SRP-2 Is a Cross-class Inhibitor That Participates in Postembryonic Development of the Nematode Caenorhabditis elegans

Abstract

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as ␣ 1 -antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.Serpins are a unique class of proteinase inhibitors that irreversibly neutralize target proteinases by a mechanism involving the conformational distortion of the proteinase. Serpins have been identified in animals, plants, insects, and certain viruses (1, 2). More recently, serpins have been detected in prokaryotes (3). A search of genome data bases provides evidence for Ͼ500 serpins that are grouped into 17 clades (plus Ͼ10 unclassified orphans) based upon phylogenetic relationships (4). In humans, ϳ35 serpins have been identified. These serpins are distributed among nine clades (A-I), and most are secreted and function in the circulation or extracellular spaces. These serpins regulate proteinases involved in blood coagulation, fibrinolysis, complement activation, inflammation, and extracellular matrix remodeling. In contrast, serpins belonging to clade B reside predominantly intracellularly and have been implicated in regulating apoptosis, tumor progression, and metastasis (5). However, their biological functions in terms of an intact organism have not been well defined. To date, no naturally occurring mutations with an identifiable phenotype...

Control of granzymes by serpins

“…The nematode Caenhorabditis elegans has also been shown to encode a potent gzmB inhibitor, Srp-2. 102 The P1 residue is a Glu, suggesting that caspases are not the intended target proteases, and the kinetics seem to suggest a physiologically relevant interaction with gzmB (SI ¼ 1.75, k ass ¼ 2.3 Â 10 4 M À1 s À1 ). 102 However, it must be asked why C. elegans, which neither produces endogenous gzmB nor infects an organism that produces gzmB, and feeds on bacteria that do not produce gzmB, would evolve a potent inhibitor of this protease.…”

“…102 The P1 residue is a Glu, suggesting that caspases are not the intended target proteases, and the kinetics seem to suggest a physiologically relevant interaction with gzmB (SI ¼ 1.75, k ass ¼ 2.3 Â 10 4 M À1 s À1 ). 102 However, it must be asked why C. elegans, which neither produces endogenous gzmB nor infects an organism that produces gzmB, and feeds on bacteria that do not produce gzmB, would evolve a potent inhibitor of this protease. The inhibition is more likely coincidental, with Srp-2 actually inhibiting a bacterial subtilisin-like acidic protease, as subtilisin is efficiently inhibited by SERPINB9.…”

The Caenorhabditis elegans Muscle Specific Serpin, SRP-3, Neutralizes Chymotrypsin-like Serine Peptidases

“…In addition, SRP-3 was expressed predominately in both larval and adult muscles cells. In a previous study we showed that SRP-2 was a dual cross-class inhibitor of granzyme B serine peptidases and papain-like cysteine peptidases and that this inhibitor was expressed in intestinal and hypodermal cells (5). Thus, these two serpins IC complement one another by extending the family's overall inhibitory profile and tissue expression pattern in C. elegans.…”

“…While in silico analysis is helpful in predicting the overall repertoire of serpins IC in different species, these assessments are imperfect and require confirmation by experimentation. To date, only srp-2 has been shown to encode for a bona fide inhibitory type serpin IC in C. elegans (5). SRP-2 is a dual cross-class inhibitor and neutralizes granzyme Blike serine peptidases and cathepsin-L-like lysosomal cysteine peptidases and is expressed highly in the hypodermal and intestinal cells of larval and adult worms.…”

Inhibition Of Proteolysis In Plant And Animal Tissues

“…Следовательно, контроль серпинами за маршрутами эндоцитоза и экзоцитоза является древним способом защиты основных внутриклеточных путей экспорта и импорта от неконтролируемой эндогенной или чужеродной протеолитической активности (Van Gent D. et al, 2003;Pak et al, 2004;Kumar and Ragg, 2008). Серпины найдены у разных видов царства растений и в зеленых водорослях.…”