The Caenorhabditis elegans muscle specific serpin, SRP-3 neutralizes chymotrypsin-like serine peptidases

Serpins1 are key regulators in the multitude of biological processes that require precise control of peptide bond hydrolysis. This activity is most evident in the procoagulation, anticoagulation and fibrinolytic cascades where plasma peptidases such as thrombin, protein C and plasmin are tightly regulated by antithrombin III (SERPINC1), protein C inhibitor (SERPINA5) and plasminogen activator inhibitor type 1 (SERPINE1), respectively (reviewed in (1, 2)). Although many of the circulating serpins have been well characterized structurally, biochemically and biologically, humans and other vertebrate species also express an enigmatic subset of serpins whose functions are not well defined. This subset of serpins can be distinguished from other serpin family members by the lack of a cleavable N-terminal signal peptide, the absence of N- or C-terminal extensions, an inhibitory profile that targets serine and/or papain-like cysteine peptidases and a cytosolic or nucleo-cytosolic subcellular distribution (reviewed in (3)). These intracellular serpins (serpinsIC) are present also in all metazoan species examined to date. The broad distribution of serpinsIC provides an opportunity to study their function by a comparative genomics using simpler model systems. To this end, we embarked upon studies to assess serpinsIC activity using the genetically tractable species, Caenorhabditis elegans. The C. elegans genome encodes for 9 serpinIC (srp) genes (4). However, only five of the srp genes (srp-1, -2, -3, -6 and -7) appear to encode for peptidase-inhibitory type serpins, whereas the remaining genes encode for transcripts without a reactive site loop (RSL) or premature termination codons that would activate mRNA decay or lead to synthesis of a truncated protein without an RSL (4). While in silico analysis is helpful in predicting the overall repertoire of serpinsIC 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 serpinIC in C. elegans (5). SRP-2 is a dual cross-class inhibitor and neutralizes granzyme B-like serine peptidases and cathepsin-L-like lysosomal cysteine peptidases and is expressed highly in the hypodermal and intestinal cells of larval and adult worms. The goal of this study was to determine whether another putative inhibitory-type serpinIC, srp-3, actually encodes for a functional molecule that could participate in larval development or adult homeostasis. These studies showed that SRP-3 was a true inhibitory-type serpin that neutralized chymotrypsin-like serine peptidases that was expressed primarily in the anterior body wall muscles. This inhibitory activity confirmed that C. elegans expressed at least two functional serpinsIC whose expression patterns and inhibitory activity were shared with higher vertebrates.