Chromogranins from Cell Biology to Physiology and Biomedicine

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Immunocytochemistry studies of AtT20 cells showed that serpinin immunoreativity
is localized in the cell body, along cell processes and at the tips of the processes
where it is co-localized with the hormone, adrenocorticotropin (ACTH), in secre-
tory granules (Fig. 1B; Koshimizu et al. 2011b). Furthermore, serpinin is secreted
in a regulated manner with high K+ stimulation from 6T3 cells transfected with
bovine CgA (Fig. 1C; Koshimizu et al. 2011a).
MALDI-TOF analysis of immunoreactive serpinin peptides in secretion medium
from AtT20 cells after HPLC purification revealed several peptides with m/z values
shown in Fig.  2. Of these, a major peptide with a mass at m/z 2864 was detected and
MS/MS fragmentation of this peptide confirmed that the amino acid composition of
this peptide matched that of mouse serpinin, and therefore AtT20 cells synthesize
and secrete serpinin. Additionally, the analysis revealed a pyro-glutaminated form
of serpinin (pGlu-serpinin). Taken together, the identification of the various peptides
found in the medium led to a proposed biosynthesis pathway for mouse serpinin

kDa

230

Serpinin ACTH Merge

180

116

66

40

12

AtT20

+ - Abs^

serpinin

M

Serpinin

Endogenous Serpinin (pg/mL)6T3-WT 6T3-bcgA Endogenous Serpinin (pg/mL)Basal HK

ND

00

500

1000

1500

250

500

750

* *

Serpinin-IR

A. B.

C.

Fig. 1 Analysis of serpinin in AtT20 cells. (A) AtT20 cell lysate analysis by Western blot for
serpinin-immunoreactivity (IR) using the ProteinSimple WES system. Serpinin peptide was added
to the lysate as a positive control (+). As a negative control, the immunostaining was eliminated by
pre-incubating the serpinin antibody with serpinin peptide as an absorption control (Abs). The
results show (lane -) the presence of serpinin as well as a serpinin-containing chromogranin pep-
tide at an apparent molecular mass of 40 kDa. M, molecular mass markers. (B) Immunocytochemical
staining of serpinin-IR in AtT20 cells. Note the punctate staining within the cell body (arrow
heads) and the co-localization with ACTH accumulated at the tips of the processes (From
Koshimizu et al. 2011b). (C) Enzyme immunoassay of serpinin-IR in media from 6T3-AtT20 cells
devoid of chromogranin A (CgA) and 6T3-AtT20 cells transfected with bovine CgA (left).
Serpinin-IR was also released from AtT20 cells in a regulated manner by high potassium mem-
brane depolarization (HK) (From Koshimizu et al. 2011a)

Serpinin Peptides: Tissue Distribution and Functions