Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 31 (2), 284-90

Isolation, Identification and Biological Activity of Gastrin-Releasing Peptide 1-46 (oGRP 1-46), the Primary GRP Gene-Derived Peptide Product of the Pregnant Ovine Endometrium

Affiliations

Isolation, Identification and Biological Activity of Gastrin-Releasing Peptide 1-46 (oGRP 1-46), the Primary GRP Gene-Derived Peptide Product of the Pregnant Ovine Endometrium

A S Giraud et al. Peptides.

Abstract

We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP 1-46, the primary product of this gene in the pregnant endometrium. Full thickness 125-140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 muBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP 1-46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP(1-46) would require preferential cleavage at the Glu-Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP(18-27) and GRP(1-27) in other tissues. GRP 1-46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP 1-46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP 1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.

Figures

Fig. 1
Fig. 1
(A) Gel filtration chromatography of protein extract of pregnant ovine endometrium. Open circles indicate the total protein elution profile (A280nM) while filled rectangles show the immunoreactive GRP profile. The elution positions of human (h) GRP18-27 and hGRP1-27 as well as oGRP1-46 are indicated. (B) Reverse-phase C18 chromatography (μBondpak) of pooled fractions 40 and 41 from gel filtration step. The immunoreactive profile is indicated by the open bars with the peak fraction used subsequently for Maldi-TOF indicated with an arrow. Total protein is shown by the solid line.
Fig. 1
Fig. 1
(A) Gel filtration chromatography of protein extract of pregnant ovine endometrium. Open circles indicate the total protein elution profile (A280nM) while filled rectangles show the immunoreactive GRP profile. The elution positions of human (h) GRP18-27 and hGRP1-27 as well as oGRP1-46 are indicated. (B) Reverse-phase C18 chromatography (μBondpak) of pooled fractions 40 and 41 from gel filtration step. The immunoreactive profile is indicated by the open bars with the peak fraction used subsequently for Maldi-TOF indicated with an arrow. Total protein is shown by the solid line.
Fig. 2
Fig. 2
Mass spectroscopy of the RP-HPLC purified peptide on the Maldi MS spectrum with major mass ion of 4963 and minor ions at 4934, 4737 and 5023 Da The most abundant mass ion corresponds to GRP1-46 (APVTAGRAGALAKMYTRGNHWA VGHLMGKKSVAESPQLREEESLKE).
Fig. 3
Fig. 3
Western blot of the partially purified material after reverse-phase chromatograpy of pregnant ovine endometrium extract. Immunoreactive GRP has a mass of 5–6 kDal with a slightly smaller and faster migrating form also being apparent. Elution position of the oGRP1-27 standard (1 μg) is shown for comparison.
Fig. 4
Fig. 4
Comparison of processing events required to produce GRP1-46 or GRP18-27, 1-27 or GRP18-27gly, GRP1-27gly from the proGRP precursor.

Similar articles

See all similar articles

Cited by 1 article

Publication types

MeSH terms

Feedback