Autoimmunity in Scleroderma
CENP-C and B-cell Epitopes
CENP-C is an essential component of the
inner plate of the trilaminar structure of
the kinetocore. It is required for main-
taining proper kinetochore size and is
implicated in the metaphase-to-anaphase
transition in mitotic cells by stabilizing
microtubule attachments. It also has a
DNA binding domain and a region that
contributes to the formation of the ho-
modimer as described above for CENP-B.
CENP-C has 943 amino acids and an
apparent molecular mass of 140 kDa in
SDS-PAGE, quite larger than the molec-
ular weight derived from the sequence.
Autoepitopes of CENP-C were mainly re-
stricted to three regions that correspond
to functional domains (Fig. 4). Epitope 1,
the ±rst major epitope, resides at the N-
terminus (74–244 a.a.) that corresponds
to the presumptive instability domain re-
ported by
in vivo
analysis. Epitope 7 located
at the central part (396–552 a.a.) con-
tains the DNA binding domain, which
was recently shown to bind the alphoid
DNA selectively – the CENP-C region be-
tween amino acids 410 and 537. Together
with CENP-B, CENP-C associates with
the centromeric alphoid DNA
in vivo
de±nes a highly structured organization
within the human centromere. Epitope 9,
the other major epitope, locates in the
C-terminal region that contributes to the
formation of the homodimer. This epitope
spanning a 124 amino acid stretch from
476 to 599 is the second major epitope.
Autoimmunity to this region was quite
By immunoblotting, the reactivities be-
tween monomer and homodimer formed
by chemical cross-linking were compared.
The reactivities as well as the frequen-
cies of the antibodies against the dimer
form were obviously higher in both IgG
and IgM responses. Interestingly, GroEL
protein, which is a heat-shock chaperon
protein of
E. coli
, was copuri±ed with the C-
terminal region. When puri±ed antibodies
against the dimer in a liquid phase contain-
ing GroEL were applied to various assays
to detect ACA, they did not react to the
recombinants but very weakly recognized
cellular CENP-C by immunoblotting. Fur-
thermore, they did show centromere stain-
ing in IIF analysis. These results suggest
that these puri±ed antibodies recognize
predominantly native forms of centromere
structures. Therefore, there are assumed
537 584
Instability domain
binding domain
binding domain
Fig. 4
Functional domains and autoepitopes of CENP-C. The top
rectangle represents the full-length human CENP-C antigen. Major
autoepitope, which is recognized by more than 50% of ACA, is shown as
a black bar. Minor epitope, which is recognized by less than 50% of ACA,
is shown as a white bar. For the epitope at the C-terminus, the dimer
form is recognized by around 50% of ACA (see text).
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