Identification of a novel mammalian post-translational modification, phosphocholine, on placental secretory polypeptides

Abstract

Placental neurokinin B appears to be post-translationally modified by phosphocholine (PC) attached to the aspartyl side chain at residue 4 of the mature peptide. Corticotrophin releasing factor (CRF) was found to be expressed by the rat placenta with the main secreted forms being phosphocholinated proCRF+/- one or two polysaccharide moieties. A combination of high-pressure liquid chromatography (HPLC) and two-site immunometric analysis suggested that PC was also attached to the placental precursors of adrenocorticotrophin, hemokinin, activin and follistatin. However, the fully processed forms of rat placental activin and CRF were free of PC. Formerly, the parasitic filarial nematodes have used PC as a post-translational modification, attached via the polysaccharide moiety of certain secretory glycoproteins to attenuate the host immune system allowing parasite survival, but it is the PC group itself which endows the carrier with the biological activity. The fact that treatment of proCRF peptides with phospholipase C but not endoglycosidase destroyed PC immunoreactivity suggested a simpler mode of attachment of PC to placental peptides than that used by nematodes. Thus, it is possible that by analogy the placenta uses its secreted phosphocholinated hormones to modulate the mother's immune system and help protect the placenta from rejection.

Figure 1

(a) Detection of proCRF, PC-containing proCRF and PC/PC-containing material in rat (day 20 of pregnancy) and human (term) placental and rat brain extracts. Open histograms are the ELISA results from the proCRF/CRF two-site immunometric assay, black proCRF/PC assay and grey PC/PC assay. Data plotted are the means of duplicate determinations. (b) Diagram depicts the structure of proCRF and the epitopes of the antibodies used in this study.

Figure 2

Affinity purified rat (a) placenta and (b) plasma CRF-immunoreactive material subjected to SDS-PAGE. Western tracks were immunobloted separately with either anti-PC-, anti-CRF- or anti-proCRF- IgGs (Fig. 1b).

Figure 3

SDS-PAGE of selected fractions resulting from the preparative C3 HPLC of a rat placental extract. Coomassie blue-stained gel is shown in (a), the anti-PC IgG western immunoblot in (b) and the merged image in (c) in which only a limited number of coomassie stained bands are PC immunoreactive.

Figure 4

Comparison of PC containing neuropeptide with neuropeptide immunoreactivity in fractions from the HPLC of the rat placental extract. Fractions 30–65 from the preparative HPLC step in Fig. 3 were submitted to a number of two-site ELISAs. –◊– represents two-site anti-neuropeptide/anti-neuropeptide assay results in each case. All ELISA protocols were the same as described in Materials and methods; (a) proCRF, in (b) NKB, in (c) activin A and (d) hemokinin. –•– represents the results of two-site immunometric assays with the anti-neuropeptide antibody being in (a) anti-proCRF (1–21) IgG, (b) anti-NKB(1–5) IgG, (c) anti-proβA subunit IgG and (d) anti-HK(1–5) IgG in combination with anti-PC IgG in each case.

Figure 5

The effect of enzyme treatment on the immunoreactivity of pro-CRF-PC and pro-βA-PC. Peak one represents a pool of fractions 36–37 and peak two, a pool of fractions 41–43 from Fig. 4, which were freeze-dried separately before being reconstituted in assay buffer. Open histogram represents ELISA assay result with no enzyme treatment, black histogram after phospholipase C digestion and grey histogram after endoglycosidase digestion. Values are the means of duplicate determinations in the respective two-site immunometric assay.