Human Fetal Brain cDNA Library Research Articles

A novel splice variant of human geneNPL, mainly expressed in human liver, kidney and peripheral blood leukocyte

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.

Identification of dynein light chain 2 as an interaction partner of p21-activated kinase 1

p21-activated kinase 1 (PAK1), a member of the evolutionarily conserved PAK family of serine/threonine kinases, is essential for a variety of cellular functions. Our previous studies showed that PAK1 participated in the apoptotic pathway mediated by p110C. To further investigate its functions, we used the yeast two-hybrid system to screen a human fetal brain cDNA library and identified dynein light chain 2 (DLC2)/myosin light chain (MLC) as an interacting partner of PAK1. The association of PAK1 with DLC2 was further confirmed by in vitro binding assay. With the stimulation of EGF, PAK1 interacted with HA-DLC2 in vivo and relocalized in cytoplasm near the perinuclear location in confocal microscope analysis. The deletion analysis showed that the interaction of DLC2 with PAK1 occurred within the residues 210–332 of PAK1. For that studies showed that DLC2 was a subunit of myosin complex, so it is possible that PAK1 binds to DLC2 and transports by myosin complex.

Cloning and characterization of a novel human homolog* of mouse U26, a putative PQQ-dependent AAS dehydrogenase

Mouse U26 has been defined as a 2-aminoadipic 6-semialdehyde dehydrogenase. It was speculated to be a PQQ-dependent AAS dehydrogenase due to the research of demonstrating PQQ as a new B vitamin. We isolated a novel human cDNA from the human fetal brain cDNA library we constructed. Its deduced protein was most related to mouse U26. Thus, we termed it human U26. This putative protein contains an AMP-binding domain, a Phosphopantetheine-binding domain and six PQQ-binding motifs. Human U26 mRNA is ubiquitously expressed in adult tissues and is highly expressed in colon adenocarcinoma (CX-1) and colon adenocarcinoma (GI-112) cell lines. Further study should be made to clarify the precise function of human U26.

αPIX Associates with Calpain 4, the Small Subunit of Calpain, and Has a Dual Role in Integrin-mediated Cell Spreading

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

Physical Interaction and Functional Coupling between ACDP4 and the Intracellular Ion Chaperone COX11, an Implication of the Role of ACDP4 in Essential Metal Ion Transport and Homeostasis

Divalent metal ions such as copper, manganese, and cobalt are essential for cell development, differentiation, function and survival. These essential metal ions are delivered into intracellular domains as cofactors for enzymes involved in neuropeptide and neurotransmitter synthesis, superoxide metabolism, and other biological functions in a target specific fashion. Altering the homeostasis of these essential metal ions is known to connect to a number of human diseases including Alzheimer disease, amyotrophic lateral sclerosis, and pain. It remains unclear how these essential metal ions are delivered to intracellular targets in mammalian cells. Here we report that rat spinal cord dorsal horn neurons express ACDP4, a member of Ancient Conserved Domain Protein family. By screening a pretransformed human fetal brain cDNA library in a yeast two-hybrid system, we have identified that ACDP4 specifically interacts with COX11, an intracellular metal ion chaperone. Ectopic expression of ACDP4 in HEK293 cells resulted in enhanced toxicity to metal ions including copper, manganese, and cobalt. The metal ion toxicity became more pronounced when ACDP4 and COX11 were co-expressed ectopically in HEK293 cells, suggesting a functional coupling between them. Our results indicate a role of ACDP4 in metal ion homeostasis and toxicity. This is the first report revealing a functional aspect of this ancient conserved domain protein family. We propose that ACDP is a family of transporter protein or chaperone proteins for delivering essential metal ions in different mammalian tissues. The expression of ACDP4 on spinal cord dorsal horn neurons may have implications in sensory neuron functions under physiological and pathological conditions.

Cleavage of Apolipoprotein E by Membrane-Type Matrix Metalloproteinase-1 Abrogates Suppression of Cell Proliferation

Apolipoprotein E (apoE) in a human fetal brain cDNA library was identified, using the expression cloning method, as a gene product that formed a complex with latent matrix metalloproteinase (MMP)-2. Co-expression of membrane-type MMP-1 (MT1-MMP) with apoE in HEK293T cells reduced the amount of apoE secreted into the culture medium, whereas cell-associated apoE core protein was not affected. Incubation of native apoE protein with recombinant MT1-MMP resulted in the cleavage of apoE. Recombinant apoE protein fused to glutathione S-transferase (apoE-GST) was cleaved by MT1-MMP at the following peptide bonds; T(85)-M(86), K(93)-S(94), R(246)-L(247), A(255)-E(256) and G(296)-L(297). HT1080 cells transfected with the apoE gene, which express endogenous MT1-MMP, secreted a low level of apoE protein and its cleaved fragments, and treatment with MMP inhibitor BB94 induced accumulation of apoE and retardation of cell proliferation. Addition of apoE-GST protein to the culture of HEK293T cells suppressed cell proliferation, and stable transfection of the MT1-MMP gene partly abrogated the suppression. These results suggest that cleavage of apoE protein by MT1-MMP abrogates apoE-mediated suppression of cell proliferation.

A Novel Human Gene (WDR25) Encoding a 7-WD40-Containing Protein Maps on 14q32

Members of the large family of WD-repeat proteins are involved in diverse functions such as RNA-procession, signal transduction, vesicular trafficking, cytoskeletal assembly, and cell cycle control. By large-scale-sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA encoding a 7-WD40-repeat protein. This cDNA is 2004 bp in length and it codes for a 544-amino-acid protein. We term it human WD40-repeat-containing gene 25 (WDR25) and this gene shows significant similarity with human pre-mRNA splicing factor 17. The WDR25 gene is mapped to chromosome 14q32 and contains seven exons. RT-PCR analysis shows that the WDR25 gene is widely expressed in human tissues and the expression levers in heart, muscle, testis, ovary, uterus, and prostate are relatively high.

Molecular cloning and characterization of a novel human protein phosphatase 2C cDNA (PP2C ε)

We have isolated a novel cDNA from the human fetal brain cDNA library with homology to the Mg2+ -dependent serine/threonine protein phosphatase 2C (PP2C) family. The cDNA is 3055 bp in length, and the predicted coding region encodes a 360-amino-acid protein, which shows 99% identity to the PP2C epsilon from rat and mouse. Then we term it human PP2C epsilon gene. The gene is mapped to chromosome 3q26.1 and contains 4 exons. RT-PCR analysis shows that the PP2C epsilon is widely expressed in human tissues and the expression levels in heart, placenta, lung, liver, kidney, and pancreas are relatively high.

Characterization of a cDNA encoding a protein with limited similarity to β1, 3-N-acetylglucosaminyltransferase

Glycosyltransferases constitute a large group of enzymes that are involved in a wide range of functions in all living organisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human putative glycosyltransferase gene named beta3GnTL1. Its cDNA is 1372 base pair in length, encoding a predicted protein with 361 amino acid residues. The human beta3GnTL1 is located to chromosome 17q25.3 by comparison of its cDNA with human gemome database. RT-PCR result shows the beta3GnTL1 is expressed at various levels in most of tissues examined.

Reduction of the transcription level of the mitochondrial genome in human glioblastoma

Screening of human fetal brain cDNA library by glioblastoma (GB) and normal human brain total cDNA probes revealed 80 differentially hybridized clones. Hybridization of the DNA from selected clones and the same cDNA probes confirmed this difference for 38 clones, of which eight clones contained Alu-repeat inserts with increased levels in GB. Thirty clones contained cDNAs corresponding to mitochondrial genes for ATP synthase subunit 6 (ATP6), cytochrome c oxidase subunit II (COXII), cytochrome c oxidase subunit III (COXIII), NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 4 (ND4), and mitochondrial 12S rRNA. The levels of all these mitochondrial transcripts were decreased in glioblastomas as compared to tumor-adjacent histologically normal brain. Earlier we found the same for cytochrome c oxidase subunit I (COXI) Serial Analysis of Gene Expression (SAGE) showed lower content of the tags for all mitochondrial genes in GB SAGE libraries and together with our experimental data could serve as evidence of general inactivation of the mitochondrial genome in glioblastoma—the most malignant and abundant form of human brain tumor.

Reciprocal signaling between the transcriptional co-factor Eya2 and specific members of the Galphai family.

As part of a program to elucidate signaling processes controlled by the heterotrimeric G protein Galphaz, a human fetal brain cDNA library was screened for proteins that specifically interact with the activated form of Galphaz. One of the most-encountered molecules in this screen was Eya2, a member of the Eyes absent family of proteins. Mammalian Eya proteins are predominantly cytosolic proteins that are known to interact with members of the Sine oculis (Six) family of homeodomain transcription factors. This interaction facilitates the translocation of Eya into the nucleus, where the Eya/Six complex regulates transcription during critical stages of embryonic development. In vitro binding studies confirmed that Galphaz interacts with Eya2 in an activation-dependent fashion; furthermore, most other members of the Galphai family including Galphai1, Galphai2, and Galphai3 were found to interact with Eya2. It is interesting that one of the most abundant Galphai proteins, Galphao, did not interact with Eya2. Coexpression of the activated forms of Galphai1, Galphai2, and Galphai3, but not Galphao, with Eya2 recruited Eya2 to the plasma membrane, prevented Eya2 translocation into the nucleus, and abrogated Eya2/Six4-mediated transcription. In addition, Eya2 impinged on G protein-mediated signaling, as evidenced by its ability to relieve Galphai2-mediated inhibition of adenylyl cyclase. These results demonstrate that the interaction between the Galphai proteins and Eya2 may impact on seemingly disparate regulatory events involving both classes of proteins.

A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript.

The Down's syndrome candidate region 1 (DSCR1) protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD) was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD). Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT). UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATa) expressing AD-UXT with the strain Y187 (MATalpha) expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

Cloning and characterization of a novel human cDNA encoding a J-domain protein (DNAJA5) from the fetal brain

J-domain proteins are involved in protein importing and sorting, protein translocation into the endoplasmic reticulum and interaction with hsp70, cell cycle regulation and exocytosis. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a putative 531 amino acid protein with a DnaJ domain and C2H2-type zinc finger motif, which was named as DNAJA5 (human DnaJ homology subfamily A member 5). The putative protein of DNAJA5 gene was homologous to Caenorhabditis elegans DNJ-17 (identity 33.2% similarity 45.1%). By mapping, we located DNAJA5 gene to human chromosome 5p12-5p13. The DNAJA5 gene contained at least 12 exons that spanned more than 25.6 kb of the genomic sequence. The reverse transcription-polymerase chain reaction analysis indicated that it was expressed in the human brain, placenta, kidney and pancreas. DNAJA5 might play a role in co-operating with Hsp70.

Molecular cloning and characterization of a novel dual-specificity phosphatase 23 gene from human fetal brain

Most of dual-specificity protein phosphatases (DSPs) play an important role in the regulation of mitogenic signal transduction and controlling the cell cycle in response to extracellular stimuli. In this study, a novel human dual-specificity protein phosphatases gene named dual-specificity phosphatase 23 ( DUSP23) was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. Its cDNA was 726 bp in length, encoding a 150-amino acid polypeptide which contained a dual-specificity phosphatase catalytic (DSPc) domain but not a CDC25 homology (CH2) domain. Reverse transcription-PCR (RT-PCR) revealed that the DUSP23 was expressed in most fetal tissues and two adult tissues: testis and colon. Transient transfection experiment suggested that DUSP23 was localized in the cytoplasm of HEK293 cells. DUSP23 showed distinctive phosphatase activity toward p-nitrophenyl phosphate ( pNPP), as well as oligopeptides containing phospho-tyrosine and phospho-threonine residues. Furthermore, DUSP23 could dephosphorylate p44ERK1 but not p38 and p54SAPKβ in vitro. All the results indicated that DUSP23 was a novel protein phosphatase with dual substrate specificity.

Molecular cloning and characterization of SGT1.2, a novel splice variant of Homo sapiens SGT1.

SGT1.2, a novel splice variant of Homo sapiens SGT1 was isolated from a human fetal brain cDNA library. This cDNA is 1404 bp and contains an open reading frame from 68 to 1165 encoding a putative protein of 365 amino acids (SGT1.2) that share 90% identities to Homo sapiens SGT1, suppressor of G2 allele of SKP1 at protein level. RPS-BLAST searching revealed that SGT1.2 have a TPR domain, a p23 domain, a SGS domain and a CS domain. According to the search of the human genome database, SGT1.2 was mapped to human chromosome 13q14.13. Reversed transcription-polymerase chain reaction analysis indicated that it widely expressed in human adult tissues.

Cloning and identification of a novel human gene PDLIM5, a homolog of AD-associated neuronal thread protein (AD7c-NTP).

A novel human gene cDNA was successfully cloned from the human fetal brain cDNA library constructed by our lab, and this gene was termed PDLIM5 after acquiring the agreement of HUGO. BLASTX searching revealed that the hypothetical protein is a homolog of AD-associated neuronal thread protein (AD7c-NTP), which is over-expressed in Alzheimer disease (AD) beginning early in the course of disease, and over-expression of the AD7c-NTP gene would cause neuritic sprouting and cell death. SMART analysis showed that both our predicted protein and AD7c-NTP comprise BCL domain (only contains BH1 and BH2 regions). RT-PCR experiment revealed that the expression level of PDLIM5 in brain, skeletal muscle, prostate, colon and leukocyte is obviously higher than that in other tissues.

CDNA cloning and expression analysis of a novel human F-box domain containing gene.

F-box proteins are a large family of eukaryotic proteins that are characterized by an approximately 40 amino acid motif. Some F-box proteins are critical for the controlled degradation of cellular regulatory proteins. Here we report that a novel member of F-box proteins, FBXO35 gene, was cloned and identified during the large-scale sequencing analysis from a human fetal brain cDNA library. FBXO35 gene shares amino acid similarity with several putative mouse genes not only in F-box domain but also in the rest of the sequence, which indicates that FBXO35 might also contain some other unknown conserved domain. RT-PCR analysis indicated that FBXO35 gene had a ubiquitously low expression pattern in most human adult tissues. According to bioinformatics analysis, the FBXO35 gene was found located in chromosome 3p21.

The cloning and preliminarily functional analysis of the human neurotrimin gene.

Proteins of the immunoglobulin superfamily (IgSF) are involved in a variety of specific cell-cell interactions in the developing nervous system. To identify and characterize new members of this protein family in human nervous system, we screen the human fetal brain cDNA library and isolate a full-length cDNA clone which contains a 1032 bp open reading frame encoding a protein of 344 amino acids. Sequence analysis reveals that it is a glycoprotein comprised of three C2-like immunoglobulin domains and is anchored to the plasma membrane via a post-translationally attached glycosyl-phosphatidylinositol (GPI) moiety. The protein shows high sequence similarity to the rat Ntm (97%), so we term it human neurotrimin (NTM). Northern blot analysis reveals that (HUMAN)NTM has three different transcripts with the length of 3.2 kb, 4.0 kb and 9.0 kb respectively. It has a wider expression pattern than that of (RAT) Ntm. Notably, the expression of NTM in fetal brain is higher than that in mature brain and is stronger in nervous tumors than that in normal brain tissues. We insert an HA epitope tag between the third Ig-like domain of NTM and the site of GPI attachment, then construct it into the eukaryotic expression vector pcDNA3.1+/Zeocin. The pcDNA3.1-HA-NTM is transfected into the Chinese Hamster Ovary (CHO) cells. The results demonstrate that HA-NTM is expressed on the surface of CHO cells and could strengthen the aggregation of CHO-NTM cells.

Immunomics in inflammatory rheumatic diseases

Autoimmune diseases such as rheumatoid arthritis (RA) are characterized by autoantibodies to different autoantigenic proteins. Using proteomic 2D immunoblots, we identified a new 40 kDa autoantigen – hnRNP A3 – from HeLa nuclear extracts, which is frequently (30%) detected by RA. Moreover, we used a set of protein arrays of about 50 000 proteins derived from a human foetal brain cDNA expression library for screening with patient sera. Additionally, we utilized a human foetal brain cDNA library in a robot-based T7 phage display screening system with RA patient sera. To determine the diversity of the enriched library, we amplified the cDNA inserts and hybridized them onto the custom-made human ENSEMBL cDNA array. By these methods, over 80 clones were identified to bind patient immunoglobulins. Moreover, nine clones show only IgA-specific reactivity. We have now evaluated two different clones thoroughly: the carboxyl-terminal half of the nucleolar phosphoprotein p130 (NOPP 130) and a clone representing a 41-amino-acid mimetic peptide showing homology to calreticulin, a previously reported autoantigen. The remaining proteins are still undergoing thorough investigation. Applying state-of-the-art proteomic techniques, such as protein array and phage display, we have succeeded in identifying more than 80 potentially autoantigenic marker molecules, of which we have characterized a subset for RA specificity by screening with large numbers of patient and control sera. However, none of the molecules characterized thus far is exclusively discriminatory for RA and they all exhibit overlap with other autoimmune diseases.

CDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type VI.

Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.