Peroxinectin, a cell adhesive protein associated with the proPO system from the black tiger shrimp, Penaeus monodon

Abstract

Upon activation of the prophenoloxidase activating system in the shrimp, Penaeus monodon, a cell adhesion activity in the haemolymph is generated. A cell adhesion assay showed that a high number of granular cells (60%) adhered to coverslips coated with a shrimp haemocyte lysate supernatant, whereas a very low number of cells adhered to coverslips coated with bovine serum albumin. Inhibition of adhesion by an antiserum against crayfish peroxinectin, a cell adhesion protein, revealed that the cell adhesion activity detected in shrimp haemocyte lysate supernatant might result from a peroxinectin-like molecule in shrimp. A cDNA clone encoding shrimp peroxinectin was isolated, which had an open reading frame of 2337 nucleotides, with a polyadenylation sequence and a poly A tail. It encodes a protein of 778 amino acids including a 20 amino acid signal peptide. The mature protein (758 amino acids) has a predicted molecular mass of 84.8 kDa and an estimated pI of 9.0. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and KGD (Lys-Gly-Asp), were found in shrimp peroxinectin. Sequence comparison shows that the shrimp protein is similar to crayfish peroxinectin (69%) and to various peroxidases and putative peroxidases from invertebrates and vertebrates. The shrimp peroxinectin cDNA also shows similarity (51%) to both Drosophila peroxinectin-related protein (AAF78217) and peroxidasin (S46224), an extracellular matrix protein combining an active peroxidase domain as well as immunoglobulin domains, leucine rich repeats and procollagen-like motif. However, the sequence similarity to both Drosophila molecules are mostly within the peroxidase domain. Northern blot analysis, using a non-peroxidase region in peroxinectin as a probe, revealed that peroxinectin is constitutively expressed in shrimp haemocyte and was reduced significantly in shrimp injected with a β-1,3-glucan, laminarin, to mimic an infection with a fungus.