Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith

Abstract

SUMMARYGastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

Expression of cytoskeletal and molt-related genes is temporally scheduled in the hypodermis of the crayfish Procambarus clarkii during premolt

“…Some of them include, in addition to the carbohydrate esterase 4 CDA-like 1 domain identified here, the chitin-binding peritrophin A domain (CBM; Pfam ID PF01607) and/or the low density lipoprotein (LDL) receptor domain class A (Pfam ID PF00057). The sequences of these 10 transcripts are found in TSA GADE01000000 (Glazer et al, 2013) and TSA GAFS01000000 , and are also characterized at the bottom of supplementary material Table S1 here. A division of the CDAs may be done on a putative functional basis, namely, determining the contigs that contain the essential aspartic acid residue in motif 1 and the two histidine residues in motif 2, as described by Dixit et al (Dixit et al, 2008).…”

“…The structural proteins and the enzymes responsible for the partial degradation of the old cuticle and the construction of the new one are synthesized in, or transported through the underlying hypodermis, which is composed mainly of epithelial cells supported by structural and storage connective tissues (Glazer et al, 2013;Johnson, 1980;Roer and Dillaman, 1984). The cuticle is composed of four layers ordered: epicuticle, exocuticle, endocuticle and membranous layer (Roer and Dillaman, 1984;Skinner, 1985;Travis, 1965).…”

Barnacle biology before, during and after settlement and metamorphosis: a study of the interface

“…The bacterial staining showed that the ROS was largely distinct from the bacteria and from features of the barnacle. We hypothesize that these ROS at the cuticle layers also contribute to oxidative crosslinking (Okay et al, 1995;Dijkgraaf et al, 2003;Lattuada et al, 2013), as seen in other crustaceans (Willis, 1999;Glazer et al, 2013), and are critical to cuticle development and hardening, as seen in insects (Hopkins and Kramer, 1992;Suderman et al, 2006;Andersen, 2010). Further work is needed to define the specific ROS present and show the extent of their presence during development as only post-metamorphic juveniles were examined.…”

“…Arthropod cuticle is not solely a physical barrier to the environment but can contribute to critical processes such as immune responses and cross-linking for cuticle hardening and calcification of exoskeleton (Ashida and Brey, 1995;Sugumaran and Nelson, 1998;Adachi et al, 2005;Suderman et al, 2006;Glazer et al, 2013). In crustaceans, cuticle tearing leaks hemolymph and releases proteins, which coagulate sealing the wound and preventing infection (Iwanaga et al, 1998;Li et al, 2002;Jiravanichpaisal et al, 2006).…”

A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix c properties

“…In silico confirmation of Cq-M15 sequence and transcription pattern Reference C. quadricarinatus transcriptome was constructed from sequencing of four sets of samples as described previously (Tynyakov et al, 2015): (1) RNA extracted from hypodermis-, gastrolith-and molarforming epithelium sequenced by 100 bp paired-end sequencing (Illumina HiSeq2000, USA); (2) RNA extracted from molar-and gastrolith-forming epithelium from different molt stages (intermolt, early pre-molt, late premolt and post-molt), barcoded and sequenced by 50 bp single-end sequencing (Illumina HiSeq2000, USA); (3) RNA sequenced by 454 technology, as described by Glazer et al (2013); and (4) RNA sequenced 'single pass' by Sanger technology (expressed sequence tags, ESTs) as described by Yudkovski et al (2010). All sequences (also called 'reads') were de novo assembled with CLC Genomics Workbench 6.51 (CLC Bio, Aarhus, Denmark) using default parameters.…”