Alkaline Phosphatase Reporter Transposon for Identification of Genes Encoding Secreted Proteins in Gram-Positive Microorganisms

We describe the construction of TnFuZ, a genetic tool for the discovery and mutagenesis of proteins exported from gram-positive bacteria. This tool combines a transposable element (Tn4001) of broad host range in gram-positive bacteria and an alkaline phosphatase gene (phoZ) derived from a gram-positive bacterium that has been modified by removal of the region encoding its export signal. Mutagenesis of Streptococcus pyogenes with TnFuZ (“FuZ” stands for fusions to phoZ) identified genes encoding secreted proteins whose expression was enhanced during growth in an aerobic environment. Thus, TnFuZ should be valuable for analysis of protein secretion, gene regulation, and virulence in gram-positive bacteria.

     

Characterization of Novel Secreted and Membrane Proteins Isolated by the Signal Sequence Trap Method

We recently described a method, called the signal sequence trap (SST) method, to clone cDNAs of secreted proteins and/or type I transmembrane proteins containing N-terminal signal sequences by using an epitope-tagging expression plasmid vector. In this paper we describe the summary of a large-scale screening of approximately 5900 clones of an SST cDNA library constructed from mouse bone marrow stromal cell line ST-2 cells. Of 26 positive clones obtained and sequenced, 11 clones appeared to contain authentic signal sequences. Five of the clones corresponded to the 5′ ends of the cDNA of known genes containing N-terminal signal sequences. The full-length cDNA clones of the 6 other unknown clones were isolated and sequenced. One clone, termed SDF3, encoded a mouse counterpart of human pigment epithelium-derived factor. Another clone, termed SDR1, had considerable homology with basigin, a member of the immunoglobulin superfamily. A third clone, termed SDF5, had partial homology with aDrosophilatissue polarity genefrizzled(fz) and its rat homologues,fz-1andfz-2.The other three clones had no significant homology with sequences in the databases. These results indicate that the SST method is effective and useful for the isolation of secreted and membrane proteins without knowledge of their functions.     

Identification and Chromosomal Location of Two Human Genes Encoding Enzymes Potentially Involved in Proteolytic Maturation of Farnesylated Proteins

Two human cDNAs encoding proteins similar to yeast enzymes involved in proteolytic processing of farnesylated proteins like a-factor mating pheromone and Ras2p have been cloned from an ovary cDNA library. These proteins have been tentatively called Face-1 and Face-2 (farnesylated protein-converting enzymes 1 and 2), respectively, and are integral membrane proteins, belonging to distinct families of metalloproteinases. Northern blot analysis of poly(A)+ RNAs isolated from a wide variety of human tissues demonstrated that both genes are expressed in all examined tissues, which suggests that these enzymes play housekeeping roles in normal processes. Fluorescence in situ hybridization experiments showed that the human FACE-1 gene maps to 1p34, whereas FACE-2 is located at 11q13, a region frequently amplified in human carcinomas and lymphomas. On the basis of these results, we suggest that inhibition of Face-1 and/or Face-2 could be part of strategies directed to block the functioning of prenylated proteins activated in oncogenic processes, including Ras proteins.     

一类新的编码PRPs基因的分离及其在棉花纤维等组织细胞中的表达

富含脯氨酸的细胞壁蛋白(proline-rich proteins,PRPs)在植物中广泛分布,它们在建造围绕特定细胞类型的细胞壁结构上起着很重要的作用.从棉花的cDNA文库中分离了5个编码富含脯氨酸的细胞壁蛋白质基因,这5个基因推断的氨基酸序列最普遍的特点就是脯氨酸含量非常高.根据氨基酸组成、富含脯氨酸的重复单元和结构域组织的特点,将这5个蛋白质分成2个亚类:一类(包括GhPRP3-6)与典型的PRPs结构相似,由N端疏水区(或信号肽)与不同富含脯氨酸的重复序列组成;另一类(GhPRPL)与典型的PRPs结构不同,这个蛋白质的N端为亲水序列,GhPRPL在靠近C端有8个5肽(类似PPKKE)的重复基序,与典型PRPs所含有的重复序列PPVYK非常相似.实时RT-PCR(Real-time RT-PCR)分析表明,GhPRP3和GhPRP5在10dpa纤维中特异表达,而GhPRPL在子叶中优势表达.GhPRP4和GhPRP6在所分析的组织中都有表达,GhPRP4mRNA在下胚轴中最丰富,在花药中次之,而GhPRP6在10dpa纤维中表达最强,在10dpa胚珠中次之.此外,GhPRP3,GhPRP5基因表达受纤维发育调节,表明它们可能在棉纤维发育中起重要作用.     

Characterization of Streptococcus suis Genes Encoding Proteins Homologous to Sortase of Gram-Positive Bacteria

Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptidoglycan by a specific enzyme called sortase. Using the type strain of Streptococcus suis, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated srtA, was linked to gyrA, as were the sortase and sortase-like genes of other streptococci. Three genes, designated srtB, srtC, and srtD, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated srtE, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of Streptococcus gordonii and Staphylococcus aureus, except that SrtA of S. suis had 65% identity with that of S. gordonii. Isogenic mutants deficient for srtA, srtBCD, or srtE were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with N-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the srtA mutant compared with that of the parent strain, and this phenotype was completely complemented by srtA cloned from S. suis. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the srtA mutant appeared in the profiles of the srtBCD and srtE mutants. These results provide evidence that the cell wall sorting system involving srtA is also present in S. suis.     

Transmembrane-Domain Trapping: A Novel Method for Isolation of cDNAs Encoding Putative Membrane Proteins

We have developed a method that enables us to isolate cDNAsof putative membrane proteins. The system is designed to isolatea cDNA which can provide the transmembrane domain to the extracellularpart of the IL-2 receptor chain. We constructed a p18Mac vectorby putting part of the IL-2 receptor chain cDNA that encodedits signal sequence and extracellular domain, a cDNA cloningsite and a poly(A) additional signal after a strong promoterSR. If a cloned cDNA provides a transmembrane domain in-frame,the extracellular domain of the IL-2 receptor chain will beexpressed on the surface of the transfected cells. Otherwise,the chimeric protein will be either secreted or retained insidethe transfected cells. We made a cDNA library using p18Mac andscreened for cDNA clones which allowed the expression of theextracellular domain of the IL-2 receptor chain on the cellsurface. Of the 2000 clones screened, 5 clones were scored aspositive. Partial sequence analysis revealed that one cloneencoded the amyloid precursor protein, two others encoded mitochondrialproteins and the rest were new. These results suggest the systemis effective in isolating cDNAs encoding putative membrane proteins.     

High Throughput Identification of Monoclonal Antibodies to Membrane Bound and Secreted Proteins Using Yeast and Phage Display

Antibodies are ubiquitous and essential reagents for biomedical research. Uses of antibodies include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and determining how proteins function under normal or pathological conditions. Specific antibodies are only available for a small portion of the proteome, limiting study of those proteins for which antibodies do not exist. The technologies to generate target-specific antibodies need to be improved to obtain high quality antibodies to the proteome at reasonable cost. Here we show that renewable, validated, and standardized monoclonal antibodies can be generated at high throughput, without the need for antigen production or animal immunizations. In this study, 60 protein domains from 24 selected secreted proteins were expressed on the surface of yeast and used for selection of phage antibodies, over 400 monoclonal antibodies were identified within 3 weeks. A subset of these antibodies was validated for binding to cancer cells that overexpress the target protein by flow cytometry or immunohistochemistry. This approach will be applicable to many of the membrane-bound and the secreted proteins, 20–40% of the proteome, accelerating the timeline for Ab generation while reducing the cost.     

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.     

Molecular Characterization of Genes Encoding a Novel ABC Transporter in Thermoanaerobacterium thermosulfurigenes EM1

The nucleotide sequence of two open reading frames (ORFs) from Thermoanaerobacterium thermosulfurigenes EM1 was determined that encode proteins with similarity to components of ATP-binding cassette (ABC) transport systems. Sequence analysis suggests that the deduced proteins AbcA and AbcB consist of an NH₂-terminal membrane-spanning domain and a COOH-terminal ATP-binding domain. The deduced proteins AbcA and AbcB showed highest similarity to proteins of the MsbA subfamily of ABC transporters. AbcA and AbcB probably function as a heterodimer. An ORF predicted to encode the primary sigma factor SigA was identified downstream of abcB. Received: 11 March 1997 / Accepted: 14 April 1997     

生物信息学预测与实验验证相结合策略筛选鉴定新的人分泌蛋白基因

使用生物信息学预测结合实验验证的策略筛选鉴定人新的分泌蛋白基因。用SignalP、SOSUI、PSORT和BLAST等程序对UniProt蛋白数据库进行生物信息学分析 ,筛选出用于实验验证的 1 4个功能未知基因。采用RT PCR方法 ,克隆得到 1 4个基因的全长编码序列 ,并构建到真核表达载体pcDNA3.1 ( - ) Myc His质粒。采用蛋白质印迹与免疫荧光分析 ,检测到其中 7个基因的表达。除其中一个在细胞核表达外 ,其余 6个只在细胞质中表达 ;其中的 4个基因的表达产物在细胞培养液中可被检测到 ,鉴定为 4个新的分泌蛋白基因。     

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein “multisubstrate glycosidase A” (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest K_m with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest k_cat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α₁-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.     

The Effect of Various Stresses on the Expression of Genes Encoding the Secreted Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

The effects of various stresses (osmotic, salt, low-temperature, high-temperature, and high-light stress) on the amount of mRNA of eight genes encoding the secreted proteins of Synechocystis sp. PCC 6803 were studied. Osmotic stress (0.5 M sorbitol) reduced the amount of all mRNAs, with the exception of slr0924. Supposedly, this gene encodes Tic22, a polypeptide involved in the formation of the transport system for proteins crossing the internal thylakoid membrane on the way to the lumen. Salt stress (0.5 M NaCl) inhibited the expression of all genes for secreted proteins almost completely. Low temperature (20°C) did not affect the expression of the sll1891 gene of an unknown function and the slr0924 gene. The high temperature (44°C) suppressed the expression of all genes tested. A detailed study of the expression of the sll1694 (pilA1) gene, which encodes the main structural protein of cyanobacterial pili, pilin PilA1, demonstrated that virtually all stresses suppressed its expression. Thus, various stresses were shown to suppress the expression of most genes encoding Synechocystis secreted proteins.     

Identification and Characterization of a Novel Human Phosphatidylinositol 4-kinase

The extensive sequence homology that exists among the catalyticdomains of phosphatidylinositol 3- and 4-kinases allowed usto clone a novel human gene encoding a putative phosphatidylinositolkinase, NPIK. Among other known phosphatidylinositol 3- and4-kinases, NPIK was most closely related to yeast PIK1 phosphatidylinositol4-kinase. Several forms of NPIK cDNAs were isolated, and expressionof NPIK message was detected in a wide variety of tissues. Fluorescencein situ hybridization and radiation hybrid analyses assignedthe NPIK gene to human chromosome 1. Recombinant NPIK proteincatalyzed a conversion from phosphatidylinositol to phosphatidylinositol4-phosphate. The catalytic activity of NPIK was augmented byTriton X-100, and was reduced in the presence of adenosine.Using green .uorescent protein system we determined that NPIKis localized in the cytoplasm. Taken together, the data suggestthat NPIK may play a pivotal role in regulating the synthesisof phosphatidylinositol 4-phosphate at the site(s) accessiblefrom cytoplasm.