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 coagulation 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 protein 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 fibrinogen, 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 disulfide 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