Multiple Tissue cDNA Research Articles

Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Remarkable Expression in the Colon Adenocarcinoma ofHmat-Xa, a Human Mannosyltransferase-Like Gene, That Is Homologous to Drosophila Gene GC15914

We cloned a novel human mannosyltransferase-like gene, designated Hmat-Xa, as a gene homologous to the Drosophila GC15914 gene encoding the 9QVXN0 protein: see "Project Report for FY2002 on the 'Construction of Libraries of Human Genes Participating in Glycosylation' project" 43-45 (2003), New Energy and Industrial Technology Development Organization (NEDO), NEDO and Research Association for Biotechnology, Tokyo, Japan (in Japanese). After that, the GTDC1 gene, as reported by Zhao et al., DNA Cell Biol., 23, 183-187 (2004), was found to be the same as the Hmat-Xa gene. Domain EXFGI/L/VX(2)L/VE in the Hmat-Xa protein, also present in both human mannosyltransferase II/III and mannosyltransferase IV/V, which are involved in the synthesis of lipid-linked oligosaccharides, and some bacterial mannosyltransferases. A real-time PCR study of Hmat-Xa mRNA expression in human normal and tumor multiple tissue cDNA identified its tissue-specific expression and its remarkable expression in colon adenocarcinoma as compared to the normal counterpart. Thus the elevated expression of Hmat-Xa might serve as a candidate marker for colon adenocarcinoma.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Identification of a Novel Family of Laminin N-terminal Alternate Splice Isoforms

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5′-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) α3 and LaNt α5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt α3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt α3 and LaNt α3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt α3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt α3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1

Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.

Generation of CD8+ Cytotoxic T Cell Clones Recognizing BMI1-Derived Peptides

Leukemic stem cells (LSCs) resistant to conventional chemotherapeutic drugs can cause relapse of disease even after intensive treatment. Therefore, novel therapeutic strategies targeting LSCs are needed. The polycomb group gene BMI1 was initially identified as a cooperating oncogene in Eμ/myc transgenic mice. The protein product of BMI1 has been shown to repress the products of the CDKN2 tumor suppressor gene, p16 (INK4A) and p14 (ARF), and is required for self-renewal of both normal hematopoietic stem cells as well as LSCs. BMI1 expression level has been correlated with prognosis in myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). Furthermore, higher BMI1 expression in the tumorigenic cancer stem cell population has been demonstrated in brain tumor, breast cancer, and head and neck cancer. Thus, BMI1 represents a potential attractive target for cancer stem cell-specific immunotherapy. To further evaluate BMI1 potential, we generated CD8+BMI1-specific cytotoxic T cell clones. Two BMI1-encoded peptides (CLPSPSTPV, BMI1271-279, and TLQDIVYKL, BMI174-82) with predicted high affinity binding to the class I MHC molecule HLA-A*0201 were identified using publicly available prediction algorithms, and high-affinity binding to HLA-A*0201 was confirmed using an MHC stabilization assay on T2 cells. CD8+ T cells were negatively enriched from peripheral blood mononuclear cells (PBMC) from three normal human HLA-A 0201+ donors and stimulated at 10-day intervals with BMI1 peptide-pulsed autologous dendritic cells or PBMC for two or three cycles. Reactivity of the resulting T cell lines for the corresponding BMI1 peptides was subsequently assessed by ELISPOT assay, and was detected in lines from two of the three donors for CLPSPSTPV and in a line from one of the three donors for TLQDIVYKL. A CTL clone specific for CLPSPSTPV was isolated from one of the donors and evaluated in further studies. A peptide titration assay demonstrated that the clone had high affinity (peptide concentration required for half maximal lysis at E/T=10:1, 0.1 nM). In standard cytotoxicity assays, this CTL clone efficiently killed CLPSPSTPV peptide-pulsed T2 cells and 6 of 9 HLA-A*0201-positive or -transduced BMI1+ myeloid leukemia cell lines, and did not kill nonmalignant HLA-A2+ cells, including fibroblasts, PHA-blasts, and EBV-transformed B cells. Quantitative RT-PCR analysis of BMI1 expression was performed on a commercial multiple tissue cDNA panel, 39 primary leukemia/lymphoma samples (4 ALL, 22 AML, 4 CLL, 4 DLBCL, 4 FL, 1 MCL), and 31 solid tumor lines (21 Renal cell ca., 1 Ovarian ca., 3 Breast ca., 4 Melanoma, 1 Colon ca., 1 Pancreas ca.). All samples had detectable levels of BMI1 expression. Among normal tissues, testis had the highest level of BMI1 expression. Of the primary leukemia/lymphoma samples, four of 22 AML cells and one of four CLL cells expressed high levels of BMI1, and all solid tumor lines had low levels of BMI1 expression. When tested for cytotoxicity against five HLA-A*0201+ primary leukemia cells (3 AML, 1 ALL, 1 CML-BC), the BMI1-specific CTL clone killed 3 of 5 efficiently. The clone was also tested for recognition of 14 HLA-A*0201+ solid tumor lines (6 Renal cell ca., 2 Breast ca. 2 Melanoma, 1 Ovarian ca., 1 Colon ca., 2 Medulloblastoma), but only killed 2 of the 14 lines. These results suggest that CTL therapy targeting BMI1 may have utility as a novel strategy for eliminating LSCs. Future studies will determine whether this clone can specifically recognize leukemic stem cells and inhibit the engraftment of human AML in NOD/SCID mice.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E(2)-induced production of cAMP were significantly decreased. A remarkable increase in [(35)S]guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of G(i/o) proteins. Involvement of GPR20 in the activation of G(i/o) proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [(35)S]GTPgammaS incorporation and inhibition of the prostaglandin E(2)-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [(3)H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates G(i) proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling.

Comparative Biophysical Characterization of p53 with the Pro-apoptotic BAK and the Anti-apoptotic BCL-xL

The p53 transcription-independent apoptosis in mitochondria, mediated by its interaction with the pro-apoptotic and the anti-apoptotic members of the Bcl2 family of proteins, has been described in vivo, especially in radiosensitive tissues. We have characterized the interaction of p53 with both the pro-apoptotic Bak and the anti-apoptotic Bcl-x(L) proteins, comparing their affinity and their interaction surfaces, using biophysical techniques such as fluorescence anisotropy, analytical ultracentrifugation, and NMR. We have shown that both proteins interact with only the p53 core domain and not with its N- and C-terminal regions. Further, p53 has a higher affinity for Bcl-x(L) than for Bak, which is consistent with the previously described sequential binding of Bcl-x(L) and Bak by p53. Interestingly, although the interaction with both proteins is electrostatic in character, they have different binding sites. Using NMR spectroscopy, we have determined that Bcl-x(L) interacts with the DNA binding site of p53, but Bak does not interact with this site. A new potential interaction surface for Bak is proposed.

Chicken genomics resource: sequencing and annotation of 35,407 ESTs from single and multiple tissue cDNA libraries and CAP3 assembly of a chicken gene index

Its accessibility, unique evolutionary position, and recently assembled genome sequence have advanced the chicken to the forefront of comparative genomics and developmental biology research as a model organism. Several chicken expressed sequence tag (EST) projects have placed the chicken in 10th place for accrued ESTs among all organisms in GenBank. We have completed the single-pass 5'-end sequencing of 37,557 chicken cDNA clones from several single and multiple tissue cDNA libraries and have entered 35,407 EST sequences into GenBank. Our chicken EST sequences and those found in public databases (on July 1, 2004) provided a total of 517,727 public chicken ESTs and mRNAs. These sequences were used in the CAP3 assembly of a chicken gene index composed of 40,850 contigs and 79,192 unassembled singlets. The CAP3 contigs show a 96.7% match to the chicken genome sequence. The University of Delaware (UD) EST collection (43,928 clones) was assembled into 19,237 nonredundant sequences (13,495 contigs and 5,742 unassembled singlets). The UD collection contains 6,223 unique sequences that are not found in other public EST collections but show a 76% match to the chicken genome sequence. Our chicken contig and singlet sequences were annotated according to the highest BlastX and/or BlastN hits. The UD CAP3 contig assemblies and singlets are searchable by nucleotide sequence or key word (http://cogburn.dbi.udel.edu), and the cDNA clones are readily available for distribution from the chick EST website and clone repository (http://www.chickest.udel.edu). The present paper describes the construction and normalization of single and multiple tissue chicken cDNA libraries, high-throughput EST sequencing from these libraries, the CAP3 assembly of a chicken gene index from all public ESTs, and the identification of several nonredundant chicken gene sets for production of custom DNA microarrays.

Mouse Sphingosine Kinase Isoforms SPHK1a and SPHK1b Differ in Enzymatic Traits Including Stability, Localization, Modification, and Oligomerization

Sphingosine kinases catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate. Mice have two isoforms of sphingosine kinase type 1, SPHK1a and SPHK1b. In addition to the previously reported difference in their enzyme activities, we have found that these isoforms differ in several enzymatic characteristics. First, SPHK1b is unstable, whereas SPHK1a is highly stable. Degradation of SPHK1b occurs at the membrane and is inhibited by a proteasome inhibitor. Second, only SPHK1b exhibits abnormal mobility on SDS-PAGE, probably due to its SDS-resistant structure. Third, SPHK1a and SPHK1b are predominantly detected in the soluble and membrane fractions, respectively, when their degradation is inhibited. Fourth, only SPHK1b is modified with lipid, on its unique Cys residues (Cys-4 and Cys-5). Site-directed mutagenesis at these Cys residues resulted in increased sphingosine kinase activity, suggesting that the modification is inhibitory to the enzyme. Finally, SPHK1b tends to form homo-oligomers, whereas most SPHK1a is presented as monomers. We have also determined that the lipid modification of SPHK1b is involved in its homo-oligomerization. Thus, although these two proteins differ only in a few N-terminal amino acid residues, their enzymatic traits are extremely different.

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon–intron structure as follows: (1) The L2 protein has four transmembrane regions (M1–M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.

Molecular cloning and characterization of AAAS-V2, a novel splice variant of human AAAS

Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Much initial molecular analysis supported that Triple-A syndrome was caused by mutations in AAAS, a WD-repeat protein gene. Here we report cloning and characterization of a novel splice variant of human AAAS, which we named AAAS-v2, which is located on the human chromosome 12p13. The cDNA is 1703 bp, encoding a 513-amino acid polypeptide, which contains three WD40 domains, one less than the original which we called AAAS-v1 (Gen Bank: NM_015665.3). RT-PCR analysis in our work revealed that AAAS-v2 and AAAS-v1 were ubiquitously detected in human multiple tissue cDNA (MTC) panels (CLONTECH).

Molecular cloning and characterization of a novel human BTB domain-containing gene, BTBD10, which is down-regulated in glioma

The broad-complex, tramtrack (ttk) and bric-a-brac/poxvirus and zinc finger proteins (BTB/POZ) domain is highly conserved in a large family of eukaryotic proteins and is crucial for the latter's diverse roles in mediating interactions among proteins that are involved in transcription regulation and chromatin structures. From a fetal brain cDNA library, we isolated a cDNA of 2489 base pairs (bp) encoding a novel human BTB domain-containing protein named BTBD10. The cDNA contained an open-reading frame (ORF) of 1428 bp encoding a putative 475-amino acid (aa) protein. The BTBD10 gene was located on human chromosome 11p15.2 and consisted of nine exons spanning about 75.2 kilobase pairs (kb) of the human genome. The cDNA microarray analysis showed that BTBD10 was down-regulated in all 18 glioma samples. The expression pattern of BTBD10 gene was examined by multiple tissue cDNA (MTC) panels (Clontech), which showed a ubiquitous expression pattern in the 16 tissues examined with high expression in adult brain, testis and small intestine and weak expression in the heart, lung, liver, kidney, pancreas, spleen, thymus, prostate, ovary and colon. The subcellular localization result revealed that BTBD10 was located specifically in the nucleus of HEK293 and COS7 cell lines, suggesting that it may function in transcriptional regulation. The different expression patterns of BTBD10 in different grades of glioma versus normal brain were also examined by RT-PCR and Northern blot. We also investigated the expression of BTBD10 in hepatocellular carcinoma, ovary cancer and lung cancer, and the results revealed no significant difference in these three tumors. All these data suggested that BTBD10 might play a role in glioma.

Over-expression of Bim α3, a novel isoform of human Bim, result in cell apoptosis

Bim proteins are essential factors of apoptosis. Nine isoforms of Bim have been submitted to GenBank database. In order to improve the understanding of the regulation of Bims’ proapoptotic activity, we screened a multiple tissue cDNA panels for Bim isoforms and tested their proapoptotic activity by over-expression. Two novel cDNA isoforms of Bim family are generated by alternative splicing. One isoform encodes a 79 residue putative protein with a BH3 domain, named Bim α3. There is not any significant ORF found in another isoform, named Bim β5. Subcellular localized analysis of EGFP-Bim fusion protein suggests Bim α3 distributed to both plasma and nucleus of HEK 293 cell. HEK 293 cells transfected with pcDNA-Bim α3 presented a similar proapoptotic activity (32.05±6.42%) with Bim α2 (30.14±2.66%). The proapoptotic activity of Bim α3 was obviously weaker than that of Bim S (46.52±5.07%) and Bim L (55.53±1.99%). Anti-sense over-expression of Bim in HEK 293 cells results in a weak down-regulated proapoptotic level. Expression pattern analysis reveals that both the novel cDNAs are expressed widely in normal tissue just like the other reported isoforms. The expression pattern of Bim isoforms shows tissue specific obviously. The results suggest that BH3 domain is sufficiency for proapoptotic activity of Bim proteins. The functional state of Bims might be regulated both in the transcript and post transcript process.

Genetic Evidence for a Novel Human Desmosomal Cadherin, Desmoglein 4

Desmosomes are essential adhesion structures in most epithelia that link the intermediate filament network of one cell to its neighbor, thereby forming a strong bond. The molecular components of desmosomes belong to the cadherin superfamily, the plakin family, and the armadillo repeat protein family. The desmosomal cadherins are calcium-dependent transmembrane adhesion molecules and comprise the desmogleins and desmocollins. To date, three human desmoglein isoforms have been characterized, namely desmogleins 1, 2, and 3 that are expressed in a tissue- and differentiation-specific manner. Here we have identified and characterized, at the genetic level, a novel human desmoglein cDNA sharing homology with desmogleins 1, 2, 3 and we name this desmoglein 4. The human desmoglein 4 cDNA (3.6 kb) contains an open reading frame of 3120 bp that encodes a precursor protein of 1040 amino acids. The predicted mature protein comprises 991 amino acids with a molecular weight of 107822 Da at pI 4.38. Human desmoglein 4 shares 41% identity with human desmoglein 1, 37% with human desmoglein 2, and 50% with human desmoglein 3. Analysis of the exon/intron organization of the human desmoglein 4 gene (DSG4) demonstrates that it is composed of 16 exons spanning approximately 37 kb of 18q12 and is situated between DSG1 and DSG3. We have demonstrated using RT-PCR on multiple tissue cDNA samples that desmoglein 4 has very specific tissue expression in salivary gland, testis, prostate, and skin.

Extended Core 1 and Core 2 Branched O-Glycans Differentially Modulate Sialyl Lewis x-type L-selectin Ligand Activity

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcalpha2-->3Galbeta1-->4(Fucalpha1-->3(sulfo-->6))GlcNAcbeta1--> 3Galbeta1-->3GalNAcalpha1-->Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that beta1,3-N-acetylglucosaminyltransferase-3 (beta3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either beta3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.

CDNA cloning, expression profile and genomic structure of a novel human transcript on chromosome 10q24, and its analyses as a candidate gene for infantile onset spinocerebellar ataxia

In our search for the disease gene underlying autosomally recessively inherited infantile onset spinocerebellar ataxia (IOSCA), we identified an expressed sequence tag cluster representing a previously uncharacterized transcript in the restricted genomic sequence covering the IOSCA locus on chromosome 10q24, and for mutation analyses in IOSCA patients isolated the corresponding novel human cDNA, C10orf6. Multiple tissue cDNA and Northern analyses showed that this gene is ubiquitously expressed, with expression levels highest in the skeletal muscle and less abundant in the brain, liver, and heart than in other tissues examined. C10orf6 consists of 20 exons forming a 7.3 kb cDNA which is capable of encoding a 1173 amino acid polypeptide and possesses orthologues in other mammals. Sequencing of RT and genomic PCR products of the gene revealed no alterations in IOSCA patients when compared to control subjects, and neither could differences be detected in expression levels between patient and control brain RNA samples, thus excluding mutation(s) in this novel gene as causative for IOSCA. However, this study facilitates future investigations on both the role of C10orf6 gene product in human cells as well as its possible involvement in the pathogenesis of other hereditary diseases mapped to chromosome 10q24.

ERK8, a New Member of the Mitogen-activated Protein Kinase Family

The ERKs are a subfamily of the MAPKs that have been implicated in cell growth and differentiation. By using the rat ERK7 cDNA to screen a human multiple tissue cDNA library, we identified a new member of the ERK family, ERK8, that shares 69% amino acid sequence identity with ERK7. Northern analysis demonstrates that ERK8 is present in a number of tissues with maximal expression in the lung and kidney. Fluorescence in situ hybridization localized the ERK8 gene to chromosome 8, band q24.3. Expression of ERK8 in COS cells and bacteria indicates that, in contrast to constitutively active ERK7, ERK8 has minimal basal kinase activity and a unique substrate profile. ERK8, which contains two SH3-binding motifs in its C-terminal region, associates with the c-Src SH3 domain in vitro and co-immunoprecipitates with c-Src in vivo. Co-transfection with either v-Src or a constitutively active c-Src increases ERK8 activation indicating that ERK8 can be activated downstream of c-Src. ERK8 is also activated following serum stimulation, and the extent of this activation is reduced by pretreatment with the specific Src family inhibitor PP2. The ERK8 activation by serum or Src was not affected by the MEK inhibitor U0126 indicating that activation of ERK8 does not require MEK1, MEK2, or MEK5. Although most closely related to ERK7, the relatively low sequence identity, minimal basal activity, and different substrate profile identify ERK8 as a distinct member of the MAPK family that is activated by an Src-dependent signaling pathway.

Cloning, tissue distribution, and functional expression of the human G protein β4-subunit

Heterotrimeric G proteins (Galphabetagamma) play an essential role in coupling membrane receptors to effector proteins such as ion channels and enzymes. Among the five mammalian Gbeta-subunits cloned, the human G protein beta4 has not been described. The purpose of the present study was to functionally characterize the newly identified human Gbeta4 subunit. The Gbeta4 open reading frame (ORF) was amplified utilizing PCR from brain cDNA. Amplification primers were generated following 5' rapid amplification of cDNA ends (5'-RACE) from an expressed sequence tag (EST) containing the predicted 3' end of the protein. Multiple tissue cDNA panel analysis showed that Gbeta4 mRNA was strongly expressed in lung and placenta, whereas it is weakly expressed in brain and heart. Heterologous overexpression of Gbeta4gamma2 or Gbeta4gamma4 in rat sympathetic neurons resulted in tonic modulation of N-type voltage-gated Ca(2+) and G protein-gated inwardly rectifying K(+) currents. Furthermore, coexpression of Gbeta4gamma2 and Galpha(oA) resulted in heterotrimer formation. These results show that the newly cloned Gbeta subunit shares several properties with other human Gbeta family members.