stage. Expression is not detected in the embryo during maturation.This results suggests an expression pattern correlated with dividing cells.     

A genetically engineered purpurin/retinol-binding protein hybrid that binds to transthyretin.

A mini-gene encoding rat retinol-binding protein (RBP) and a cDNA encoding chicken purpurin were separately transfected into HeLa cells. In contrast to RBP, expressed purpurin did not bind to transthyretin (TTR). A purpurin/RBP hybrid protein was constructed by substituting the cDNA sequence encoding the N-terminal 29 amino acids of purpurin for the corresponding part of RBP. The expressed hybrid molecule bound to the TTR-Sepharose. These results demonstrate that purpurin does not bind to TTR, that a functional purpurin/RBP hybrid can be constructed, and that the N-terminal coil of RBP is not required for TTR binding.     

人GRY-RBP基因的表达及其RNA结合活性的初步鉴定

ＲＲＭ ＲＮＡ 结合蛋白在基因的转录后调控中起着重要作用．人ＧＲＹ－ＲＢＰ 是我们实验室最近克隆的一个新的全长ｃＤＮＡ,编码一个ＲＲＭ ＲＮＡ 结合蛋白．ＧＲＹ－ＲＢＰ 的全编码区用ＰＣＲ扩增后,克隆到ｐＥＴ３０ａ＋ 表达载体中,在Ｅ．ｃｏｌｉ中获得了高效表达,表达的ＧＲＹ－ＲＢＰ重组蛋白占细菌总蛋白的２１％ ．利用融合在ＧＲＹ－ＲＢＰＮ 端的Ｈｉｓ．Ｔａｇ,采取亲和层析的方法纯化了表达的重组蛋白．为了检测ＧＲＹ－ＲＢＰ的ＲＮＡ 结合活性及其所结合的ＲＮＡ 性质,将表达的蛋白与ｐｏｌｙ（Ａ）－Ｓｅｐｈａｒｏｓｅ ４Ｂ结合,发现ＧＲＹ－ＲＢＰ能与ｐｏｌｙＡ （Ａ）结合,结合活性能被可溶性的ｐｏｌｙ（Ａ）、ｐｏｌｙ（Ｃ）减弱．改变ＮａＣｌ浓度和加入不同浓度的酵母ｔＲＮＡ竞争物后,ｐｏｌｙ（Ａ）结合活性不变．     

cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.     

Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations.

The interaction of bacterial mRNAs with the small ribosomal subunit is strongly promoted by Watson-Crick base pairing between a purine-rich consensus ribosomal RNA-binding sequence (RBS) on mRNA and its complementary message-binding sequence (MBS) on rRNA known as the Shine-Dalgarno interaction. To identify and characterize components of the Shine-Dalgarno interaction that contribute to translation initiation, we simultaneously and randomly mutated both the MBS of the 16S rRNA gene from Escherichia coli and the RBS of the chloramphenicol acetyl transferase (CAT) gene and selected chloramphenicol-resistant mutant combinations. Nucleotide distribution in both mutated sequences of the survivors was nonrandom and the MBSs of the surviving clones showed a preference for purines. In addition, strong interactions between specific nucleotide pairs within each of the mutated sequences were indicated. Although the contribution of free energy of duplex formation between rRNA and mRNA was highly significant (P < 0.001), only 23% of the observed activity in all of the mutants could be attributed to this variable. MBSs that were lethal upon expression were also isolated. These sequences may cause overtranslation of specific messages in the cell. These data indicate that specific sequence constraints exist (primarily within the MBS) that are necessary to establish a functional threshold for translation and that only after establishment of this threshold is the level of expression significantly affected by the free energy of MBS-RBS duplex formation.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

Distribution and molecular characterization of mRNA-binding proteins specific to the (U)15 region of 3' UTR of the mouse catalase (Cas-1).

The 3' UTR of the mouse Cas-1 mRNA, encoding the antioxidant enzyme catalase, has a U-rich motif that is conserved across species. This motif is an active site for complex and dynamic interactions involving RNA-binding proteins. The spatial, temporal, and phylogenetic distribution of the Cas-1 3'-UTR U-rich motif-specific RNA-binding proteins was evaluated by gel mobility shift and UV cross-linking assays. The specific RNA-protein complexes were observed in mouse tissue homogenates representing developmental stages as early as day 10 pc and ranged in molecular weight from approximately 38 kDa to approximately 52 kDa. These mRNA-protein complexes appeared in all vertebrate species examined (human, mouse, rat, dog, rabbit, chicken, fish, and frog) but not in insects. The approximately 38-kDa protein was the most prominent protein in vertebrates. The cDNA sequence of the mouse approximately 38-kDa protein was obtained by purification of the protein, microsequencing, and RT-PCR. The resulting 456-nt sequence, representing the partial internal cDNA sequence, and its deduced amino acid sequence were similar to the RNA recognition motif (RRM) of a protein superfamily, implicated in splicing, stability, localization, and translation of RNAs. Although the results suggest that cis element-binding activity could be a cytoplasmic regulator of Cas-1 mRNA metabolism, the significance of this binding remains to be determined.     

RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication.

Hepatitis delta antigen (HDAg) is an RNA-binding protein with binding specificity for hepatitis delta virus (HDV) RNA (J. H. Lin, M. F. Chang, S. C. Baker, S. Govindarajan, and M. M. C. Lai, J. Virol. 64:4051-4058, 1990). By amino acid sequence homology search, we have identified within its RNA-binding domain two stretches of an arginine-rich motif (ARM), which is present in many prokaryotic and eukaryotic RNA-binding proteins. The first one is KERQDHRRRKA and the second is EDEKRERRIAG, and they are separated by 29 amino acids. Deletion of either one of these ARM sequences resulted in the total loss of the in vitro RNA-binding activity of HDAg. Thus, HDAg is different from other RNA-binding proteins in that it requires two ARM-like sequences for its RNA-binding activity. Replacement of the spacer sequence between the two ARMs with a shorter stretch of sequence also reduced RNA binding in vitro. Furthermore, site-specific mutations of the basic amino acid residues in both ARMs resulted in the total loss or reduction of RNA-binding activity. The biological significance of the RNA-binding activity was studied by examining the trans-activating activity of the RNA-binding mutants. The plasmids expressing HDAgs with various mutations in the RNA-binding motifs were cotransfected with a replication-defective HDV dimer cDNA construct into COS cells. It was found that all the HDAg mutants which had lost the in vitro RNA-binding activity also lost the ability to complement the defect of HDV RNA replication. We conclude that the trans-activating function of HDAg requires its binding to HDV RNA.     

Genetic characterization of Drosophila Mi-2 ATPase

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA.     

Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin

A full-length cDNA for rat lung beta-galactoside lectin (subunit Mr approximately 14,000, lectin 14K) was cloned and the nucleotide sequence determined. The deduced amino acid sequence agrees with the amino acid composition and direct amino acid sequence analysis of purified rat lung lectin peptides. We found that the amino-terminal alanine is blocked with an acetyl group. Comparison of the amino acid sequence with other proteins shows a high degree of homology only with other vertebrate lectin sequences, supporting the suggestion that these lectins may constitute a unique class of vertebrate proteins. The amino acid composition and sequence of lectin peptides, the sequence of lectin cDNA, and isoelectric focusing of purified lectin indicate that rat lung lectin 14K is composed predominantly of a single protein. In addition, rat uterus lectin 14K was found to be the same protein as that present in lung. We characterized the secondary and tertiary structure of rat lung lectin 14K by circular dichroism, by analytical ultracentrifugation, and by computer analysis of its primary structure. Results of these experiments suggest that lectin 14K is primarily a hydrophilic protein with an asymmetric, elongated structure consisting of approximately equal amounts of alpha helix, beta sheet, beta turn, and random coil. We found that Cys-2 and Cys-130 react most rapidly with iodoacetamide; one or both of these residues may be primarily responsible for the thiol requirement of lectin activity.     

RNA-binding properties of the mitochondrial Y-box protein RBP16

We have previously identified a mitochondrial Y-box protein in Trypanosoma brucei that we designated RBP16. The predicted RBP16 amino acid sequence revealed the presence of a cold-shock domain at its N-terminus and a glycine- and arginine-rich C-terminus reminiscent of an RGG RNA-binding motif. Since RBP16 is capable of interacting with different guide RNAs (gRNAs) in vitro and in vivo primarily via the oligo(U) tail, as well as with ribosomal RNAs, possible functions of RBP16 may be in kinetoplastid RNA editing and/or translation. Herein, we report experiments that further define the RNA-binding properties of RBP16. RBP16 forms a single stable complex with the gRNA gA6[14] at low protein concentration, while at higher protein concentration two stable complexes that possibly represent two different conformations are observed. Both complexes are stable at relatively high salt and moderate heparin concentrations indicating that the binding of RBP16 to gA6[14] does not rely primarily on ionic interactions. Phenylglyoxal treatment of the protein indicates that arginine residues are important in RNA binding. The minimal length of RNA sequence necessary for the binding of RBP16 was assessed by gel retardation and UV cross-linking competition assays using oligo(U) ribonucleotides of varying lengths (4–40 nt). Although RBP16 can bind to oligonucleotides as small as U₄, its affinity increases with the length of the oligo(U) ribonucleotide, with a dramatic increase in binding efficiency observed when the length is increased to 10 nt. Gel retardation assays employing T.brucei mRNAs demonstrated that, although it acts as a major binding determinant, a 3′ U tail is not an absolute requirement for efficient RBP16–RNA binding. Experiments with oligonucleotides containing U stretches embedded at different positions in oligo(dC) indicated that high-affinity binding requires both a uridine stretch, as well as 5′ and 3′ non-specific sequences. These results suggest a model for the molecular interactions involved in RBP16–RNA binding.     

Chromosomal localization of the retinol binding protein gene and its elimination as a candidate gene for the repeated epilation (Er) mutation in mice.

The repeated epilation (Er) mutation is an autosomal defect that blocks differentiation in stratified epithelia and appendages in mice. Plasma retinol binding protein (RBP) was tested as a possible candidate gene for the Er defect because of the importance of retinol as a modulator of epithelial morphogenesis and differentiation. Two approaches were used: (1) cloning and sequencing of the RBP cDNA from normal and mutant mice, and (2) the chromosomal localization of the mouse RBP gene. The mouse RBP sequence differs slightly from that of the rat RBP, but mutant and normal mouse RBP have identical sequences. The mouse RBP gene was localized by in situ hybridization to the distal portion of chromosome 19. This physical mapping confirms the recent assignment of the gene to chromosome 19 by linkage analysis. These results eliminate the RBP gene as a candidate gene for the defect in the Er mutation that maps to chromosome 4.     

Hamster contraception associated protein 1 (CAP1)

Based on cDNA and amino acid sequence, we demonstrate that hamster contraception associated protein 1 (CAP1) protein (an homolog of DJ-1 in mouse, CAP1/SP22/RS in rat and DJ-1/RS in human) is conserved during evolution. Through solubilization studies, it was demonstrated that hamster CAP1 has a peripheral membrane localization. SDS-PAGE analysis revealed that the migration pattern for hamster CAP1 compared to the other rodent counterparts, rat and mouse was different; indicating species-specific differences in the protein (possibly due to post-translational modifications). This protein also shows a ubiquitous presence in both somatic and germ tissues, and has been localized to the sperm tail. It was noticed that hamster CAP1 was lost from the mid piece of spermatozoa during capacitation. Interestingly, following in vitro treatment with ornidazole, CAP1 was lost from the spermatozoa and immunofluorescence studies showed that the major loss was from the mid piece of the spermatozoa. Another interesting feature highlighted about hamster CAP1 is its tendency to exist in two pI isoforms. Summarily, hamster CAP1 appears to exhibit species-specific differences compared to its rodent counterparts with respect to its unique peripheral localization, its size, two pI isoforms, and fate during capacitation, which may have implications in its functions.     

Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.