Biosynthesis of a γ₃‐Melanotropin‐like Peptide in the Pars Intermedia of the Amphibian Pituitary Gland

Abstract

This study reveals the biosynthesis of a y3-melanotropin-like peptide in the pars intermedia of the pituitary gland of the aquatic toad Xenopus luevis. Pulse-chase experiments in vitro showed that this product is synthesized through processing of a prohormone, pro-opiomelanocortin, and that it is released into the incubation medium. The peptide immunoprecipitated with antiserum to y3-melanotropin, appeared to be a glycopeptide and displayed melanotropic activity. This last observation together with the results of tryptic and chymotryptic peptide mapping of the newly synthesized product indicates that Xenopus y3-melanotropin is structurally different from the proposed mammalian y-melanotropins.It is well established that in the adrenocorticotropic cells of the pituitary gland corticotropin and P-lipotropin are produced through processing of a common prohormone (for a review, see [I]). In the pars intermedia the same prohormone functions as the precursor for a-melanotropin, corticotropinlike intermediate lobe peptide [corticotropin-(I 8 -39)], and /I-endorphin ; it was, therefore, named pro-opiomelanocortin [2]. Nakanishi et al. [3] revealed the nucleotide sequence of the cloned cDNA, which is complementary to the mRNA that codes for pro-opiomelanocortin in bovine pituitary glands. The primary structure deduced from this nucleotide sequence showed three repetitive units consisting of the tetrapeptide His-Phe-Arg-Trp, which sequence is characteristic for melanotropic peptides. One of these units constituted a segment of the sequence corresponding to a-melanotropin, a second unit was part of the region corresponding to P-melanotropin. A segment of 12 amino acids, containing the third unit and flanked on either side by pairs of basic amino acids, was named y-melanotropin. Subsequently three forms of y-melanotropin were postulated, y1, y2 and y 3 [4]. Of these forms, y2 corresponds to the segment of 12 amino acids; y l represents the sequence 1 -11 of yz with an amidated C terminus; y3 constitutes a segment of 27 amino acids, namely y2 C-terminally extended with a sequence of 15 amino acids. The putative y3-melanotropin contains the sequence AsnGly-Ser and, thus, could be a glycopeptide.Immunological investigations [5,6] as well as analyses of amino acid sequences [7-91 indicate the occurrence of y-melanotropin-like peptides in the pituitary gland of several species. Biosynthetic studies showing the processing of proopiomelanocortin to yield y-melanotropin have, however, not been reported. Indeed, while considerable information is available concerning the enzymatic conversion of the C-terminal portion of pro-opiomelanocortin [which contains the sequences of a-melanotropin, corticotropin-( 18 -39), p-melanotropin and P-endorphin] little is known about the significance of the N-terminal 'cryptic region' containing the sequence of y-melanotropin.Using pulse-chase incubations and high-performance liquid chromatographic analysis, we recently demonstrated that in the neurointermediate lobe of Xenopus laevis proopiomelanocor...

Effect of tunicamycin on biosynthesis, processing and release of proopiomelanocortin-derived peptides in the intermediate lobe of the frog Rana ridibunda

“…There appears to be considerable species differences both in the number of oligosaccharide side-chains attached to POMC and in the position of their attachment within the precursor molecule. POMC of another amphibian species, Xenopus laevis, has been shown to have a single glycosylation which is in the 7-MSH region [33,34], identical to the situation we now report for the frog prohormone. Characterization of POMC-related peptides of the salmon [23] and dogfish pituitary gland [32] suggests that the prohormones of these species are not glycosylated.…”

“…trypsin digestion. It has been applied previously in characterization of POMC-related peptides [18,19,33,34,44,45].…”

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

“…In the early secretory pathway of Xenopus melanotrope cells, 37-kDa POMC is processed by PC1 into 18-kDa POMC, representing the N-terminal portion of the POMC molecule and containing the only N-glycosylation site in the POMC molecule (Martens et al , 1982; Ayoubi et al , 1990). We decided to use bafilomycin A1 because the action of this inhibitor toward the V-ATPase has been well characterized (Bowman and Bowman, 2002; Huss et al , 2002; Bowman et al , 2006).…”

A Cell-Specific Transgenic Approach inXenopusReveals the Importance of a Functional p24 System for a Secretory Cell

“…This product represents the N-terminal portion of 37-kDa POMC, is generated by the first endoproteolytic cleavage step during POMC processing and contains the only N-linked glycosylation site present in the POMC molecule (Martens, 1986). The amount of the 18-kDa POMC protein was lower for the 2.5-h than for the 1.5-h time point, because during the chase period this newly synthesized product is processed further (to ␥-MSH; Martens et al, 1982b) (Figure 6A, top left). During the 10-min pulse, the POMC cleavage enzyme PC2 was synthesized as a 75-kDa proenzyme form that in the course of the subsequent 1.5-h and 2.5-h chase incubations was processed to a 69-kDa mature form of PC2 that represents the end product of proPC2 processing ( Figure 6A, top right, and B, lanes 1 and 2).…”