Purification and cDNA cloning of a four‐domain Kazal proteinase inhibitor from crayfish blood cells

Abstract

A cDNA with an open reading frame of 684 base pairs was isolated from a library from blood cells of the crayfish Pacifastacus leniusculus. It codes for a signal sequence and a mature protein of 209 amino acids with a predicted molecular mass of 22.7 kDa. The amino acid sequence consists of four repeated stretches (45-73% identical to each other), indicating that the protein has four domains. The domains have significant sequence similarity to serine proteinase inhibitors of the Kazal family. The three first domains have a leucine residue in the putative reactive site, suggesting that the protein is a chymotrypsin inhibitor.A monomeric 23-kDa proteinase inhibitor, which by amino terminal sequencing of the mature protein was confirmed to be the cloned Kazal inhibitor, was purified from crayfish blood cells. It inhibited chymotrypsin or subtilisin, but not trypsin, elastase or thrombin. The inhibitor seemed to form a 1 : 1 complex with chymotrypsin or subtilisin.This protein seems to be the first described Kazal inhibitor from blood cells of any animal and the first one with four domains.Invertebrates do not have antibodies or other features of the vertebrate adaptive immune system but have innate hostdefence reactions [l]. As many of them have an open circulatory system, they need efficient immediate and constitutive defence mechanisms. Proteinase inhibitors in the blood are believed to be important in this context, either aimed against proteinases of microorganisms or parasites, or being regulators of host-defence reactions [2-41.In our laboratory, several proteinase inhibitors from the blood of freshwater crayfish have been isolated. From crayfish plasma, an inhibitor homologous to mammalian a2-macroglobulin has been purified [ 5 ] and partially sequenced [6]. Also, a 155-kDa trypsin inhibitor that controls activation of the so-called prophenoloxidase activating system has been purified [7, 81 (for a review on the prophenoloxidase system, a proposed recognition and defence system in arthropods, see [2, 91). From the blood cells, a subtilisin inhibitor that inhibits fungal proteases was isolated [lo] and, recently, a proteinase inhibitor of the serpin family has been cloned (Liang, Z. and Soderhall, K., unpublished results).In this report, we present the cDNA cloning and sequencing as well as the purification and characterization of a fourdomain proteinase inhibitor of the Kazal family from crayfish blood cells.

Purification and cDNA Cloning of Ferritin from the Hepatopancreas of the Freshwater Crayfish Pacifastacus leniusculus

“…The crayfish ferritin was analyzed by native PAGE and SDS/ PAGE under reducing conditions. For determination of the Nterminal sequence, 10 pg ferritin was run on 15% SDSPAGE and then transferred to poly(viny1idene difluoride) membrane (Millipore), as described in Johansson et al (1994). The 20-kDa ferritin band was excised for determination of the N-terminal sequence.…”

Antilipopolysaccharide Factor Interferes with White Spot Syndrome Virus Replication In Vitro and In Vivo in the Crayfish Pacifastacus leniusculus

“…Some of these genes code for proteins known to be involved in defense reactions, such as VMO-I, MBP, and proteinase inhibitors (1,3,13,15,29), although there is little information available about to which extent, if any, they are effective in an antiviral response. Our experiments have, so far, not indicated that these proteins, with the exception of ALF, have a specific role in reducing viral replication.…”

cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

“…The monospecific antiserum was used to screen a crayfish hemocyte cDNA library (23). An Northern Blot Analysis.…”