Corticotrophin-related peptides in the intermediate lobe of the rodent pituitary gland: characterization by high performance liquid chromatography and radioimmunoassay

High performance liquid chromatography (HPLC) and radioimmunoassay have been used to characterize corticotrophin-related peptides in extracts of the intermediate lobe of the rat and mouse pituitary gland. Multiple peaks have been observed, which resemble corticotrophin-like intermediate lobe peptide (CLIP) in that they cross-react with antisera raised against the COOH-terminal region of corticotrophin (ACTH) but not against NH2-terminal directed antisera. These CLIP-like peptides were released from the incubated neurointermediate lobe and their secretion was inhibited in the presence of dopamine. Heterogeneity of peptide species was also observed with antisera raised against alpha-MSH. Multiple peaks of CLIP and alpha-MSH-like material were identified in pituitary extracts from the mouse and levels were elevated in the genetically obese (ob/ob) animal. The nature and possible functions of multiple forms of intermediate lobe peptides are discussed.     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs)

We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and alpha-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1-39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18-39))-like immunoactivity. Acetylated alpha-MSHs were not detectable at Day 50. alpha-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl alpha-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1-39) levels were negligible when compared to increasing alpha-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of alpha-N- and alpha-N,O-acetylated forms of alpha-MSHs were detected during middle and late stage fetal development. Diacetyl alpha-MSH was the predominant form of alpha-MSH in adult NIL extracts. No acetylated alpha-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1-39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and alpha-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Characterization of eight forms of corticotropin-like intermediary lobe peptide from the rat intermediary pituitary

The isolation and characterization of eight forms of corticotropin-like intermediary lobe peptide (CLIP, adrenocorticotropin18-39) from the intermediary lobe of the rat pituitary has been accomplished by using reversed phase high performance liquid chromatography. The eight forms are the result of all combinations of the presence or absence of three post-translational modifications. These are glycosylation, phosphorylation, and removal of the carboxyl-terminal amino acid. The sites of phosphorylation and glycosylation are at serine 31 and asparagine 29, respectively. The eight forms (in order of elution from the reversed high performance liquid chromatography column) are glycosylated, phosphorylated CLIP18-38; glycosylated, nonphosphorylated CLIP18-38; nonglycosylated, phosphorylated CLIP18-38; nonglycosylated, nonphosphorylated CLIP18-38; glycosylated, phosphorylated CLIP18-39; glycosylated, nonphosphorylated CLIP18-39; nonglycosylated, phosphorylated CLIP18-39; and nonglycosylated, nonphosphorylated CLIP18-39.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Molecular forms of alpha-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe.

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of alpha-melanocyte-stimulating hormone (alpha-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of alpha-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-alpha-MSH, while monoacetyl-alpha-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-alpha-MSH was the major form of alpha-MSH. The profile of alpha-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-alpha-MSH and diacetyl-alpha-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of alpha-MSH predominate in PI tissue, while nonacetylated alpha-MSH is the major form in AL tissue. It appears, however, that acetylation of alpha-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.     

Biosynthesis and characterization of adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone, and an NH2-terminal fragment of the adrenocorticotropic hormone/beta-lipotropin precursor from rat pars intermedia.

Isolated intermediate lobe cells from 40 rat pituitaries were incubated for 3 h with [35S]methionine + [3H]-phenylalanine, [35S]methionine, [3H]valine, and [3H]leucine. The cell extracts were purified by carboxymethyl-cellulose chromatography (CMC) and the fraction eluting with ovine adrenocorticotropic hormone (ACTH) was further purified either by another CMC under the same conditions or by high performance liquid chromatography (HPLC). Microsequencing of the product from the second CMC allowed the identification of a peptide containing methionine 4 and phenylalanine 7, as expected for the NH2 terminus of ACTH. Purification by HPLC of a similar peptide obtained from the three other incubations gave three main raoactive peaks which were further characterized by their migration rates on polyacrylamide gels, molecular weight, and microsequencing. Results indicated that intact ACTH (residues 1-39) is present in extracts of rat intermediate lobe, but in very small quantities (less than 1% of the beta-endorphin content). ACTH is probably broken down into smaller fragments, e.g. alpha-melanocyte-stimulating hormone (alpha-MSH) (ACTH, 1-13) and corticotropin-like intermediate lobe peptide (CLIP) (ACTH, 18-39). These studies also revealed with existence of a peptide having identical sequence with the (N-1) terminus of the ACTH/lipotropin (LPH) precursor.     

TRH stimulates the release of POMC-derived peptides from goldfish melanotropes

The release of immunoreactive (ir) alpha-MSH and ir ACTH from goldfish (Carassius auratus) melanotropes was investigated using superfused isolated dispersed neurointermediate lobe cell columns. Stimulation of neurointermediate lobe cell columns with pulses of TRH evoked dose-dependent increases in the concomitant release of ir alpha-MSH and ir ACTH. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to characterize the alpha-MSH and ACTH immunoreactivities released from a neurointermediate cell column under spontaneous release conditions. Six peaks of ir alpha-MSH were revealed. Three of these peaks were identified as des-acetyl alpha-MSH, mono-acetyl alpha-MSH and di-acetyl alpha-MSH. Seven peaks of ir ACTH were revealed. Four of these peaks were tentatively identified as ACTH variants. These studies suggest that TRH stimulates the release of peptide hormones from teleost melanotropes and that the goldfish neurointermediate lobe in vitro releases numerous peptides derived from POMC.     

Hypnogenic effects of des-acetyl-alpha-MSH and CLIP (ACTH 18-39) in the rat

In the rat, the intraventricular administration of ACTH (adrenocorticotropic hormone) has no effect upon the sleep-waking cycle. However, administration of two ACTH-derived peptides is followed by a selective and significant increase of each sleep state: Des-alpha-MSH (Des-acetyl-alpha-melanocyte stimulating hormone) administration induces an increase of slow wave sleep, while CLIP (corticotropin-like intermediate lobe peptide) is followed by an increase of paradoxical sleep.     

Characterisation of N-terminally extended met-enkephalin Arg6Gly7Leu8 variants in the porcine upper digestive tract

Proenkephalin A-derived peptides are known to occur in the gut, but their precise identity is uncertain. We report here the isolation of N-terminally extended forms of Met-enkephalin Arg6Gly7Leu8 from porcine upper digestive tract monitored by radioimmunoassay. A single major form was identified in pyloric antral muscle and mucosa, but in the duodenum two major forms were detected. Microsequence analysis together with immunological data revealed that the antral mucosal peptide and the most acidic duodenal peptide had identical amino-acid sequences, corresponding to a 5.3 kDa peptide terminating in Met-enkephalin Arg6Gly7Leu8. The data indicate that high-molecular-weight peptides may constitute a major proportion of gut opioid peptide immunoactivity.     

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).     

Purification and Characterisation of Cerebellins from Human and Porcine Cerebellum

The primary human and porcine structure of the novel neuropeptide cerebellin is unknown. These peptides were, therefore, isolated by a combination of ion-exchange and reverse-phase chromatography using a specific radioimmunoassay against rat cerebellin. The sequences of the peptides were deduced by mass spectrometry (for both human and porcine cerebellins) and gas-phase Edman degradation (for porcine cerebellin). In both species, two molecular forms were identified. In the human, the major form corresponded to the pentadecamer [des-Ser1]-cerebellin (approximately 95% of the total) and the minor form, to the hexadecamer peptide. In the pig, however, both molecular forms were present in approximately equal amounts. The finding that the sequences of human and porcine cerebellin are identical to that of the rat suggests that strong evolutionary pressure has acted to conserve this sequence.     

The immunolocalization of ACTH and alpha MSH in human and rat pituitaries.

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.     

Evidence for gonadotropin-releasing hormone peptides in the ovary and testis of rainbow trout.

GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA is present also in the ovary and testis. However, isolation of the peptide from testis has not been reported. We used HPLC and specific RIAs to determine whether the GnRH peptide can be detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH that are present in the ovary and testis. Extracts of immature and mature ovarian and testicular tissue were examined from 17- to 21-mo-old rainbow trout (Oncorhynchus mykiss). For the first time, GnRH peptides were isolated from testis and identified by HPLC-RIA with specific antisera and by elution position compared with synthetic standards. GnRH peptides were also present in the ovary. In addition, multiple forms of GnRH, including a form not normally detected in the brain of trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages in the gonads, which may explain the inability to detect and isolate the GnRH peptides from gonads in earlier studies.     

Removal of Arg1 and Phe22 from CLIP (ACTH18-39) by rodent pituitary and blood peptidases

The major product on incubation of CLIP (ACTH18-39) with rat and mouse serum, rat plasma and whole blood, and soluble extracts of rat pituitary is [des-Arg1]-CLIP (ACTH19-39) while [des-Phe22]-CLIP (ACTH18-38) is the major product with pituitary particulate fraction. In both cases, p-chloromercuribenzoate-sensitive, metal-dependent peptidase activity appears to be responsible for the cleavage. The serum enzyme may be related to proline aminopeptidase. Material coeluting with [des-Arg]-CLIP on two HPLC solvent gradients is present in the superfusion media from neurointermediate lobes of genetically obese (ob/ob) mice but is not present in acid extracts of the lobe. This suggests that postsecretory processing of CLIP may involve removal of the N-terminal Arg residue.     

Pro-opiomelanocortin-derived peptides in the pig pituitary: alpha- and gamma 1-melanocyte-stimulating hormones and their glycine-extended forms

Pro-opiomelanocortin (POMC)-related peptides in extracts of anterior and neurointermediate pituitary lobes from pigs were characterized by gel chromatography, reversed-phase chromatography and radioimmunoassays. The peptide content was ca. 3-fold greater in the anterior lobe compared to the neurointermediate lobe (19.8 nmol POMC/anterior lobe vs 7.0 nmol/neurointermediate lobe). In the neurointermediate lobe 93% of POMC was processed to alpha-melanocyte-stimulating hormone (alpha-MSH) and analogs exclusively of low molecular weight. Most of the remaining adrenocorticotropic hormone (ACTH)-related material consisted of the glycine-extended intermediate ACTH-(1-14) and analogs. In contrast only one fourth to one third of the N-terminal part of POMC (N-POMC) was processed to amidated gamma-MSH and its C-terminal glycine-extended precursor. The relative amount of amidated gamma-MSH was the same as alpha-MSH and analogs (94%). However, more than 95% of these peptides were of high molecular weight. In the anterior lobe 2.3% of N-POMC was processed and 94% was amidated gamma-MSH of only high molecular weight. These results show that gamma-MSH and alpha-MSH are amidated to the same extent and that gamma 1-MSH and gamma 2-MSH immunoreactivity are present in both the anterior lobe and the neurointermediate lobe. The results suggest that the production of amidated peptides is not regulated by the amidation process itself but at an earlier step (e.g. at the proteolytic cleavage).     

alpha-MSH analogues and adrenal zona glomerulosa function

Recent studies on the control of adrenal zona glomerulosa function and aldosterone secretion have focussed attention on the role of MSH-like peptides. In particular, at low concentrations, alpha-MSH has a specific stimulatory effect on rat adrenal glomerulosa cells. The synthesis of alpha-MSH analogues which have potent and prolonged effects on melanocyte systems offers new methods of examining the specificity of this response. Two peptides were tested in which potential for a beta-turn configuration was stabilised. These were: [Nle4, D-Phe7]-alpha-MSH and the cyclic [Cys4, Cys10]-alpha-MSH. In contrast to their effects on melanocyte systems, only [Cys4, Cys10]-alpha-MSH stimulated glomerulosa cells, and it was equipotent with alpha-MSH, while [Nle4, D-Phe7]-alpha-MSH and shorter fragments had no effect when added alone. [Nle4, D-Phe7]-alpha-MSH, however, augmented the response of cells already maximally stimulated with alpha-MSH and in this respect its actions resembled those of gamma-MSH and related peptides. The augmentation produced by [Nle4, D-Phe7]-alpha-MSH and gamma 3-MSH was not additive when the two peptides were added together with alpha-MSH. The results suggest that the specificity of the alpha-MSH receptors in rat adrenal glomerulosa cells and the peptide structure-function relationships in this system are different from those described for melanocytes.     

Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.     

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.