Isolation and primary structure of novel neurointermediate pituitary peptides derived from the C-terminal of the rat vasopressin-neurophysin precursor (propressophysin)

Abstract

Four novel peptides were isolated from rat neurointermediate lobes by gel filtration and high-pressure liquid chromatography. Analyses of amino acid composition and sequence showed that all four peptides were derived from the C-terminal portion of propressophysin (CPP); they were identified as the glycopeptides CPP 1 -19, CPP 1 -20, CPP 22 -37 and CPP 22 -39. Processing of CPP 1 -39 could thus produce the four isolated peptides by specific post-Arg or post-Leu cleavages.Arginine vasopressin in the posterior lobe of the pituitary gland is produced by magnocellular neurons of the hypothalamus. It is synthesized together with neurophysin and a glycopeptide (CPP) in the form of a precursor called propressophysin [l-41. Part of this glycopeptide was first encountered in a posterior pituitary extract by Holwerda [5, 61 and later isolated as a 39-amino-acid peptide from porcine, bovine, sheep, rat, and human pituitaries [7-91. Based on size and tissue distribution, it has been proposed by Seidah et al. [8] to be a fragment of propressophysin. Analysis of cDNA and genes encoding propressophysin has elucidated the composition of propressophysin and confirmed the hypothesis that the glycopeptide region is at the C-terminus of the precursor [lo -121; it has therefore been termed the C-terminal peptide of propressophysin, hence CPP [13]. No biological function of this glycopeptide is known. While searching for novel peptides in rat neurointermediate lobe extracts, we detected considerable amounts of CPP-derived peptides. Here we report the isolation and structure of a series of related CPP fragments. The structure indicates processing sites in propressophysin including Arg-Xaa and Leu-Xaa bonds. Although we cannot exclude the artifactual formation of these peptides, it is suggested that these peptides are endogenous forms of CPP, possibly having biological activities for central or peripheral functions. METHODS ExtractionNeurointermediate lobes from 200 male Wistar rats were dissected immediately after death and stored at -80 "C until extracted by boiling in 3 M acetic acid and ultrasonic disruption at 0°C as described previously [14]. ChromatographyThe neurointermediate lobe extract was subjected to gel filtration on a column (104x 1.6 cm) of Sephadex G50 superfine eluted in 50% (v/v) acetic acid at 4 ml/h. Fractions were pooled according to molecular mass range guided by the distribution of endorphins of the fractions as in [14].Pooled gel filtration fractions were dried in vacuo and subjected to HPLC. The first step involved reverse-phase HPLC on a pBondapak CIS column eluted by a linear gradient running from 30% to 75% acidified methanol (0.15%, vjv, acetic acid) in 10 mM ammonium acetate, pH 4.15, over 45 min at 2.0 ml/min [15]. For final purification in some cases (see Results) paired-ion reverse-phase HPLC was used. The system employed a linear gradient of 0.10% (v/v) heptafluorobutyric acid in methanol and 0.1 3% heptafluorobutyric acid in water at 1.5 ml/min [14]. The eluate was monitored by absorbance at 210 nm...

Mass spectrometric charting of bovine posterior/intermediate pituitary peptides.

“…Full-length CPP has been reported to stimulate the release of prolactin (40). Biological activity of the CPP fragments may also be anticipated because their in vivo biosynthesis has recently been shown for the rat (41,42) [CPP (1-19/20) and CPP (22-37/39)]. By analogy, the bovine counterparts found in this study are likely to be processing products rather than artifacts of the isolation procedure.…”

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization

“…This type of cellular processing has been implicated in the generation of bioactive peptides such as ␣-and ␥-endorphin (4), the C-terminal glycopeptide fragment 1-19 of provasopressin (5), platelet factor 4 (6), the metalloprotease ADAM-10 (7), site 1 cleavage of the sterol regulatory element-binding proteins (SREBPs) (8), as well as in the production of the Alzheimer's amyloidogenic peptides A␤40, -42, and -43 (9). Processing of this type occurs either in the endoplasmic reticulum (ER) (8), late along the secretory pathway, within secretory granules (4,5), at the cell surface, or in endosomes (6,7,9). The proteinases responsible for these cleavages are not yet identified.…”

The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

“…This represents about 5% of the total CPP immunoreactivity. The proportion of fragments is less than that reported for a previous isolation and characterization of CPP fragments from acetic acid extracts of the rat NIL [3] which contained approximately 20% the molar ratio of fragments. The amount of radioactivity in each fraction was determined by liquid scintillation counting of one-tenth (i.e., 100/xl) of each sample, c: The immunoreactivity of each fraction was determined by RIA for CPP using antiserum M as described in the Method section, d: Radioactivity in each fraction after a similar experiment five weeks after injection of 25 /xCi of3H-fucose.…”

“…The amount of labelled N-terminal fragment increased slowly to reach a maximum of about 5% after 5 weeks which equalled the level of C-terminal fragment immunoreactivity found in the tissue. The amount (up to 5%) was less than the recovery of the CPP fragments purified from large batches of NIL extracted by a different procedure [3]. However, the nature of the fragments formed in viva is similar to those found in these previous extracts of rat pituitaries.…”

“…CPP was originally encountered as a 17-amino acid fragment isolated from porcine pituitaries [10]. Subsequently, other investigators iso-lated peptides having the sequence of CPP 1 to 10 and 1 to 19 in acid acetone extracts of the pituitary [26], and acetic acid extracts of rat neurointermediate lobes (NIL) were found to contain fragments derived from the C-terminal glycopeptide [3]. These peptides included the N-terminal fragments CPP1-20 and CPPl-19, and the C-terminal fragments CPP22-39 and CPP22-37.…”