Family Of Serine Protease Genes Research Articles

Genome-wide identification and analysis of Subtilisin-like serine protease gene family in banana (Musa accuminta L.) and their expression under abiotic stresses

Genome-wide identification and analysis of Subtilisin-like serine protease gene family in banana (Musa accuminta L.) and their expression under abiotic stresses

Genomic insights into the serine protease gene family and expression profile analysis in the planthopper, Nilaparvata lugens.

BackgroundThe brown planthopper (Nilaparvata lugens) is one of the most destructive rice plant pests in Asia. N. lugens causes extensive damage to rice by sucking rice phloem sap, which results in hopper burn (complete death of the rice plants). Despite its importance, little is known about the digestion, development and defense mechanisms of this hemimetabolous insect pest. In this study, we aim to identify the serine protease (SP) and serine protease homolog (SPH) genes, which form a large family in eukaryotes, due to the potential for multiple physiological roles. Having a fully sequenced genome for N. lugens allows us to perform in-depth analysis of the gene structures, reveal the evolutionary relationships and predict the physiological functions of SP genes.ResultsThe genome- and transcriptome-wide analysis identified 90 putative SP (65) and SPH (25) genes in N. lugens. Detailed gene information regarding the exon-intron organization, size, distribution and transcription orientation in the genome revealed that many SP/SPH loci are closely situated on the same scaffold, indicating the frequent occurrence of gene duplications in this large gene family. The gene expression profiles revealed new findings with regard to how SPs/SPHs respond to bacterial infections as well as their tissue-, development- and sex-specific expressions.ConclusionsOur findings provide comprehensive gene sequence resources and expression profiles of the N. lugens SP and SPH genes, which give insights into clarifying the potentially functional roles of these genes in the biological processes including development, digestion, reproduction and immunity.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-507) contains supplementary material, which is available to authorized users.

Cloning, expression, and immunological evaluation of two putative secreted serine protease antigens of Mycobacterium tuberculosis.

Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp. infection is discussed.

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29-53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.

The organization of the human complement factor I gene (IF): a member of the serine protease gene family.

The human complement factor I gene (IF) was cloned from a flow-sorted cosmid library. The gene spans 63 kb and comprises 13 exons. The first exon, which encodes the leader sequence and 5' untranslated region, is separated from the body of the gene by a large intron of 36 kb. Factor I is a mosaic protein, and there is a correlation between the genomic organization and the modular structure of the protein. The second exon encodes a module found only in complement C6 and C7 (FI/C6/C7); the third and fourth exons encode a single CD5 domain; and the fifth and sixth exons each encode a low-density lipoprotein receptor module. Two very small exons, 21 and 36 bp, then separate the first six exons from the last five that encode the serine protease domain of factor I. Within the serine protease gene family factor I has a unique genomic structure, but it bears a much closer resemblance to trypsin than it does to the other complement system serine proteases, factor B, C2, and C1r/C1s.

An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina).

A large and diverse family of serine protease genes was identified in first-instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first-instar larval cDNA, or from third-instar larval salivary glands or cardia, generated using a microscale RT-PCR method, were cloned into a plasmid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue-specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty-nine randomly selected clones from entire first-instar larvae revealed forty-nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first-instar larvae of L. cuprina. DNA sequence analysis of ten randomly-selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first-instar total cDNA and third-instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Drosophila melanogaster larval gut cells.

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 bp to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes.

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene.

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.

A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human alpha/delta T-cell receptor locus.

The chymotrypsin-like family of serine protease genes includes several members that are expressed exclusively in subsets of hematopoietic cells. For example, human neutrophil elastase and cathepsin G are expressed only in myelomonocytic precursors, and cytotoxic-T-cell serine proteases are found only in cytotoxic lymphocytes. We have used a cathepsin G cDNA probe to clone two cathepsin G-like genes (designated CGL-1 and CGL-2) from a human genomic library. We have determined that CGL-1 is identical to a previously identified gene (known as CCPI, CTLA I, or cytotoxic serine protease B) that is expressed only in activated cytotoxic T lymphocytes. We show here that cathepsin G, CGL-1, and CGL-2 are linked on an approximately 50-kilobase locus found on human chromosome 14 at band q11.2. This gene cluster maps to the same chromosomal band as the alpha and delta T-cell receptor genes; this region is involved in most chromosomal translocations and inversions that are specifically associated with T-cell malignancies.

Levels of RNA from a Family of Putative Serine Protease Genes Are Reduced in Drosophila melanogaster dunce Mutants and Are Regulated by Cyclic AMP

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.

Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.

The isolation and characterization of a family of serine protease genes expressed in activated cytotoxic T lymphocytes.

The isolation and characterization of a family of serine protease genes expressed in activated cytotoxic T lymphocytes.

Characterization of the human blood coagulation factor XII gene. Intron/exon gene organization and analysis of the 5'-flanking region.

A human genomic phage library was screened using a human factor XII cDNA as a hybridization probe. Two overlapping phage clones were isolated which contain the entire human factor XII gene. DNA sequence and restriction enzyme analysis of the clones indicate that the gene is approximately 12 kilobase pairs in size and is comprised of 13 introns and 14 exons. Exons 3-14 are contained in a genomic region of only 4.2 kilobase pairs with introns ranging in size from 80 to 554 base pairs. The coding sequence of factor XII consists of multiple putative domains that are homologous to putative domains found in fibronectin and tissue-type plasminogen activator. These regions were found as separate exons in the gene. The intron/exon gene organization is similar to the serine protease gene family of plasminogen activators and not to the clotting factor family. Analysis of the 5' end of the gene shows that it does not contain the typical TATA and CAAT sequences found in other genes. This is consistent with the finding that transcription of the gene is initiated at multiple sites.