Primary structure and cellular localization of callinectin, an antimicrobial peptide from the blue crab.

We report the complete amino acid sequence of callinectin, a 32 amino acid, proline-, arginine-rich antimicrobial peptide (AMP) with four cysteines and having the sequence WNSNRRFRVGRPPVVGRPGCVCFRAPCPCSNY-amide. The primary structure of callinectin is highly similar to arasins, AMPs recently identified in the small spider crab (Hyas araneus). Callinectin exists in three isomers that vary in the functional group on the tryptophan (W) residue. The most prevalent isomer had a hydroxy-N-formylkynurenine group, while the other two isomers had either N-formylkynurenine or hydroxy-tryptophan. Using a sequence highly similar to native callinectin, we chemically synthesized a peptide which we called callinectin-like peptide (CLP). Via immuno-electron microscopy, affinity-purified rabbit antibodies raised to CLP successfully localized the site of callinectin in blue crab hemocytes to the large electron-dense granules that are found primarily in large granule hemocytes.

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics.

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.