Identification and characterization of KLK-L4, a new kallikrein-like gene that appears to be down-regulated in breast cancer tissues

Kallikreins are a subgroup of serine proteases and these proteolytic enzymes have diverse physiological functions in many tissues. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins constitute a large multigene family, but in humans, only three genes were identified. By using the positional candidate gene approach, we were able to identify a new kallikrein-like gene, tentatively named KLK-L4 (for kallikrein-like gene 4). This new gene maps to chromosome 19q13. 3-q13.4, is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes. KLK-L4 is expressed in a variety of tissues including prostate, salivary gland, breast, and testis. Our preliminary results show that KLK-L4 is down-regulated, at the mRNA level, in breast cancer tissues and breast cancer cell lines. Its expression is regulated by steroid hormones in the breast cancer cell line BT-474. This gene may be involved in the pathogenesis and/or progression of breast cancer and may find applicability as a novel cancer biomarker.     

Molecular characterization, tissue expression, and mapping of a novel Siglec-like gene (SLG2) with three splice variants.

The sialic acid binding immunglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within the Siglec family, there exists a subgroup, which bears a high degree of homology with the molecule CD33 (Siglec-3), and has thus been designated the CD33-like subgroup of Siglecs. Members of this subgroup have been localized to chromosome 19q13.4. Through the positional candidate approach, we identified a novel potential member of this subgroup of Siglecs. We have characterized the complete genomic structure of this gene, determined its chromosomal localization, its homology to other members of the Siglec family, and its tissue expression profile. This new Siglec-like gene is comprised of 11 exons, with 10 intervening introns, and is localized 278 kb telomeric to Siglec-9 and 35 kb centromeric to Siglec-8 and on chromosome 19q13.4. The coding region consists of 2094 base pairs, and encodes for a putative 76.6 kDa protein. All Siglec-conserved structural features, including V-set domain, three C-set domains, transmembrane domain, ITIM and SLAM motifs, were found in this Siglec-like gene. Also, it has the conserved amino acids essential for sialic acid binding. The Siglec-like gene has 40-66% homology with members of the CD33-like subgroup, including Siglecs 5-9. Through RT-PCR we have examined the expression profile of this new gene in a panel of human tissues and found it to be primarily expressed in the bone marrow, spleen, brain, small intestine, colon, and spinal cord. We were also able to identify three different splice variants of the new gene. This gene may represent the latest novel member of the CD33-like subgroup of Siglecs, and, given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec-7.     

Genomic organization, mapping, tissue expression, and hormonal regulation of trypsin-like serine protease (TLSP PRSS20), a new member of the human kallikrein gene family

The cDNA for the trypsin-like serine protease gene (TLSP, HGMW-approved symbol PRSS20) has been recently identified. TLSP is expressed in brain and skin tissues but little else is known about this new serine protease gene. In this paper, we describe the complete genomic organization and precise mapping of the TLSP gene. This gene spans 5.3 kb of genomic sequence on chromosome 19q13.3-q13. 4. The gene consists of six exons, the first of which is untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of other kallikrein-like genes, including zyme (PRSS9), NES1 (PRSSL1), and neuropsin (PRSS19). Fine-mapping of the area indicates that TLSP lies downstream from the PSA, zyme, neuropsin, and NES1 genes. Significant sequence homologies were found between TLSP and other human kallikreins. Furthermore, there is conservation of the catalytic triad (histidine, aspartic acid, serine) and of the number of coding exons (five; the same in all members of the kallikrein gene family). We thus suggest that TLSP is a new member of the human kallikrein gene family. TLSP is expressed in many tissues including cerebellum, prostate, salivary glands, stomach, lung, thymus, small intestine, spleen, liver, and uterus. TLSP expression appears to be regulated by steroid hormones in the breast carcinoma cell line BT-474.     

Molecular characterization, expression and mapping of porcine LMP2 and MECL-1 genes.

Low molecular mass polypeptides 2 (LMP2) and Multicatalytic endopeptidase complex subunit (MECL-1) are two of three catalytic beta-type subunits of the 20s proteasome and upon interferon gamma-induction. LMP2 is critical for the production of major histocompatibility complex (MHC) class I ligand and T-lymphocytes. The LMP2 gene is located in the MHC region, but MECL-1 is located outside the MHC region. They are involved in the antigen presentation and are important candidate genes for an initial exploration of relationships between the antigen processing genes and disease resistance. In this report, the porcine LMP2 and MECL-1 cDNA were cloned and A 5099 bp LMP2 genomic DNA structure was identified, then two single nucleotide polymorphisms were detected in the exon2 and exon5 of LMP2 gene in 367 individuals. The LMP2 and MECL-1 genes putative protein included 219,274 amino acids, respectively. Alignment and phylogenetic of predicted porcine LMP2 and MECL-1 amino acid sequence with their homologies were analyzed. Tissues expression of LMP2 and MECL-1 mRNA were observed by real time quantitative PCR (Q-PCR) method, the results revealed MECL-1 expressed widely in all tissues, but LMP2 was not detected in muscle. The porcine MECL-1 gene was mapped to chromosome 6, closely linked to microsatellite SW1108 (LOD = 4.09, 84cR) by radiation hybrid panel.     

Molecular cloning,expression, and characterization of bovine tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.     

The Arabidopsis XET-related gene family: environmental and hormonal regulation of expression

Enzymes that modify cell wall components most likely play critical roles in altering size, shape, and physical properties of plant cells. Regulation of such modifying activity is expected to be important during morphogenesis and in eliciting developmental and physiological alterations that arise in response to environmental conditions. Previous work has shown that the Arabidopsis TCH4 gene encodes a xyloglucan endotransglycosylase (XET) which acts on the major hemicellulose of the plant cell wall. The expression of TCH4 is dramatically upregulated in response to several environmental stimuli (including touch, wind, darkness, heat shock, and cold shock) as well as the growth-enhancing hormones, auxin and brassinosteroids. This paper reports the presence of an extensive X ET ,related (XTR) gene family in Arabidopsis. In addition to TCH4, this family includes two previously identified genes, EXT and Meri-5, and at least five additional genes. The cDNAs of the XTR family share between 46 and 79% sequence identity and the predicted XTR proteins share from 37 to 84% identity. All eight proteins include potential N-terminal signal sequences and most have a conserved motif (DEIDFEFLG) that is also found in Bacillusβ-glucanase and may be important for enzyme activity. The members of the XTR gene family are differentially sensitive to environmental and hormonal stimuli. Magnitude and kinetics of regulation are distinct for the different genes. Differential regulation of expression of this complex gene family suggests a recruitment of related, yet distinct, cell wall-modifying enzymes that may control the properties of cell walls and tissues during development and in response to environmental cues.     

Mouse IL-1 receptor antagonist protein. Molecular characterization, gene mapping, and expression of mRNA in vitro and in vivo.

The IL-1R antagonist protein (IL-1RN/IL-1rn) is a member of the IL-1 family of inflammatory mediators. We have isolated and analyzed a mouse IL-1rn cDNA clone and established that the derived mouse IL-1rn protein sequence is highly homologous to the human counterpart molecule. Mouse IL-1rn mRNA may be induced in P388D1 monocytic cells with PMA and in mouse liver in vivo by development of an experimental inflammation via s.c. injection of azocasein. This latter observation implies the existence of an autocrine hepatic negative feedback loop that down-regulates the acute phase response and is itself induced at the same time as the major acute-phase proteins. The mouse Il 1rn gene was mapped to the proximal region of chromosome 2 between the centromere and Spna2; the other known members of the mouse IL-1 gene family, Il 1a and Il 1b, both map to the same chromosome, although not in close linkage.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Molecular cloning, sequence characterization, SNP detection, and tissue expression analysis of duck FMO3 gene

Flavin-containing monooxygenase 3 (FMO3) is an important monooxygenase for catalytic oxygenation of many harmful xenobiotics. Mutations in the FMO3 gene have been identified as causing trimethylaminuria in human and fishy off-flavor in cow milk and chicken eggs. In this study, the full-length cDNA sequence of Pekin duck FMO3 gene was cloned, sequenced, and characterized. The full-length cDNA sequence consisted of 1,846 bp and contained a 1,599 bp open-reading frame encoding 532 amino acids. Duck FMO3 gene shared a similar nine exon–eight intron structure with chicken and human. The duck FMO3 putative protein sequence showed high identity with that of chicken (82 %), and relative low identity with those of mammals (61–66 %). We also found that the duck FMO3 gene was dramatically expressed in liver, lung, and kidney compared to that in other tissues in the ducks, indicating the possible roles the FMO3 gene could play in the three tissues. By bidirectional sequencing, we also found one nonsense mutation, 5 nonsynonymous, and 21 synonymous mutations in the coding region of the FMO3 gene in 11 duck breeds and some of them were predicted to be potentially associated with the activities of FMO3 protein.     

The expanded human kallikrein gene family: locus characterization and molecular cloning of a new member, KLK-L3 (KLK9)

In rodents, kallikreins are encoded by a large multigene family but in humans, only three kallikrein genes were thought to exist. Based on the homology between the human and the rodent kallikrein loci, we defined a 300-kb human kallikrein gene region on chromosome 19q13. 3-q13.4. By using linear sequence information, restriction analysis, PCR, and blotting techniques, we were able to construct the first detailed map of the human kallikrein gene locus. Comparative analysis of genes located in this area enabled us to expand the human kallikrein multigene family with some recently identified serine proteases and establish common structural features. We further identified a new kallikrein-like gene, named kallikrein-like gene 3 (KLK-L3; HGMW-approved symbol KLK9). We describe the structural characterization of the KLK-L3 gene, together with its precise chromosomal localization in relation to other kallikreins and its tissue expression pattern and hormonal regulation.     

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene.

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.     

Tissue specific hormonal regulation of the rat angiotensinogen gene expression

Angiotensinogen is the precursor of the most potent pressor substance, angiotensin. Angiotensinogen levels are increased in some forms of human hypertension. Its levels are modulated by various factors including glucocorticoids, estrogens, and prostaglandins. We have recently reported the isolation of a human angiotensinogen cDNA clone and provided evidence for the presence of its mRNA in rat liver, brain, and heart. In this communication we report the effect of dexamethasone and estradiol on angiotensinogen mRNA levels in rat liver, brain, and heart. Our results indicate that angiotensinogen levels are increased to different extents in these three tissues as a result of glucocorticoid or estrogen administration.