First Page (Title Page)
Title
Peptidylarginine deiminase type 4, anti-citrullinated peptide antibodies, and
rheumatoid arthritis
Author
Ryo Yamada
Affiliation
Laboratory for Rheumatic Diseases, SNP Research Center, The Physical and Chemical
Research Institute (RIKEN), Yokohama, Japan
Supported by The Japanese Millennium Project
Corresponding Author
Ryo Yamada
1-7-22 Suehirocho, Tsurumiku, Yokohama, Kanagawa 230-0045, Japan
Tel: +81-45-503-9569
Fax: +81-45-503-9590
Email: ryamada@src.riken.go.jp
Second Page
Abstract
Anti-citrullinated peptide antibodies seem to be one of the most clinically reliable
serologic markers for rheumatoid arthritis (RA). A genetic approach revealed that one
of the citrullinating enzymes has a RA-susceptible variant. Peptidyl citrullination
alters the chemical character of peptides and subsequently their antigenicity as well.
This change in antigenicity of self-peptides seems to invoke citrulline-related
autoimmunity. Although the precise physiologic role of citrullination is still unknown,
accumulating data indicates that citrullination has a definite role in biologic
phenomena, along with other post-translational protein modifications, such as
methylation and phosphorylation. In RA synovial tissue, two of five PADI isotypes are
known to be expressed and their expression is regulated at multiple steps:
transcription, translation, intra-cellular localization, and activation/inactivation of
PADI proteins. Further investigations on citrulline and PADIs from various aspects
will provide a more profound understanding of RA-related autoimmunity.
Key Words
(1) Rheumatoid arthritis
(2) Citrullination
(3) Anti-CCP antibody
(4) PADI
Take-Home messages
(1) Anti-citrullinated peptide antibodies are the most reliable autoantibody for
rheumatoid arthritis.
(2) Peptidylarginine deiminase (PADI) enzymes produce peptidyl citrulline by
deimination/demethylimination of arginine/methylarginine residues in proteins.
(3) The physiologic role of peptidyl citrullination is still unknown.
(4) PADI2 and PADI4 and citrullinated proteins are present in synovial tissue from
patients with rheumatoid arthritis.
(5) SNP-based whole genome surveys have identified rheumatoid arthritis-associated
variants in PADI4.
(6) Enzymatic activity of PADIs takes place over several steps: transcription,
translation intracellular localization, and Ca2+-dependent enzyme activation.
1. Introduction
Rheumatoid arthritis (RA) is one of the most common human systemic autoimmune
diseases and is characterized by autoantibody production and synovial inflammation.
The fact that autoantibody production against citrullinated proteins is highly specific
to RA (1) and that peptidylarginine deiminase type 4 (PADI4), a gene encoding one of
the citrullinating enzymes, is associated with RA (2), leads us to believe that
citrullination by PADI is a fundamental phenomenon in RA. This paper reviews the
role of anti-citrullinated peptide antibody, citrullination, and PADI in RA.
2. Anti-citrullinated peptide antibodies in RA
Although various autoantibodies can be detected in the sera of patients with
RA, several of these autoantibodies have been reported to be more specific and to have
higher positive predictive value for RA than others. Many highly RA-specific
autoantibodies, such as antiperinuclear factor (3), anti-keratin antibody (4-6), and
anti-Sa antibody (7) have been found to recognize citrullinated peptides (8-12) Based on
these findings, an enzyme-liked immunosorbent assay, recognizing anti-cyclic
citrullinated peptide antibody, was developed. Clinical evaluations of this assay have
shown it is one of the best diagnostic tests in rheumatology clinics (Table 1). (13) (14)
(15) (16) (17) This assay is not only very specific for RA (up to 98%), but the production
of these autoantibodies can be detected very early in the disease—even several years
before disease onset—and titre values tend to correlate with an erosive subtype of RA.
(10, 13-19)
3. Citrulline and peptidyl arginine deiminase enzymes
3.1 Citrulline and arginine
Citrulline is a non-coding native amino acid. It is a deiminated form of
arginine. The biggest difference between arginine and citrulline is that arginine is one
of the most basic amino acids and citrulline lacks the charged feature. The enzyme
responsible for the conversion of peptidyl arginine to peptidyl citrulline is
peptidylarginine deiminase (PADI) (20). Although some biological events, such as
inflammation, apoptosis, trauma, aging, and gene expression regulation by histones
have been reported to be associated with post-translational citrullination (21, 22), the
precise physiologic role of citrullination is still unknown (23-26). Although the
physiologic role of citrulline in peptides is not clear, what is known about citrullination
and its consequences is that various proteins are citrullinated with subsequent changes
in their conformation that lead to breakdowns in immunologic tolerance (18).
3.2 Enzymatic reaction and isozymes of PADIs
PADI enzymes catalyze the conversion of arginine residues to citrulline
residues in proteins. Five isotypes of PADI—PADI1, 2, 3, 4, and 6—have been cloned
from several mammals, including humans (20). All these isotypes possess a Ca-binding
motif, and they depend on high concentrations of Ca2+ for their enzymatic activity. The
tissue distribution of the isotypes of PADIs varies. PADI2 and PADI4 were reported to
be expressed in synovial tissue and intranuclear localization seems to characterize
PADI4 (20).
3.3 PADIs in RA synovial tissue
3.3.1
PADI2 and PADI4
PADI2 and PADI4 were reported to be present in RA synovial tissue. As
anticipated from the fact that PADI4 has an NLS but PADI2 does not, PADI4 was
detected in the nucleus (20) and cytoplasm and PADI2 was detected only in cytoplasm.
SNP-based whole genome surveys have identified RA-associated variants in PADI4
(27). Increases in PADI4 activity due to polymorphisms in the gene seem to accelerate
RA development. (18) Although one study from the United Kingdom did not report
statistically significant results (28), it was not contradictory to the original study (2)
and the association was replicated in another Japanese study (29). A genetic
contribution to RA of PADI4 variants, but not PADI2 variants, seems to be true, at
least for the Japanese population, for the following reasons: all five PADI genes,
including PADI2 and PADI4, cluster in chromosome 1p; genetic analysis discriminates
PADI4 from PADI2; and the association with RA was only detected in PADI4 but not
in PADI2.
3.3.2
Control of expression and activation of PADI2 and PADI4
PADI2 and PADI4 have different profiles of tissue distribution. PADI2 is
relatively ubiquitously expressed and PADI4 is more specifically expressed in
leukocytes (27) The differences in tissue distribution, transcription, and translation of
PADI2 and PADI4 are also regulated separately. Depending on cell lineages and their
stages of differentiation, transcription and translation are regulated differently (30).
Moreover, enzymes of PADI2 and PADI4 are inactivated in steady state. Because a
much higher concentration of intracellular Ca2+ is required for PADIs to be active as
enzymes, regulation of Ca2+ seems to play a pivotal role in the regulation of PADI
enzyme activation. A structural study of PADI4 revealed that binding of Ca2+ to PADI4
molecules produced catalytic sites in homodimers (31). Figure 1 illustrates this
multi-step regulation of PADI activity.
4 Summaries
Studies on RA-specific autoantibodies have revealed that autoimmunity against
citrulline-containing self-peptides is highly specific to RA and measurement of the
quantity of anti-CCP, one of the anti-citrullinated peptide antibodies, is useful and
reliable in rheumatology clinics. Along with the immunologic findings on citrullination,
a genetic approach has identified a genetic connection between RA and PADI4, a
specific gene encoding one of the citrullinating enzymes. Two mutually independent
approaches highlighted citrulline in self-proteins as a breakthrough for understanding
the precise pathophysiology of RA. For the last few years, new insights on PADIs have
been presented, such as the crystal structure of PADI4 enzyme (31) and involvement of
citrullination in a physiologic role in histone-related gene expression regulation (32, 33),
along with expressional evidence on PADI isozymes, as well as validating data on the
use of anti-CCP antibodies in clinics (16) (17). Further investigations on citrulline and
PADIs from various aspects will provide a more profound understanding of RA-related
autoimmunity.
Acknowledgment
We thank all the members of the Laboratory for Rheumatic Diseases, SRC, RIKEN,
particularly Ms. K. Komakine for providing illustrations.
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