Current Genomics, 2004, 5, 000-000 1 Current Genome-Wide Analysis on Serine Proteases in Innate Immunity

Jeak L. Ding1,*, Lihui Wang1 and Bow, Ho2

Departments of Biological Sciences1 and Microbiology2, National University of Singapore, 14, Science Drive 4, Singapore 117543

Abstract: Recent studies on host defense against microbial pathogens have demonstrated that innate immunity predated adaptive immune response. Present in all multicellular organisms, the innate defense uses genome-encoded receptors, to distinguish self from non-self. The invertebrate innate immune system employs several mechanisms to recognize and eliminate pathogens: (i) blood to immobilize the invading microbes, (ii) lectin-induced complement pathway to lyse and opsonize the pathogen, (iii) melanization to oxidatively kill invading microorganisms and (iv) prompt synthesis of potent effectors, such as antimicrobial peptides. Serine proteases play significant roles in these mechanisms, although studies on their functions remain fragmentary, and only several members have been characterized, for example, the serine protease cascade in Drosophila dorsoventral patterning; the Limulus blood clotting cascade; and the silk worm prophenoloxidase cascade. Additionally, serine proteases are involved in processing Späetzle, the Toll ligand for signaling in antimicrobial peptide synthesis. The recent completion of the Drosophila and Anopheles genomes offers a tantalizing promise for genomic analysis of innate immunity of invertebrates. In this review, we discuss the latest genome-wide studies conducted in invertebrates with emphasis on serine proteases involved in innate immune response. We seek to clarify the analysis by using empirical research data on these proteases via classical approaches in biochemical, molecular and genetic methods. We provide an update on the serine protease cascades in various invertebrates and map a relationship between their involvement in early embryonic development, blood coagulation and innate immune defense. Key Words: Serine proteases; proteolytic cascades; innate immunity; genomics; DNA microarray; empirical approaches.

INTRODUCTION THE PHYLOGENY OF SERINE PROTEASE CASCADES IN DEVELOPMENT, HEMOSTASIS & Serine proteases are indispensable in various fundamental INNATE IMMUNITY biological processes including cellular and humoral immunity and embryonic development. Remarkably, a growing body In mammalian hemostasis, the tissue factor dependent of information has accumulated on the serine protease pathway is the primary initiator of blood coagulation. Upon networks in various aspects of innate immunity, which are injury, Factor VII complexes with the exposed mature tissue evolutionarily conserved from invertebrates to vertebrates. factor on the cell surface, to activate Factor X. Activated Although mapping of individual serine proteases in all the Factor Xa forms an activation complex with other cofactors pathways in innate immune response is far from direct to convert prothrombin to thrombin, which eventually leads predication of annotated genome databases on all the model to fibrin clot formation [1-3]. Factor VII and Factor X along organisms, mass transcript profiling by cDNA microarray, with other factors are serine proteases, which are well- enabled by the availability of the genome sequence data has characterized in the tissue-factor dependent blood coagula- greatly shortened the time and effort to locate and isolate tion pathway in mammals. In addition, serine proteases are many ‘candidate’ serine protease genes individually. For also major components of complement pathway in mammals: example, a set of microarray experiments can provide a basic C1r, C1s and C2 in the classical complement pathway; definition of the innate immune genes for study in additional MASP 1-3 (mannose binding associated serine functional genomics projects. Through huge efforts made in protease) in lectin-induced pathway; and Factor B, Factor D data collection and bioinformatics, diverse data sets may be and Factor I in the alternative pathway; all of which are combined to accurately define the function of each gene and responsible for the formation of C3 convertase in the to transform this knowledge into genetic networks. In this complement system [4]. During inflammatory response, in review, we provide a comprehensive update on the genome- addition to the complement pathway, blood coagulation can wide analysis of serine proteases involved in innate also be modulated by bacterial endotoxin (or lipopolysaccha- immunity, with particular reference to invertebrates. The ride, LPS) and various cytokines through regulation of the significance and importance of empirical experimental expression of coagulation-related factors [5, 6]. These two design to complement the genome database information is fundamental biological processes: inflammation and blood critically discussed. coagulation, are integrated into an essential part of the host immune response in mammals. The simultaneous activation of the innate immune *Address correspondence to this author at the Departments of Biological response and the blood coagulation system after injury is a Sciences, National University of Singapore, 14, Science Drive 4, Singapore phylogenetically-ancient adaptive response that can be traced 117543; Tel: (65) 6874-2776; Fax: (65) 6779-2486; back to early evolution of eukaryotes. Having survived E-mail: [email protected]

1389-2029/04 $45.00+.00 ©2004 Bentham Science Publishers Ltd. 2 Current Genomics, 2004, Vol. 5, No. 2 Ding et al. virtually unchanged for 400 million years of evolution, the Easter (GD/SNK/Ea) cascade results in the cleavage of Limulus, which is also known as a “living fossil” has Späetzle into its active form [10]. Toll receptor recognizes developed a remarkably sensitive LPS-activated blood the activated Späetzle to elicit a signal response, resulting in coagulation cascade. By binding LPS, Factor C which is the the translocation of dorsal transcription factor from the initiating serine protease zymogen in the serine protease cytoplasm into the nucleus leading to the generation of a cascade, cleaves Factor B which in turn activates proclotting dorsal concentration gradient to transcriptionally regulate the enzyme, resulting in the conversion of coagulogen, a dorsoventral polarity-related genes in the early Drosophila functional homolog of , to coagulin, which embryo [11-13]. Späetzle is the only reported invertebrate polymerises to form an insoluble clot [7, 8]. Factor G, homologue to the Limulus coagulogen, sharing the another serine protease, which recognizes b-1,3 glucan from distinguished cysteine knot motif and NGF domain that is fungal cell wall, can also enzymatically process Factor B originally considered to be unique in higher vertebrates [14, leading to the formation of blood clot (Fig. 1 and 2). 15]. The upstream serine proteases of the two pathways, Although no sequence and structure similarity is Easter and Snake, are homologs to Factor B and proclotting enzyme, respectively [16]. All the serine proteases involved observed in the components of the serine protease cascades in both pathways share a unique clip domain, which is in the Limulus and mammalian blood clotting, the Limulus knitted together by three bonds to form a very LPS- and b-glucan -sensitive blood coagulation cascade compact structure [17, 18]. Later on, Späetzle/Toll/Dorsal displays an interesting parallel to the Drosophila pathway was also found to be involved in the expression of dorsoventral polarity determination pathway [9] (Fig. 1). In this developmentally-related serine protease cascade, the antimicrobial peptides during fungal and gram-positive sequential proteolysis of Gastrulation Defective, Snake and bacterial infection in Drosophila [19]. Thus, the dorsoventral

Drosophila Limulus Innate immunity Development Innate immunity / Blood coagulation

GPB Fungi Dorsoventral GNB Fungi PRR:PGRP-SA PRR? signal?Nudel Factor C ? ? Gd ? Factor G ? Snake Factor B

? Persephone Easter Proclotting enzyme

Späetzle Coagulogen Coagulin ?

Toll TLR ? Dorsal LimuluskB

Fig. (1). The phylogeny of serine protease cascades in invertebrates. The serine proteases in the blood coagulation cascade and innate immunity of Limulus is shown on the right panel. These serine proteases are activated by gram negative bacteria (GNB) and fungi, respectively. Factor G is the upstream serine protease in the alternate blood coagulation pathway which is triggered by b-1,3-glucan. Discontinuous arrows annotate the putative LPS-mediated signaling pathway. On the left are the serine protease cascades in the dorsoventral determination of Drosophila and in innate immune response against gram positive bacteria (GPB) and fungal infection in Drosophila. Dotted arrows with ‘?’ indicate unidentified /uncharacterised components in the postulated signaling pathways. Homologues of individual components from the two pathways are boxed. PRR, pathogen recognition receptor; PGRP-SA, peptidoglycan recognition protein-SA isoform. Gd, Gastrulation defective; TLR, Toll-like receptor. Adapted with modifications from Wang et al. [56]. Current Genome-Wide Analysis on Serine Proteases Current Genomics, 2004, Vol. 5, No. 2 3

Limulus Drosophila Bacteria LPS/Bacteria b-1,3- glucan/fungi Fungi/Bacteria

PGRP-SA PGRP-LE PGRP-LC Factor C Factor G Factor B Proclotting enzyme Persephone proPOAE unidentified Hemocyanin (proPOAE) coagulogen Serpins cascade? Phagocytosis unidentified Spätzle Melanization Melanization Blood coagulation cascade?

TLR Toll IMD

Limulus-kB Dorsal/Dif Relish

Synthesis of defense molecules

Fig. (2). Network of serine protease cascades in innate immunity exemplified by Limulus and Drosophila. The Limulus blood clotting and melanization are connected via hemocyanin as indicated in the diagram on the left panel. In Drosophila, various facets of innate immunity, for example, production of defense molecules, melanization and phagocytosis are weaved into a network by serine proteases, as explained in the text. Dotted arrows represent pathways supported by preliminary evidence. determination pathway and the antifungal & antibacterial microbial challenge. The intermediary compounds generated responses converge on the proteolytic processing of during the process of melanization as well as the end Späetzle. However, another uncharacterized serine protease product, melanin itself, are toxic to the microorganisms. In cascade, which is distinguishable from GD/SNK/Ea cascade, arthropods, ProPO is proteolytically cleaved into an active has been suggested to cleave Späetzle into its active form phenoloxidase through prophenoloxidase activating enzyme [9]. Remarkably, Persephone, a newly discovered serine pro- (proPOAE). ProPOAEs are also serine proteases like the tease involved in signal transduction of anti-gram positive potential candidates, Factor B and proclotting enzyme in the bacteria and anti-fungal immune responses, is deduced to Limulus. To date, proPOAEs have been isolated and well- contain structural domains homologous to those in Easter, characterized from crayfish [22, 23] and silkworm [24]. Snake and proclotting enzyme [20]. Figure 1 illustrates some key members of the serine protease cascades which are RESOLUTION OF THE SERINE PROTEASE multifunctionally involved in development, blood coagula- NETWORKS FROM THE GENOME DATABASES tion and innate immunity. Thus, it is widely acknowledged All the serine proteases characterized so far were purified that a common ancestral serine protease cascade has given rise to the serine proteases in controlling early development, and studied through traditional biochemical procedures and classical molecular techniques. By these means, serine blood coagulation and innate immune responses. This can be proteases in mammalian blood coagulation and complement further evidenced by the complicated and sophisticated pathways have been well-characterized. In contrast, although interconnection of individual serine protease cascades res- progress has been made steadily over the years on research ponsible for different defense mechanisms. Table 1 outlines on invertebrate serine proteases involved in innate immunity the serine proteases and serine protease inhibitors identified in innate immunity and development of invertebrates. and other important cellular mechanisms, complete resolu- tion of all the components in the serine protease cascades has Biochemical studies have revealed an intricate network been achieved in only two processes: the Limulus blood of innate immune-related serine proteases in the Drosophila coagulation and Drosophila dorsoventral polarization. and Limulus (Fig. 2). In the Limulus, Factor B and proclot- Nevertheless, the advent of genome-wide analysis, ting enzyme are proteases involved in the proteolytic proces- combining the power of genomic sequence data and DNA sing of the prophenoloxidase activity of hemocyanin [21]. microarray analysis has greatly revolutionized the way we Prophenoloxidase (ProPO) acts as the upstream activating study genes. With the availability of annotated genome enzyme of phenoloxidase, which catalyses the oxidation of sequence data from model organisms and other species, phenols to quinones. Non-enzymatic polymerization of quinones to melanin is the basis of the melanization process thousands of putative genes with identifiable open reading frames can be coated on a single chip to study a genome- widely observed in invertebrates, which occurs upon 4 Current Genomics, 2004, Vol. 5, No. 2 Ding et al.

Table 1. Serine Proteases and Serine Protease Inhibitors Identified in Innate Immunity and Development of Invertebrates

Serine proteases with Clip domain Serine protease inhibitors (serpins)

Members encoded Innate immunity (blood Members encoded Development characterized in genome coagulation/melanization) in genome

Nudel; Gastrulation Defective; Necrotic (Spn43C) Fruit fly Thirty five Persephone (20) Thirty Snake; Easter (10, 11, 61) (36, 62); Spn27A (46, 51)

Mosquito Forty one Sp22D (48, 63) Fourteen

Factor C; Factor B; Limulus NA NA LICI (64, 65) Factor G; Proclotting enzyme (7, 8, 64)

LICI, limulus intracellular coagulation inhibitor NA, not applicable Numbers in parentheses refer to the corresponding references wide transcription profile by hybridization with mRNA or offer the much needed insights into such systems in the cDNA from certain tissues or cells. By comparing the gene vertebrates. expression level under different physiological conditions or environmental stimuli, genes that are in control of important DNA MICROARRAY SHOWS THAT DROSOPHILA biological processes can be easily identified simultaneously IMMUNE-RELATED SERINE PROTEASES ARE in large numbers. Therefore, deciphering complicated CONTROLLED BY IMD AND/OR TOLL PATHWAYS biological pathways has been tremendously accelerated for example, (a) after the completion of the genome sequence of As Drosophila melanogaster has served as an invaluable model for biological research conducted in invertebrates, the Saccharomyces cerevisiae, virtually every annotated gene was analyzed by DNA microarrays to investigate the completion of its annotated genome sequence has provided a solid foundation for a new era of downstream sophisticated temporal gene expression profiles in metabolic shift, from fermentation to respiration [25]; (b) the LPS-induced innate functional studies. Combined with the advantage of having immune responses in neutrophils were investigated from accumulated a large research community and powerful both the genomic and proteomic capabilities by DNA research tools developed from many years of intensive research on the fruitfly, many important biological processes microarray of 7070 genes coated on Affymetrix chips, revealing the principal role of p38 mitogen-activated protein that have commonly evolved from invertebrates and are kinase upon LPS simulation [26]. Hence, it is highly highly conserved in the whole animal kingdom were being anticipated that investigations on the innate immune-related deciphered and resolved, in particular, the potent innate immune response of this tiny fly. serine proteases in both higher vertebrates and invertebrates would also benefit from genome and proteome analysis, To combat microbial infection, Drosophila adopts which will provide a very promising future in this area of various cellular and humoral responses like proteolytic research. Indeed, the Drosophila genome contains a high cascades leading to blood coagulation and melanization that number of genes encoding putative proteases, including 199 facilitate further phagocytosis, leading to the mass members that appear to code for trypsin-like serine proteases production of antimicrobial peptides (AMPs), which are and many others that code for with fibrinogen- and small molecules that kill microbes directly. Excellent complement-like domains, suggestive of a complicated research has been published within this decade to dissect the innate immune system connected by the putative serine cell signaling pathways in control of the synthesis of AMPs proteases [27]. Another 178 chymotrypin-type serine [30, 31]. Different AMPs can be produced according to the proteases are also reported to be encoded in the Drosophila types of invading microbes. Bacterial infection induces the genome [28]. Although it is highly likely that many of the expression of antibacterial peptides such as attacin, ceropin, annotated serine proteases might control other important defensin, diptericin and drosocin, while fungal infection processes like digestion and embryonic differentiation, the strongly induces the expression of antifungal peptides like genome data-mining approach has provided a solid platform drosomycin and metchnikowin [32]. It has been discovered for an efficient downstream analysis on the innate immunity- through genetic analysis that upon microbial infection, the related serine proteases. It is noteworthy that the latest Toll and Imd signaling pathways control the transcription of finalized genome sequencing of Anopheles gambiae also these AMP genes [33, 34]. However, the Imd and Toll revealed high numbers of serine proteases involved in the pathways do not appear to share any intermediate innate immunity of the mosquito [29]. The availability of the components to mediate differential transcriptions of AMPs Anopheles genome database will undoubtedly contribute via distinct NF-kB/Rel proteins. Depending on the type of towards further understanding and resolution of serine infection, Toll signaling pathway largely responds to protease networks in the invertebrates, which will ultimately infection by gram positive bacteria and fungi, while the Imd Current Genome-Wide Analysis on Serine Proteases Current Genomics, 2004, Vol. 5, No. 2 5 pathway is mainly in control of the expression of effectors On the other hand, the Imd pathway seems to be induced by gram negative bacteria. independent of serine proteases and is less well-characterized Toll signaling pathway, which constitutes Tube, Pelle compared to the Toll pathway. PGRP-LC, another peptido- and Cactus was originally identified to regulate the nuclear glycan recognition receptor, has been shown to act upstream gradient of Dorsal in the early embryonic dorsoventral of the Imd pathway for the final activation of Relish, a Dorsal homologue, to transcribe AMP upon gram negative determination of Drosophila [12]. From an uncharacterized signal for differentiation, it was proposed that an extra- bacterial infection. In contrast to PGRP-SA, the PGRP-LC is a transmembrane receptor that lacks a cytoplasmic signal cellular protease, Nudel, is activated (Fig. 1). In turn, Nudel triggers the serine protease cascade including Gastrulation transduction domain [41, 42]. Interestingly, by using functional RNAi genomic analysis, PGRP-LC was found to Defective, Snake, and Easter on the ventral surface of newly fertilized eggs [35]. The final outcome of this proteolytic play a significant role in phagocytosis in Drosophila besides processing of serine proteases is the cleavage of Späetzle its roles in Imd pathway [43]. into its activated form. Activated Späetzle recognizes and The completion of the Drosophila genome in year 2000 binds to Toll receptor to trigger the downstream signaling [44] has greatly accelerated research on innate immunity of pathway leading to the degradation of Cactus, a dorsal this model organism. Over the recent years, through transcription factor inhibitor (functionally homologous to nucleotide microarray analyses, 400 immune-related genes ikB in the vertebrates) resulting in the subsequent transloca- have been identified in the Drosophila [45]. The genes were tion of Dorsal from cytoplasm to nucleus. Whilst the classified to function in innate immune responses including Späetzle/Toll/Dorsal pathway was discovered to be in AMP synthesis, pathogen recognition, blood coagulation, control of the innate immune response for AMP synthesis, melanization, phagocytosis, and production of reactive another distinct serine protease cascade in the hemolymph oxygen species, wound healing and iron sequestration. To was suggested to trigger the processing of Späetzle. Perse- elucidate the roles of Toll and Imd pathways in immune phone and Necrotic (a serpin), have been reported to be responses other than the prompt production of AMP, the involved in this serine protease cascade to process Späetzle same group of researchers used cDNA microarray to [20, 36]. The discovery of Toll in Drosophila has prompted a compare the effects of mutations affecting the Toll and Imd parallel search for mammalian genes related to Toll. pathways on the transcriptional profiles of the 400 immune Allegedly, at least ten Toll-like receptors are believed to be response-related genes induced by septic injury [46]. By coded in the human genome. Amongst these, only TLR4, comparing mRNA samples of Toll and/or Imd mutants with TLR2 and TLR9 are biochemically and genetically charac- the mRNA of the wild type adult fly on a DNA chip capable terized [37, 38]. Interestingly, TLRs in mammals recognize of measuring mRNA levels for nearly every single gene in and bind various microbial products directly without the the Drosophila genome, the majority of the 400 immune- involvement of proteolytic processing to elicit the immune related genes has been shown to be targets of the Toll and responses similar in the Drosophila Toll signaling pathway, Imd pathways. Clearly, the microarray studies have demons- culminating in the activation and translocation of trated that the Toll and Imd pathways are the major regula- mammalian NFkB transcription factors into the nucleus. tors of immune response in the Drosophila adults, besides Recognition of infection by exploiting microbial features AMP production. that distinguish microbes from their infected host organisms Microarray studies have implicated many serine is the most central strategy developed in the evolution of proteases and serpins in both the Toll and Imd pathways. innate immunity in both invertebrates and vertebrates. LPS The Toll pathway was found to control genes encoding three of gram- negative bacteria, and lipoteichoic acid of gram- uncharacterized serine proteases, two serpins, one kunitz- positive bacteria, peptidoglycan of both gram-negative and - type serine protease inhibitor, and three putative proPOAEs positive bacteria and b-1,3-glucan of fungi are well-known that may play a role in regulating the prophenoloxidse pathogen associated molecular ‘patterns’ (PAMPs) commonly cascade. However, whether other serine proteases identified observed in these microorganisms. Receptors capable of in the Toll mutant are directly involved in the Toll signaling detecting these PAMP molecules are defined as pattern pathway, for example, the cleavage of Späetzle in different recognition receptors, PRRs [39]. Thus, in contrast to types of infection, blood coagulation or complement Drosophila Toll, mammalian TLRs are considered to be pathway, still needs further investigation. Interestingly, the amongst the prominent members of PRRs. Certainly, there Imd signaling pathway was also shown to control a putative must be PRRs acting further upstream of all the serine proPOAE and other components of a potential melanization process, three other uncharacterized serine proteases and one protease cascades in the Toll pathway. Recently, an extra- serpin. Furthermore, five uncharacterized serine proteases cellular recognition protein, PGRP-SA, which belongs to a and two serine protease inhibitors have been found to be large protein family characterized as peptidoglycan binding regulated by both the Imd and Toll pathways. A putative proteins, has been implicated in the activation of Toll coagulation factor was also discovered by this method, pathway [40]. Although the direct connection and interaction albeit, independent of the two pathways. Compared to the between PGRP-SA and its downstream serine protease large repertoire of potential serine proteases from direct cascade remain unknown, it is highly likely that the Toll prediction of genome sequence, serine proteases that are pathway is triggered by soluble recognition molecules like involved in regulating the Toll and Imd pathways identified PGRPs through distinct proteolytic cascades converging on through the microarray analysis of the Drosophila innate the production of the activated Späetzle. immune response are relatively limited. 6 Current Genomics, 2004, Vol. 5, No. 2 Ding et al.

COMPARATIVE GENOME-WIDE ANALYSIS analysis and classical biochemical procedures, viz, the SUGGESTS NUMEROUS INNATE IMMUNE- empirical approaches. RELATED SERINE PROTEASES IN ANOPHELES GAMBIAE EMPIRICAL STUDIES ON SERINE PROTEASES IN THE POST-GENOMIC ERA - COMBINING BIO- Due to its significance in malaria control, Anopheles INFORMATICS WITH EXPERIMENTAL EFFORTS: , the disease transmission vector has also long been gambiae LESSONS FROM THE DROSOPHILA AND C. one of the intensively studied invertebrates. Recently, its ELEGANS SERPINS AND THE LIMULUS SERINE robust innate immune response against the parasite PROTEASE Plasmodium has become the hot topic of study on parasite transmission process. In search for genes related to malaria In addition to the predominant role that serine proteases transmission in the mosquito, several serine proteases have play in various aspects of innate immune response in also been discovered by cDNA differential display and RT- invertebrates, peptidases other than serine proteases are also PCR techniques. These were found to be involved in indispensable in important biological processes in all immune responses. cDNA differential display was initially organisms. The highly conserved amino acid sequence of the adopted to screen for specific genes that were differentially distinct active sites of the proteases render themselves easily expressed upon bacterial challenge in the larval stage of the searched against the genome database, which is the basis for mosquitoes [47]. The number of innate immune-related a latest review paper based on data-mining of proteases in all genes identified and characterized by this method was the currently known genome sequences, from yeast to human relatively limited. Among 19 distinct cDNA clones, only one [50]. As for serine proteases, by using the identifier motif was reported to be a non-trypsin serine protease. In another described within MEROPS (a peptidase database based on study on the serine proteases in mosquitoes, RT-PCR using the statistically significant similarities in amino acid degenerate primers of conserved serine protease catalytic sequence), the genomes of human, yeast, fly, worm and cress domain isolated five cDNA clones of serine proteases, which putatively encode 140, 16, 225, 62 and 197 serine proteases, were further characterized [48, 49]. However, the precise respectively. Nevertheless, direct identification and predic- physiological functions of these serine proteases have not tion of genes and gene family from mere genome sequences, been well charted yet. particularly peptidase gene family, has inherent flaws. Inevitably, the interpretation of a genome sequence has to be Apparently, knowledge obtained so far from the mos- performed largely by comparing against a collection of quito is rather limited and scattered. With the completion of available databases in two ways: computer algorithms and the annotated sequencing of the Anopheles genome, it has human manual annotation. Consequently, direct predictions become possible to compare the genome sequences of the might be completely erroneous or insufficiently analyzed two dipteran insects (Drosophila and Anopheles), thus because of human biasness. Investigations on the true revealing conserved genes that remain unchanged in innate functions of all the putative immune response-related serine immune response inspite of the dramatic differences that proteases are further complicated by the large amounts of stem from their respective selective pressure. By compara- repetitive sequences and pseudogenes in the eukaryotic tive bioinformatics and manual annotation, Christophides et genomes. Therefore, precise characterization of the serine al [29] have identified 242 immune-related genes from 18 gene families and observed manifested diversification of protease gene and its product in the context of genetics and biochemical functions relies on sophisticated and complex these genes from Drosophila. The authors classified the genes into several categories reflecting different aspects of downstream empirical studies. innate immunity: recognition of pathogens, signal modula- A recent study on a serpin gene in Drosophila has served tion and amplification, signal transduction and effector as an excellent model for combining the genome data and response. Despite the divergence and excessive gene expan- classical empirical experimental efforts. Based on the sion from Drosophila homologs, the Toll signaling pathway genome-wide analysis of innate immune-related genes in is significantly conserved. Anopheles has 11 Toll genes and a Drosophila [45], several serpins involved in the melanization single gene each for the downstream signaling components: cascade were further characterized [51]. From 30 candidate MyD88, Tube, Pelle and Cactus. More interestingly, only serpin genes revealed in cDNA microarray analysis that one invertebrate NFkB homolog, Dorsal, was identified in showed upregulation upon infection of a mixture of gram the genome. It is noteworthy that serine proteases associated negative and gram positive bacteria, five were selected for a with the Toll signaling modulation or other immune effector thorough downstream genetic and biochemical analysis. responses in innate immunity are often characterized with a These researchers discovered that one of the five serpins, clip domain of a compact disulfide-bridged structure Spn27A, is regulated by the Toll pathway and is involved in putatively regulating the catalytic protease domain. The the melanization response via inhibition of a prophenol- Anopheles genome encodes 41 clip domain serine proteases oxidase activating enzyme. The cognate serine protease and 14 serpins, the protease inhibitors, which also play inhibited by Spn27A is currently under further investigation. crucial roles in the regulation of immune responses. The Recently, a PGRP protein (PGRP-LE) was reported to be genome-wide analysis has tremendously accelerated the involved in both Imd/Relish-mediated antibacterial pathway discovery of immune-related serine proteases compared to and prophenolxidase cascade in Drosophila larvae [52]. the cDNA differential display and RT-PCR. However, as Taking all the experimental data together, it is interesting to none of these serine proteases has been well-characterized, it observe that the antimicrobial defense system controlled necessitates researchers to revert to the traditional genetic largely by Toll and Imd pathways are closely linked with melanization through serine proteases and serpins (Fig. 2). Current Genome-Wide Analysis on Serine Proteases Current Genomics, 2004, Vol. 5, No. 2 7

Caenorhabditis elegans is another genetically tractable one putative prophenoloxidase activating enzyme and two sample model with a fully sequenced genome. Unexpec- serpins were isolated and found to be induced upon infec- tedly, the majority of components in Toll and Imd pathways tion. As discussed in previous sections, cDNA differential of Drosophila have not been found in the worm genome [53, display is less efficient in obtaining a complete gene map of 54]. The existence of the two conserved pathways in the a certain biological process due to the restrictions on tissue innate immune response of C. elegans is highly unlikely. and stimuli types. We anticipate that many other serine Nevertheless, by cDNA microarray technique, a genome proteases involved in the recognition of pathogens, scale analysis of genes responsive to gram negative bacteria transduction and amplification of the ‘danger’ signal leading strongly suggests that this nematode possesses an inducible to the production of defense molecules to direct the killing of antibacterial defense mechanism [55]. Although genomic the invader will be unveiled by the availability of genome studies have so far not reported any innate immune-related sequence data of the Limulus and its further annotation by serine proteases in the C. elegans, nine serpin genes were comparison with the known genome databases of identified in its genome [54]. Predictably, the C. elegans Drosophila, Anopheles and C. elegans. genome ought to contain serine proteases that are regulated by their cognate serpins. Nevertheless, the specific biological CONCLUSION AND FUTURE PERSPECTIVES roles of the worm serpins definitely demands further empirical studies. Serine protease cascades are intertwined and pervasive in almost every stage of innate immunity, comprising a Limulus, the “living fossil”, has served as a useful animal complex serine protease network in invertebrates (Fig. 2). model for the study of the phylogenetic development of Much hope, expectations and recent research efforts have innate immune response. Its LPS-sensitive blood coagulation focused on genome-wide analyses using microarrays to is considered to represent an ancient form of serine protease analyse up- and down-regulated immune gene expression cascades that combines the function of hemostasis and innate resulting from pathogen-infection and injury [45, 46, 57-59]. immune defense. Factor C, the first serine protease zymogen This new approach has accelerated the discovery of immune- of the blood coagulation cascade, is activated by gram related serine proteases in an unprecedented pace. However, negative bacterial LPS leading to the activation of the despite using classical model organisms such as Drosophila, downstream serine proteases in the blood coagulation Anopheles and C. elegans, many limitations persist with the cascade, see Fig. 1). The close interrelationship between small size of these model animals which require collections innate immune response and blood coagulation is further of large numbers, resulting in heterogeneity of phenotype corroborated by our recent finding that the Limulus Factor C thus, obscuring gene expression results. The large amount of gene expression is intricately regulated by a potential NFkB RNA required for microarray analysis also restricts the type signaling pathway [56]. In this work, we studied the trans- of questions asked. A further limitation with microarray is criptional profile of a single serine protease gene in response imposed by frequent alterations of the annotations of to gram negative bacterial challenge. The transcriptional predicted protein-encoding genes, in an effort to increase the expression was studied by traditional Northern analysis and accuracy of predictions through the alignment of ESTs and real time PCR after gram-negative bacterial infection. also through personal curation on a gene-by-gene basis [60]. Subsequently, the promoter was isolated and characterized in However, these alterations have generated confusion transfection assays. Monitoring the expression changes of amongst those who have embarked on functional genomics the reporter gene driven by different deletion constructs of projects. As an example, the creation of a DNA microarray the Factor C promoter sequences and combined with data on requires the selection of a set of PCR amplification primers the mutagenesis of potential cis-elements have revealed that based on the current gene annotations. This selection makes an NFkB-like motif is delicately modulated upon LPS the DNA microarray a relatively static entity, unless challenge. Such intensive experimental investigations have considerable attention is paid to the updated names, and new enabled us to establish the involvement of a potentially primers are ordered for genes that were “mis-named” during conserved NFkB in the transcriptional regulation of the key earlier annotations. To keep abreast with such updating serine protease of blood coagulation and pathogen would call for major commitment on the part of the recognition. researcher. Other technical problems also exist with DNA It is interesting to note that many essential biological microarray – the possibility of cross-hybridization of related processes are conserved in Arthropods, within Drosophila, transcripts to multiple spots on the array; danger of large Anopheles and Limulus, although great diversity is also amount of data that are being generated and propagation of observed in all these species. The knowledge we gain from the unfounded information, particularly in the case of gene research in one organism will greatly enhance and provide annotation [60]. clues for potential in-depth investigations in other species, in Therefore, to chart the precise position of the gene particular, on the potent innate immunity that invertebrates product in the intricate map of serine protease cascades, possess. Although efforts are ongoing with sequencing the traditional biochemical, molecular biological and genetic Limulus genome, cDNA differential display has been approaches are still crucial. Thus, functional genomics employed to isolate genes responsive to gram negative should combine investigations at two levels: (i) genome- bacterial infection (Tan et al., unpublished data; personal wide analysis to locate the target gene and (ii) empirical communication). By comparing the sequences of cDNA experimental approaches to characterize the target gene and clones from subtractive libraries with known gene sequences its cognate proteins and protein partners in order to fully deposited in Blast and Swiss-plot databases, clones unravel the modular biology of the serine proteases and their putatively coding for two immune-related serine proteases, multifunctional capabilities in early developmental biology 8 Current Genomics, 2004, Vol. 5, No. 2 Ding et al. of the organism, innate immunity and blood coagulation. [23] Chosa, N., Fukumitsu. T., Fujimoto, K., Ohnishi, E. Activation of Such collaborative levels of studies in the so called ‘post- prophenoloxidase A1 by an activating enzyme from crayfish blood cells. Insect Biochem. Mol. Biol., 1997, 27: 61-68. genomic’ era will undoubtedly resolve many fundamental [24] Ashida, M., Brey P.T. 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