Structural and Functional Analyses of a Strong Chitin-Binding Protein-1 (SCBP-1) from the Exoskeleton of the Crayfish <i>Procambarus clarkii</i>

Abstract

The organic matrices in the exoskeleton of the crayfish Procambarus clarkii are classified into three groups depending on solubility; acid soluble, acid insoluble-SDS/dithiothreitol (DTT) soluble, and acid insoluble-SDS/DTT insoluble fractions. In our previous studies, Casp-1 and -2 were identified in the acid soluble fraction, and CAP-1 and -2 were identified in the acid insoluble-SDS/DTT soluble fraction. In this study, acid insoluble-SDS/DTT insoluble materials were digested with proteases and the resulting peptides were purified and sequenced. Based on the sequences, a cDNA encoding this protein was cloned. The whole primary sequence of the matrix protein named strong chitinbinding protein-1 (SCBP-1), was deduced. SCBP-1 consisted of 155 amino acid residues and had a Rebers-Riddiford consensus sequence for chitin binding. A recombinant protein of SCBP-N corresponding to the N-terminal part of SCBP-1 showed no chitin-binding ability, while SCBP-C corresponding to the C-terminal part of SCBP-1, showed weak affinity to chitin. These results suggest that the primary sequence of SCBP-1 does not have strong chitin-binding ability. Therefore, SCBP-1 probably binds covalently to chitin through a particular residue contained in the peptide part that was not obtained by protease digestion.

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

“…The newly identified Cq-M15 was found to share significant similarity with known proteins from arthropod cuticles and its sequence was more closely related to crustacean proteins from mineralized parts of the cuticle (Gosline, 1980;Inoue et al, 2008;Suzuki et al, 2013) and more distantly related to proteins found in crustacean non-mineralized arthrodial membranes (Gosline, 1980) and insect cuticles (Kim et al, 2005;Futahashi and Fujiwara, 2008;Werren et al, 2010). Such close homology further supports the involvement of Cq-M15 in biomineralization in the crayfish.…”

“…Moreover, several crustacean proteins associated with calcification processes present amino acid composition profiles similar to that of Cq-M15 (Inoue et al, 2004;Glazer et al, 2010). In fact, such shared patterns of amino acid composition were also found in proteins from organic matrices involved in invertebrate calcium carbonate precipitation (Faircloth and Shafer, 2007;Gayathri et al, 2007;Marie et al, 2007;Inoue et al, 2008;Suzuki et al, 2013), as well as in protein extracted from vertebrate enamel (Robinson et al, 2005).…”

“…Signal peptide and phosphorylation sites were predicted using SignalP 4.0 (Petersen et al, 2011) and NetPhos 2.0 (He et al, 2005), respectively. Identification of putative domains in the deduced protein sequence was predicted by SMART (Schultz et al, 1998;Suderman et al, 2006) and Motif Scan (Suzuki et al, 2013) and its type by a CuticleDB server (Karouzou et al, 2007). Physico-chemical parameters of the Cq-M15 protein sequence, including molecular weight and amino acid composition, were predicted using the ProtParam tool (Fujisawa and Tamura, 2012).…”

Identification and characterization of a matrix protein (PPP‐10) in the periostracum of the pearl oyster, Pinctada fucata

“…The fibers of the exoskeletons of crustaceans contain chitin and matrix proteins that have a Rebers–Riddiford (RR) chitin‐binding concensus motif [23]. CAP‐1, CAP‐2, Casp‐2 and SCBP‐1 have the RR consensus motif and were identified from the exoskeleton of the crayfish [24–27]. The aromatic amino acids in the β‐sheet structure of the RR consensus motif bind to chitin through hydrophobic interactions [28].…”

The Molecular Mechanism of Calcification in Aquatic Organisms

“…Recently, a matrix protein that binds very strongly to chitin and cannot be extracted with a detergent solution, was identified. 41) As to the binding manner of this protein, there remains a possibility of covalent binding.…”