Calcium Biochemistry
139
sites, but rich in aspartic acid and as-
paragine residues. Recently, it was shown
that replacing a single aspartic acid residue
by either lysine or alanine in one of these
sites renders the protein more susceptible
to protein degradation due to the loss of
Ca
2
+
binding and abolishes the adhesive
function of the molecule.
Bone-forming cells, osteoblasts, synthe-
size and secrete a number of noncol-
lagenous Ca
2
+
binding proteins such as
osteocalcin, osteopontin, and osteonectin
that bind to bone minerals such as hydrox-
yapatite in a calcium-dependent manner.
Osteocalcin belongs to the class of Ca
2
+
binding proteins rich in the unusual amino
acid
γ
-carboxyl glutamic acid mediating
the Ca
2
+
binding property, whereas osteo-
pontin, a glycoprotein, is rich in sequences
of aspartic or glutamic acid residues re-
sponsible for Ca
2
+
binding. In addition,
both proteins contain arginine-glycine-
aspartic acid sequence domains known
to be important in mediating binding
to cell-surface receptors. Osteonectin, on
the other hand, is an extracellular Ca
2
+
binding protein that can contain at least
one EF-hand type of high-af±nity Ca
2
+
binding sites that are normally typical
for
intra
cellular Ca
2
+
binding proteins.
One interesting abnormality of this EF-
hand is the fact that it is stabilized
by an S–S bridge that normally does
not occur in EF-hand type proteins (see
below).
Similar to the intracellular Ca
2
+
bind-
ing proteins that are composed of EF-hand
domains (see below), most extracellular
proteins are assembled from a limited
number of domain structures or mod-
ules. This is well documented for the
calcium binding domains of a number
of extracellular proteins like the
γ
-carboxyl
glutamic acid–rich Gla-domain in the vi-
tamin K–dependent clotting factors, the
cadherin module, or the epidermal growth
factor (EGF)-like domain. The latter is one
of the best-studied examples and should
be described in more detail.
3.1
The Calcium Binding EGF-domain
The epidermal growth factor or EGF-like
domain is one of the most common mod-
ules identi±ed in extracellular proteins. It
plays a general role in cell–cell adhesion,
blood coagulation, and receptor–ligand in-
teraction. EGF modules contain about 40
to 45 amino acids including 6 cysteine
residues that normally build S–S disul-
±de bond bridges. A distinct subset of
EGF-domains containing a calcium bind-
ing domain (cbEGF) has been identi±ed
and its structure studied in detail. A con-
sensus sequence associated with calcium
binding has been identi±ed as D/N-
x
-D/N-
E/Q-
y
-D/N-
y
-Y/F, where
x
symbolizes a
variable amino acid,
y
av
a
r
i
a
b
l
es
e
-
quence of amino acids, and D/N, E/Q, and
Y/F represent the usual one-letter codes
for
aspartic
acid/asparagine,
glutamic
acid/glutamine, or tyrosine/phenylalanine
respectively.
CbEGF domains have been identi±ed in
a variety of proteins such as different coag-
ulation factors, the low-density lipoprotein
receptor (LDLR), the cell-surface signaling
receptor Notch or the extracellular ma-
trix protein ±brillin-1. In most of these
proteins, the cbEGF domains exist as
multiple tandem repeats. In a number
of proteins, mutations within cbEGF do-
mains have been linked to genetic diseases
affecting blood clotting owing to factor
de±ciency, familial hypercholesterolaemia
owing to mutations in the LDLR, inherited
forms of cerebrovascular disorder such as
CADASIL owing to mutations or deletions
in the Notch signaling receptor, or the
