Cloning and characterization of a novel human SPRYD4 gene encoding a putative SPRY domain-containing protein.

We report here the cloning and characterization of a novel human SPRYD4 gene which encodes a SPRY domain containing protein. The SPRYD4 gene is isolated from the human brain cDNA library, and mapped to 12q13.2 by searching the UCSC genomic database. The SPRYD4 cDNA is 1201 base pairs in length and contains an open reading frame encoding 207 amino acids. The SPRYD4 gene consists of two exons and encodes a putative protein with a SPRY domain ranging from 86 to 203 amino acids. The RT-PCR analysis reveals that SPRYD4 is ubiquitously expressed in 18 human tissues. However, it is strongly expressed in kidney, bladder, brain, thymus and stomach, while weakly expressed liver, testis, uterus, spleen and lung. Subcellular localization demonstrates that SPRYD4 protein is localized in the nuclear when overexpressed in COS-7 cell.     

A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus

Hepatoma-derived growth factor (HDGF) and HDGF-related proteins (HRP) belong to a gene family with a well-conserved amino acid sequence at the N-terminus (the hath region). A new member of the HDGF family in humans and mice was identified and cloned; we call it HRP-3. The deduced amino acid sequence from HRP-3 cDNA contained 203 amino acids without a signal peptide for secretion. HRP-3 has its 97-amino-acid sequence at the N-terminus, which is highly conserved with the hath region of the HDGF family proteins. It also has a putative bipartite nuclear localizing signal (NLS) sequence in a similar location in its self-specific region of HDGF and HRP-1. Northern blot analysis shows that HRP-3 is expressed predominantly in the testis and brain, to an intermediate extent in the heart, and to a slight extent in the ovaries, kidneys, spleen, and liver in humans. Transfection of green fluorescent protein (GFP)-tagged HRP-3 cDNA showed that HRP-3 translocated to the nucleus of 293 cells. GFP-HRP-3 transfectants significantly increased their DNA synthesis more than cells transfected with vector only. The HRP-3 gene was mapped to chromosome 15, region q25 by FISH analysis. These findings suggest that a new member of the HDGF gene family, HRP-3, may function mainly in the nucleus of the brain, testis, and heart, probably for cell proliferation.     

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.     

Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene

We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3′-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals. Received: 5 May 1999 / Accepted: 11 February 2000     

Human endo-alpha1,2-mannosidase is a Golgi-resident type II membrane protein

The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity analysis of this amino acid sequence, as well as proteolytic degradation studies, indicate that the enzyme is a type II protein, anchored in the membrane via a 19 amino-acid long apolar sequence close to the N-terminus. Human endo-alpha1,2-mannosidase displays a high degree of sequence identity with the catalytic domain of the homologous rat liver endo-enzyme, but differs substantially in the N-terminal peptide region, which includes the transmembrane domain. No sequence similarity exists with other processing alpha-glycosidases. Based on sequence information provided by the 2837 bp construct, the cDNA consisting of the complete 1389 bp ORF was amplified by RT-PCR using human fibroblast RNA. Incubation of E. coli lysates with this cDNA, previously modified for boost translation by codon optimization, resulted in the synthesis of an approximately 52 kDa protein which degraded [(14)C]Glc(3)-Man(9)-GlcNAc(2) efficiently, indicating that the catalytic domain of the enzyme folds correctly under cell-free conditions. Transfection of the endo-alpha1,2-mannosidase wild-type cDNA into COS 1 cells resulted in a moderate (approximately 1.5-fold) but reproducible increase of activity compared with control cells, whereas >18-fold increase in activity was measured after expression of a chimera containing green-fluorescent-protein (GFP) attached to the N-terminus of the endo-alpha1,2-mannosidase polypeptide. This, together with the observation that GFP-endo-alpha1,2-mannosidase is expressed as a Golgi-resident type II protein, points to enzyme-specific parameters directing folding and membrane anchoring, as well as Golgi-targeting, not being affected by fusion of GFP to the endo-alpha1,2-mannosidase N-terminus.     

Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.     

人骨髓基质细胞中糖基化磷脂酰肌醇特异性磷脂酶DcDNA的克隆

为探讨人糖基化磷脂酰肌醇特异性磷脂酶D(GPI PLD)cDNA的结构及功能 ,应用RT PCR从人骨髓基质细胞中克隆了长约 2 6kb的GPI PLDcDNA ,包含完整阅读框架 ,编码 2 3个氨基酸的信号肽及 817个氨基酸的成熟肽 .该cDNA与人胰腺GPI PLDcDNA几乎百分之百同源 ,与人肝脏GPI PLDcDNA同源性为 95 %,氨基酸同源性为 94 %,3者对应的结构基因只有 1个 ,位于人类第 6号染色体上 ,基因组序列长约 80kb ,包括 2 5个外显子 .构建克隆的GPI PLDcDNA的真核表达载体 ,通过脂质体转染能表达GPI锚定的胎盘型碱性磷酸酶 (PLAP)而无GPI PLD活性的G9细胞 ,同时设立对照组检测GPI PLDcDNA的功能 .结果显示 ,对照组细胞几乎检测不到GPI PLD活性 ,其表达的PLAP主要位于细胞膜上 ;而转染GPI PLDcDNA的G9细胞能检测到较高水平的GPI PLD活性 ,而且大部分酶活性存在于培养液中 ,其表达的PLAP也主要被释放入培养液 .结果证实 ,从人骨髓基质细胞中克隆的GPI PLDcDNA有生物学功能 ,它能释放细胞膜上GPI锚定蛋白质 .     

Characterization, chromosomal assignment, and tissue expression of a novel human gene belonging to the ARF GAP family

We have identified and characterized a novel human ADP-ribosylation factor GTPase-activating protein (ARFGAP1) gene that is related to other members of the ARF GAP family. The full-length cDNA for human ARFGAP1 was cloned following the identification of an EST obtained by large-scale cDNA library sequencing through a Blast search of public databases. Structurally, ARFGAP1 encodes a polypeptide of 516 amino acids, which contained a typical GATA-1-type zinc finger motif (CXXCX(16)CXXC) with the four cysteine residues that are highly conserved among other members of the ARF GAP family. The conserved ARF GAP domain may emphasize the biological importance of this gene. The ARFGAP1 gene, which contained 16 exons ranging from 0.5 to 9.3 kb, was mapped to human chromosome 22q13.2-q13.3 using radiation hybridization and in silico analyses. ARFGAP1 is strongly expressed in endocrine glands and testis. Interestingly, the expression of ARFGAP1 in testis is about sixfold higher than that in ovary, indicating a possible role of ARFGAP1 in the physiological function of sperm. Expression of ARFGAP1 in four human fetal tissues and seven cancer cell lines was also detected.     

From mosquito to man: Identification of a novel protein kinase, HsHPK, which is highly expressed in human hepatoma tissues

Protein kinases play an important role in the signaling pathway of growth factors in most of the higher organisms. During the study of protein kinase profiles of mosquitoes using RT-PCR and degenerate primers for consensus catalytic domain motifs to amplify protein kinase genes, we have noticed that a novel mosquito kinase, AaPK-38, shares a stretch of amino acids identical to the corresponding domain in Tousled gene ofArabidopsis thaliana that is required for leaf and flower development. A 2.1-kb cDNA encoding human HsHPK gene, which is a homolog of AaPK-38, was isolated from human testis cDNA library. This cDNA contains an open reading frame of 563 amino acids, with a complete kinase domain in its carboxyl terminus. The expressed Flag-tagged HsHPK was shown to have kinase activity based on in vitro autophosphorylation. Northern blot analysis revealed that human HsHPK mRNA is most abundant in testes, much less in heart and skeletal muscle and almost undetectable in liver and lung. Finally, we found that the expression of HsHPK in 4 out of 6 human hepatoma tissues is much higher than that in the adjacent normal counterpart. This result suggests HsHPK may play a role in the development of human hepatoma.     

Isolation and characterization of a novel liver-specific gene, hepassocin, upregulated during liver regeneration

By differential cDNA cloning coupled with Xenopus oocyte expression screening, we isolated a cDNA encoding a novel protein, termed 'hepassocin', the expression of which is upregulated in the regenerating rat liver. The cDNA contained a single open reading frame encoding a protein of 314 amino acids (ca. 34 kDa), including 24 amino acids of signal sequence. The protein expressed from the cDNA in Verots cells had activity to stimulate DNA synthesis in primary rat hepatocytes and was of 66 kDa or 34 kDa, under non-reducing or reducing conditions, respectively. Using an affinity column conjugated with the antibody raised against a peptide in a hydrophilic region, we purified hepassocin from the rat liver: it had a DNA synthesis-stimulating activity in hepatocytes. The hepassocin obtained here was 66 kDa, and the 34 kDa protein obtained under reducing conditions contained five cysteine residues, indicating that hepassocin is active as a homodimer. Northern blot analysis revealed that hepassocin mRNA (1.4 kb in length) occurred only in the liver, and in situ hybridization studies revealed its presence in parenchymal hepatocytes but not in endothelial cells. Furthermore, the expression of hepassocin mRNA was upregulated during compensatory hyperplasia after partial hepatectomy and regeneration after galactosamine treatment in the rat liver. These results suggest that hepassocin plays an important role in stimulating liver cell growth, through an autocrine mechanism.     

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.