Cloning and characterization of a novel gene,WS-3, in human chromosome 8p11-p12

A novel human gene referred to as the WS-3 gene, in the short arm of human chromosome 8, was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR (TAIL-PCR) and the Marathon-Ready cDNA amplification method. The gene consists of 7 exons separated by 6 introns, and is at the telomere side of the STS marker, D8S1055. The full-length WS-3 gene contains 1052 nucleotides and codes for a protein of 190 amino acids with a calculated mol. wt. of 20 747. Southern blot experiments showed that the WS-3 gene exists as a single copy in the human genome. A protein encoded by the WS-3 gene has an R-G-D (Arg-Gly-Asp) motif in the N-terminal region, which seems to confer adhesive properties to macromolecular proteins like fibronectin. Although WS-3 is a small gene with unknown biological function, its ubiquitous expression in various tissues and organs suggests that the encoded protein is one of the essential components of all organs and tissues.     

Molecular characterization of a novel mammalian DnaJ-like Sec63p homolog.

We identified a human cDNA sequence encoding a polypeptide of 760 amino acids that shares 53% homology and 25.6% identity with the yeast DnaJ-like endoplasmic reticulum (ER) translocon component Sec63p. Three epitope-specific antisera revealed a protein of an apparent molecular mass of 83 kDa, both in human cell extracts and in dog pancreatic microsomes. Biochemical analyses show that it is an integral membrane protein of the rough ER, which has the DnaJ domain located in the ER lumen. The novel Sec63 protein could thus represent a key component of the mammalian ER protein translocation machinery.     

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.The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY314787.     

Cloning and functional characterization of GNPI2, a novel human homolog of glucosamine-6-phosphate isomerase/oscillin

The enzyme, glucosamine-6-phosphate isomerase (GNPI) or deaminase (GNPDA) (EC 5.3.1.10), catalyzes the conversion of GNP to fructose-6-phosphate and ammonia, with an aldo/keto isomerization and an amination/deamination. A hamster sperm-derived protein (Oscillin) with high similarity to bacterial GNPI has been proved to be capable of inducing calcium oscillation in eggs at fertilization. GNPI/Oscillin was supposed to be an important factor in starting embryonic development. From the cDNA library of human dendritic cells (DC), we isolated a novel full-length cDNA encoding a 276-amino acid-residue protein that shares high homology with human GNPI/Oscillin. So, the novel molecule is named as GNPI2. The GNPI2 gene consists of seven exons and six introns. It is mapped to chromosome 4. Northern blot analysis indicated that the tissue distribution of GNPI2 mRNA is different from that of human GNPI or Oscillin mRNA. GNPI2 is ubiquitously expressed in most of human tissues with high expression in testis, ovary, placenta, and heart. Like GNPI, the recombinant GNPI2 has been proved to have the enzymatic activity to catalyze the conversion of GNP to fructose-6-phosphate. Our results indicated that GNPI2 is a novel protein with definite function as a GNPI.     

Cloning, expression and characterization of a novel human REPS1 gene.

Ral is a member of the small GTPase-binding protein (G protein) family, and plays an important role in the Ras-RalGDS signal transduction pathway. A series of recent findings reveal several important downstream target proteins of Ral, such as RalBP1, Reps1, and others. Here we report another binding partner for RalBP1, which we have isolated from the human fetal brain library. The human REPS1 protein shares 83% amino acid identity with the mouse Reps1 protein. Northern blot analysis shows that the REPS1 is expressed in a variety of tissues, with the strongest expression in the heart and testis.     

Cloning and characterization a novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase gene AGPAT7.

The 1-Acylglycerolphosphate acyltransferase is crucial enzyme for synthesis of glycerolipids as well as triacylglylcerol biosynthesis in eukaryotes. Six members of 1-acyl-sn-glycerol-3-phosphate acyltransferase family in human have been described, which were AGPAT1, 2, 3, 4, 5 and 6. Here we report the cloning and characterization of another novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase member AGPAT7 (1-acyl-sn-glycerol-3-phosphate acyltransferase 7) gene, which was mapped to human chromosome 15q14. The AGPAT7 cDNA is 1898 bp in length, encoding a putative protein with 524 amino acid residues, which contains an acyltransferase domain in 123-234 aa. RT PCR amplification in 18 human tissues indicated that human AGPAT7 gene was widely expressed in uterus, thymus, pancreas, skeletal muscle, bladder, stomach, lung and testis. AGPAT7 protein was mainly localized to the endoplasmic reticulum (ER) in Hela cells.     

Cloning,expression and characterization of a novel human VMP gene*

We report here cloning and characterization of a novel human gene, termed VMP, which is a vesicular membrane protein. RT-PCR analysis shows that VMP is expressed exclusively in brain of the 16 tissues examined, suggesting that it is a neuron-specific membrane protein. The cDNA encodes 195 amino acid with a putative molecular weight of about 24 KDa. VMP contains two putative membrane spanning domains and a hydrophilic tail homologous to the microtubule-binding domain of MAPs. So it is speculated that VMP may associated with microtubules through its C-terminal and plays an important role in vesicular organelles transport and nerve signals.     

Cloning and characterization of human syndecan-3.

Syndecans are cell-surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co-receptors for growth factors and mediate cell-cell and cell-matrix interactions. Four syndecans (syndecan 1-4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan-3. The human syndecan-3 sequence has high homology to the rat and mouse sequences, with the exception of the 5'-region. Syndecan-3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full-length syndecan-3 cDNA, it was localized to the membrane and induced the formation of long filopodia-like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re-organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co-localizing with the syndecan-3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan-deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan-3 could perform comparable functions to those described for syndecan-3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan-3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism.     

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.     

A novel iron uptake mechanism mediated by GPI-anchored human p97.

The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI)-linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

Cloning and characterization of CPVL, a novel serine carboxypeptidase, from human macrophages

Carboxypeptidases are proteases that cleave single amino acids from the carboxy termini of proteins or peptides. In addition to degradative functions in the gut, carboxypeptidases activate or inactivate bioactive peptides such as angiotensin, bradykinin, and endothelin I. Using differential display PCR, we cloned a novel carboxypeptidase expressed in human macrophages but not in other leukocytes. The 476-amino-acid gene product has a putative signal sequence but no transmembrane domain and has striking sequence similarity to serine carboxypeptidases, a large family of enzymes in eukaryotes. Only one serine carboxypeptidase, lysosomal protective protein, has previously been reported in mammals. Among known proteins, this gene is most similar (43% amino acid identity) to vitellogenic carboxypeptidase, a serine carboxypeptidase expressed in mosquito ovaries. Therefore, we have named this new gene carboxypeptidase, vitellogenic-like (CPVL). In addition to monocyte/macrophage-rich sources such as spleen, leukocytes, and placenta, CPVL mRNA is abundantly expressed in heart and kidney, suggesting a separate role for CPVL outside the immune system. The CPVL gene contains at least 13 exons spread over more than 150 kb on human chromosome 7p14-p15. An affinity-purified polyclonal antiserum recognized a protein of approximately 57 kDa in macrophage lysates, but not in lysates from lymphocytes, neutrophils, or monocytes. CPVL protein expression was induced during maturation of monocytes into macrophages. Possible functions for CPVL in macrophages include digestion of phagocytosed particles in the lysosome, participation in an inflammatory protease cascade, and trimming of peptides for antigen presentation.