Endogenous peptide PDCAP counteracts CAP's inhibition of enteric neural crest cell colonization in Hirschsprung disease

Hirschsprung disease (HSCR) is a severe congenital malformation characterized by the absence of the enteric nervous system (ENS) in the distal colon. This aganglionosis stems from defective colonization of enteric neural crest cells (ENCCs) during development, leading to functional obstruction. The precise molecular mechanisms underlying HSCR remain incompletely understood, limiting targeted therapeutic strategies. Current treatments primarily involve surgical resection, which can be associated with significant morbidity. Identifying novel endogenous regulators of ENCC development could pave the way for non-surgical or adjunctive interventions.

Researchers investigated the pathogenesis of HSCR, initially identifying c-Cbl-associated protein (CAP), also known as SORBS1, as upregulated in aganglionic colon tissues from children with HSCR. Functional studies explored CAP's mechanism, revealing its binding to flotillin-1 via its sorbin homology (SoHo) domain and subsequent recruitment of vinculin via its SH3 domain, which suppresses ENCC colonization. Using mass spectrometry, an endogenous PDCAP (CAP-derived peptide) was identified in these same aganglionic colon tissues. ELISA was then used to quantify PDCAP levels in diseased colon tissues of HSCR children, and its protective role was validated in Caplsl/lsl;Nestin-Cre and Ednrb-/- mouse models.

The study found that c-Cbl-associated protein (CAP) is significantly upregulated in the aganglionic colon tissues of children with Hirschsprung disease. This overexpression of CAP was shown to suppress enteric neural crest cell (ENCC) colonization by forming a complex: CAP binds the lipid raft protein flotillin-1 through its SoHo domain, which then recruits the focal adhesion protein vinculin via its SH3 domain. Mass spectrometry identified an endogenous CAP-derived peptide, PDCAP, within these same aganglionic colon tissues. Subsequent ELISA analysis revealed that PDCAP levels were reduced in the diseased colon tissues of HSCR children compared to controls. Mechanistically, PDCAP exerts a protective effect by directly competing with its precursor protein, CAP, for binding to flotillin-1. This competitive binding effectively reverses the CAP-mediated inhibition of ENCC colonization.

This protective function of PDCAP was further validated in two distinct mouse models of HSCR, Caplsl/lsl;Nestin-Cre and Ednrb-/-, where PDCAP administration promoted ENCC colonization and enhanced ENS development.