(Received for publication, October 4, 1994; and in revised form, November 9, 1994)
From the
Triple-stranded laminin molecules assemble via an -helical
coiled-coil structure spanning approximately 600 amino acid residues of
each chain. We reported that the C termini of the
1 and
1
chains direct the specific dimer and trimer assembly (Utani, A.,
Nomizu, M., Timpl, R., Roller, P. P., and Yamada, Y.(1994) J. Biol. Chem. 269, 19167-19175). In this
study, we focused on the mechanism of trimer formation of the
2
chain utilizing three different approaches. First, competition assays
using mutated recombinant
2 chain defined a 25-amino acid sequence
at the C terminus of the long arm as an essential site for assembly
with
1 and
1 chain. Site-specific mutations and synthetic
peptides of this site revealed that both positively charged amino acid
residues and the
-helical structure within this site were
critical. Second, overexpression studies of recombinant
2 chain
long arm confirmed that the C-terminal end was critical for the trimer
assembly within NIH 3T3 cells. Third, circular dichroism spectroscopic
examination of the complexes reconstituted in vitro revealed
dynamic conformational changes of the
2 and
1 chains in the
process of assembly. These studies also revealed that the proper
folding of the extreme C terminus of
2 chain was critical for the
stability of trimer. From these data, it is concluded that the C
terminus of
2 chain long arm is required for the effective
initiation of laminin heterotrimer assembly.
Laminin comprises a family of glycoproteins specific to basement
membrane. Laminin plays an important role in both development and
maintenance of tissues. In culture, laminin has been shown to have a
number of biological activities such as promoting cell adhesion,
migration, growth, and neurite outgrowth (Martin et al., 1988,
Beck et al., 1990). The prototypical laminin-1 molecule from
mouse Engelbreth-Holm-Swarm (EHS) ()tumor consists of three
different chains:
1,
1, and
1 chains. Two other laminin
chains have been identified:
2 chain (Am chain) in muscle, nerve,
and placenta (Ehrig et al., 1990) and
2 chain (B1s chain)
originally in neuromuscular junctions (Hunter et al., 1989).
The
2 and
2 chains are also able to form a heterotrimer
(Engvall et al., 1990; Sanes et al., 1990). More
recently, the epithelial cell-specific laminin, kalinin/nicein
(laminin-5,
3/
3/
2) and k-laminin (laminin-6,
3/
1/
1), has been described (Rousselle et al.,
1991; Kallunki et al., 1992; Marinkovich et al.,
1992).
Electron microscopy showed that laminin has a cross-like
shape with one long arm and three short arms (Timpl, 1989; Engel,
1993). The long arm of laminin chain consists of about 600 amino acids
of -helical structure that participates in a heterotrimer
formation. The
-helical region contains numerous heptad repeats in
which hydrophobic and charged amino acids are often located at regular
intervals (Paulsson et al., 1985; Beck et al., 1990,
1993). Hunter et al.(1990, 1992) reported that the proteolytic
fragment (E8) of EHS laminin containing the C-terminal one-third of the
long arm could be reconstituted into a heterotrimer after denaturation.
Recently, we demonstrated that the specific region and the charged
amino acids of the C termini of the
1 and
1 chains were
essential for the specific dimer and trimer assembly (Utani et
al., 1994).
In this report, we expressed recombinant laminin
2 chain fragments in bacterial cells to determine the active
region for trimer assembly. In addition, we expressed recombinant
2 chain fragments in eukaryotic cells that express endogenous
1 and
1 chains and monitored trimer assembly. Additional
analysis using competitive affinity chromatography assays, in vitro reconstitution assays, and thermal stability studies were employed
to analyze the active site for trimer formation. Circular dichroism
spectroscopy revealed that the drastic changes of
-helical
structure of
2 and
1 monomers after assembly into dimeric and
trimeric structure. Furthermore, the activities of the
1 and
2 chains for trimer formation were compared. The data in this
report support the model that only a limited region is necessary and
sufficient to initiate the interaction with corresponding regions of
the
1 and
1 chains, resulting in the entire assembly of the
laminin long arm portion.
Figure 1:
Trimer
formation of recombinant 2 chain with
1 and
1 chains in
the affinity bead assays. Left, the schematic picture of
laminin-2 and the bacterially expressed recombinant proteins of
truncated
2 chain long arm. The long arm of
2 chain spans 575
amino acid residues between the last cysteine residue of the short arm
region and the first cysteine residue of the globular region. The
recombinant proteins contain the following region of the
2 chain:
2-400, residues 1,575-1,974;
2-221,
residues 1,929-2,149;
2-101, residues
1,929-2,029;
2-120, residues 2,030-2,149. Right, the recombinant proteins immobilized on CNBr-activated
Sepharose beads were incubated with
[
S]methionine-labeled RD cell lysates. The bound
proteins were analyzed by 4% SDS-PAGE under reducing conditions
followed by fluorography. Lane 1, immunoprecipitates of RD
cell lysates with anti-EHS laminin antibody showing
1 and
1
chains; lane 2, bound materials to the
2-221
affinity beads; lane 3, the
2-400 affinity beads; lane 4, the
2-101 affinity beads; lane 5,
the
2-120 affinity beads. Molecular markers are shown on the left in kDa.
Figure 2:
Inhibitory activity of various mutants and
synthetic peptides of 2 chain on trimer formation with
1 and
1 chains. A, a list of the deletion and substitution
mutants, and synthetic peptides of
2 chain long arm and the values
of IC
. The C-terminal amino acid sequence is shown at the top. IC
(nanomolar) indicates the concentration
of the competitor that inhibited 50% of the binding of the
1 and
1 chains to the
2-221-coupled beads. n.d., not
determined. The values of IC
were > 6,000 nM for
2-S3 and >3,300 nM for
2-p48 and
2-p40. B, inhibitory activities of deletion mutants as
soluble competitors. x axis shows the concentration of the
competitors. y axis indicates the percent (%) binding of
1 and
1 chains to the
2-221-coupled affinity
beads. The amounts of those bound to affinity beads in the absence of
competitors were taken as 100%. Each point shows the mean value
obtained by densitometric scanning of the x-ray films (as seen in Fig. 7A) of the two or three independent experiments.
The competitors are listed at right.
Figure 7:
Comparison of the activity for trimer
formation of 2 and
1 chain. A, binding of
1 and
1 chains to the
2-221-coupled affinity beads were
competed with the increasing amounts of the
2-221 (lanes
1-5) or those of the
1-211 (lanes6-10). The concentrations of the competitors
are:(-) in lanes 1 and 6, 25 nM in lanes2 and 7, 50 nM in lanes3 and 8, 250 nM in lanes4 and 9, and 1.25 µM in lanes5 and 10. The materials bound to the
2-221-coupled affinity beads were analyzed by SDS-PAGE and
subsequent fluorography. B, amino acid sequence homology among
the
2,
1, and
3 chains. The numbers of amino acid
residues of mouse
2,
1, and human
3 chain are from
Bernier et al.(1995), Sasaki et al.(1989), and Ryan et al.(1994), respectively. Dottedboxes show identical sequences, and d indicates the position of
the heptad repeats of the hydrophobic residues within the C termini. Arrow depicts the 25-amino acid region, and asterisks indicate 3 amino acids critical for trimer formation of
2
chain defined by competition assay.
To verify this observation, a proline substitution within
this 12-amino acid region was introduced at the hydrophobic residue to
disrupt the -helical structure.
2-S1 did not significantly
reduce the activity in this competition assay, since the IC
value was 2.4 nM (Fig. 2A). A different
proline substitution,
2-S2, reduced the activity by approximately
3-fold with an IC
value of 6.2 nM. These data are
consistent with those presented above, in which truncated mutants from
this region showed little loss of trimer assembly activity. Finally, a
proline substitution at isoleucine 2,126 (
2-S3) was completely
inactive as a competitor (Fig. 2A). These data strongly
suggest that the conformation surrounding isoleucine 2,126 is critical
for trimer assembly, consistent with the precipitous drop in activity
seen with
2-
3. Based on the results from the
2-
4
and
2-S1, it was concluded that the C-terminal 25-amino acid
sequence (residues 2,119-2,143) was the essential site for trimer
assembly.
We prepared the synthetic peptides consisting of C
terminus of the 2-120 to further delineate the active region (Fig. 2A). The activity for trimer assembly of the
synthetic peptides in the competition assay was surprisingly weak
compared with that of
2-120 (Fig. 2A). For
example, peptide
2-p55, which is the strongest competitor of the
synthetic peptides, was approximately 1,700-fold weaker than
2-120. The shorter peptide
2-p40 was completely
inactive, despite containing the critical 25-amino acid region
determined by the competition assays using recombinant proteins. As
discussed later, the low contents of
-helical structure of the
short synthetic peptides may explain the poor activity as competitor.
Thus, synthetic peptides were not helpful in identifying the active
site by the competition assay.
Figure 3:
Inhibitory activities of substitution
mutants of the two positively charged amino acids. A list of the
alanine substitution mutants of the positively charged amino acids
derived from the 2-
1. d indicates the hydrophobic
residue in the heptad repeat (abcdefg)
.
The values of IC
(nanomolar) are shown at right.
Figure 4:
Trimer formation with transfected 2
chains in NIH 3T3 cells. A, the entire and mutated long arm of
the
2 chains were constructed using the cytomegalovirus promoter
driven mammalian expression vector. Each construct has an extracellular
domain of the 55-kDa IL2R at N terminus as a tag (shown as a dottedcircle). Asterisks show the substitution sites
within
2-V and
2-VI. B, 24 h after transfection with
LipofectAMINE, cells in a six-well dish were labeled with
[
S]methionine. Expressed proteins were
immunoprecipitated from cell lysates with monoclonal anti-IL2R antibody
and protein G beads and analyzed by 5% SDS-PAGE under reducing
conditions. Immunoprecipitates from the cell lysate transfected with
2-I (lanes 1 and 7), untransfected cells (lane 2),
2-II (lane 3),
2-IV (lane
4),
2-V (lane 5),
2-VI (lane 6), and
2-III (lane 8). Upper panel, long exposure of
the fluorography to show
1 and
1 chains. Lower
panel, short exposure of the same x-ray film of upper panel. Middle panel, Western blotting probed with anti-
2
antibodies. Arrow indicates doublets of
1 and
1
chains. Asterisks show nonspecifically precipitated
fibronectin. Molecular markers are shown at
left.
To
verify the active site identified by the competition assays, three more
mutant cDNAs were prepared. 2-IV included nearly the whole
-helical region but with the same C-terminal deletion as the
2-
1.
2-V and
2-VI had the entire
-helical
region but with the same proline substitution as the
2-S3 and
2-S2, respectively (Fig. 4A). Among these three
proteins, only
2-VI showed a weak activity for trimer assembly
with the
1 and
1 chains (Fig. 4B, lane6, upperpanel). These results are
consistent with the observations that the
2-S2 and
2-
1
reduced the activity in the competition assays by approximately 3- and
8-fold, respectively (Fig. 2A). Thus, the C terminus of
the
2 chain long arm, including the residues surrounding
isoleucine 2,126, is critical for trimer assembly in the transfected
cells.
Figure 5:
In
vitro reconstitution assays of trimer assembly. A set of recombinant
2 chains or synthetic peptides were mixed with equimolar amounts
of
1-217 and
1-217. The products were analyzed by
4% non-denaturing electrophoresis and stained with Coomassie Brilliant
Blue. The
2 components of the mixture are listed on the top of each lane. The monomer of the
2 chain components could not
enter the gel except
2-101 (lane 3) (Utani et
al., 1994). Upperarrow indicates the trimer
complexes, and lowerarrow indicates
1-217/
1-217
heterodimer.
The thermal stability of the trimeric complexes was evaluated
by monitoring the []
by CD spectroscopy
during the heat change from 25 °C to 80 °C (Table 1). The
three different kinds of trimers with the
2-221,
2-120, and even with the
2-p55 showed high T
values of 63-64 °C. This is in good
agreement with the T
value obtained with the
proteolytic fragment E8 of laminin-1 (Paulsson et al., 1985;
Hunter et al., 1992), suggesting these trimer formed a
coiled-coil trimeric structure. These results indicated that the
C-terminal 55 amino acid residues of the
2 chain long arm was able
to form a stable trimer with
1-217/
1-217. On the
contrary, the T
values of the reconstituted
trimers with
2-
1,
2-S1, and
2-S2 decreased more
than 10 °C. These significantly lower T
values
suggest that the 12-amino acid sequence deleted in
2-
1
greatly contributes to the formation of the stable trimer utilizing its
-helical structure. As for the three alanine substitutions of the
charged amino acid residues of
2-
1, these mutants showed T
values only 3 °C and 4 °C lower than
that of the trimer with
2-
1 (51 °C). The slightly reduced T
value may indicate that these 2 positively
charged residues contribute to some degree of stability. Furthermore,
these data indicate that the drastic changes of the activities induced
by the truncation or substitution mutants in the competition assay did
not directly correlate with the thermal stability once the trimers were
formed.
Figure 6:
Circular dichroism analysis of
-helical structure. CD spectra of monomer and reconstituted
complexes were monitored in PBS at protein concentrations of 1.5
µM. A, spectra of monomer,
1-217,
1-217, and
2-221. B, spectra of dimer
(
1-217/
1-217) and mean of monomer
s(
1-217 +
1-217). C, spectra of
trimer (
1-217/
1-217/
2-221), mean of
the dimer and monomer (
1-217/
1-217 +
2-221), and mean of the monomers (
1-217 +
1-217 +
2-221).
Laminin chains have a large -helical region of about 600
amino acids in their long arm where the three chains interact and form
a triple-stranded coiled-coil structure (Paulsson et al.,
1985). One of the major questions that we wished to address in the
present studies was what region of the
2 chain long arm was
required for laminin assembly. Furthermore, we discussed mechanisms for
the initiation of laminin heterotrimer formation.
Here, we employed
three different approaches to evaluate the essential region of the
2 chain for trimer assembly: competition assays, transfection
assays, and in vitro reconstitution assays. Each of these
assays has unique properties and different sensitivity. The competition
assay for trimer assembly proved most useful to map precisely the
active region of the
2 chain. Deletion and substitution mutants
allowed us to define a sequence within the C terminus of the
2
chain long arm that is essential for trimer assembly. The data
indicating progressive loss of activity as more amino acid residues
were deleted from the C terminus suggest that all of these amino acids,
but especially a 25-amino acid sequence (residues 2,119-2,143),
act in unison to promote trimer assembly. The transfection assay showed
that the
2-IV construct with a 12-amino acid deletion at the C
terminus had little assembly activity. However, the
2-
1
construct with the same deletion at the C terminus retained some
activity in the competition assays. Therefore, it seems impracticable
to determine the precise activity of the mutated
2 chain with less
activity for the assembly in the transfection assays. However, the
observation that the 12-amino acid deletion and the disruption of the
-helical structure within the C terminus abrogated the activity
for trimer formation reinforces the significance of the C-terminal
25-amino acid sequence as an essential site even under physiological
conditions.
In the in vitro reconstitution assay, the
absence of activity using 2-
3 (a 22-amino acid deletion from
C terminus) and
2-S3 (a proline substitution at isoleucine 2,126)
demonstrated again the importance of the C-terminal region of the
2 chain long arm. However, differences in assembly activity
observed in the competition assays could not be detected. For example,
2-
1S3 with a very low activity in the competition assays
showed activity for trimer formation in the in vitro reconstitution assays. These results may be due to the high
concentrations of the substrates used in the in vitro reconstitution assays, which make it difficult to distinguish the
relevant activity of the mutant
2 chains and synthetic peptides
with less activity. Nonetheless, the reconstituted trimer complexes
gave us substantial information about the conformation and thermal
stability requirements of laminin chain assembly as discussed below.
Charged amino acids at ``e'' and ``g'' positions
of the heptad repeat have been shown to play an important role in
heterodimer of the b-Zip family of transcription factors (Schuermann et al., 1991; O'Shea et al., 1992). By
calculating the electrostatic interactions between the charged amino
acids of each laminin chain, Beck et al. (1990, 1993) have
suggested that ionic interactions may play a crucial role in
chain-specific laminin assembly. Alanine substitutions for the
positively charged amino acids within the critical site of the 2
chain markedly reduced its trimer formation activity. These results,
together with our earlier observations that alanine substitution of the
glutamate residue at the C terminus of the
1 chain abolishes
trimer formation (Utani et al., 1994), provide evidence that
electrostatic interactions play a critical role in triple-stranded
coiled-coil formation of laminin.
It is noteworthy that 2-p40,
a short peptide, was inactive in the competition assays and in the in vitro reconstitution assays, despite that it encompasses
the essential 25-amino acid sequence for trimer assembly. Furthermore,
2-p55 is a poor competitor (IC
= 3,300
nM) when compared with
2-120 (IC
= 2.0 nM) in the competition assays. CD analysis
revealed that the synthetic peptides with length shorter than 40 amino
acids from the C terminus of the
2 chain long arm did not retain
-helical structure but had a random coil structure (data not
shown). The observation that the C terminus of
2 chain long arm
requires long stretches of amino acid residues for the
-helical
structure could explain the low activity of the short peptides in the
competition assays.
The thermal stability studies provided
additional insights into the mechanism of trimer assembly. Although
2-p55 was a poor competitor in the competition assay,
2-p55
was able to form a trimer as well as
2-120 or
2-221. It is likely that once the initial interaction occurs
with
1 and
1 heterodimer,
2-p55 undergoes a
conformational change on assembly into the stable trimer structure,
which is indistinguishable from that of the trimer seen with
2-120 or
2-221. The drastic increase of the
-helix content of
2-221 after trimer formation may
support this hypothesis. Based on the data from CD analysis, both
1-211 and the C-terminal 223-amino acid residues of
2
chain (Utani et al., 1994) showed a low
-helix content of
27% and 29%, respectively. Since the
1 and
2 chains also have
a low
-helix content as monomers, the drastic increases of an
-helix content of the
and
chains after assembly may be
a common feature to laminin dimer and trimer formation. The stability
of the trimer with the
2-S1 (a proline substitution at valine
2,144) significantly decreased despite that it retained almost complete
competition activity. These observations may be also explained by the
conformational change of
2 chain after trimer formation. The
-helical structure at the extreme C terminus does not affect the
initiation of trimer formation, which in turn becomes essential for
maintaining the stability with an increased
-helix structure after
trimer formation. A similar mechanism may also explain the equivalent T
values of the trimers with three different
alanine substitution mutants. Thus, electrostatic interactions are an
essential part of the assembly process but lose their significance
after drastic conformational change as a result of trimer formation.
A recently identified 3 chain also forms a trimer with
1
and
1 chain as a laminin-6 molecule (Marinkovich et al.,
1992). Alignment of the C-terminal sequence of human
3 chain long
arm (Ryan et al., 1994) reveals striking sequence identity
within the essential 25-amino acid site for assembly (Fig. 7B). Furthermore, three essential residues for
the activity are conserved, i.e. isoleucine at 2,126, lysine
at 2,127, and arginine at 2,134. These findings suggest that
3
chain may also use this region for trimer formation. Recently, Harbury et al.(1993, 1994) reported that synthetic peptides with
isoleucine residues at ``a'' and ``d'' positions of
leucine zipper motif of b-Zip protein could form a coiled-coil
homotrimer. When leucine substitutions were introduced at all
isoleucine residues of
2-p55, this mutant reduced its trimer
formation activity (Nomizu et al., 1994). Therefore, the two
conserved isoleucine residues at ``a'' and ``d''
positions (2,126 and 2,130) within the essential site may contribute to
form a trimer with the isoleucine residues concentrated in the C
terminus of
1 chain. In this context analysis of the active site
of
1 and
3 chains will deepen our understanding of the
mechanisms by which laminin form a triple-stranded coiled-coil
structure.