217purified and sequenced serpinin from mouse. Of note is the sequence of bovine and
human serpinin differs by 4 amino acids from mouse (see Fig. 2C). A bovine CgA
fragment consistent with cleavage at the RR↓P site (Fig. 2C) and liberating serpinin
has been reported (Wohlfarter et al. 1988 ). However, cleavage at that site to liberate
human serpinin remains to be determined.
3 Expression and Secretion of pGlu-Serpinin in AtT20 Cells
Examination of the cellular localization of pGlu-serpinin using a specific antibody
showed that this peptide is expressed in AtT20 cells, but not in 6T3 cells lacking
CgA (Fig. 3B; Koshimizu et al. 2011b). pGlu-serpinin was localized primarily in
the tips of the processes and co-localized with ACTH in AtT20 cells (Fig. 3A;
Koshimizu et al. 2011b). This is not surprising since pyro-glutamination is a late
step in the biosynthetic pathway and therefore this peptide resides mainly in
Fig. 3 Analysis of pGlu-serpinin in AtT20 cells. (A) Immunocytochemical staining of pGlu-
serpinin- immunoreactivity (IR) in AtT20 cells. The punctate staining of pGlu-serpinin-IR co-
localized with ACTH accumulated at the tips of the processes. Note the absence of pGlu-serpinin
staining when the antibody was pre-absorbed with the pGlu-serpinin peptide. (B) Enzyme immu-
noassay of pGlu-serpinin-IR in 6T3-AtT20 cells devoid of chromogranin A (CgA) and WT AtT20
cells (top panel). (B, lower panel) pGlu-serpinin-IR was also detected in non-stimulated culture
media (2 h. basal) of AtT20 cells and was secreted in a stimulated manner by high potassium
membrane depolarization (10 min stimulation) (From Koshimizu et al. 2011b)
Serpinin Peptides: Tissue Distribution and Functions