The 65-kDa phorbol-diester hydrolase in mouse plasma is esterase 1 and is immunologically distinct from the 56-kDa phorbol-diester hydrolase in mouse liver

Esterase 1, a well-characterized mouse plasma protein of unknown function, has activity against a wide range of ester substrates including beta-alanine nitrophenyl esters and 17 beta-esters of estradiol. In this article, we report that esterase 1 is also responsible for a majority of the phorbol-12-ester hydrolase activity in mouse plasma. Incubation of homogeneous esterase 1 with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) at either 4 or 37 degrees C for up to 18 h yielded phorbol 13 alpha-acetate as the only hydrolysis product. Specific polyclonal antibodies to esterase 1 inhibited 95% of PMA hydrolysis by a purified esterase 1 preparation and 65% of PMA hydrolysis by mouse plasma. Perfused mouse liver homogenates contain two distinct phorbol diester hydrolases with apparent molecular masses of 65 kDa and 56 kDa, respectively. The 65-kDa protein appears to be immunologically identical to the plasma enzyme, while the 56-kDa protein, found in liver but not in plasma, is immunologically distinct. Phorbol 12-myristate, phorbol 12,13-dibutyrate, and PMA were found to be competitive inhibitors of the beta-alanine-nitrophenyl esterase activity of esterase 1 with Ki values of approximately 7 microM. Phorbol 13-acetate and phorbol itself were less effective with Ki values of 37 and 140 microM, respectively. Sodium salts of valeric and myristic acids did not inhibit at 10 microM. The above results indicate that efficient substrate binding requires a phorbol 12-ester. Similar results were obtained with estradiol 17 beta-valerate which is a better substrate for esterase 1 than is PMA. Our results strongly suggest that esterase 1 and a recently described phorbol ester hydrolase isolated from mouse serum (Saito, M., and Egawa, K. (1984) J. Biol. Chem. 259, 5821-5826) are the same and are immunologically and kinetically distinct from the 56-kDa phorbol 12-ester hydrolase in mouse liver.     

Nucleotide sequence of mouse Tcp-1a cDNA

We have isolated complete cDNA clones encoding the mouse t-complex polypeptides 1A and 1B (TCP-1A and TCP-1B) from t-haplotype and wild-type (wt) mice, respectively. The complete nucleotide (nt) sequence of the Tcp-1a cDNA was determined. The Tcp-1a cDNA has an open reading frame (ORF) encoding a 60-kDa protein of 556 amino acids (aa). A comparison of nt sequences between the Tcp-1a and Tcp-1b cDNAs revealed that the 1786-bp regions upstream from their polyadenylation signals differed by 17 substitutions and that Tcp-1a had different polyadenylation sites from Tcp-1b. In these ORFs, 15 bp were substituted between the two alleles, occurring in 14 codons and resulting in eleven single-aa substitutions. Among these 15 substitutions, twelve were nonsynonymous (aa change) and three were synonymous (no aa change). The aa substitution in TCP-1 has occurred at least 20 times faster between t-haplotype and wt than between mouse and human or mouse and Drosophila.     

New mouse 5-HT2-like receptor. Expression in brain, heart and intestine.

A novel member of the family of G protein-coupled receptors has been isolated from a mouse brain cDNA library by screening with polymerase chain reaction (PCR) generated fragment of mouse genomic DNA amplified using degenerated primers. Sequence comparison demonstrates that the encoded protein sequence shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from COS cells transfected with this cDNA, corresponds to a new 5-HT2-like receptor that we propose to call 5-HT2C. Its major sites of expression are in the mouse intestine and heart, also with detectable expression in brain and kidney. We speculate that it could account at least in part for the 'atypical' functions attributed to the 5-HT1C/5-HT2 receptors.     

Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase is involved in their interaction

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.     

Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain.

Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.     

Cloning and characterization of a family C orphan G-protein coupled receptor

Members of the family C receptors within the G-protein coupled receptor superfamily include the metabotropic glutamate receptors, GABA(B) receptors, the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and a small group of uncharacterized orphan receptors. We have cloned and studied the mouse GPRC6A family C orphan receptor. The open reading frame codes for a protein with highest sequence identity to the fish 5.24 odorant receptor and the mammalian CaSR. The gene structure shows a striking resemblance to that of the CaSR. Results from RT-PCR analyses showed that mouse GPRC6A mRNA is expressed in mouse brain, skeletal muscle, heart, lung, spleen, kidney, liver, and in the early stage mouse embryo. Immunocytochemical analysis of the cloned mouse GPRC6A cDNA expressed in human embryonic kidney 293 cells demonstrated that GPRC6A was present on the plasma membrane, as well as in the endoplasmic reticulum and nuclear envelope membranes of transfected cells. A chimeric cDNA construct in which the extracellular ligand binding domain of the fish 5.24 amino acid-activated odorant receptor was ligated to the complementary downstream sequence of the mouse GPRC6A receptor indicated that GPRC6A is coupled to phosphoinositol turnover and release of intracellular calcium. Further studies with mouse GPRC6A expressed in Xenopus laevis oocytes demonstrated that this receptor possesses a pharmacological profile resembling that of the fish 5.24 odorant receptor. These findings suggest that GPRC6A may function as the receptor component of a novel cellular transmitter system in mammals.     

Prediction of the coding sequences of mouse homologues of KIAA gene: I. The complete nucleotide sequences of 100 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a human cDNA project to predict protein-coding sequences in long cDNAs (> 4 kb) since 1994. The number of these newly identified human genes exceeds 2000 and these genes are known as KIAA genes. As an extension of this project, we herein report characterization of cDNAs derived from mouse KIAA-homologous genes. A primary aim of this study was to prepare a set of mouse. KIAA-homologous cDNAs that could be used to analyze the physiological roles of KIAA genes in mice. In addition, comparison of the structures of mouse and human KIAA cDNAs might enable us to evaluate the integrity of KIAA cDNAs more convincingly. In this study, we selected mouse KIAA-homologous cDNA clones to be sequenced by screening a library of terminal sequences of mouse cDNAs in size-fractionated libraries. We present the entire sequences of 100 cDNA clones thus selected and predict their protein-coding sequences. The average size of the 100 cDNA sequences reached 5.1 kb and that of mouse KIAA-homologous proteins predicted from these cDNAs was 989 amino acid residues.     

The gene for the beta-subunit of retinal transducin (Gnb-1) maps to distal mouse chromosome 4, and related sequences map to mouse chromosomes 5 and 8

The heterotrimeric G protein transducin releases cGMP-phosphodiesterase from inhibition in retinal rod photoreceptor cells when stimulated by light-activated rhodopsin. As a result the level of cGMP goes down, the rod plasma membrane hyperpolarizes, and the release of neurotransmitter is modified. We have used a bovine cDNA for the beta-subunit of transducin (G beta 1) to map its gene Gnb-1 to distal mouse chromosome 4. This cDNA also identified two other homologous sequences in the mouse genome. One of the sequences was on chromosome 5 which we identified as the locus of Gnb-2, a second G protein beta-subunit gene. The other sequence was on chromosome 8 and is either a pseudogene or an as yet undiscovered third G beta-subunit gene, here termed Gnb-3.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Identification and characterisation of a homolog of an activation gene for the recombination activating gene 1 (RAG 1) in amphioxus

Expression of recombination activating genes (RAG) involved in the V (D) J recombination is regulated by the RAG1 gene activator (RGA) in mammals. The sequence of a cDNA clone from an amphioxus cDNA library was found to be homologous to that of RGA from mouse stromal cells with 45% identity. The full-length cDNA sequence comprises 1119 bp and encodes a putative protein of 210 amino acid residues. Characterisation of the amino acid sequence revealed that two MtN3 domains and seven transmembrane spans are present in this protein, indicating a potential role as a plasma membrane protein. This gene is expressed in many tissues and at differential developmental stages. A high expression level of RGA is detected in gonad tissues, and gastrula embryo and adult stages. The presence of the RGA gene in amphioxus suggests that the signal pathway required for the expression of RAG could exist in this primitive protochordate. It also implies that in the related molecules, primitive adaptive immunity may have existed in cephalochordate although the complete machinery of VDJ rearrangement may not be formed.     

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.     

Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse,a Model of Krabbe's Disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.     

C3G, a guanine nucleotide exchange factor bound to adapter molecule c-Crk, has two alternative splicing forms

Two types of C3G cDNA were isolated from mouse 3T3-L1 adipocyte cDNA library. A 114-bp sequence in the middle of C3G cDNA is deleted in the short type cDNA. By RT-PCR analysis, it was found that these two types of C3G mRNA existed in all the mouse tissues. Sequence comparison revealed 88% nucleotide sequence identity between mouse and human C3G cDNA. Comparison of mouse C3G cDNA with the human genome database suggested that this 114-bp sequence comprised an entire exon, and it is confirmed by PCR analysis using mouse genomic DNA and cDNA template. These results indicate that two C3G mRNAs and proteins result from alternative RNA splicing.