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domains, one activity of FKBP proteins is to accelerate
cis-trans isomerization of Xaa (amino acid)-proline pep-
tide bonds in newly synthesized proteins. This enzymatic
activity is an important step in the energy demanding
process of protein folding and thus is necessary for the
synthesis and trafficking of extracellular matrix proteins.
FKBP65 was first identified in mouse NIH3T3 cells.
9
It has ten coding exons, proposed alternate transcripts and
encodes a 582 amino acid protein. The molecular structure
contains four PPIase-FKBP type domains, two EF hand
domains, two potential calcium-binding domains and
a HEEL motif (an ER retention sequence) (
Figure 15.1
).
Though very early work speculated that it had a cytoplas-
mic role, it was shown to be a luminal rough endoplasmic
reticulum resident protein. It was then shown to co-local-
ize with tropoelastin, a proline-rich extracellular molecule,
and it was suggested that the PPIase activity of FKBP65
was important to the folding of the molecule, especially
to prevent formation of intracellular aggregrates.
10
In a
lung injury model of disease, Patterson and colleagues
showed that induction of FKBP65 expression coincided
with increased expression of tropoelastin and type I col-
lagen expression.
11
Zeng et al. showed that FKBP65 could
be isolated from rER from chick embyros using a gelatin-
Sepharose column indicating some involvement in the
biosynthesis of procollagens (type III) as well as showing
that one of the four FKBP domains had peptidyl-prolyl
cis-trans isomerase activity.
12
The role of ER-resident chaperones is fundamen-
tal to the biosynthesis and secretion of collagens. There
are numerous rough ER proteins involved in post-
translational modification of the growing polypeptide
chains of procollagen. One of the first steps includes
proline residues undergoing 4-hydroxylation by prolyl
4-hydroxlase, which ultimately stabilizes the triple helix.
Numerous ER chaperones are involved in preventing pre-
mature chain association such as PDI, BiP/GRP78, GRP94
and HSP47. Additional modifications are made to proline
and lysine residues including hydroxylation and glyco-
sylation. Trimers of selected chains form by association
of C-terminal propeptides. Disulfide bonds form between
the chains and this is likely mediated by protein-disul-
fide isomerase (PDI) and the chains wind from the C- to
N-terminus. The rate-limiting step involved in this wind-
ing is cis-trans isomerization of peptide bonds and this
can be catalyzed by both cyclophilins and FKBPs.
Appreciation of the complexity of the formation and
secretion of stable procollagen molecules has identi-
ied numerous collagen-specific and nonspecific chap-
erones. Procollagen molecules are unstable and easily
form aggregates, and this needs to be prevented within
the cell. Extracellularly, procollagen forms insoluble
complexes that provide strength to tissues. Thus, iden-
tification of specific chaperones for procollagen biosyn-
thesis is not unexpected. HSP47 is considered a specific
type I procollagen chaperone,
13
and loss of HSP47 leads
to type I procollagen aggregates
14
and osteogenesis
imperfecta (OI) in humans and dachshunds.
15,16
Zeng
et al.
12
showed, using a gelatin-Sepharose column, that
when rER from chick embryos is eluted with a low pH
buffer, the two most abundant proteins recovered are
HSP47 and FKBP65, suggesting perhaps that they have
similar functions. Further work by Ishikawa et al.
17
established that FKBP65 interacts with both folded and
HEEL
FKBP65
Ca
Ca
PPiase
PPiase
PPiase
PPiase
EF handEF hand
62-150
174-262
286-1374
399-486
497-532
542-577
579
amino acids
PPiase - peptidyl cis/trans isomerase
domain
EF hand - calcium binding domain
Ca - calcium
HEEL - endoplasmic reticulum retention
sequence
FIGURE 15.1
Cartoon of FKBP65 showing the different domains of the molecule.
