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1 Discovery of Serpinin Peptides: An Historical Prospective
Serpinin is a 26 amino acid peptide derived from proteolytic cleavage of the
penultimate and last pair of basic residues at the C-terminus of chromogranin A
(CgA). This peptide was discovered through studies on the role of CgA in secretory
granule biogenesis using the mouse pituitary endocrine cell line, AtT20 as a model
system (see section below). We first found that expression of bovine CgA in 6T3
cells, a variant of the AtT20 cells, which lacks CgA and dense core secretory gran-
ules (DCG), induced DCG biogenesis and restored regulated secretion in these cells
(Kim et al. 2001 ). Furthermore, CgA-knockout mice (Mahapatra et al. 2005 ) and
CgA-antisense transgenic mice (Kim et al. 2005 ) had significantly decreased num-
bers and size of DCG in chromaffin cells in the adrenal medulla, compared to wild
type mice. These studies indicated a major role of CgA in granule biogenesis. To
understand the mechanism of action of CgA in granule biogenesis, a DNA microar-
ray analysis was carried out on 6T3 cells with and without CgA transfection. Results
indicated a significant increase in mRNA encoding a serine protease inhibitor (in the
serpin family), protease Nexin-1 (PN-1) in cells transfected with CgA. PN-1 was
localized to the Golgi apparatus and stabilized the degradation of DCG proteins
required for DCG biogenesis (Kim and Loh 2006 ). We hypothesized that the increase
in PN-1 expression was mediated by secreted CgA or a smaller processed fragment
of it. We then synthesized and tested several peptide fragments of CgA based on
predicted paired basic residue cleavage sites. A peptide of <3 kD from the C-terminus
of CgA was found to increase PN-1 mRNA expression when added to the medium
of 6T3 cells. Concomitantly, a<3 kD fraction from conditioned media of AtT20 cells
was found to have the same action. The active fragment from conditioned medium of
AtT20 cells was then analyzed by high performance liquid chromatography (HPLC)
and enzyme immunoassay (EIA) using an anti-serpinin polyclonal antibody which
we developed to detect serpinin (Koshimizu et al. 2011b). Subsequently, the serpinin
immunoreactive fractions from the HPLC were subjected to matrix assisted laser
desorption/ionization time of flight (MALDI- TOF) analysis to identify the peptides.
A peptide identical to the sequence of the 26 amino acid synthetic peptide shown to
upregulate PN-1 expression was identified. Hence, this endogenous peptide was
named serpinin. Subsequent studies showed that serpinin was secreted by AtT20
cells and there are two additional forms, pGlu- serpinin and serpinin-RRG. In this
review we describe the tissue and cellular distribution of the serpinin peptides and
the distinct biological functions of the different members of this peptide family.
2 Expression and Characterization of Serpinin Peptides
in AtT20 Cells
Western blot analysis using an antibody that cross reacts with both CgA and ser-
pinin indicate that AtT20, a mouse cell line express a serpinin size immunoreactive
peptide, as well as CgA with an apparent molecular weight of 40 kD (Fig. 1A).
Y. PengfiLoh et al.