(Received for publication, May 19, 1995; and in revised form, July 20, 1995)
From the
Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptad-less full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.
The macromolecular composition of the matrix of cartilage
results from the expression of a unique repertoire of genes by
chondrocytes. The matrix macromolecules synthesized by the chondrocytes
have multiple domains that permit interactions with other matrix
molecules or with cell surface components. These complex interactions
determine the structure and the integrity of cartilage. The major
components of the cartilage extracellular matrix are collagens,
proteoglycans, and noncollagenous proteins. Cartilage matrix protein
(CMP) ()is one of the most abundant noncollagenous
extracellular proteins in cartilage (1, 2) and has
been shown to associate with the cartilage collagen fibril that
consists of collagen types II, IX, and XI (3) as well as with
proteoglycans(1) .
The deduced amino acid sequence of CMP
reveals that a CMP monomer is made up of a unique combination of
structural domains(4, 5, 6) . Two highly
homologous domains, CMP-A1 and CMP-A2, are separated from each other by
a domain with homology to epidermal growth factor (EGF). The last
domain is the carboxyl-terminal tail, which contains a series of heptad
repeats(7) . Each domain has significant sequence or structural
homology to portions of other proteins. Homology to the CMP-A domains
is found in soluble proteins including von Willebrand factor, the
complement components C2 and B, matrix proteins such as collagen types
VI, VII, XII, and XIV, undulin, transmembrane proteins such as the
-chains of the integrins VLA-1, VLA-2, LFA-1, Mac-1, p150,95, and
a Caenorhabditis elegans protein involved in muscle attachment
as well as the dihydropyridine-sensitive calcium channel and the
inter-
-trypsin inhibitor (reviewed in (8) and (9) ). The A domains of several proteins have been shown to
bind extracellular matrix molecules such as collagen(10, 11, 12, 13, 14, 15) and
the glycosaminoglycans heparin and hyaluronic
acid(16, 17) . EGF-like domains are found in many
classes of molecules, and some of them have been shown to bind calcium
ions(18) . The function of the EGF-like domain in CMP is not
established. The carboxyl-terminal heptad repeat domain of CMP (7) has structural similarity to the coiled-coil domains of
fibrinogen and members of the laminin, tenascin, and thrombospondin
families(18, 19, 20, 21) . The
function of the heptad repeat domains in these molecules has been shown
to be the formation of hydrophobic interactions between three adjacent
and similar coils, in effect forming homo- or heterotrimers (22) .
CMP monomer contains 12 cysteine residues. Each of the two CMP-A domains has two cysteines, one at the amino-terminal end and one at the carboxyl-terminal end of each domain. The EGF-like domain has six cysteine residues. The final two cysteines are at the beginning of the carboxyl-terminal heptad repeats(4, 5, 6) . In this communication we test the hypotheses that CMP-A domains become stabilized by intradomain disulfide bonds, that the cysteine residues in the carboxyl-terminal tail are involved in homotrimer formation(5, 23) , and that the heptad repeats in the carboxyl-terminal tail are involved in the initiation of trimer formation(7) . These hypotheses were tested by the creation of a variety of CMP mutants and the expression of these mutant CMP molecules in COS-7 cells that do not normally express CMP.
Figure 1: CMP constructs. Full-length CMP consists of a signal peptide (open boxes), two CMP-A domains (cross-hatched boxes) separated by an EGF-like domain (stippled boxes), and a COOH-terminal tail containing four heptad repeats (solid line). The 12 cysteines in mature CMP are numbered starting from the amino terminus. The disulfide bridges are drawn based on the assumption that each domain folds independently. Mini-CMP is lacking the CMP-A1 and the EGF-like domains and contains only four cysteines. The heptad-less CMP has the tail domain deleted immediately after the two cysteine residues and therefore lacks the heptad repeats.
Figure 2:
Construct production and cysteine
mutations. The relative locations of the primers used to produce the
various CMP constructs are shown underneath the schematic model of CMP
in A. Cysteines were mutated to the amino acids indicated by single letters. The introduced stop codons are shown as stars. B shows the sequence of all primers used in 5`
to 3` orientation. The primers are numbered as in A. Lower
case letters indicate mismatches with the template sequence. The
mutated codons are listed in bold, and underlining indicates the stop codons introduced for the heptad-less
constructs. Nonadjacent areas of overlap with template, used to connect
the signal sequence to the CMP-A2 domain in the recombinant PCR, are
separated by a tilde (). The primer pairs, templates, and
introduced mutations for the single or recombinant PCRs are listed in C. The delta symbol (
) indicates deletion of the
corresponding domains. N/A, not
applicable.
Figure 3:
Mini-CMP behaves as full-length CMP.
Conditioned medium of COS cells transfected with mini-CMP (lanes 1 and 4), full-length CMP (lanes 2 and 5), or co-transfected with the cDNAs of both constructs (lanes 3 and 6) was separated on a 8% gel, blotted to
a polyvinylidene difluoride membrane, and incubated with antiserum
D2/1476 against the CMP-A2 domain. Bound antibodies were detected with
a peroxidase-coupled secondary antibody and a chemiluminescence
detection kit. Lanes 1-3 were run under reducing
conditions (+), and lanes 4-6 were run under
nonreducing conditions(-). Molecular weights of the protein
standards are shown on the left 10
.
Schematic drawings of the obtained mixed multimers between mini- and
full-length CMP are shown in B. The total number of CMP-A
domains/multimer is indicated in parentheses and corresponds
to the numbers shown on the right side of A.
Figure 4:
C11 and C12 are necessary and sufficient
for interchain disulfide bond formation in mini-CMP. Conditioned medium
of cells expressing mini-CMP (lanes 1 and 3),
mini-CMP with mutated cysteines C11 and C12 (lanes 2 and 4), and mini-CMP with mutated cysteines C9 and C10 (lanes
5 and 6) were run under nonreducing (-) and
reducing (+) conditions on a 11% gel and analyzed by Western
blotting as described in the legend of Fig. 3. Molecular weights
of the protein standards are shown on the left
10
. Above each lane in the blot is a figure
representing the mini-CMP construct used. The open box represents the CMP-A2 domain, and the solid line represents the COOH-terminal tail domain. The cysteines (C) and the mutated cysteines (X) are shown.
The(-) and (+) symbols indicate the absence or presence of
reducing agent during electrophoresis.
Figure 5:
Cysteines C11 and C12 are also necessary
for interchain disulfide bond formation in full-length CMP. Conditioned
medium of cells expressing full-length CMP (lanes 1 and 2) and full-length CMP with mutated cysteines C11 and C12 (lanes 3 and 4) were run under nonreducing (lanes
1 and 3) and reducing (lanes 2 and 4)
conditions on a 11% gel and analyzed by Western blotting as described
in the legend of Fig. 3. Molecular weights of the protein
standards are shown on the left 10
. Above each lane in the blot is a figure representing the CMP
construct used. The open boxes demonstrate the CMP-A1 and -A2
domains, the black box represents the EGF-like domain, and the solid line indicates the COOH-terminal tail. The cysteines (C) and the mutated cysteines (X) are shown.
The(-) and (+) symbols indicate the absence or presence of
reducing agents during electrophoresis.
Figure 6:
The heptad repeats are needed for the
initiation of disulfide-bonded trimerization. Conditioned medium of
cells expressing heptad-less CMP (lanes 1 and 3) and
heptad-less CMP with mutated cysteines C11 and C12 (lanes 2 and 4) were run under nonreducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions
on a 11% gel and analyzed by Western blotting as described in the
legend of Fig. 3. Molecular weights of the protein standards are
shown on the left 10
. Above each lane in the blot is a figure representing the CMP construct
used. The open boxes demonstrate the CMP-A1 and -A2 domains,
the black box indicates the EGF-like domain, and the short
solid line represents the truncated COOH-terminal tail. The
cysteines (C) and the mutated cysteines (X) are
shown. The (-) and (+) symbols indicate the absence or
presence of reducing agents during
electrophoresis.
An important aspect of the macromolecular organization of the extracellular matrix is that some of its components spontaneously assemble into homotypic or heterotypic oligomers. The formation of triple helices of collagens and the assembly of these trimers in supramolecular structures are examples of such assemblies, as are the formation of laminins, thrombospondin, and tenascin(18, 19, 20, 21, 22) .
The organization of CMP into a functional trimer requires that a
number of co- or post-translational modifications occur both within and
between monomers. We have postulated that intramonomeric changes
include the formation of disulfide bridges within the A domains and the
EGF-like domain(5, 23) . The intermonomeric changes
include the initiation of the trimer formation through the
establishment of coiled-coil -helices followed by the
stabilization of the trimer through disulfide bonds involving the two
cysteines in the heptad repeat-containing tail. Although reducing
agents are necessary to obtain CMP monomers in denaturing gels, the
interchain disulfide bonds are not necessary for the maintenance of the
trimeric structure under otherwise nondenaturing
conditions(7) .
To test this model, we engineered a mini-CMP in which the CMP-A1 and the EGF domains were deleted, thus reducing the number of cysteine residues in the mini-CMP from 12 to 4. Two of the cysteines are in the CMP-A2 domain and two are in the heptad repeat-containing tail domain. When the length of the tail domain of the mini-CMP was intact, we showed clearly that C11 and C12 are both necessary and sufficient for interchain disulfide bond formation that results in a stable trimer. If, however, the heptad repeats of the tail domain are deleted, trimers will not form even if C11 and C12 are present. Therefore, the heptad repeats act as nucleation sites in the formation of the CMP trimer, and the trimers are subsequently stabilized through disulfide bonds involving C11 and C12. The establishment of trimers through the interaction of heptad repeats have been proposed for the formation of heterotrimers of laminin and homotrimers of tenascin, fibrinogen, and thrombospondin. As in CMP, closely spaced cysteine residues are at the amino and/or carboxyl end of the heptad repeats of these proteins (19, 20, 21, 22) . We also present evidence that intradomain disulfide bonds form between residues C9 and C10 within CMP-A2, suggesting that a similar bridge may exist between C1 and C2 in the CMP-A1 domain. Based on the presence of two intra-A domain disulfide bridges, along with the three disulfide bridges in the EGF-like domain, a very compact and globular CMP structure can be predicted. This is consistent with electron microscopic observations that native CMP trimer isolated from cartilage consists of three compact ellipsoids connected at one end(3, 7) .
Not
all A domains of other proteins have cysteine residues at their amino
and carboxyl ends as seen in CMP. Some A domains have no cysteines and
others have one, two, three, or five cysteines(8) . When there
are two cysteine residues in an A domain, they are most often situated
at the amino and carboxyl ends of the domain. In those A domains, it is
possible that the two cysteine residues form intradomain disulfide
bonds as we have shown to exist in the CMP-A2 domain. However, the
crystal structure of the A domain of an -chain of the CR3 integrin
suggests that the ends of an A domain may be juxtaposed even in the
absence of an intradomain disulfide bridge(9) .