Molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.     

Molecular cloning,expression and chromosomal localization of a human gene encoding a 33 kDa putative metallopeptidase (PRSM1)

The zincins are a superfamily of structurally-related Zn²⁺-binding metallopeptidases which play a major role in a wide range of biological processes including pattern formation, growth factor activation and extracellular matrix synthesis and degradation. In this paper we report the identification and complete primary structure of a novel 33 kDa protein which contains the zinc-binding HEXXH motif found in the zincin superfamily. We have named this novel protein PRSM1 (PRoteaSe, Metallo, number 1). The gene was identified by the immunoscreening of a human placental cDNA library using polyclonal antibodies raised to the 70 kDa human matrix metalloendopeptidase, type III procollagen N-proteinase [Halila, R. and Peltonen, L. (1986) Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum. Biochem. J. 239, 47–52]. The protein is found in placenta and cultured osteosarcoma cells. PRSM1 could share sequence homology with the type III procollagen N-proteinase. The prsml gene is represented once in the human genome and is localized on chromosome 16 (q24.3).     

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.     

Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene.

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the lysine residue of the lipoate-dependent enzymes. We isolated human lipoyltransferase cDNA and genomic DNA. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids. Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate-protein ligase A, respectively. Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb. mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydrogenase complexes and H-protein of the glycine cleavage system which accept lipoic acid as a prosthetic group. The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA. Some alternatively spliced mRNA species were found by 5'-RACE analysis, and the most abundant species lacks the third exon. The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization.     

Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase.

Murine macrophages have previously been shown to secrete a zinc-dependent proteinase that can degrade elastin. In this report, we identify murine macrophage elastase (MME) cDNA and show that it is a distinct member of the metalloproteinase gene family. Small amounts of MME were purified to homogeneity, and N-terminal amino acid sequence was obtained. This sequence was used to obtain a partial cDNA clone by the polymerase chain reaction; a cDNA library derived from a mouse macrophage-like cell line (P388D1) was screened with this probe. A full-length MME cDNA spanning approximately 1.8 kilobases contained an open reading frame of 1386 base pairs; the predicted molecular mass of the MME proenzyme is 53 kDa. The gene encoding MME is represented only once in the mouse genome and is located on chromosome 9. Despite a size that is similar to other metalloproteinases, MME is distinct, sharing only 33-48% amino acid homology with other metalloproteinases. In contrast to other metalloenzymes, MME appears to be rapidly processed to an active truncated form (N-terminal and C-terminal cleavage). We expressed recombinant MME in Escherichia coli and demonstrated that it has significant elastolytic activity that is specifically inhibited by the tissue inhibitor of metalloproteinases. MME is therefore a true metalloproteinase that may be involved in tissue injury and remodeling.     

Molecular cloning, functional expression and chromosomal localization of an amiloride-sensitive Na(+) channel from human small intestine

Amiloride-sensitive Na(+) channels belonging to the recently discovered NaC/DEG family of genes have been found in several human tissues including epithelia and central and peripheral neurons. We describe here the molecular cloning of a cDNA encoding a novel human amiloride-sensitive Na(+) channel subunit that is principally expressed in the small intestine and has been called hINaC (human intestine Na(+) channel). This protein is similar to the recently identified rodent channel BLINaC and is relatively close to the acid sensing ion channels (ASICs) (79 and 29% amino acid identity, respectively). ASICs are activated by extracellular protons and probably participate in sensory neurons to nociception linked to tissue acidosis. hINaC is not activated by lowering the external pH but gain-of-function mutations can be introduced and reveal when expressed in Xenopus oocytes, an important Na(+) channel activity which is blocked by amiloride (IC(50)=0.5 microM). These results suggest the existence of a still unknown physiological activator for hINaC (e.g. an extracellular ligand). The presence of this new amiloride-sensitive Na(+) channel in human small intestine probably has interesting physiological as well as physiopathological implications that remain to be clarified. The large activation of this channel by point mutations may be associated with a degenerin-like behavior as previously observed for channels expressed in nematode mechanosensitive neurons. The hINaC gene has been mapped on the 4q31.3-q32 region of the human genome.     

Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor.

The sequencing of endopeptidase-generated peptides from the peripheral binding site (PBS) for benzodiazepines, purified from a Chinese hamster ovary (CHO) cell line, produced internal sequence information, and confirmed and extended the NH2-terminal PBS sequence that we previously reported. Since the sequences were highly similar to the corresponding rat PBS sequences, we investigated whether they were also conserved in human PBS. Scatchard analysis of [3H]PK11195 (a derivative of isoquinoline carboxamide) binding and photoaffinity labeling with [3H]PK14105 (a nitrophenyl derivative of PK11195) revealed that CHO PBS and human PBS are closely related. Furthermore a rabbit antiserum raised against three peptides synthesized on the basis of the CHO PBS sequence immunoprecipitate the solubilized U937 PBS and also recognize the human protein in an immunoblot analysis. Based on these results, we screened a U937 cell cDNA library with four oligonucleotide probes derived from the CHO sequence. Two of the probes hybridized with several clones that we isolated and sequenced. One of these, h-pPBS11, is 831 nucleotides and contains a full-length representation of human PBS mRNA. The amino acid sequence of human PBS deduced from the cDNA is 79% identical to that reported for rat PBS, however, human PBS contains two cysteines while rat PBS is characterized by the absence of this amino acid. Using the cDNA of human PBS as a probe, the PBS gene was located in the 22q13.3 band of the human genome.     

Molecular cloning, chromosomal localization, and expression of the murine SALL1 ortholog Sall1

SALL1 has been identified as one of now three human homologs of the region specific homeotic gene spalt (sal) of Drosophila, which encodes a zinc finger protein of characteristic structure. Mutations of SALL1 on chromosome 16q12.1 cause Townes-Brocks syndrome (TBS, OMIM no. 107480). In order to facilitate functional studies of this gene in a model organism, we searched for the murine homolog of SALL1. Here we report the genomic cloning, chromosome mapping, and partial expression analysis of the gene Sall1. Sequence comparison, Northern blot hybridization as well as the conserved chromosome location on the homologous mouse chromosome indicate that we have indeed isolated the murine homolog of SALL1.     

Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

Macroautophagy/autophagy, which is one of the main degradation systems in the cell, is mediated by a specialized organelle, the autophagosome. Purification of autophagosomes before fusion with lysosomes is important for both mechanistic and physiological studies of the autophagosome. Here, we report a simple method to accumulate undigested autophagosomes. Overexpression of the autophagosomal Qa-SNARE STX17 (syntaxin 17) lacking the N-terminal domain (NTD) or N-terminally tagged GFP-STX17 causes accumulation of autophagosomes. A HeLa cell line, which expresses GFP-STX17ΔNTD or full-length GFP-STX17 under the control of the tetracycline-responsive promoter, accumulates a large number of undigested autophagosomes devoid of lysosomal markers or early autophagy factors upon treatment with doxycycline. Using this inducible cell line, nascent autophagosomes can be easily purified by OptiPrep density-gradient centrifugation and immunoprecipitation. This novel method should be useful for further characterization of nascent autophagosomes.     

Molecular cloning, expression, and chromosomal localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/ fructose-2, 6-bisphosphatase gene (PFKFB3)

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15-->p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.     

Molecular cloning and chromosomal assignment of a human perforin (PFP) gene

Human perforin cDNA was isolated and the complete nucleotide sequence of the gene determined. The deduced amino acid sequence of human perforin showed 68.4% similarity to that of mouse perforin. RNA blot analysis of the human perforin gene revealed that the gene product is expressed preferentially in killer-type cells among cell lines tested, and in large granular lymphocytes among the peripheral blood mononuclear cells. In situ hybridization analysis with a human perforin cDNA probe revealed that the human perforin (PFP) gene is located on chromosome17q11-21. The nucleotide sequence data reported in this paper have been submitted to the GeBank nucleotide sequence database and have been assigned the accession number M28393.