Fig. 2. Flow diagram showing the putative post-translational processing of sulfakinins and sulfakinin precursor-related peptides (SPRPs) from the deduced Homarus americanus prepro-sulfakinin. Translation of the nucleotide sequence of the cDNA encoding H. americanus prepro-sulfakinin predicts a 120 amino acid prepro-hormone (top sequence). The first 24 amino acids of the prepro-hormone are predicted to be a signal peptide (SignalP 3.0 analysis) (Bendtsen et al., 2004), with a cleavage site between Ser²⁴ and Ala²⁵ (red residues and arrowhead). Processing between these residues by signal peptidase would produce a 96 amino acid pro-sulfakinin (second sequence). Via homology to known insect pro-hormone cleavage sites (Veenstra, 2000), two Lys-Arg and two Arg-X_n-Arg (where X is a variable amino acid and n is either 0, 2, 4 or 6 residues) processing sites were identified in Hoa-pro-sulfakinin (yellow residues and arrowheads). Proteolytic processing by a prohormone convertase at these sites would liberate five peptides (third line of sequences); the basic residues on four of these are predicted to be the targets of carboxypeptidase action (green residues and arrowheads). In three of these four peptides, carboxypeptidase action would expose a glycine residue (fourth line of sequences), which likely serves as a target for -amidation by peptidyl-amidating monooxygenase [blue residues and arrowheads; homology to known sulfakinin isoforms (e.g. Johnsen et al., 2000; Torfs et al., 2002)]. Action by this enzyme would result in the amidation of the carboxy termini of these three peptides (fifth line of sequences). Additional post-translational processing of tyrosine residues by tyrosylprotein sulfotransferase in two of the peptides is predicted to result in the addition of sulfate groups to them [purple residues and arrowheads; homology to known sulfakinin isoforms (e.g. Johnsen et al., 2000; Torfs et al., 2002) and prediction via Sulfinator software (Monigatti et al., 2002)]. Likewise, based on homology to known sulfakinin isoforms (e.g. Johnsen et al., 2000; Torfs et al., 2002), the amino (N)-terminal glutamic acid in one peptide and the N-terminal glutamine in another (purple residues and arrowheads) are hypothesized to undergo enzymatic or spontaneous cyclization, resulting in the formation of pyro-residues in the mature forms (final line of sequences). The five resulting peptides (two SKs and three SPRPs) are shown and labeled in white.