Posttranslational processing of proopiomelanocortin family molecules in sea lamprey based on mass spectrometric and chemical analyses

In gnathostomes, adrenocorticotropic hormone (ACTH), melanophore-stimulating hormones (MSHs), and β-endorphin (β-END) are derived from a common precursor, proopiomelanocortin. In sea lamprey, ACTH and two forms of MSHs are contained in independent precursors, proopiocortin (POC), and proopiomelanotropin (POM), respectively, together with a distinct β-END. Here, we characterized products from POC and POM. An analysis of previously purified ACTH by mass spectrometry (MS) detected four peptides with a molecular weight of 6469.4, 6549.6, 6556.6, or 6636.1. The sequence analysis of an ACTH preparation following enzymatic and chemical cleavage revealed the presence of ACTH1–59 and ACTH1–60 corresponding to a molecular weight of 6469.4 and 6556.6, respectively, and of ACTH1–59 and ACTH1–60 modified at Ser35 by a group having a mass of 80, giving the molecular weight 6549.6 and 6636.1, respectively. The modification could be due to phosphorylation based on the increase in molecular weight of 80. Analyses of frozen pituitary slices with MALDI-TOF MS detected several mass numbers corresponding to POC-derived peptides such as ACTH1–60, modified ACTH1–60, and POCβ-END, and those corresponding to POM-derived peptides such as MSH-A, MSH-B, and the C-terminal fragment of POMβ-END lacking a Met-enkephalin segment. The present results together with previous characterizations show that in sea lamprey pituitary the major products derived from POC in the PD by posttranslational processing are ACTH and β-END as in gnathostomes. The posttranslational processing of POM in the PI is similar to that in gnathostomes in the sense of the occurrence of MSH, however, it differs in that β-END is further cleaved, thus generating Met-enkephalin.