Serpin Gene Research Articles

Down-regulated expression of SERPIN genes located on chromosome 18q21 in oral squamous cell carcinomas

Serpins (serine protease inhibitors) are known as a diverse family of protease inhibitors; however, various other biological activities including tumor suppression, have been recently reported for these molecules. To clarify whether members of the serpin family are involved in OSCC (oral squamous cell carcinoma), global gene screening using microarray analysis was performed with OSCC-derived cell lines. A trend toward diminished expression was shown for some SERPIN genes located on 11q12-q13.1 and 18q21. mRNA expression of SERPIN genes at these chromosome regions was therefore analyzed using real-time quantitative RT-PCR (qRT-PCR) in 55 OSCC samples and matched normal tissue. Statistically significant decreases in expression were found for SERPINB12 (P=0.001), SERPINB13 (P=0.001), SERPINB4 (P=0.042), SERPINB3 (P<0.001), SERPINB11 (P<0.001), SERPINB7 (P=0.021) and SERPINB2 (P=0.018). All of these genes are located on 18q21, the known location of the serpin gene cluster. The results strongly suggest that this chromosome region plays a crucial role in OSCC. Some serpin members in the region might be involved in tumor suppression, or there might be unidentified tumor suppressor genes within or near the chromosome region.

Haemonchus contortus: Cloning and characterization of serpin

The serpin gene of Haemonchus contortus ( hc- serpin) was cloned and characterized in this study. Specific primers for rapid amplification cDNA ends (RACE) were designed based on the expression sequence tag (EST, BM173953) to amplify the 3′- and 5′-ends of hc- serpin. The full length of the cDNA of this gene was obtained by overlapping the sequences of 3′- and 5′-extremities and amplification by reverse transcription-PCR. The biochemical activities of the recombinant protein (rHc-Serpin), which was expressed in prokaryotic cells and purified by affinity chromatography and size-exclusion chromatography, were analyzed by assays of trypsin inhibition, anti-coagulation activity, and stability to temperature and pH. The results showed that the cloned full-length cDNA comprised 1317 bp and encoded a peptide with 367 amino acid residues which showed sequence similarity to several known serpins. The rHc-Serpin inhibited trypsin activity effectively and prolonged the coagulation time of rabbit blood in vitro. The rHc-Serpin was stable from pH 2.0–10.0 and kept activity at high temperature until 75 °C. Optimal pH of rHc-Serpin protein to inhibit trypsin activity was at pH 7.6. The natural serpin of H. contortus detected by immunoblot assay was about 63 kDa, and the rHc-Serpin was recognized strongly by serum from naturally infected goats. By immunohistochemistry, the serpin was localised exclusively in the epithelial cells of gastrointestinal tract in adult H. contortus. The results indicated that the cloned gene was serpin and that the protein may play important roles in the biological functions of H. contortus.

Molecular cloning and characterization of a novel serpin gene of Clonorchis sinensis, highly expressed in the stage of metacercaria

The serpins are a superfamily of proteins (350-500 amino acids in size) that fold into a conserved structure. From about 3,475 unigenes of Clonorchis sinensis metacercaria, a novel gene-encoding serpin was identified and characterized. The opening reading frame is 1,149 bp encoding 382 amino acids. The deduced amino acid sequence shows high identity to previously reported serpins from C. sinensis and other helminthic parasites. A typical serpin signature was found by motif search. The recombinant C. sinensis serpin protein (rCsproSERPIN) was produced and purified. Semiquantitative analysis revealed that the transcripts of this serpin gene in metacercaria were much higher than that in adult worms and that the corresponding band of serpin protein in the crude soluble antigen of metacercaria probed by rat anti-CsproSERPIN serum was also much clearer compared with that of adult, suggesting that it plays an important role in the stage of C. sinensis metacercaria. Although we are not much clear about the detailed function of this serpin protein, the study that proteinase initiates metacercaria excystment gives a clue that it may participate in the encystment of cercaria.

Identification and analysis of serpin-family genes by homology and synteny across the 12 sequenced Drosophilid genomes

BackgroundThe Drosophila melanogaster genome contains 29 serpin genes, 12 as single transcripts and 17 within 6 gene clusters. Many of these serpins have a conserved "hinge" motif characteristic of active proteinase inhibitors. However, a substantial proportion (42%) lacks this motif and represents non-inhibitory serpin-fold proteins of unknown function. Currently, it is not known whether orthologous, inhibitory serpin genes retain the same target proteinase specificity within the Drosophilid lineage, nor whether they give rise to non-inhibitory serpin-fold proteins or other, more diverged, proteins.ResultsWe collated 188 orthologues to the D. melanogaster serpins from the other 11 Drosophilid genomes and used synteny to find further family members, raising the total to 226, or 71% of the number of orthologues expected assuming complete conservation across all 12 Drosophilid species. In general the sequence constraints on the serpin-fold itself are loose. The critical Reactive Centre Loop (RCL) sequence, including the target proteinase cleavage site, is strongly conserved in inhibitory serpins, although there are 3 exceptional sets of orthologues in which the evolutionary constraints are looser. Conversely, the RCL of non-inhibitory serpin orthologues is less conserved, with 3 exceptions that presumably bind to conserved partner molecules. We derive a consensus hinge motif, for Drosophilid inhibitory serpins, which differs somewhat from that of the vertebrate consensus. Three gene clusters appear to have originated in the melanogaster subgroup, Spn28D, Spn77B and Spn88E, each containing one inhibitory serpin orthologue that is present in all Drosophilids. In addition, the Spn100A transcript appears to represent a novel serpin-derived fold.ConclusionIn general, inhibitory serpins rarely change their range of proteinase targets, except by a duplication/divergence mechanism. Non-inhibitory serpins appear to derive from inhibitory serpins, but not the reverse. The conservation of different family members varied widely across the 12 sequenced Drosophilid genomes. An approach considering synteny as well as homology was important to find the largest set of orthologues.

P.1.d.004 Effects of green tea extract on behaviour in chronic lead intoxicated rats

Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter.Short SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT.

Replication and narrowing of gene expression quantitative trait loci using inbred mice

Gene expression quantitative trait locus (eQTL) mapping has become a powerful tool in systems biology. While many authors have made important discoveries using this approach, one persistent challenge in eQTL studies is the selection of loci and genes that should receive further biological investigation. In this study we compared eQTL generated from gene expression profiling in the livers of two panels of mouse strains: 41 BXD recombinant inbred and 36 Mouse Diversity Panel (MDP) strains. Cis-eQTL, loci in which the transcript and its maximum QTL are colocated, have been shown to be more reproducible than trans-eQTL, which are not colocated with the transcript. We observed that between 9.9 and 12.1% of cis-eQTL and between 2.0 and 12.6% of trans-eQTL replicated between the two panels depending on the degree of statistical stringency. Notably, a significant eQTL hotspot on distal chromosome 12 observed in the BXD panel was reproduced in the MDP. Furthermore, the shorter linkage disequilibrium in the MDP strains allowed us to considerably narrow the locus and limit the number of candidate genes to a cluster of Serpin genes, which code for extracellular proteases. We conclude that this strategy has some utility in increasing confidence and resolution in eQTL mapping studies; however, due to the high false-positive rate in the MDP, eQTL mapping in inbred strains is best carried out in combination with an eQTL linkage study.

Serpin genes AtSRP2 and AtSRP3 are required for normal growth sensitivity to a DNA alkylating agent in Arabidopsis.

BackgroundThe complex responses of plants to DNA damage are incompletely understood and the role of members of the serpin protein family has not been investigated. Serpins are functionally diverse but structurally conserved proteins found in all three domains of life. In animals, most serpins have regulatory functions through potent, irreversible inhibition of specific serine or cysteine proteinases via a unique suicide-substrate mechanism. Plant serpins are also potent proteinase inhibitors, but their physiological roles are largely unknown.ResultsSix Arabidopsis genes encoding full-length serpins were differentially expressed in developing seedlings and mature tissues. Basal levels of AtSRP2 (At2g14540) and AtSRP3 (At1g64030) transcripts were highest in reproductive tissues. AtSRP2 was induced 5-fold and AtSRP3 100-fold after exposure of seedlings to low concentrations of methyl methanesulfonate (MMS), a model alkylating reagent that causes DNA damage. Homozygous T-DNA insertion mutants atsrp2 and atsrp3 exhibited no differential growth when mutant and wild-type plants were left untreated or exposed to γ-radiation or ultraviolet light. In contrast, atsrp2 and atsrp3 plants exhibited greater root length, leaf number and overall size than wild-type plants when exposed to MMS. Neither of the two serpins was required for meiosis. GFP-AtSRP2 was localized to the nucleus, whereas GFP-AtSRP3 was cytosolic, suggesting that they target different proteinases. Induction of cell cycle- and DNA damage-related genes AtBRCA1, AtBARD1, AtRAD51, AtCYCB1;1 and AtCYCD1;1, but not AtATM, was reduced relative to wild-type in atsrp2 and atsrp3 mutants exposed to MMS.ConclusionExpression of specific serpin genes (AtSRP2 and AtSRP3 in Arabidopsis) is required for normal responses of plants following exposure to alkylating genotoxins such as MMS.

The serpin gene family in Anopheles gambiae

Serpins (serine protease inhibitors) regulate some innate immune responses of insects by inhibiting endogenous proteases. In this study, we characterized the serpin ( SRPN) gene family in the mosquito Anopheles gambiae, the major malaria vector in Sub-Saharan Africa. We identified 18 A. gambiae SRPN genes, all on chromosomes 2 and 3, through searches of genomic DNA and EST databases. In addition to SRPN10, previously documented to exhibit alternative splicing, we found three splicing isoforms of SRPN4. We completed sequencing of cDNAs for the A. gambiae serpins to obtain complete coding sequence information and to verify or improve gene predictions. The predicted SRPN9 and 15 in the initial genome annotation were determined to be a single gene ( SRPN9). Sixteen of the serpins contained putative secretion signal sequences. Multiple sequence alignments showing conserved residues important in structural conformation, including the consensus pattern within the hinge region, indicated that most of the A. gambiae serpins may be inhibitory. Phylogenetic analyses confirmed that SRPN1, 2, 3, 8, 9 and 10 formed phylogenetic clusters with known inhibitory serpins from Drosophila melanogaster and Manduca sexta. Many of the A. gambiae serpins were expressed during all life stages. However, SRPN7, 8, 12, and 19 were expressed at very low levels in the adult stage. SRPN13 was expressed mostly in eggs and young larvae, whereas SRPN5 and 14 were expressed mostly in adults. Such differences in expression pattern suggest that the serpins are involved in multiple physiological processes. Determining the biological functions of the mosquito serpins will require future work to identify the proteases they inhibit in vivo.

Molecular Cloning and Biochemical Characterization of Manduca sexta Serpin‐7, a Regulator of Prophenoloxidase Activation

Serine protease inhibitors (serpins) are involved in various physiological reactions in humans and insects. Six serpins, SPN1‐SPN6, have been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a complete DNA sequence of another Manduca serpin gene, SPN7. The putative mature SPN7 protein was 385 amino acid residues long (MW 42.90 kDa, pI 5.32) with a predicted 15 residue signal sequence. Multiple sequence alignment of the reactive center loop region of the M. sexta serpins indicated that SPN7 contained Arg and Ile residues at P1′‐P1 positions, similar to SPN4 and 5. RT‐PCR results demonstrated an up‐regulation of SPN7 expression in the fat body at 24 h after injection of larvae with Micrococcus. The recombinant SPN7 protein was expressed in E. coli and purified using nickel‐affinity chromatography. Addition of recombinant SPN7 significantly inhibited both 1) spontaneous melanization of the 5th instar larval plasma, and 2) phenoloxidase activity of the plasma challenged by Micrococcus bacteria. SPN7 inhibited the enzymatic activity of the active prophenoloxidase‐activating protease‐3 by forming a stable serpin‐protease complex as indicated by SDS‐PAGE under reducing conditions. SPN7 inhibited bovine trypsin but not chymotrypsin. We conclude that SPN7 is inhibitory and likely involved in regulation of innate immunity in the insect. (supported by NIH grant GM41247)

A comparative analysis of serpin genes in the silkworm genome

Serine protease inhibitors (serpins) are a superfamily of proteins, most of which control protease-mediated processes by inhibiting their cognate enzymes. Sequencing of the silkworm genome provides an opportunity to investigate serpin structure, function, and evolution at the genome level. There are thirty-four serpin genes in Bombyx mori. Six are highly similar to their Manduca sexta orthologs that regulate innate immunity. Three alternative exons in serpin1 gene and four in serpin28 encode a variable region including the reactive site loop. Splicing of serpin2 pre-mRNA yields variations in serpin2A, 2A′ and 2B. Sequence similarity and intron positions reveal the evolutionary pathway of seven serpin genes in group C. RT-PCR indicates an increase in the mRNA levels of serpin1, 3, 5, 6, 9, 12, 13, 25, 27, 32 and 34 in fat body and hemocytes of larvae injected with bacteria. These results suggest that the silkworm serpins play regulatory roles in defense responses.

Ixodes scapularis tick serine proteinase inhibitor (serpin) gene family; annotation and transcriptional analysis

BackgroundSerine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option.ResultsA sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL) and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K) residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG) and/or midguts (MG) of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively.ConclusionGiven the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of advances on the molecular physiology of I. scapularis. Although the paper is descriptive, this study provides the first step toward a comprehensive understanding of serpins in tick physiology.

Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

BackgroundIntron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes.ResultsHere we investigated intron dynamics in the serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing.ConclusionMultiple intron acquisitions were identified in serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.

Identification and Characterization of a Serpin From Eimeria acervulina

Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.

Deciphering the phylogenetic history of neuroserpin orthologs across metazoans by analysis of synteny and rare genomic characters

The superfamily of serine proteinase inhibitors (serpins) is involved in wide arrays of fundamental biological processes such as blood coagulation, complement activation, fibrinolysis, angiogenesis, inflammation and tumor suppression. The average protein size of a serpin family member is 350-400 amino acids, but gene structure varies in terms of number and position of exons and introns. All known serpins can be grouped into 16 clades and 10 orphan sequences. Vertebrate serpins can be conveniently classified into six sub-groups, based on three independent biological features - genomic organization, diagnostic amino acid sites and rare indels.The objective of this study was to elucidate the phylogenetic kinships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. This study illustrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Rare genomic characters/changes (RGC) are used to decipher that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans.

Deciphering the phylogenetic history of neuroserpin orthologs across metazoans by analysis of synteny and rare genomic characters

AbstractThe superfamily of serine proteinase inhibitors (serpins) is involved in wide arrays of fundamental biological processes such as blood coagulation, complement activation, fibrinolysis, angiogenesis, inflammation and tumor suppression. The average protein size of a serpin family member is 350-400 amino acids, but gene structure varies in terms of number and position of exons and introns. All known serpins can be grouped into 16 clades and 10 orphan sequences. Vertebrate serpins can be conveniently classified into six sub-groups, based on three independent biological features - genomic organization, diagnostic amino acid sites and rare indels.The objective of this study was to elucidate the phylogenetic kinships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. This study illustrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Rare genomic characters/changes (RGC) are used to decipher that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans.

A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species.

Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation

In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.

Cloning and detection of serpin-like protein encoding gene inBifidobacterium longum strains

Bifidobacterium longum has immunostimulant activity thatin vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin ofB. longum (DD087853). In order to createBifidobacterium serpin transformants, aB. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, anEscherichia coli — Bifidobacterium shuttle vector, to construct pRM3. Several protocols forB. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dot-blot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. Our research study is aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line.

Functional diversification of a protease inhibitor gene in the genus Drosophila and its molecular basis

The mutually exclusive use of alternative reactive site loop (RSL) cassettes due to alternative splicing of serpin (serine protease inhibitor) gene transcripts is a widespread strategy to create target-selective protease inhibitors in the animal kingdom. Since molecular basis and evolution of serpin RSL cassette exon amplification and diversification are unexplored, the exon–intron organization of the serpin gene spn4 from 12 species of the genus Drosophila was studied. The analysis of the gene structures shows that both number and target enzyme specificities of Spn4 RSL cassettes are highly variable in fruit flies and includes inhibitor variants with novel antiproteolytic activities in some species, indicating that RSL diversity is the result of adaptive evolution. Comparative genomics suggests that interallelic gene conversion and/or recombination events contribute to RSL cassette exon amplification. Due to an intron that is located at the most suitable position within the RSL region, multiple inhibitors can be formed in an economic manner that are both efficient and target-selective, allowing fruit flies to control an astonishing variety of proteases with different cleavage chemistry and evolutionary ancestry.

Ancestry and evolution of a secretory pathway serpin

BackgroundThe serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity.ResultsHere, rare genomic characters are used to show that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans. A C-terminal address code assigning association with secretory pathway organelles is present in all neuroserpin orthologs, suggesting that supervision of cellular export/import routes by antiproteolytic serpins is an ancient trait, though subtle functional and compartmental specialisations have developed during their evolution. The results also suggest that massive changes in the exon-intron organisation of serpin genes have occurred along the lineage leading to vertebrate neuroserpin, in contrast with the immediately adjacent PDCD10 gene that is linked to its neighbour at least since divergence of echinoderms. The intron distribution pattern of closely adjacent and co-regulated genes thus may experience quite different fates during evolution of metazoans.ConclusionThis study demonstrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Serpins with the capacity to defend the main cellular export/import routes against uncontrolled endogenous and/or foreign proteolytic activity represent an ancient trait in eukaryotes that has been maintained continuously in metazoans though subtle changes affecting function and subcellular location have evolved. It is shown that the intron distribution pattern of neuroserpin gene orthologs has undergone substantial rearrangements during metazoan evolution.